EVALUATION OF THE HYDROPHOBIC AND OLIGOSACCHARIDE BINDING

FUNCTIONS OF THE GM2 ACTIVATOR PROTEIN BY FLUORESCENCE

DEQUENCHING ASSAY

Natasha Smiljanic-Georgijev

Thesis subrnitted in conformity with the requirements

for the degree of Master of Science

Department of Clinical Biochemistry

in University of Toronto

@ Copyright by Natasha Smiljanic-Georgijev 1997

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TABLE OF CONTENTS

........................................................................................ AB STRACT v ... .......................................................................... ACKNOWLEDGMENT vri ... ................................................................................ AB B REVIATIONS viu

.................................................................................. PUB LICATIONS ix

CHAPTER 1 GENERAL INTRODUCTION

........................................................................ 1.1. GM2 Gangliosidosis 2 ............................................ 1.2. Structure of the HEXA and HEXB Genes. - 2

1.3. Hexosarninidase Isozymes ................................................................ - 5 1.4. The Interaction Between the Activator . GM2 Ganglioside and ........................ 6

............................................................................... Hexosaminidase A - 6

1 .5 . Genomic Structure of the GM2A Gene Encoding the GM2 Activator Protein ...... - 9

1.7. AB Variant Form of GM2 Gangliosidosis ................................................ 14

1.8. Other Lysosomal Sphingolipid Activator Proteins ........... .... ................... 17

1.9. Other Possible Functions of the GM2 Activator Protein ................................ 18

................................................................................ 1.10. Gangliosides 19

1.10.1. Ganglioside Biosynthesis ............................................. 22

............................................... 1.10.2. Ganglioside Catabolism 25

.......................................................................................... References 26

CHAPTER n DEVELOPMENT OF THE FLUORESCENCE DEQUENCHING ASSAY FOR STRUCTURE / FUNCTION STUDIES OF THE G M ~ ACTIVATOR PROTEIN

2.1 Introduction ................................................................................... 34

2.2 Materials and Methods ...................................................................... - 38 ................................................................. 2.2.1. Materials 3 8

2.2.2. Production of the His6-GM2 Activator Fusion Protein from E-Coli

.................................................................................. 3 8

2.2.3. Preparation of Phosphatidylcholine Containing Large Unilamellar

.......................................................................... Vesicles -42

2.2.4. Preparation of R- 18 labeled liposomes .............................. -42

.................................... 2.2.5. Fluorescence Dequenching assay -42

....................................................................................... 2.3. Results -43

2.3.1. Fluorescence dequenching of R- 18 labeled liposomes in the presence of

recombinant human GM2 activator protein ............. .. ................ 43

2.3.2. Detemination of the optimal time course range for the fluorescence ............................................................. dequenching assay -50

2.3.3. Detemination of the optimal GM2 activator protein amount for the

fluorescence dequenching assay ................ .. .................... 50

2.3.4. Determination of the optimal pH ..................................... -59

................................................................................... 2.4. Discussion -59 .......................................................................................... References 61

CHAPTER nI EVALUATION OF THE HYDROPHOBIC AND OLIGOSACCHARIDE BINDING FUNCTIONS AND IDENTIFICATION OF A SECRETORY FORM OF THE G M ~ ACTNATOR PROTEIN

................................................................................. 3.1. Introduction -64 ..................................................................... 3.2. Materials and Methods -67

3.2.1. Materials .......................... .,.. ................................... 67

3.2.2.Expression and Purification of His6-GM2 Activator Protein Constmcts

from E . Coli ...................................................................... 68

3.2.3.The Expression and Purification of the His6-Activator Containine a ....................................................... Cys 138Arg Substitution -68

..................................... 3.2 4 . Fluorescence Dequenching Assay 69

3.2.5 Analysis of the Distribution of Activator Molecules with a High Mannose

................................. or a Complex Type Oligosaccharide Moiety 69

3.2.5.1. Isolation of a Semi-Purified Sarnple of Newly Synthesized Activator from

Normal Human Fibroblasts ............................................................. 69

........................................................ 3.2.5.2. Glycosidase Digestion 70

........................................................ 3.2.5.3. Western Blot Analysis 71

3.3. Results ........................................................................................ 71

3.3.1. Determination of the Recombinant Hurnan GM2 Activator Protein .............. Binding Affinity for Various Glycolipids and Gangliosides -71

3.3.2. Analysis of the Hydrophobic Function of Different Recombinant

Human GM2 Activator Protein Constmcts .............. .... ............ 81

3.3.3. Hydrophobic Function of the Activator Protein Containhg a Point Mutation

Linked to the AB Variant Form of G M ~ Gangliosidosis by Ruorescence Dequenching

.............................................................................. Assay 86

3.4. Discussion.. ................................................................................. -86 References ......................................................................................... -92

CHAPTER N FUTURE WORK

4.1. X-ray Crystallographic Studies of the GM2 Activator Rotein ......................... 96 4.2. Determination of the R-18 / Ganglioside Binding Site of the GM2 Activator Protein by

Mu tagenesis ....................................................................................... -96

4.3. Identification of the Hexosaminidase A Binding Site of the GM2 Activator Protein Through the Expression of the Mouse / Human Fusion Protein ........................... -97

R e f e r e n c e s .......................................................................................... 98

ABSTRACT

G M ~ activat~r protein is a substrate specific cofactor for degradation of GMZ ganglioside by

phexosaminidase A. It solubilizes individual molecules of the ganglioside by interacting with both

its hydrophilic oligosaccharide and hydrophobic ceramide moieties. However, the specificity of

binding is pnmarily determined by the oligosaccharide moiety of the glycolipid. Thus, the activator

protein contains at least three functional elements; a hydrophobic binding pocket, an oligosaccharide

binding site, and an area that interacts with hexosarninidase A. Mutations in the gene encoding the

activator result in the AB-variant form of G M ~ gangliosidosis.

The goal of this thesis is to evaluate the hydrophobic and oligosaccharide binding functions of

the G M ~ activator protein. Recentiy, the investigation of endosomeAysosome fusion hy fluorescence

dequenching assay, suggested that G M ~ activator protein c m act as a transfer protein of the

fluorescent lipid probe. octadecylrhodamine, between egg phosphatidylcholine (PC) liposomes. as

weil as isolated endosornes and lysosomes. Based on these findings rny first objective was to develop

a fluorescence dequenching assay that could be used to evaluate the hydrophobic binding function of

the activator protehi. This assay was based on the concentration dependent fluorescence dequenching

of the fluorescent Lipid probe in presence of the G M ~ activator protein. The optimal time course was

determined to be from 5th to 10th minute after the initiation of the assay (first five minutes were

required for fluorescence stabilization). The optimal amount of the activator protein and pH used in

this assay, were found to be from 0.75 pg to 4 pg and pH 5. respectively.

The second objective of this thesis was to determine the activator protein binding affinity for

various glycolipids and gangliosides by the fluorescence dequenching method. 1 determined the

degree to which various glycolipids could inhibit the transport of the octadecylrhodamine. between

liposomes. by the activator protein. The levei of inhibition produced by each glycolipid was used to

charactenze the oligosaccharide-binding specificity of the activator: G M ~ » G ~ l b » G ~ l 2 -2

G M ~ > GA^-

The fluorescence dequenching assay was also used to evaluate the hydrophobic function of the

C-terminal part of the G M ~ activator protein. 1 investigated the functionality of three truncated

activator protein consmcts, lacking different number of C-terminal amino acids (36, 11 and 9 amino

acids, respectively). AU three constructs were inactive in releasing fluorescence, compared to the wild

type activator, indicating that they lacked a functional hydrophobic binding site. Thus, it is possible

that the C-terminal part of the activator protein could be involved in the ganglioside binding function.

Finally. the hydrophobic hnction of the activator protein containing a point mutation Linked to

the AB variant f o m of G M ~ gangliosidoses (T412c (Cp 138Arg )) was analyzed by the fluorescence

dequenching assay. This protein caused an increase of fluorescence which was 96% of that caused

by the wild type. This function was suongIy inhibited by Gh.12 ganglioside, suggesting that the

mutated activator had a fully functional ganglioside binding site. These data suggest that ~~s~~~ may

not be involved in the ganglioside binding function of the activator protein. but only in the interaction

with Hex A, possibly through the formation of disulfide bond with another cysteine.

ACKNOWLEDGMENT

The work presented in this thesis was done in the Division of Neurosciences at the Hospital

for Sick Children, Toronto, under the supervision of Dr. Don Mahuran. 1 would like to extend rny

sincere gratitude to Dr. Mahuran for allowing me the opporninity to improve my scientific knowledge

under his continued guidance and support. 1 also wish to acknowledge Dr. Joan Boggs and Dr. John

Callahan for their advice and help over past two years, as well as Dr. Fred Keeley for his participation

in evaluating my work.

1 am very indebted to people in the laboratory for their friendship and assistance. 1 would like

to thank Mr. Roderic Tse for helpful discussions and friendly collaboration. 1 very much appreciate

advises and assistance of Mrs. Euijung JO. Mr. Jude Pereira a ~ d Dr. Brigitte Rigat. Special thanks to

Ms. Amy Leung and Mrs. Godha Rangaraj for their great technical assistance. 1 would also like to

acknowledge Dr. Sunqu Zhang, Dr. Benny Chen, Mrs. Irene Warren. Mrs. Marie-Anne

Skomorowski. Mrs. Kathleen Sparacino. Mrs. Caroline Bruce and al1 members of Dr. Cecil Pace-

Asciak's and Dr. FIavio Coceani's laboratories for making the Neurosciences an enjoyable place to

work.

Most of al1 1 rhank my husband, Zvonimir Georgijev, for his love and support during the

time. as well as our two wonderful sons. Nikola and Peter. for making my Iife joyous and

worthwhile. 1 specidly acknowledge families Smiljanic and Georgijev whose help and continuous

encouragemznt made the beginning of my Life and work in Canada more successful and easier.

LIST OF ABBREVIATIONS

Act 183 Act 185 cDNA Cer CHO CMF' EDTA ER FCS Ga1 GaiNAc Glc GlcNAc GM2A GM2A P GSL HEXA W E X B Hex rPTG kDa Man MEM mRNA M-6-P MPR 4-MUGS NeuNAc Ni-NTA a3 PC-LUV PCR R-18 S A P SDS-PAGE SFM UDP

muicated activator protein ma& by point mutation at codon 183 mmcated activator protein made by point mutatior; at codon 185 complementary DNA ceramide Chinese hamster ovary cytidine-5'-diphosphate ethylenediaminetetraacetate Endoplasmic reticulum fe tai calf senun Gala tose N-acetylgalac tosamine Glucose N-acetylglucosamine gene encoding G M ~ activator protein pseudogene related to GMU g~ycosphingolipids gene encoding a-subunit of Hex A

gene encoding ksubunit of Hex A

isopropyl- l -thio-PD-galactoside kdodaltons mannose minimum essential media messenger RNA mannose-6-phosphate mannose-&phosphate receptor Qmethylumbelliferyl P-N-acetylglucosarnine 6-sulfate N-ace ty lneuraminic acid (sialic acid) nickel-nitrilo tri acetic acid optic density phosphatidilcolin conraining large unilarnellar vesicle polymerase chah reaction octadecilrhodarnine B chloride sphingolipid activator protein sodium dodecil sulphate polyacrylamide gel electrophoresis semm free medium uridine-5'-monophpsphate

PUBLICATIONS

Natasha Smiijanic-Georgijev. Brigitte Rigat, Bei Xie, Wei Wang and Don J.

Mahuran. "Characterization of the Affinity of the G m Activator Protein for Glycolipids by

Fluorescence Dequenching Assay" Biochim.Biophys.Acta, in press.

C H A P T E R 1

GENERAL INTRODUCTION

The G M ~ gangliosidoses are a group of inherïted disorders caused by excessive

intralysosomal accumulation of ganglioside G M ~ and related glycolipids. particularly in neuronal

cells. These disorders result from defective G M ~ catabolism, which physiologicaIIy involve the

action of a lysosomal enzyme, f3-hexosaminidase A (Hex A). which is composed of two subunits, an

a and a p, and its substrate specific CO-factor the G M ~ activator protein. Since the a and the B subunit of Hex A are encoded by two unlinked structural genes. HEXA and HEXB, G M ~

degradation requires the participation of three geneticaily distinct polypeptides (Fig 1.1 .) (reviewed in

(Fürst and Sandhoff 1992)). Mutations in any of the three genes encoding these proteins may give

rise to G M ~ gangliosidosis. The three forms of GMZ gangliosidosis are; Tay-Sachs disease (a

defects). Sandhoff disease (P defects), and AB variant f o m of G M ~ gangliosidosis ( G M ~ activator

protein defects) (reviewed in (Gravel et al. 1995). Tay-Sachs disease occurs most frequently in

Ashkenazi Jews (1/3600 birth) (Kaback et al. 1977). primarily as a result of two very frequent

mutations (Gravel et al. 1995). To date. nearly 60 H E M gene mutations leading to this disease in

various populations have been identified. Sandhoff disease and the AB variant form of gangliosidosis

are rarer than Tay Sachs disease. with a total of 17 HEXB mutations and two GMZA mutations

k i n g Linked to disease states (reviewed in (Gravel et al. 1995)).

1.2. STRUCTURE OF THE HEXA A N D HEXB GENES

The HEXA gene maps to chromosome 15q23-q24 (Nakai et al. 1991) while the HEXB gene

maps to chromosome 5q13 (Bilcker et al. 1988). The HEXA gene is 35 kb long and contains 14

exons (Proia and Soravia 1987). The HEXB gene has about 45 kb and also contains 14 exons

(Neote e t al. 1988; Proia 1988). The structures of HEXA and HEXB genes show a striking degree

of homology in both the number and the placement of exon / intron junctions. As well. a cornparison

of the deduced primary sequences from their cDNAs, reveals an overall57% identity.

Figure 1.1 .:

The ~hexosaminidase gene system (from (Grave1 et al. 1995)).

Gene

Polypeptide

aa Hex S

Su bstrates

HEXA HEXB Chr 1 Sq23-24 Chr Sq13

4 Hex A

QQ Hex 8

GMZA Chr 5q32-33

Physiotog ic Glycosaminogl ycans? Glycoproteins G!ycoproteins Oligosaccharides OIigosaccharides GIycosaminoglycans GIycosarninoglycans GlycoIipids Gl ycolipids

I ~ a n ~ l i o s i d e GM~\ - cornplex formation

These data suggested, that the HEXA and HEXB arose from a common ancestor (Proia 1988;

Komeluk et al. 1986)- Thus, functional domains are Lkely to be conserved within aligned structures

of the two subunits. This hypothesis has b e n confirmed expetimentally (Brown et al. 1989: Brown

and Mahuran 1993; De Gasperi et ai. 1996).

1.3. HEXOSAMINIDASE ISOZYMES

PN-acetylhexosaminidase. Hex, is a lysosornal hydrolase that cleaves texminal non-reducing

P- 14-Iinked N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues frorn

oligosaccharides. glycolipids, gangliosides* glycoproteins and glycosarninoglycans. There are three

human P-hexosaminidase isozymes which are made up of al1 possible dimeric combinations of a

andor p subunits: A (ap) , B (PP) and S (aa) (Srivastava and Beutler 1973; ikonne et al. 1975).

Only dimer forms of Hex are functional, however each subunit is believed to contain one potentially

active site or two partial active sites. Active sites associated with eilher a or B subunits are able to

hydrolyse some sarne neutral artificial (Hou et al. 1996) and natural substrates (Mahuran et al. 1985).

But only the catalytic site associated with the a subunit c m hydrolyse negatively charged substrates,

e - g . CMUGS (Bayleran e t al. 1984). P-linked glucosamine 6-sulphate containing

glycosaminoglycans (Kresse et al. 1981). and the most important G M ~ ganglioside (Meier et al.

1991). Recently. based on the structural homology between a and P subunits and the common

evolutionary origin of the H E U and HEXB genes. the functional areas within two subunits were

located by cellular expression of a@ fusion proteins joined at adjacently aligned residues (Tse et al.

1996). It was concluded that: a) The least homologous amino terminal sections of a and D

polypeptides are only needed for proper folding in endoplasmic reticulum (ER). b) The most

homologous middle sections contain the substrate binding / active sites. c) The carboxy terminal

sections are likely involved in subunit-subunit interactions.

1.4. TEE INTERACTION B E T W E E N THE ACTIVATOR, G M ~ GANGLIOSIDE AND

Hex A can not directly attack the membrane associated substrate (Conzelmann and Sandhoff

1978) but can degrade G M ~ in the activator-GM~ complex in vivo (Conzelmann et al. 1982), or in

vitro in the presence of detergent. This suggests that the activator acts primarily as a substrate

specific CO-factor of Hex A. instead of " activating" the enzyme (Sandhoff et al. 1989). The G M ~

activator protein itself, appears unable to penetrate the liposomal membrane, as only lipid moIecules

on the outer leaflet of the membrane are accessible to the activator (Conzelmann et al. 1982). It binds

G M ~ and "presents" it to Hex A for degradation (Sandhoff et al. 1989). Figure 1.2. presents

possible models of the activator assisted G M ~ hydrolysis by Hex A (reviewed in (Fürst and Sandhoff

1992)).

The activator interacts with both the hydrophilic oligosaccharide and hydrophobic ceramide

moieties of gangliosides. The hydrophobic binding site for the ceramide portion of gangliosides has

ken suggested to be composed of a pocket forrned by amphiphilic a helices predicted from the arnino

acid sequence of the activator (Fürst et al. 1990). The spectmm of glycolipids that interacts with the

activator is primarily determined by their oligosaccharide moieties. In vitro binding studies have

indicated that the terminal N-acetylgalactosamine (GalNAc) and interna1 sialic acid (NeuAc) residues

of GMZ play an important role in deterinking binding specificity, Le. GM2>> GM 1> GD la= GM3=

GA2. The resulting 1 to 1 activator: ganglioside complex must then specifically interact with Hex A

(reviewed in (Sandhoff et al. 1995))-

Studies conceming the interactions of the G M ~ activator protein with Hex A suggest that the

molar ratio of Hex A to the activator 1 ganglioside complex neccssary for obtaining the maximal

hydrolysis of G M ~ is close to 1: 1 (Li et al. 198 1). The degradation of CMUGS (a negatively

charged artificial substrate that is bound by the active site associated with the a subunit) by Hex A

(ap) and Hex S (aa) cm be competitively inhibited by the G M ~ activator protein, indicating that the

a subunit of Hex A also contain an activator-binding site (Kytzia and Sandhoff 1985). As the

Figure 1.2.:

Two models presenting the interaction of G M ~ activator with Hex A (from (Fürst and

Sandhoff 1992)).

Model 1: The formation of G M ~ , G M ~ activator and Hex A complex can take place on the

lysosomal membrane radier than in the free solution, and the function of the G M ~ activator is to lift

G M ~ a few angstroms out of the membrane which eliminates the stenc hindrance of adjacent lipid

molecules and gives the enzyme access to the Blinked GaNAc residue.

Model 2: The G M ~ activator vansports G M ~ from membrane to free solution of the lysosome,

presenhg it to Hex A for hydrolysis.

Either or both models may exist-

activator does not promote degradation of G M ~ by Hex S. it is possible that Hex S does not bind to

the GMZ activator 1 G M ~ complex in the correct orientation and that the subunit of Hex A may also

interact with the activator (Kytzia and Sandhoff 1985) .

1.5. GENOMIC STRUCTURE OF THE G M 2 A GENE ENCODING THE G M ~ ACTIVATOR

PROTEIN

Recently, the fful length cDNA clones encoding the GMZ activator protein have been isolated

by ourselves and others (Xie et al. 199 1 ; Klima et al. 1991 ; Nagmjan et al. 1992). The GM2A gene

has been rnapped to chromosome 5q32-33, while a pseudogene. CMZAP, has been mapped to

chromosome 3 (Xie et al. 1992; Swallow et al. 1993). The structure of -95% of the human GMZA

gene has been characterized and four exons idenûfied. The 2.4 kb cDNA codes for a prepro-protein

of 193 arnino acids. The signal peptide, required for the entry into the rough endoplasmic reticulum

(23 amino acids) and the mature protein (162 amino acids) are connected by 8 amino acids which are

removed during maturation in the lysosome (Fürst et al. 1990). The deduced primriry sequence. also

contains one consensus site for Asn-linked glycosylation (Asn63-Val-Thr).

A second alternatively spliced mRNA product containing exons 1-3 and intron 3 has been

identified (Fig. 1.3.) (Nagarajan et al. 1992). Due to the presence of a STOP codon early in the

retained inuon 3 sequence, the product of the alternatively spliced mRNA is a truncated form of the

activator missing residues 142-193 (counting from the initial Met residue) and containing an additional

three residues encoded by the inuon 3, Val-Ser-Thr.

Figure 1.3.:

Gene structure of G M ~ activator protein and G M ~ A protein (from (Wu et al. 1996)).

El, E2, E3 and E4 represent exon 1, exon 2, exon 3 and exon 4, respectively. I l , 12 and 13

represent intron 1. intron 2 and intron 3. respectively. The word "stop" means stop codon. VST is

the last three amino acids encoded by intron 3. The sizes of the gene fragments are not in the exact

proportion.

G , , activaror inRNA %,A

1 El 1 ES i E3 1 E4 1 1 El ES 1 E3 1 13 1

G,, ac~ivaior protein

[ ~ l r E2 1 E3 1 E4 1 CM,, proiein

The G M ~ activator protein as well as the a and i3 chains of hexosarninidase A are

glycoproteins synthesized on polysomes attached to the rough endoplasmic reticulum (ER). To enter

the rough ER glycoproteins require a signal peptide (residues 1-23 in the G M ~ activator protein). The

signal peptide is cleaved by signal peptidase in the lumen of the ER. This event may be followed by

the addition of an oligosaccharide c h a h to the asparagine residues contained in the consensus

sequence (Am-X-Ser/Thr). This glycosylation step involves the en block transfer of large preformed

oligosaccharide. GalNAc2 Mang Gk3, from a lipid-linked intennediate to the nascent polypeptide

(Komfeld and Kornfeld 1985). Before exiting the ER glycoproteins lose their Glcg groups from the

oligosaccharides and fold to their near native conformation. In some cases, like Hex. oligomerization

must also occur; however, the activator protein is a monomer. A group of resident ER proteins,

chaperones, bind to the elongating nascent polypeptide, preventuig aggregation of the polypeptide and

accelerate its correct folding. Another chaperone. disulfide isomerase, assists in the correct placement

of disulfide bonds (Hendrick and Hartl 1993). If nascent polypeptides are not released from

chaperones, e-g. due to mutations, their degndation rate is increased.

After the newly synthesized glycoproteins are properly folded, they enter Ihe cis Golgi where

transport continues unidirectionally via bulk flow. For most lysosomal proteins. mannose-6-

phosphate (M6P) markers are added to one or more high mannose type oligosaccharides. The M6P

recognition marker is generated by the sequential action of two Golgi enzymes. First, N -

acetylglucosamine-phosphotransferase transfers N- acetyIglucosamine-1-phosphate from the

nucleotide sugar uridine diphosphate-N-acetylglucosamine to selected mannose residues on lysosomd

enzymes. to $ive rise to a phosphodiester interrnediate. nien. N- Acetylglucosamine phosphodiester

glycosidase removes N-acetylglucosamine residue to expose the recognition signal (Lang et al. 1984).

The phosphotranspherase is the enzyme defective in patients with 1-ce11 disease. This disease is

characterized by the tissue specific intracellular deficiency of many lysosomal proteins which are

elevated in the plasma of the patients. Fibroblasts from these patients fail to phosphorylate mannose

residues on their newly synthesized lysosomal proteins and secrete a large percentage of thern into the

culture medium, as a precursors. (Hasilik and von Figura 198 1; Burg et al. 1985; Varki et al. 198 1).

The phosphorylation of lysosomal glycoproteins specifically targets them to the lysosome

through the interaction with one of two mannose-6-phosphate receptoa (MPRs) in the trans Golgi

network (Griffith et al. 1988). MPRs are tram-membrane proteins which c m be concentrated in

clathrin coated vesicles on the membrane of the tram Golgi network or the plasma membrane by

adaptor proteins (Glickman et al. 1989). Adaptors recognize specific protein motifs in the

cytoplasmic domain of transmembrane proteins and promote polyrnerization on clathrin triskelions

ont0 the membranes (Pearse and Robinson 1990). The receptor-ligand binding in the Golgi diverts

lysosomal proteins away from the default route followed by secretion proteins containing complex

oligosaccharide structures. After these interactions. the membrane of the clathrin coated vesicles buds

from the trans Golgi membrane forming srnall cytosolic vesicles. These vesicles translocate

lysosomal glycoproteins into the late endosome where acidic pH causes uncoupling of the MPRs and

ligands. Ligands are then transported via vesicles to lysosomes or the late endosome fuses with or

becomes a mature lysosome (Croze et al. 1989). The recepton recycle back to the Golgi.

In the lysosome or late endosome a series of proteolytic and glycosidic processing events

occur to the pro-Hex isozymes forming their mature subunit stnicturcs (Mahuran et al. 1988; Stirling

et al. 1988; Hubbes et al. 1989; O'Dowd et al. 1988; Mahuran 1990). Proteolytic processing of the

single 67 kDa pmpolypeptide chain results in the generation of the pp ( 1 1- 14 kDa). Pb (22-24 kDa)

and Pa (26-28 kDa) chains. Similarly, the 65 kDa a propolypeptide results in genention of the a p (7

kDa) and a m (56 kDa) chains. comprising the mature P and a chains. respectivcly. Each set of

mature a or are held together by disulfide bonds in the mature subunits.

Transportation of the G M ~ activator protein to the lysosome is still not completely understood.

Two reports have suggested that the activator is transported to the lysosome via MPR. First, there

was a 2.5 fold increase in the amount of activator in the s e m of a single 1-ce11 patient (Banerjee et al.

1984) and secondly. there was a similar increase in the secretion of the activator from normal

fibroblasts grown in the presence of NH4CI (which prevents acidification of both endosornes and

lysosomes. causing the newly synthesized precursors to be treated as secretory proteins) (Burg et al.

1985). However. these data are far from conclusive a s the secretion of other MPR-targeted enzymes

are increased 10-50 fold with a concomitant loss of intrace11ular enzyme in b o t . circumstances (Creek

et al. 1983; Wiesmann et al. 197 1))-

After entering the lysosome, the pro G M ~ activator protein. 24 D a . is processed by

proteolytic and glycosidic enzymes, foming the 22 kDa mature form. The pro activator can be

precipitated from the culture medium, while only mature form is detected in cells. suggesting a rapid

processing compared to the low biosynthetic rate (Burg et al. 1985). The molecular mass of the

polypeptide c h a h without its single oligosaccharïde is 17.6 kDa (Fürst et al. 1990). The mature G M ~

activator has eight cysteine residues in its 162 amino acid polypeptide chain. Presumably the

formation of 4 cystines. from these cysteines. is responsible for the hert stability of the protein (Fürst

et al. 1990).

AB variant form of gangliosidosis is extremely rare and is caused by mutations in the gene

encoding G M ~ activator protein. To date, four mutations have k e n reported by ourselves and others;

a T4 12 to C (Cys 138 to Arg ) (Xie et al. 1992), a G506 to C (Arg 169 to Pro) (Schroder et al. 1993),

a deletion ~ ~ ~ 2 6 * - 2 6 4 (deletion of ~ ~ ~ 8 8 ) . a deletion ~ 4 1 0 (frarne shift) (Schepers et al. 1996).

These mutations were associated with the infantile acute form of the disease. This form of G M ~

gangliosidosis. AB variant. is clinically indistinguishable frorn the infantile onset of Tay-Sachs and

Sandhoff diseases. The symptoms, involving the l o s of motor skills, ophthalmologic problems and

the increase of seizure activity, begin in the first 3 to 5 months of life and progress rapidly with a fatal

ending between 2 to 4 years of age. AU G M ~ gangliosidoses are histopathologically charactenzed by

the accumulation of G M ~ ganglioside, mostly affecting neuronal lysosomes. which appear by electron

Figure 1.4.:

Concenvic lamellar bodies in G m gangliosidosis (from (Agamanolis 1995)).

microscopy as numerous unilarnellar bodies, consisting of dense concenttic membranes (Fig. 1.4.)

(reviewed in (Agamanolis 1995)). However, in the AB variant f o m of G M ~ gangliosidosis, changes

including prominent inclusions in glial ceiis and large conglornerates of lipid inclusions, are found to

be significantly different from those in Tay-Sachs and Sandhoff diseases. These differences indicate

that the activator may have some other, as yet uncharacterised. function(s) Le. the involvement in the

general glycolipid transportation.

G M ~ activator is one of five known lysosomal sphingolipid activator proteins (SAPs)

(reviewed in (Fürst and Sandhoff 1992)). Four of these, sap A-D. are encoded by a single gene

mapped on chromosome 10. They enter the lysosome as a single 65-73 kDa precursor chain. The

large precursor, prosaposin, is processed into four - 13 kDa polypeptide chains that share a fair

degree of structurai hornology. Sulphatase activator. Saposin-B or SAP- 1, activates the hydrolysis of

cerebroside sulphate, GM 1 and globotnaosylceramide by arylsulphatase A, B-galactosidase and a-

galactosidase, respectively. Glucosylcerarnidase activator, Saposin-C or SAP-2, stimulates the

hydrolysis of glucosylceramide, galactosylcerarnide and sphingornyelin by O-glucosylceramidase. O-

galactosidase and sphingomyelinase, respectively. Other two potential activator proteins are Saposin-

A and Saposin-D. In vitro. Saposin-A activates glucosylcerarnidase and galactosylceramidase, while

Saposin-D shows some stimulatory effect on the degndation of cerarnide in vitro and in vivo.

h comparison to other SAPs. the G M ~ activator protein is unique in several aspects: a) it is

encoded by an unrelated gene on chromosome 5 (Xie et al. 1992), b) it shares no significant deduced

primary structure homology with Sap A-D (Schroder et ai. 1989). c) it functions as a monomer (sap

A-D are hornodirners) and d) its only proven in vivo function is as a substrate specific cofactor for the

degradation of G M ~ ganglioside by Hex A, a rolc that can not be filled by any of the other SAPs

(Sandhoff et al. 1995).

Recently, in addition to its role as a lysosomal precursor of sap A-D. prosaposin was found to

exist as a secretory protein in human milk, cerebrospinal fluid and seminal plasma (Hiraiwa et al.

1993). It was further demonstrated that prosaposin can bind gangliosides with high affinity and

facilitate their transfer from micelles to membranes (Hiraiwa et al. 1992). Taken together, these in

vitro and in vivo studies of prosaposin, suggest it as a possible intraceilular ganglioside binding and

nansfer protein. Furthemore. prosaposin was identified as a neurotrophic factor, stimulahg neurite

outgrowth by binding to a high-affinty receptor (O'Brien et al. 1994).

1.9. OTRER POSSIBLE FUNCTIONS OF THE G M ~ ACWATOR PROTEIN

The only proven in vivo function of the G M ~ activator protein is to stimulate the breakdown

of G M ~ to G M ~ ganglioside by Hex A (Conzelmann et al. 1979). In vitro. it also promotes the

conversion of G M ~ to GAZ by clostridial sialidase, the hydrolysis of GA^ by Hex A and the release

of Ga1 from GM 1 by B-galactosidase when added in high concentrations (Kytzia and Sandhoff 1985;

Wu et al. 1994). As weI1, in the absence of Hex A, the activator c m act in vitro as a general

sphingolipid transport protein. The rate of the ganglioside transfer between liposomes is: G M b

GM2> GDla>> GM3= GA2 (Conzelmann et al. 1982). Thus, one of the most abundant brain

gangliosides. GM 1, is transferred at a preferential rate. Furthemore, the activator was also shown to

transport a fluorescent lipid probe between labeled and unlabeled liposomes (Kuwana et al. 1995).

Thus, as with prosaposin (Hiraiwa et al. 1992). it is possible that the activator can act in vivo as a

generd glycolipid sorting / transport protein. The retention of the activator's transport function at

neutral pH in vitro (Conzelmann et al. 1982; Kuwana et al. 1995) also supports this hypothesis.

Recently, the function of the G M ~ A protein produced by alternative splicing of the activator

mRNA. which is truncated at residue 142 and contains 3 unrelated amino acids, was investigated (Wu

et al. 1996). This protein, synthesized in bactena and refolded. was reported to retain its hydrophobic

binding pocket and NeuAc recognition site based on; a) its ability to stimulate the hydrolysis of

to GA^ by clostridial sialidase, and b) its inability to stimulate the removal of NeuAc from the

oligosaccharide of the G M ~ ganglioside alone. i. e. without the ceramide moiety , in the presence of the

same enzyme. The GM~A protein also lacked the ability to stimulate the removal of the GaiNAc

residue from by Hex A. suggesting it lacked either the GalNAc recognition site or its domain for

interacting with Hex A.

Glycosphingolipids (GSL) are amphiphilic molecules that constitute the basic Iipid core

structure of ce11 membranes. Whereas they are present at relatively high concentrations in neuronal

plasma membranes, they are also present in the plasma membrane of essentially ail other mammalian

cells. The essential molecular features of glycosphingolipids are a hydrophobic backbone, ceramide

(Cer). which consists of a long chah aliphatic amino alcohol (sphingosine) which is attached to a fatty

acid via an amide linkage and a polar carbohydrate head group which protmdes into the extracellular

environment. Neutra1 glycosphingolipids have one or more neutral sugars, covalently attached via a

glycoside bond to the ceramide. Gangliosides are a group of acidic glycosphingolipids, containing in

addition to the neutral sugar. one or more N-acetylneurarninic acids (NeuAc or sialic acid) (reviewed

in (Hoekstra and Kok 1992; Ledeen and Yu 1982)). The major gangliosides in the body are GM 1,

GD l a and GD 1 b. Others, G M ~ . G M ~ and GT, are intermediates in both the synthesis and

degradation of these more complex gangliosides and consequently are found in lower concentrations.

G M ~ ganglioside (Fig 1.5.) consists of a cerarnide. made of sphingosine and stelinc acid, and the

teuasaccharide made of glucose (Glc), galactose (Gal), N-acetylneuraminic acid (NeuNAc) and N-

acetylgalactosarnine (GaiNAc) (Zeller and Marchase 1992).

The precise biological role(s) for gangliosides has yet to be elucidated. However. recent data

has suggested some important roles that gangliosides may play in the physiologie operations of the

nervous system, in particular that of brain. Their potential roles are in the developmental. ce11

adhesion and signal transduction processes (reviewed in (Zeller and Marchase 1992)). Sorne

Figure 1.5.:

Structure of G M ~ ganglioside.

GalNAc G al GIc ceramide

gangliosides have k e n identified as binding sites on ce11 surfaces For viruses and bacterial toxins.

For example, the cholera toxin subunit B binds to cells specifically through GM 1. allowing the A 1

subunit of the toxin to enter the cells and activate adenylate cyclase (Fishman et al. 1993).

Furthermore, the levels of individual gangliosides are significantly changed in some human tumors.

e.g. the disialoganglioside GD^ is synthesized in abundance by pnmary untreated neuroblastoma and

can be detected in the plasma of patients with this type of tumor (Valentino et al. 1990; Sariola et al.

1991). Finaily, the growth of ectopic dendrites of cortical pyramidal neurons has heen shown to

specifically correlate wiih the arnounts of accumulated G M ~ , suggesting an unique in vivo function

for G M ~ ganglioside (Siegel and Walkley 1994).

Ganglioside biosynthesis starts in the ER where the ceramide portion is synthesized frorn

serine and paimitoyl CoA. After addition of the amide-linked fatty acid. the c e m i d e is transferred to

the Golgi apparatus by an as yet unidentified mechanism. Ceramide is glycosylated in a stepwise

manner by the transfer of the individual sugar from the respective uridine-5'-diphosphate (UDP)

derivatives and sialyl residues from the cytidine-5'-monophosphate (CMP) - Neu NAc. First step of

glycosphingolipids synthesis is the linking of glucose to cerarnide and formation of glucosyiceramide

(GlcCer) in the presence of glucosyltransferase. The coupling of galactose to GlcCer yields

lactosylceramide (LacCer). the precursor of more complex glycosphingolipids. This reaction is

catalyzed by galactosyltransferase. The sequential addition of s id ic acid residues to the growing

oligosaccharide chain. yielding G M ~ , GD^ and more complex ganglioside is catalyzed by

sialyltransferases which exhibit relatively high levei of substrate discrimination. On the other hand,

there is a single N-acetylgalactosarninyltransferase that catalyses the synthesis of GAZ. G M ~ . GD2

and G T ~ by the addition of the third neutral sugar GalNAc. Similarly, the synthesis of terminal

carbohydrate structures s h m d by other glycolipids is aiso catalyzed by this set of enzymes.

Figure 1-6.:

Degradation pathway of ganglioside G M ~ in lysosome showing the type of ganglioside. its

structure and the glycohydrolases involved in its breakdown.

Gai-GalNAc-Gai-GIc-Ceramide

4 I

G M r ganglioside NANA

Gal GMi ganglioside O-galactosidase

GaINAc-G 1-Glc-Ceramide a GM2 ganglioside

Gaf NAc

GM3 ganglioside

NANA

lactosylceramide

Gal

glucosylceram ide

Glc

1 NANA

1 f3-hexosaminidase A

Gal-Glc-Ceramide i

NANA

neuraminidase

Gal-Glc-Ceramide i

"1 "B-galactosidase"

glucocerebroside O-glucosidase

Ceramide

i ceramidase

fatty acid + sphingenine

After their synthesis in the Golgi, gangliosides are transported to the plasma membrane by vesicular

flow and anchored to the outer leaflet of the membrane by their ceramide moieties, with their

oligosaccharides extending into the extracellular space (Van Echten and Sandhoff 1993). Despite an

abundance of studies, detailed locdization of ganglioside biosynthesis as well as mechanisms of

intracellular transfer are still to be elucidated.

1.10.2. Clandioside Catabolism

The catabolism of gangliosides occurs through the action of specific lysosomal hydrolases. In

a stepwise manner they remove individual sugar residues from the non-reducing end of the

oligosaccharide (Fig. 1.6.).

Whereas lysosomal exohydrolases directly attack membrane bound GSLs with hydrophilic

head groups extending far enough into the aqueous space, they need the assistance of srnaIl

glycoprotein cofactors. the " sphingolipid activator proteins " (SAPS) to degrade GSLs with short

oligosaccharide chahs made of 3 or less monosaccharide residues (Van Echten and Sandhoff 1993).

It is not yet clear how gangliosides are uansponed to the lysosomes for degradation. It is

generally assumed that components of plasma membrane reach the lysosomes mainly by an

endocytotic membrane flow dong the early and latc endosomal compartments. This mode1 proposes

that parts of the endosomal membranes. possibly those ennched in plasma membrane components.

bud off into the endosornal lumen forming intraendosomal vesicles. Those vesicles could finally be

delivered directly into the lysosol, the lumen of lysosomes. for degndation (reviewcd in (Fürst and

Sandhoff 1992)). However. it is possible that some other mechanisms could be invclved in the

glycolipid transfer to lysosomes. In vitro expenments showed that the G M ~ activator protein can

remove labeled gangliosides from one liposome and insert them to another. transfemng the most

abundant brain gangiioside, GM 1, at a preferential rate (Conzelmann e t al. 1982). Furthemore. the

fluorescent lipid probe, octadecylrhodamin B chloride. can be transferred from donor to acceptor

membranes in vitro by the activator protein (Kuwana et al. 1995). The activator retained its transport

25

ability at neutral pH in both mentioned cases. Thus, it is possible that the G M ~ activator protein could

have other extra and intracellular functions in vivo , involving its ability to transport a variety of

glycolipids.

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C H A P T E R II

DEVELOPMENT OF THE FLUORESCENCE DEQUENCHING

ASSAY FOR STRUCTURE 1 FUNCTION STUDIES OF THE GM2

ACTIVATOR PROTEIN

2.1 INTRODUCTION

The G m activator protein is a substrate specific cofactor for degradation of GMZ ganglioside

by lysosomal B-hexosaminidase A. It solubilizes individual molecules of the ganglioside by

interacting with both its hydrophilic oligosaccharide and hydrophobic ceramide moieties. However,

the specificity of binding is pnmarily deterrnined by the oligosaccharide moiety of the glycolipid.

From the deduced primary structure. the activator protein is predicted to contain as many as three a

helices, frorn which two are predicted to have a significant amphiphilic character. These functions

may be involved in the formation of a lipid binding cavity (Fürst et al. 1990). In vitro. the activator

can also act as a general sphingolipid transport protein (Conzelrnann et ai. 1982).

The formation of the G M ~ activator protein : glycolipid complex has been investigated by a

variety of techniques (gel filtration. electrophoresis. isoelectric focusing and ultracentrifugation)

(Conzelrnann et al. 1982). However. not only are these techniques complex and require radio-labeled

glycolipids, but none have allowed the direct kinetic measurements of glycolipid hinding. i.e. Kd and

"n". The difficulty in such measurements in part resides in the inability to immohilize gangliosides

through either their ceramide or oligosaccharide moieties without affecting activator hinding. Indirect

measurements through functional assays with Hex A have indicated a "n" value of 1 (number of

binding sites) and a Kd of 3.5 pM (reviewed in (Sandhoff et al. 1995)).

Recently. a fluorescence dequenching assay sugpstcd itself as a new method to aid in

structure / function studies of G M ~ activator protein. Originally, it was used to investigate the fusion

of sub-cellular organelles ((Hoekstra et al. 1984) and reviewed in (Hoekstra 1990; Mullock and Luzio

1992)). The principle of the assay relies upon the efficient self-quenching of the fiuorophore

rhodamine, conjugated to a saturated C 18 hydrocarbon chah (R- 18). This probe is readily inserted

into biological membranes by exogenous addition of an ethanolic solution of the fluorophore. At

concentrations up to 9 mol% with respect to total lipid, the efficiency of the self quenching is

proportional to its surface density. After membrane insertion, the localization of the probe is such that

the rhodarnine head group likely resides at the lipid-water interface, while the hydrocarbon moiety is

Figure 2.1 .:

The principIe of the fluorescence dequenching assay:

1) R- 18 (octadecylrhodamine B chloride) added to a suspension of membranes becornes

incorporated in the membranes at selfquenching concentrations.

2) The selfquenching is relieved by fusion of these membranes with unlabeled membranes.

3) Fusion results in probe dilution and increase in fluorescence which c m be rneasured.

anchored in the hydrophobic interior of the membrane. Once inserted into the membrane. the probe

does not dissociate from it by either a spontaneous transfer of free monomers through the aqueous

phase or by a collision mediated transfer process. Upon fusion of R-18 containing membranes with

membranes devoid of the probe. the surface density of the fluorophor decreases. resulting in an

increase in fluorescence that can be monitored continuously (Fig.2.1.) (reviewed in (Hoekstra 1990;

Mullock and Luzio 1992)).

Using fluorescence dequenching assay for monitoring the fusion of endosomes and

lysosomes, Kuwana et al . (Kuwana et al. 1993; Kuwana et al. 1995) isolated a protein that increased

fluorescence in their assay. It was purified and found to be 22 kDa molecule with sequence,

immunological and functional characteristics consistent with the rat homologue of human G M ~

activator protein. However, Our lahoratory showed that the human activator was not involved in

endosomal-Iysosomal fusion (Xie and Mahuran 1994). Further wark suggested that the rat protein

had leaked from the lysosome preparation (Kuwana et al. 1995). For both, the recombinant human

and rat homologue of human G M ~ activator protein, these data were consistent with the activator

acting as an R- 1 8 transfer protein.

The simplicity of the R- 18 assay and the finding that the activator c m act as R- 18 transfer

protein between egg phosphatidylcholine (PC) liposomes. as well as isolated endosomes and

lysosomes (Kuwana et al. 1995). suggested to us the possibility of using this fluorescence assay for

structure / function investigations of the G M ~ activator protein.

2.2 MATERIALS AND METHODS

R-18 was obtained from Molecular Probes. Egg phosphatidylcholine (PC) in chloroform was

from Avanti Polar Lipids. Unless otherwise stated al l other reagents were from Sigma Chemicai Co.

2.2.2. Production of the H i s a Activator Fusion Protein from E. Coli

With minor modifications we used the sarne basic methods for the synthesis by transformed

E. Coli, purification. and refolding of a Hi%-Gm activator fusion protein (Hi%-activator) as reported

by Klima et al. (Klirna et al. 1993). The method for the synthesis of the G M ~ activator fusion protein

and its vuncated form (see below) was set up by Dr.Brigitte Rigat in our laboratory. Bnefly. PCR

was used to amplify the coding sequence of the mature activator (residues 32-193) lrom pActl (Xie et

al. 199 1) in such a manner a s to allow the product to he inserted in frarne and down Stream from the

encoded N-terminal His6 in the E. Coli expression vector pQE-8 (Qiagen). A cloned insert was

confirmed by sequencing to encode the proper amino acid sequence. MRGS(H)~GSIEGR-S~~-~~~~.

In one other clone a Taq-error was identified chat produced a frame shift after codon 157 followed by

the generation of 13 new codons (MRGS(H)~GSIEGR-~32-LIST-WSCPVGSPPGTTA) before a

STOP codon was encountered. This protein (referred to as the truncated-activator) was purified and

refolded into a water soluble form in the sarne rnanner as the wild type protein.

The purifkation procedure started with the preparation of E-Coli pre-culture. One day before

main culture. 100 pl of E.Coli cells in 10 ml Super media (25 g bactotryptone, 15 g bactoyeast

extract and 5 g NaCl. per liter) were grown in the presence of ampicillin (100 pgml) and kanamycin

(50 pg/rnl), ovemight. The next day. the pre culture (IO ml) was grown in 100 ml of Super media

with ampicillin and kanamycin for lh. The expression of the cDNA insert was then induced by

adding IPTG to a fmai concentration of 3 mM. The cells were incubated for 7h and then harvested by

centrifugation (4000 g. 15 min). The pellet was kept at -70°C. ovemight

The pellet of a 100 ml culture was left on the ice for 2h to thaw and then resuspended in 10 ml

of Extraction buffer (8 M Urea 0.1 M Sodium Phosphate. 0.01 M Tris. 3% v/v f3 mercaptoethanol.

pH 8) for 2 h by gentle mixing. After extraction the ce11 debris was removed by cenuifuging (6000 g.

15 min). The crude supematant was used for further procedures.

The crude supematant was added to 5 ml of Ni-NTA resin (Quiagen Inc.. Hilden. Gemany)

and gently mixed ovemight The hexahistidine residues of the recombinant G M ~ activator protein

bound with high affinity to ~ i 2 + ions. The next day. the proteinfresin complex was loaded ont0 a

column for the washing and elution steps. The colurnn was washed with two column volumes of the

Washing buffer 1 (Extraction buffer at pH 7). and two column volumes of the Washing buffer II (6 M

Guanidine. 0.1 M Sodium Phosphate. 0.01 M Tris. 3% v/v beta mercaptoethanol, pH 7). it was then

eluted with two column volumes of the Elution buffer (Washing buffer II at pH 5). Elution fractions

were collected in different volumens. consecutively (5 ml. 20 ml, 10 ml and 15 ml). A sample of 100

pl from each fraction and each experirnental step was rnixed with 1 ml of precipitating solution (99%

ethanol, 1% acetic acid) at - 20°C ovemight and then cenuifuged and washed with 70% ethanol. To

determine the punty, as well as the molecular weight of the H i s g - G ~ 2 activator protein in sarnples

from al1 purification steps. the pellet was analyzed by SDSPAGE using the Laemrnli system

(Laemmli 1970) and a 12.5% running gel and 4.52 stacking gel. The protein was visualized by

Coomassie blue staining (Fig 2.2).

Refolding of the recombinant G M ~ activator protein was accomplished by the dilution of the

Elution buffer containing the activator protein (fraction 2 (20 ml)). with five volumes of the Folding

buffer A (2 m M reduced Glutathione, 0.2 mM oxidized Glutathione, 50 mM Tris pH 8 and 0.0038

v/v Tween 20) and stored at 4'C for 18 h. After that period, the solution was dialyzed against folding

buffer B (100 mM NaCI. 50 m M Tris pH 8 and 0.003% v/v Tween 20) for 48 h at room temperature.

Figure 2.2:

SDS-PAGE separation of the Hisfj -G~2 activator protein, in samples from different activator

protein purification steps: lysate (lane 2)- unbound fraction (lane3). wash 1 (lane 4), wash II (lane 5).

elution 1 (lane 6), elution 2 (lane 7), elution 3 (lane 8) and elution 4 (lane 9). Standard molecular

weight markers are presented in the lane 1.

The protein was concentrated in a Stirred ce11 (Arnicon. U.S.A.) fitted with Diafio ultra filter [(YM

10.43 mm) Amicon. U.S.A.]. Concentration of the protein was carried out at CC and the protein

level was detemined by the Lowry method (Lowry et al. 195 1).

2.2.3. Pre~aration of Phos ohatidvlcholine Con_- 1 Inilamel . . lar Vesicla

Phosphatidylcholine containing large unilamellar vesicles (PC LW,). i.e. Liposomes, were

prepared with some modifications a s described by Kuwana et al. (Kuwana et al. 1995). Egg PC in

chloroform was dned under nitrogen, dehydrated under high vacuum for 1-2 h and kept at -20°C until

use. The lipid was resuspended at 5 mg/ml in NHE huffer (0.15 M NaCI. 10 mM Hepes. O. 1 mM

EDTA. pH 5) and freeze -diawed ten times to ensure entrapment of the huffer in the inner space of

vesicles fomed. The vesicles vere consecutively exmded through two polycarbonate filters (pore

diameter 200 nm and 100 nm), fitted in a mini-extruder (Avestin. Ottawa, Ont., Canada) with 19

passes (MacDonald et al. 1991). The PC-LUV, were made fresh for each experiment

2.2.4. Prepmtion of R- 18 labeled liposomes

In a total volume of 0.5 ml of NHE buffer. the PC-LUV, (0.44 pmol) were rapidly and

thoroughly mixed nt room temperature with 0.04 pmol of R- 18 (20 m M in 1 0 % cthanol). then

incubated for 15 min with gentle mixing and protected from light. Free R-IR was removed by

Sephadex G-50 gel tiltration. The 1 x 4 cm column was cluted with NHE buffer. This preparation of

R- 18 labeled liposomes was sufficient for ten assays.

2.2.5. Fluorescence de au en ch in^

Fluorescence dequenching assays were performed with some modifications as described

(Kuwana et al. 1995). In each assay, 630 nmol of unlabeled liposomes were mixed with appropriate

42

arnounts of recombinant human G M ~ activator protein with or without gangliosides. Gangliosides

were added in the 2 p l total volume of 2:l chloroform : methano1 standard solution- The assay was

initiated by the addition of 44 m o l of R- 18 labeled liposomes. The assay mixture was made up to 1

ml with SPM buffer (0.25 M Sucrose, IO m M Na phosphate buffer, 1 mM MgCl2, pH 5).

Fluorescence emission was read at 590 nm using an excitation wavelength of 560 nm. on a 650-40

fluororneter (Perkin-Eimer) in the thermostated (37') quartz cuvette.

The fluorescence dequenching of the R- 18 liposome preparations was measured and calculated

with some modifications as described by Epand et al. (Epand et al. 1995). The fluorescence intensity,

5 min after addition of R- 18 PC-LW,, was taken as Fo. At the end of the reaction, 60 pl of 20% v/v

Triton X-100 was added to the assay mixture to obtain the fluorescence value at infinite dilution of the

probe (Flm). Fluorescence was taken at any tirne point (FJ and dequenching calculated as; % R-18

dequenching = 100 (Ft-FO )/ F ~ w . The fluorescence dequenching units (Fu) were calculated by

subtraction of the baseline percentage of R- 18 fluorescence dequenching (obtained from the sample

containing al1 assay components except the activator) from each sample. Slopes. [A Fu I A T (min)].

were calculated by subtraction of the baseline dope from the dope of each sample.

2.3. RESULTS

2.3.1. Fluo rescence deauench in^ of R-18 labelcd 1'- ioosomes in the presence of recombinant

human Gm activator protein

Incubation of R- 1 8 labeled and unlabeled liposomes in the presence of 4pg of the recombinant

human activator (0.21pM), resulted in a time dependent loss of fluorescence quenching. i .e. an

increase in fluorescence over time (Fig.2.3.). Baseline fluorescence. corresponding to spontaneous

fluorescence dequenching (possibly due to dissociation from the liposomes) in R- 18 liposomes

Figure 2.3-:

Fluorescence dequenching. expressed as 5% of total (see Methods) of R- 18 labeled liposomes.

in presence of 4 pg of recombinant human G M ~ activator protein, open triangles, fit in a bat-f i t

straight iine by least squares analysis (dashed h e ) or absence of the activator (negative control. solid

squares) presented as a solid best fitted line.

2 3 4 Time (min.)

Figure 2.4a:

Fluorescence dequenching uni& Fu (see Methods), developed over 60 minutes with 1-3 pg

of recombinant human G M ~ activator protein. The best fit curves [fitted to the equation "FU=Clx T/

(C2+T)" by non-linear least squares] are shown with the actual data points.

Time (min)

Figure 2.4b.:

The time range for the linear rate of fluorescence dequenching by 1-3 pg of activator. Data

points are fitied to the bat-fit line by least squares analyses.

2 3 4

Time (min)

system without the recombinant human activator. was -2 fold lower (Fig.2.3.). After the subtraction

of the baseline slope. the resulting activator slope was 0.365. The tnincated f o m of the G M ~

activator protein (see Methods) was used as a negative control (Fig.3.2.). Unlike the full length

mature protein produced in bacteria which readily enhances Hex A's ability to hydrolyse G m (Hou et

al. 1996). the truncated protein was totally non-functional (unpublished results). In the fluorescence

dequenching assay 4pg of the tnincated activator (see Chapter III) produced a dope of 0.018. after

baseline subtraction. suggesting that it does not contain a functional hydrophobic binding site.

2.3.2. Determination of the optimal time course range for the fluorescence dequenchin~ assu

The optimal time course for the fluorescence dequenching assay was determined for three

arnounts of activator (1 pg. 2 pg. 3 pg). Fluorescence dequenching was fint measured over a 60 min

lime penod. Data from al1 samples fit the mode1 equation Fu = (C l)x (T)/ (C2+T), where C 1 and C2

are constants. with a correlation coefficient A.996. The first five minutes were required for

fluorescence stabilization; thus the fifth minute was taken a s time zero (Fig.2.4a.). The fluorescence

dequenching was linear for next 5 minutes (Fig.2.4b.).

2.3.3. Determination of the optimal G u activator protein amount for the fluorescence

gieauenchin~ assav

To determine the optimal range of activator concentrations used in the fluorescence

dequenching assay. seven levels of the activator (0.75 pg- 12 pgl ml) were tested. Slopes from each

sample were calculated and plotted against the amount of activator added (Fig.23.) . The best

concentration range for the fluorescence dequenching assay was from 0.75 pg/ ml to 3 pg/ ml

(FigSb.); however linear results werc obtained for up to 5 pg./ ml (Fig.2.5a.).

Figure 2%:

The slope of fluorescence dequenching (see Methods), versus amount of activator presenf

(pg), over an extended range of activator concentrations with data points fit to the equation

"AFu/AT=C l x pg activatod (C2yrg ac tivator)" b y non-linear least squares (dashed Line).

pg Activator

Figure 2.5b.:

The linear range of fluorescence dequenching (dashed he) . The amount of the activator (pg),

open triangles, in a total reac tion volume of 1 ml is plotted versus the dope of fluorescence

dequenching. open triangles, (data points are fitted to die bat-f i t line by least squares analyses).

pg Activator

Figure S.6a. :

Effect of pH on the initial rate (over 5 minutes) of fluorescence dequenching by 1-3 pg/ ml of

the activator. The dopes of the lines. i.e. (MU/ AT)/ (Apg of activator). were determined to be: pH

4= 0.052, pH 4.5= 0.095, pH 5= 0.1 16, pH 6= 0.086. pH 7= 0.050.

O 0.5 1 1.5 2 2.5 3 3.5 pg Activator

Figure 2.6b.:

The dopes (over 5 minutes) of fluorescence dequenching determined at four different

concentrations of the activator, 0.75. 1.5, 2.25, and 3.0 pg/ml. which were fitted to the best-fit line

by least squares analyses, i.e. (Mu/ AT)/ (Apg of activator), versus pH.

2.3.4. Determination of the ogtimal pH

To optimize pH for the fluorescence dequenching assay, fou. concentrations of activator (0.75

pg, 1.5 pg, 2.25 pg and 3 pg ) were analyzed at pHs from 4 - 7.5. At pH 7.5 (data not shown), 7

and 4 (Fig.2.6a) fluorescence dequenching was significantly lower than that observed at pH 4.5,5

and 6 (Fig 2.6a). The optimal pH was determined to be pH 5. At pH 5 the slope was about 18% and

27% higher than the those at pH 4.5 and pH 6, respectively; while the slopes at pH 7 and pH 4 were

reduced 50% (Fig.2-6a and Fig.2.6b.).

The data from the fluorescence dequenching assay were reproducible for each preparation of

R- 18 labeled liposomes. However, it was noted that the slope for the same sarnple size assayed under

the same conditions slowly decreased with the age of the R-18 used to prepare the liposomes. For

example, one of the experiments shown in Fig.2.4b.. produced a slope of -0.6 for 3 pg of activator

at pH 5. while an identical experiment performed latter produced a slope of -0.35 (Fig.2.5b.).

2.4. DISCUSSION

Our expenments were designed to optimize the conditions for the fluorescent dequenching

assay with the G M ~ activator protein. This assay measures the activator's ability 10 transport the self-

quenching fluorescent lipid probe octadecylrhodarnine (R- 18). between liposomes.

When labeled and unlabeled liposomes were mixed, the increase of fluorescence was observed

only in the presence of recombinant human G M ~ activator protein (tïg.2.3.). A tmncated form of the

activator, prepared from the transformed bacteria in a manner identical to the functional activator and

observed to be non-functional in the Hex A ganglioside assay, was also non-functional in the

transport of R- 18 between liposomes. These data confirm that the assay is mesuring hydrophobie

binding "activity" of the recombinant human G M ~ activator protein.

We initialiy confmed that this transfer reaction foliowed saturation kinetics, assuming that an

acidic pH of 5 would be close to the pH optimum for the reaction (Fig. 4a). The time period

correspondhg to the Linear range for the transfer was then detemined to be -5 min (Fig. 4b). This

time period was shown to be sufficient for the determination of the activator protein's transport

activity as weIl as for fluorescence dequenching comparison between different samples. The

concentration range of the activator protein producing a linear response was found to be 0.75-5 pg

with 3 pg resulting in the highest response that retained the best linearity (Fig. 5a and b).

Fmally, we confïrmed that the optimal pH was pH 5, and demonstrated that the rates of R- i 8

transport at both pH 4 and pH 7 were only about 2 fold Iess than that at pH 5 (Fig. 2.6.).

Investigating GM:! ganglioside transfer between liposomes by the activator protein. Conzelmann et

al.. (Conzelmann et al. 1982) reported that the activator c m perform such transfer at pH 7.1. retaining

15% of its pH 4.2 rate (early to laie endosomal pH is 6-5-55}. The transport of the R- 18 lipid probe

by the activator ~ q u i r e s its binding to the activator's hydrophobic binding site. while the transport of

the GMZ ganglioside requires binding to two different binding sites, Le . its hydrophilic

oligosaccharide and hydrophobic cerarnide binding sites. We propose, that both R-18 and the

ganglioside ceramide portion bind to the same hydrophobic binding site of the G M ~ activator protein

(see Results. Chapter III). As the rates of transport of R-18 at pH 4 and pH 7 differ by a smdler

amount than those of G M ~ ganglioside (Conzelmann et al. 1982). it is possible that the G M ~ activator

protein's hydrophobic binding function is not as pH sensitive as its oligosaccharide binding function.

Furthemore. as the activator. in vitro, partially retains its transport function for both R- 18 and G M ~

ganglioside at pH 7, it is possible that it can also function at neutral pH in vivo. We propose that, in

vivo, it could act as a general glycolipid sorting ancilor transport protein (see Discussion. Chapter m).

The development of the fluorescence assay for the lipid transport activity of the G M ~

activator, provides a wide spectrum of possibilities for the structure 1 function studies of the activator

protein.

REFERENCES

Conzelmann E, Burg J. Stephan G, Sandhoff K (1982) Cornplexing of glycolipids and their

transfer between membranes by the activator protein for degradation of lysosomal ganglioside

GM2. Eur. J. Biochem. 123:455-464

Epand RM, Nir S, Parolin M, Fianagan TD (1995) The role of the ganglioside GD l a as a receptor

for Sendai virus. Biochemistry 34: 1084- 1089

Fürst W. Schubert J, Machleidt W. Meyer HE, Sandhoff K (1990) The complete arnino-acid

sequences of human ganglioside Gm activator protein and cerebroside sulfate activator

protein. Eur. J. Biochem. l92:7O9-7 14

Hoekstra D (1990) Fluorescence Assays to Monitor membrane Fusion: Potential Application in

Biliary Lipid Secretion and Vesicle Interactions. Hepatology 12:6 1 S-66s

Hoeksua D, de Boer T. Klappe K, Wilschut 1 (1984) Fluorescence method for Measuimg the

Kinetics of Fusion between Biological membranes. Biochemistry 235675-568 1

Hou Y. Tse R, Mahuran DJ (1996) The Direct Determination of the Substrate Specificity of the a-

Active site in Heterdirnenc ~Hexosaminidase A. Biochemistry 353963-3969

Klima H. Klein A, Van Echten G, Schwanmann G, Suzuki K, Sandhoff K (1993) Over-

expression of a functionally active human GM2-activator protein in Eschenchia coli. Biochem.

J. 29257 1-576

Kuwana T, Mullock BM, Luzio JP ( 1993) Identification of a protein capable of causing fusion of

endosorne and lysosome membranes. Biochem. Soc. Trans. 2 1 :299-300

Kuwana T, Mullock BM, Luzio JP (1995) Identification of a lysosomal protein causing Lipid

transfer, using a fluorescence assay designed to monitor membrane fusion between rat liver

endosomes and lysosomes. Biochem. J. 308:937-946

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of

Bacteriophage T4. Nature 227:680-685

Lowry OH, Rosenbrough NJ, F m AL, Randall RJ (195 1) Protein measurement with the Folin

phenol reagent. I. Biol. Chem. 193:265-275

MacDonald RC, MacDonald RI, Menco BP, M., Takeshita K, Subbarao NK, Hu L-R (1991)

Smd-volume extrusion apparatus for preparation of large, unilamellar vesicles. B ioc him.

Biophys. Acta 106 1 :297-303

Mullock BM, Luzio JP (1992) Reconstitution of rat liver endosorne-lysosome fusion in vitro.

Methods Enzyrnol 2 19:52-6O

Sandhoff K, Harzer K. Fürst W (1995) Sphingolipid activator proteins. In: Scriver CR, Beaudet

AL, Sly WS. Vaiie D (eds) The Metabolic Bais of Inherited Disease. Vol. 2. McGraw-HiU,

New York, pp 2427-244 1

Xie B. Mahuran D (1994) The GM2 activator protein does not play a critical role in endosorne and

lysosome membrane fusion. Biochem. Biophys. Res. Commun. 20190-93

Xie B, M c I ~ e s B. Neote K, Lamhonwah A-M. Mîhuran D (199 1) Isolation and expression of a

fuil-length cDNA encoding the human GMZ activator protein. Biochem. Biophys. Res.

C H A P T E R III

EVALUATION OF THE HYDROPHOBIC AND OLIGOSACCHARIDE BINDING

FUNCTIONS AND THE IDENTIFICATION OF A SECRETORY

FORM OF THE GM2 ACTIVATOR PROTEIN

Full length cDNA clones encoding the G M ~ ac tivator protein (activator) have b e n isolated by

ourselves and others (Xie et al. 199 1 ; Nagarajan et al. 1992). The deduced activator sequence

predicts a prepro-polypeptide c h a h of 20,808 daltons (residues 1-193). a fully functional pro-

polypeptide of 18,463 daltons (residues 24- 193) and a mature chain of 17.53 1 daltons (residues 32-

193). The activator d s o has a single site for Asn-linked glycosylation. Asn63-Val-Thr. The structure

of -95% of the human GMZA gene has been charactenzed and four exons identified (Klima et al.

1991). A second altematively mRNA spliced product containing exons 1-3 and intron 3 has been

identified. Due to the presence of a STOP codon early in the retained intmn 3 sequence. the product

of the alternatively spliced mRNA is a tmncated form of the activator protein containing residues 1-

142 and an additional three residues encoded by intron 3, Val-Ser-Thr (Nagarajan et al. 1992; Wu et

al. 1996).

The only fully documented physiological role of the activator is to present the G M ~

ganglioside to the lysosomal Hex A for degradation. To do this, the activator first interacts with the

hydrophilic oligosaccharide and the hydrophobic ceramide moieties of the ganglioside molecule. with

its specificity of binding primaily determined by the former. The binding of various gangliosides to

the activator decrease in following order: G M ~ » GMI> GDla= G M ~ = GA^ (Conzdmann et al.

1982). In vitro binding studies have indicated that the terminal N-acetylgalactosamine (GalNAc) and

intemal sialic acid (NeuAc) residues of G M ~ play an important role in binding. The resulting 1 to 1

activator: ganglioside complex must then specificaily interact with Hex A (reviewed in (Sandhoff et al.

1995)). Thus. the functional assay (the enhancement of &2 hydrolysis by Hex A) for the activator is

actually a measure of at least three separate activator functions.

The above functional dornains, Le. oligosaccharide binding site(s). the hydrophobic binding

pocket and Hex A recognition site, may residc within different portions of the activator protein. It has

been suggested from studies of the GM~A protein (cornposed of residues 1- 142, above). that the

COOH teminal part of the G M ~ activator could be involved in the interaction with the GalNAc

residue of the G M ~ ganglioside andlor Hex A and that the G M ~ A protein contains the hydrophobic

64

binding pocket for the interaction with the oerarnide residue of the ganglioside (see Discussion) (Wu et

al. 19%). Previously it had b e n suggested that one or more amphiphilic a-helices of the activator

protein may form a hydrophobie binding pocket for the ceramide moiety (Fürst et ai. 1990).

The in vivo mechanisms for the intracellular transport of gang liosides are unclear. In vitro,

the activator can remove labeled gangliosides from one liposome and insert them to another. The rate

of transfer was indirectly detexmined to be -p -> GD~$> G M ~ = GA^. Thus. one of the most

abundant brain gangliosides. GM~. is transferred at a preferential rate. Furthemore, at pH 7.1, the

activator retains 15% of its uansport activity observed at pH 4.2 (Conzelmann et al. 1982). Thus. it

is possible that the activator functions as a general sorting / transport protein For gangliosides in vivo.

Prosaposin was also suggested to have this function (Hiraiwa et al. 1992). However. the G M ~

activator transfers GM 1 at 145-fold the rate of prosaposin (reviewed in (Fürst and Sandhoff 1992)).

suggesting itself as a better candidate for this function than prosaposin.

The progress in understanding the mechanism of action of the G M ~ activator protein was

harnpered by the difficulty in isolating sufficient quantities of the activator protein from human

tissues. The availability of large quantities of recombinant activator has overcome this obstacle. To

date, recombinant G M ~ activator has been produced using different expression systems. It was

synthesized in transfected COS-1 cells using a pSVL-B fusion vector (Xie et al. 1991). and in

transformed E-Coli using pQE-9 hexahistidine (Klima et al. 1993) and pT7-7 systerns (Wu et al.

1994). Methods for the expression and purification of the activator protein, that have been set up in

our laboratory use both transfected CHO cells and transformed E.Coli. Both the pFLAG-1

(unpublished data) and pQE-8 expression vectors (originally developed by (Klima et al. 1993)) have

k e n used for the latter. We found that the bacterial activator is less efficient at assisting Hex A in its

hydrolysis of G M ~ ganglioside than one produced in transfected mammalian cells, but its high yield

and ease of purification overcorne its slightly reduced specific activity. Properties of three different

GM2 activator protein expression systems in E.Cofi . are summarized in Table 3.1.

Table 3-1,:

Methods for the production of the recombinant G M ~ activator protein in E.Coii

1 Expression 1 pQE-sa

purification colurm

1 Refoiding 1 ves

wash inclusion bodies G75

a. Contains a cDNA insert encoding the human mature G M ~ activator protein. with a

hexahistidine rnoiety and a Factor Xa cleavage site insertcd at its amino terminus (Klima et al. 1993).

chromatograph y yes 20-30

b. Contains a cDNA insert encoding the human mature activator protein with an additional

(amino terminal), 9 non specfic amino acids encoded by the plasrnid (Wu et al. 1994).

no 0.1-0.2

c. Contains a cDNA insert encoding the precursor of the human G M ~ activator protein with an

amino termina. extension that encodes the Omp A signal peptide and the FLAG peptide (antibody

binding site) (data from our laboratory).

d. Predicted yield from IL of bacterial culture.

The deficiency of the G M ~ activator results in an accumulation of G M ~ ganglioside in

lysosomes, producing the phenotype of the AB variant form of gangliosidosis. To date. four

mutations of the GM2A gene encoding the activator have been linked with this disease (Xie et al.

1992; Schroder et al. 1993; Schepers e t al. 1996). The activator with the C ~ S ~ ~ ~ A I - ~ substitution was

produced in Our laboratory from both transfected COS cells and E.Coli (pFLAG-1 and pQE-8

systerns). A cDNA construct containing the $lk transition ( ~ ~ s ~ 3 8 A r ~ ) . caused ttansfected COS-

1 cells to vanscribe high levels of activator's rnRNA, but cell lysates contained only low levels of the

pro-activator protein which failed to signifcantly enhance Hex A activity (Xie et al.. 1992). However.

when sufficient mutant activator produced in E.Coli was added to the assay a significant enhancement

of Hex A activity towards G M ~ ganglioside was observed (- 1% of normal).

The development of the fluorescence dequenching assay (Chapter II) provides us with a

means of assessing the stmcture / function relationships in the G M ~ activator protein. Firstly. the

finding that the activator can act as an R- 18 transfer protein between egg phosphatidylcholine (PC)

liposomes. as well as isolated endosornes and lysosomes (Kuwana et al. 1995) d low us to use the

fluorescence assay to test for the presence of the functional hydrophobic binding site in the G M ~

activator protein. Furthemore, various glycolipids inhibit R- 18 transport by the activator, i.e. R- 18

and ceramide are bound by the same site. and their degree of inhibition c m be used to assess the

activator's additional binding affinity for oiigosaccharide residues presenr on the glycolipids.

3.2. MATERIALS AND METHODS

R- 1 8 was from Molecular Probes. Egg phosphatidylcholine (PC) in chloro f o m was obtained

from Avanti Polar Lipids. Al1 endoglycosidases were obtained from B o e h ~ g e r Mannheim. Unless

othewise stated al1 other reagents were from Sigma Chernicd Co.

67

3.2.2.Ex~ression and Purification of Hi

Spthesis by E. Coli , purification. and refolding of the Hi%-G~yrz activator fusion protein

(MRGS(H)~GSIEGR-~32-1193)- and its mncated form (MRGS(H)~GSIEGR-s~~-L l57-

WSCPVGSPPGTTA) have been described in Chapter II.

The synthesis of the G M ~ activator proteins with a nonsense mutation at codons for either

cys183 or ~ ~ ~ 1 8 5 , was accomplished by Dr.Rigat in Our laboratory. The synthesis. purification and

refolding of the mutant proteins (Act 138 and Act 185) were perfonned following the same protocol

as described for the wild type H i s g - G ~ 2 activator protein (Chapter Il).

3.2.3 .The Expression and Purification of the -6-Activator conta in in^ a cyS1 38&

Subshniéo . .

n

A cDNA construct encoding a missense mutation associated with the AB variant f o m of G M ~

gangliosidosis ( cys13*~rg) , was originally constructed by Dr.Bei Xie (Xie et al. 1992). Later,

Dr.Ftigat in our laboratory, subcloned the cDNA into the Hisg EsColi expression vector. pQE-8

(Hisg-AB). Bnefly. Hisg-AB variant activator protein was produced using PCR to amplify the

coding sequence of the mature mutant activator protein from the pAct 1 M (pSVL expression vector

containing the activator cDNA with the T~~ 2~ transition) (Xie et al. 1992). in such a manner as to

allow the product to be inserted in the E-Coli expression vector pQE-8 (see Chapter II). The

purification of the AB variant activator protein from the host E-Coli MWpREP4 (Klima et al. 1993),

transfomed with the Ligation product and grown as a bulk culture from individual colonies. was done

as described for the wild type w s 6 - G ~ ~ activator protein in Chapter II.

3.2 4. Fluorescence Deuuenching Assg

Preparation of the phosphatidylcholine-containing unilamellar vesicles and R- 18 labeled

Liposomes were done as described in Chapter II.

Fluorescence dequenching assay was performed using standard arnounts of labeled and

unlabeled liposomes in 1 ml of SPM buffer ( s e Chapter II). with or without (baseline fluorescence) 4

pg (0.2 1 nmol. 0.2 1 CIM) of H i s g - G ~ 2 activator protein, AB variant activator or activator constmcts

with nonsense mutations at codons 183 and 185, respectively and the 4 pg of tmncated activator

(0.22 nmol. 0.22 PM). For the inhibition of fluorescence. 2 pg (1.3 nmol. 1.3 /LM) of G M ~

ganglioside was added to the liposome suspension in the presence or absence (baseline fluorescence +

G M ~ ) of various forms of the activator protein. respectively. For testing the activator's binding

affinity for giycolipids 2 pg (-1-2 nmol. 1-2 PM) of various ligands were added to the liposome

mixture in the presence (4 pg, 0.21 nmol. 0.21 PM) or absence of H i s g - G ~ 2 activator protein.

Fluorescence dequenching was measured and calculated as descnbed in Chapter II.

3.2.5 Analvsis of the Distribution of Activator Moiecules with a Hiph Mannose or a Cornplex

Oliggsaccharide Moiety

The analyses of the distribution of the oligosaccharides on the semi-punfied activator from

human fibroblasts were done by Ms. Amy Leung in Our laboratory. Her data are included in this

chapter for completeness.

3.2.5.1. isolation of a Semi-Purified Sample of Newly Synthesbed Activator from N o m 1 Human

Fibroblasts

Normal human fibroblasts were grown in aMEM (minimum essential medium. Gibco-BRL)

containhg 10% FCS and antibioûcs (24X Pl50 dishes) until they reached confluency. Then 10 m M

m l was added to the medium. After two days the medium was replaced by CHO SFM II (senun

free medium, Gibco-BRL) medium plus 10 m M W C I . The cells were grown. the medium coliected

and replaced at day 7 and 12 of culture. The pooled media (-IL) was cleared (cenuifuged) and

applied to a 4.5 ml Octyl Sepharose (Pharmacia) column. The colurnn was washed wilh 200 ml of 10

rnM Na phosphate buffer pH 6 containing 0.1M NaCl and eluted in the same buffer containing 0.75%

octylglucoside (1 column volume was passed through and column elution stopped for at least four

hours, then the elution was continued). The pooled fractions containing semi-purified activator

protein (ODao, total yield 1.3 mg) were concentrated to 200 pl in a Centricon- 10 (Amicon).

3.2.5.2. Glyosidase Digestion

The effects on the apparent Mr of the activator after treatment with each of three glycosidases

dissolved in 10 mM sodium phosphate buffer pH 5 were determined. The enzymes used were; a) N-

Glycosidase F. b) Endoglycosidase F (N-Glycosidase F-Free), c) Endoglycosidase H. Each

digestion was conducted in duplicate as followed. First. the denaturation of 2.5 pl (16 pg) of semi-

purified G M ~ activator (above) was accomplished by addition of 5 pl of denatunng buffer (1.2%

SDS. 3% pmercaptoethanol. 128 octyl-bglycoside). Depending on the enzyme used ("a-c" above),

the denatunng buffer was either a) not diluted, b) diluted 1:2. or c) diluted 1:4 wirh 10 m M sodium

phosphate buffer pH 5. Then the samples were heated at 600C for 20 min. and an additional 8 pl of

phosphate buffer was added. Second. the appropnate amount of each glycosidase a) 0.4 Units in 12

pl, b) 0.2 Units in 10 pl or c) 0.004 Units in 10 pl was added to the cooled samples. Third. to one of

each duplicate leupeptin. lpg/pl of reaction mix was added. Finally al1 samples were incubated

ovemight at 37OC . Each digestion was then analyzed by Western blot (Xie and Mahuran 1994).

3.2.5.3. Western Biot Analysis

AU sarnples were individually mixed with sample buffer containing 3% SDS and 25rnM DTï

and were boiled for 1 min. The proteins in each sample were separated by SDS-PAGE using the

Laemmli gel systern (12.5% gel) (Laemmli 1970). The proteins were transferred to nitrocellulose

overnight (Brown et aL.1989). Nitrocellulose was blocked by exposure to 5% skim milk in

BLO'ITO (10 m M of Tris base, 150 mM of NaCl. 0.05% of Tween 20, pH 7.5) for 4 h, with gentle

shaking and then was incubated ovemight with 1: MO dilution (1% skim m i k in BLOïTO) of rabbit

anti-glutathione-S-transferase i G M ~ activator fusion protein antiserum. Nitrocellulose was washed 4

x 30 min. with 1% skim milk in BLOTTO and was incubated with a 1: I0.000 dilution (1% skim milk

in BLOTTO) of horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h. The nitrocellulose

was then washed 4 x 15 min. with 1% skim milk in BLOTTO, quickly rinsed with BLOTTO (no

skim milk). and dried between filter papers. It was then incubated in equal volumes of Detection

reagent 1 and Detection reagent 2 (Amersham ECL system) for precisely 1 min.. dried briefly on filter

papers. covered with Saran wrap. and exposed to Hypedilm-ECL (Arnersham) for 1 min.

3.3.1. Determinat . . . . ion of the Rec an G m Activator Prote n Binding Affinitv for

Various Glycolinids and Gan~liosides

Lf the site for the R-18 binding is the same site used by the activator to bind ceramide,

glycolipids should inhibit its rate of R-18 transfer. Furthemore the degree of inhibition should be

directly related to the strength of binding as determined by the oligosaccharide structure of the

glycolipid. When varying amounts of G M ~ ganglioside were added to the fluorescence dequenching

assay containing the near maximum level activator protein (4 pg, Fig.2.5a), the rate of fluorescence

dequenching (AF, / AT) was decreased in a dose dependent manner (Fig.3.1.). In order to compare t

Figure 3.1.:

The initiai rate (over 5 minutes) of fluorescence dequenching caused by 4 pg of activator in the

presence of hcreasing amounts of G M ~ ganghoside (solid squares) fitted to a best-fit straight line by

leas t squares analyses (solid line).

2 2.5 3 3.5 4 pg GU, Ganglioside

Table 3.2-:

Inhibition of the activator (0.2 lw-ass i s ted transfer of R- 18 by 1.3 p M of various

glycolipids. assessed b y fluorescence dequenc hing .

Glycolipid Oligosaccharide Structure % Inhibition

G ~ 2 GdNAcp( 1 -4)Galp(l-4)Glc- 3 fi1

GM3 Gal[a(2-3)NeuAc- JB(1-4)Glc- 17 f4

Gb4 GalNAcB( 1 -4)Gala(l-4)Galp(1-4)Glc- 24 M

1. Standard deviation of the slope. calculated from least squares analysis for the best fit line.

Figure 3.2.:

Fluorescence dequenching expressed as % of total (see Methods). of R- 18 labeled liposomes

in presence of 4 pg of recombinant human G M ~ activator protein, open triangles. fit to a best-fit

straight line by least squares analysis (dashed lane), 4 pg of truncated activator. inverted open

triangles, (dotted bat-fit straight Line) and a negative control. solid squares (solid bat-fit straight

he).

// e( /

/ A

/

A' /

/

A/ /

b* '- - I I I 1

I I

O 1 2 3 4 5 Time (min.)

Figure 3.3.:

Fluorescence dequenching expressed as 96 of total (see Methods). of R- 18 labeled liposomes

in presence of 4pg of recombinant human Ghiy activator protein, open triangles, fit to a bat-f it

straight line by les t squares analysis (dashed iine), 4 pg of Act 183, open squares. (dotted best-fit

straight line) and a negative conuol. solid squares, (solid best-fit straight lane).

2 3 4 Time (min.)

Figure 3.4.:

Fluorescence dequenching expressed as 8 of total (see Methods), of R- 18 labeled Liposomes

in presence of 4 pg of recombinant human G M ~ activator protein, open triangles, fitted to a best-fit

straight Iine by Ieast squares analysis (dashed line), 4 pg of Act 185, open crossed squares, (dotted

best fit straight line) and negative conuol, solid squares. (solid best fit straight line).

2 3 4 Time (min.)

he influence of different monosido, trisialo and asialo glycolipids on the fluorescence dequenching of

R- 18 over tirne, a constant amount of each glycolipid, 1.3 nmol (1.3 pM, -2 pg), was added to 0.2 1

nrnol(0.21 pM, 4 pg) of the activator. The least fluorescence dequenching was observed after the

addition of ganglioside; thus as expected. G M ~ was bound with the greatest affinity (Table

3.2.). Cornparing the data from dl the glycolipids tested, the binding affinity of the activator is; G m

>> G ~ l b >> 2 Gb4 2 ( 3 ~ 3 > GAZ (Table 3.1 .).

3.3.2. Anal~sis of the Hvdrophobic Function of Diffgrent Recombinant Human G m Activ;\fpr

Protein Construc&

The COOH terminal part of the G M ~ activator protein could be involved in its hydrophobic

binding function. In order to investigate this possibility, lhree different constructs of the activator

protein were exmined by the fluorescence dequenching assay. First, the truncated fonn of the

recombinant human G M ~ activator protein lacking 36 COOH terminal arnino acids and then, hvo

constnicts made by point mutations ( s e Methods). producing STOP codon at either Cysteine 183 or

Lysine 185.

When 0.22 nmol(4 pg, 0.22 pM ) of the mncated activator was added to the mixture of R-18

labeled and unlabeled liposomes, the increase of fluorescence was only 4.9% of that caused by the

wild type activator (0.2 1 nmol, 4 pg) (Fig.3.2.). The 4 pg (0.2 1 nmol, 0.2 1 PM) of Act 183 and Act

185 produced 6.3% (Fig.3.3.) and 9.04% (Fig.3.4.) of the functionality of the wild type activator

protein, respectively. All three constructs were also examined in 12.5 fold higher amounts (-2.6

nmol, 50 pg. -2.6 PM). Each produced only a -2 fold higher fluorescence dequenching slope (data

not shown). The addition of G M ~ ganglioside (1.3 nmol, 2 pg or 3.3 nmol, 5 pg, respectively) to

the liposomes mixture in the presence of each mutant constmct . did not cause inhibition of the

fluorescence dequenching (data not shown). Taken together, these results suggest that the truncated

and mutated forms of the activator protein lack a functional hydrophobic binding site.

Figure 3.5.:

Fluorescence dequenching expressed as % of total (see Methods), of R- 18 laheled liposomes

in presence of 4 pg of recombinant human G M ~ activator protein. open triangles. fitted to a best-fit

straight line by least squares analysis (dashed line), 4 pg of recombinant human GMZ activator in

presence of 2 pg of G m ganglioside, solid triangles (dotted bat-fit straight iine) and negative

conuol, solid squares, (solid bat-fit straight line).

O 1 2 3 4 5 6

Time (min.)

Figure 3.6.:

The initial rate of fluorescence dequenching (over 5 min) in presence of 4 pg of AB variant

activator protein, open circles, fitted to a bat-fit straight line by least squares analysis (dashed line), 4

pg of AB variant activator in the presence of 2 pg of G M ~ ganglioside. solid circles, (dotted best-fit

straight h e ) and negative control. solid squares. (soiid best-fit straight line)

O 1 2 3 4 5 6 Time (min.)

3.3.3. Hvdroohobic Fwction of Activator Protein Con- a Point Mutation L' . . rnked to the

Ai3 Variant Forma-ned b~ w e n c e Dequenc p Assay . . .

hin

A mutant foxm of the activator protein, synthesized in transfected COS cells from the cDNA

constmct containhg the T--sC iramition in the codon for cys l38 (cys 8 3 ~ r g ) (Xie et al. 1992). as

weli as the same mutant activator produced in two E-Coli systems (using pFLAG-1 and pQE-8

expression vectors. Table 3.1.) enhance Hex A activity in the hydrolysis of G M ~ ganglioside. by

only -1% the level achieved by the wild type protein (unpublished data). Despite this fact, it is still

possible that the mutant activator has a functional hydrophobic binding site. To test this assurnption, 1

used fluorescence dequenching assay. After the addition of 0.21 nmol(4 pg, 0.21 PM) of AB variant

activator protein to the solution of R-18 labeled and unlabeled liposomes. an increase of fluorescence

was observed corresponding to 96.88 of that seen with the same amount of wild type activator

(Fig.3.5. and 3.6.). The addition of G M ~ ganglioside (1.3 nmol, 2 pg) to the mixture of liposomes

with AB variant activator (0.21 nmol, 4 pg), caused the inhibition of fluorescence dequenching of

792 (Fig.3.6.). The inhibited fluorescence was only 0.022 fluorescence units (Fu) higher than that

observed when the AB variant activator was replaced with the recombinant human G M ~ activator

protein (0.21 nmol. 4 pg) (Fig.3.5.). From the above data we can conclude that the AB variant fom

of the activator protein has a fully functional ganghoside binding site.

The location of the activator's hydrophobic binding pocket for glycolipids has not yet been

elucidated. Our working hypothesis was that the small activator protein could only contain one

hydrophobic binding site which interacts with glycolipids. gangliosides, or R- 18. This assumption

was confirmed by demonstrating that ganglioside produced a dose dependent inhibition of

fluorescence dequenching (Fig.3.1.). Furthemore, the addition to the assay of various glycolipids

produced different degrees of inhibition. These findings provided us an opportunity to directly

measure the activator's affinity for various ligands. Previously. such determinations have been made

86

indirectly, because of the need to pre-isolate the activator : ligand (radio-labeled) complex

(Conzelmann et al. 1982). Nevertheless these procedures have demonstrated the importance of the

intemal NeuAc and the terminal GalNAc residue for maximum activator binding affinity (reviewed in

(Sandhoff et al. 1995)). Our experiments confirm and extend these fîndings. As expected, we lound

that the activator binds G~v(2 ganglioside with the highest affinity nearly totally inhibiting the transfer

reaction (Table 3.2.). Interestingly. 1.3 nmo1 of G ~ l b ganglioside which has not been previously

examined, inhibited fluorescence dequenching by almost 6096. more than 2 fold the inhibition

produced by L .3 nmol of G M ~ ganglioside. These data suggest that an extra inner sialic acid may also

increase the sirength of activator's binding. Furthemore. we found that globoside. (also never

tested), and G M ~ ganglioside produce similar degrees of inhibition. This is of interest because;

whereas GA^ like GM contains a terminal GalNAc, but no NeuAc residue. GAZ was virnially non-

inhibitory in Our assay (Table 3.2.). Thus, it appears that the addition of an extra internal a-Gai

residue in increases its affinity for the activator. In sumrnary. the degrees of inhibition produced

by varioüs glycolipids and gangliosides on the R- 18 transfer assay were: %2>> G ~ l b > > G~12

G& G M ~ > GA^. The above data validate the use of the fluorescence dequenching assay for the

evaluation of the hydrophobic binding site in the activator. and the use of gangliosides andior

glycolipids inhibition of the assay to evaluate the oligosaccharide binding site(s).

The hydrophobic C-terrninals of the human and mouse G M ~ activators are highly conserved

between the two proteins. suggesting that they are cntical for activator's function (Bellachiorna et ai.

1993). To investigate the C-terminal part of the activator protein we used three different H i s 6 - G ~ 2

activator constnicts. Firstly, we exarnined its truncated form containing residues 32-157 plus an

additional 13 unrelated amino acids at its C-terminus. This construct should have similar properties to

the - 2 ~ protein produced by alternative mRNA splicing (truncated at residue 142 and containing 3

unrelated arnino acids). The G M ~ A protein. synthesized in bacteria and refolded, was reported to

retain its hydrophobic binding pocket and NeuAc recognition site based on; a) its ability to stimulate

the hydrolysis of G M ~ to GAZ by clostridial sididase. and b) its inability to stimulate the removal of

NeuAc from the oligosaccharide of the G M ~ . alone, i.e. without the ceramide moiety. in the presence

of the sarne enzyme. The G ~ A protein also Iacked the ability to stimulate the rernoval of the GalNAc

87

residue from G m by Hex A, suggesting it lacked either the GalNAc recognition site or its domain for

interacting with Hex A (Wu et al. 1996). In agreement with the latter property of the %A protein.

Our truncated protein did not enhance the hydrolysis of G M ~ by Hex A (unpublished resuits).

However, it also proved to be inactive in the fluorescent dequenching assay. indicating that the

hydmphobic binding site was either incomplete or non-functional.

Our results indicate the importance of the residues after codon 183 for the hydrophobic

binding function of the G M ~ activator protein. Both mutant f o m s of the activator, Act 183 and Act

185. were inactive in transporthg R-18 between Liposomes (Fig.3.3. and Fig.3.4.). There is only

one Cys residue lacking in the Act 183. The role of Cys residues in proteins, panicularly those

synthesized in the endoplasrnic reticulum. is often to f o m covalent intrachain disulfide bonds and

thus to stabilize the native structure of the polypeptide (Pelham 1989). The disruption of the

stabilization mechanism could result in an inability of the protein to fold properly. Interestingly. the

AB variant activator protein. with a mutation affecting Cys13? did not lack the lipid transport

activity. However, the mutant protein's ability to assist Hex A in hydrolysis of G M ~ was 75 fold less

than the normal activator's specific activity and its heat stability was dramatically reduced

(unpublished results). Thus. it is possible that the cys13*. or the novel cystine formed by it. does

not influence the conformation odand composition of the activator's hydrophobic binding site. but is

important in fonning the recognition site for Hex A.

As with prosaposin (Misasi et al. 1996; O'Brien et al. 1994) the G M ~ activator protein may

have other extracellular in vivo functions involving its ability to transport a variety of glycolipids.

The retention of this ability at neutrd pH (Fig.2.6.) which has also been previously noted by others

(Conzelmann et al. 1982: Kuwana et al. 1995). supports this hypothesis. However any extracellular

function for the activator would quire the secretion (rather than the incorporation into lysosomes) of

a significant portion of newly synthesized activator protein. The major signal for lysosomal

incorporation is one or more mannose-6-phosphate residues (M6P) linked to a high-mannose type

oligosaccharide (Kornfeld 1990). It was suggested that the activator is transported to the lysosomes

via the mannose phosphate receptors (MPRs). which recognize the M6P signal. because there was

2.5 fold increase in the amount of activator in the serum of 1-ce11 patient (Bane rjee et al. 1984).

Despite the presence of M6P signal. ceils are induced to secrete their newly synthesized lysosomal

proteins (dong with their nomally secreted proteins) when grown in the presence of NH4Cl (it

prevents the recycling of the mannose-6-phosphate receptors). Fibroblasts grown in the presence of

NH4Cl increased their secretion of the newly formed activator protein by only 2.5 fold (Burg et al.

1985). While supporthg the assumption of the ac tivator 's MPR dependent intracellular transport, the

fact that the other MPR-targeted enzymes are increased 10-20 fold under the same circumstances

(Creek et al. 1983) suggest that other pathways for lysosomal targeting may exist. We grew nomal

human fibroblasts in serum-free media containing 10 m M NH4Cl. and concentrated and semi purifted

the activator by Octyl-Sepharose chromatography (Conzelmann and Sandhoff 1979). Three sets of

duplicate samples were incubated with one of three endoglycosidases (glycopeptidase F (cleaves al1

types of Asn-linked oligosaccharides). endoglycosidase F free of any glycopeptidase F (cleaves

biantennary. but not tri- or tetraantennary complex type oligosaccharides). and endoglycosidase H

(cleaves high mannose and hybrid. but not complex type oligosaccharides)) and analyzed by Western

blotting (Fig.3.7.). The results indicate that the majonty of the activator molecules contain a complex

rather than a high mannose type oligosaccharide (Fig.3.7.). Such molecules would not contain the

mannose-6-phosphate signal necessary to divert them from the cells' secretory pathway (Kornfeld

1990). As well. a small amount of unglycosylated activator could be seen in the untreated lane;

indicating that glycosylation is not a prerequisite for the activator's transport out of the endoplasmic

reticulum (Fig.3.7.). Thus. there is a secretory form of the activator, which may have other

extracellular functions. Other experiments done in Our laboratory have identified a mannose-6-

phosphate independent pathway for the re-capture of secreted activator by cultured cells. This

pathway may explain the low level of steady state activator seen in the 1-ce11 semm and in the media of

cells grown in NH4CI. The use of the fluorescence dequenching assay with either different whole

ce11 types or membrane fractions from different tissues acting as inhibitors, may assist in identiQing

new in vivo functions for the activator.

Western blot of semi-purified activator protein from NH&-containing medium in which

normal human fibroblasts were grown. Sarnples were treated with either a) N-glycosidase F, Le.

glycopeptidase F (Glyco-F); b) endoglycosidase F. N-glgcosidase F-free (Endo-F); or c)

endoglycosidase H (Endo-H). Two samples were incubated with each glycosidase, one contained

leupeptin (+) and the other did not (-). A untreated sample is d so shown (None) dong with the

position of the glycosylated (+Oligo. Act) and unglycosylated (-Oligo. Act) forms of the activator.

Leupeptin - + - + - + O

Enzyme Giyco-F Endo-F Endo-H None _ -- .

cOligo. Act - -0ligo. A d -

REFERENCES

Bane rjee A, Burg I, Conzelmann E, Carroll M, Sandhoff K (1984) Enzyme-linked immunosorbent

assay for the ganglioside Gm-activator protein: screening of normal human tissues and body

Buids, of tissues of Gm gangliosidosis, and for its subcellular localization. Biol. Chem.

Hoppe-Seyler 365:347-356

Bellachiorna G, Stirling JL, Orlacchio A, Beccari T (1993) Cloning and sequence analysis of a

cDNA clone coding for the mouse GM2 activator protein. Biochem. J. 294:227-230

Burg J, Bane jee A, Sandhoff K (1985) Molecular forms of GM2-activator protein: a study on its

biosynthesis in human skin fibroblasts. Biol. Chem. Hoppe-Seyler 366:887-89 1

Conzelmann E Burg J. Stephan G. Sandhoff K (1982) Cornplexing of glycolipids and their

m s f e r between membranes by the activator protein for degradation of lysosomal ganglioside

GW2. Eur. I. Biochem. 123:455-464

Conzelrnann E, Sandhoff K (1979) Purification and characterization of an activator protein for the

degradation of glycolipids GM2 and GA2 by hexosaminidase A. Hoppe-Seyler's 2. Physiol.

Chem. 360:1837- 1849

Creek KE, Fischer D, Sly W (1983) Determinanis in the uptake of lysosomal enzymes by culturcd

fibroblasts. Methods Enzymol. 98:290-300

Fürst W. Sandhoff K (1992) Activator proteins and topology of lysosomal sphingolipid catabolism.

Biochim. Biophys. Acta 1 126: 1- 16

Fürst W. Schubert I. Machleidt W. Meyer HE. Sandhoff K (1990) The complete amino-acid

sequences of human ganglioside Gw activator protein and cerebroside sulfate activator

pro tein. Eur. I. Biochem. l92:7W-7 14

Hiraiwa M. Soeda S, Kishimoto Y. O'Brien JS (1992) Binding and m s p o n of gangliosides by

prosaposin. Proc. Natl. Acad. Sci. (USA) 89: 1 1254-1 1258

mima H, Klein A, Van Echten G, Schwanmann G, Suzuki K, Sandhoff K (1993) Over-

expression of a func tionall y active human GM2-ac tivator protein in Escherichia coli. B ioc hem.

J. 292:57 1-576

&a H, Tanaka A, Schnabel D, Nakano T, Schroder M, Suzuki K, Smdhoff K (1991)

Characterization of full-length cDNAs and the gene coding for the human GM2 activator

protein. FEBS. Lett. 289:260-264

Kornfeld S (1990) Lysosomal enzyme targeting. Biochem. Soc. Trans. 18:367-374

Kuwana T. Mullock BM. Luzio JP (1995) Identification of a lysosomal protein causing lipid

transfer. using a fluorescence assay designed to monitor membrane fusion between rat liver

endosornes and lysosomes. Biochem. J. 308:937-946

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of

Bacteriophage T4. Nature 227:68û-685

Misasi R, Sonce M, Carson GS, Griggi T. Lenti L. Pontien GM, O'Brien JS (1996) Prosaposin

and prosaptide, a peptide from prosaposin, induce an increase in ganglioside content on

NS20Y neuroblastoma cells. Glycoconjugate J 13: 195-202

Nagarajan S. Chen HC. Li SC. Li YT, Lockyer JM (1992) Evidence for two cDNA clones

encoding human GM2-activator protein. Biochem. J. 282:807-8 13

O'Brien JS, Carson GS, Seo HC, Hiraiwa M, Kis himoto Y ( 1 994) Identification of prosaposin as a

neurouophic factor. Proc. Nad. Acad. Sci. (U.S.A.) 9 1:9593-9596

Pelham RB (1989) Control of protein exit from the endoplasmic reticulum. Ann. Rev. Ce11 Biol.

5: 1-23

Sandhoff K, Harzer K. Fürst W (1995) Sphingolipid activator proteins. In: Scriver CR, Beaudet

AL, Sly WS, Valle D (eds) The Metabolic Basis of lnherited Disease. Vol. 2. McGraw-Hill,

New York, pp 2427-2441

Wu W, Lockyer IM. Sugiyama E, Pavlova NV, Li YT. Li SC (1994) Expression and specificity

of human GM2 activator protein. J. Biol. Chem. 269: 16276- 16283

Wu YY, Sonnino S, Li YT, Li SC (1996) Characterization of an altematively spliced GM2 activator

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10615

Xie B, Mahuran D (1994) The GM2 activator protein does not play a critical role in endosorne and

lysosome membrane fusion. Biochem. Biophys. Res. Commun. 201:90-93

Xie B, McInnes B. Neote K. Lamhonwah A-M. Mahuran D (1991) Isolation and expression of a

full-length cDNA encoding the human GM2 activator protein. Biochern. Biophys. Ra.

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associated with the AB variant form of GW gangliosidosis. Am. J. Hum. Genet. 50: 1046-

C H A P T E R IV

FUTURE WORK

4.1. X-RAY CRYSTALLOGRAPHIC STUDIES OF THE G M ~ A CTIVATOR PROTEIN

The detailed three dimensional structure of the G M ~ activator protein could be estabüshed by

the x-ray crystallography. The large yield of the biologicdy functional His-6 activator protein (25-30

m a culture) and subsequent Octyl glucose purification. for obtaining only properly folded activator

protein molecules. should be of sufficient quantity and quality to altow the initial expenments on

forming activator crystals. If crystals can be formed, x-ray crystallographic studies will follow.

Furthemore, the different physical arrangement of the activator protein chains bound to the G M ~

ganglioside could be revealed by the sarne rnethod. facilitating the determination of the hydrophobic

and oligosaccharide binding sites of the G M ~ activator protein.

4.2. DETERMINATION OF THE R-18 / (;ANGLIOSIDE B INDING S ITE OF THE G M 2

ACTNATOR PROTEIN BY MUTAGENESIS

Once the putative ganglioside binding dornain is identified by crystailognphy. conservathe

point mutations can be made that will change charge and hydrophobicity of various residues in the

G M ~ activator protein. to confirm the identity of the amino acid c h a h critical for the activator's

hydrophobic binding function. It is expected that the binding site of the G M ~ activator protein will

have hydrophobic characier consistent with its function in solubilizing the G M ~ molecul: (reviewed in

(Fürst and Sandhoff 1992) ). Predicted secondary structure of the human activaior protein contains

three hydrophobic a helices which have been suggested as sites for ganglioside binding (Fürst et al.

1990). Recently, the secondary stmcture predictions showed that only one of these three a helices is

conserved in both mouse and hurnan sequences, suggesting that it is critical for the activator's

function. This lies in the sequence bounded by pro79 and pro98 of the human and mouse proteins

(Beliachioma et al. 1993).

In order to identify the R-18/ganglioside binding site of the human G M ~ activator protein, the

conservative point mutations affecting the hydrophobicity of this particular a helix could be made

followed by the isolation of the mutant His-6 activators from transformed E.Coli (Chapter II).

96

Fiuorescence dequenching assay (Chapter II) and if possible x-ray crystallography analyses would

detemine if this area is involved in gangfiosid&-18 binding. If any of these residues is critical to the

domain's ability to bind R- lS/ganglioside. it could be expected that the mutant protein (s) will noi be

able to cause the increase of fluorescence in fluorescence dequenching assay but would retain its

normal folding pattern.

4.3. IDENTIFICATION OF THE HEXOSAMINIDASE A BINDING SITE OF TAE G M 2

A CTIVATOR PROTEIN THROUGH THE EXPRESSION OF THE M OUSE / H U M AN

FUSION PROTEIN

Recently. a cDNA containing the complete coding sequence for the mouse G M ~ activator

protein was cloned and sequenced. Comparison of these data with those from the human cDNA.

demonstrate a high degree of identity in the deduced protein sequence (Yarnanaka et ai. 1994). The

alignment of the mouse and human deduced amino acid sequences were shown to contain 68% of

identical residues (Bellachioma et al. 1993). The mouse G M ~ activator protein function is shown to

be the same as for the human G M ~ activator protein. thus enhancing the degradation of the G M ~

ganglioside by the mouse Hex A.

Work in Our laboratory has shown that CHO ce11 Hex A can not interact with human G M ~

activator protein. thus it is possible that mouse activator andlor Hex A may not be able to intenct with

their human counterpm. In order to identify the Hex A binding site of the G M ~ activator, different

mouse/human fusion proteins could be used. Pnor to fusion. the extent to which mouse and human

activator proteins promote ganglioside G M ~ catabolism by autologous and heterologous

hexosaminidases will be examined. These wild type activator proteins could be synthesized in

transforrned E. Colli using pQE-8 hexahistidine system (Chapter II). If either one or both activator

proteins prove to be inactive (or show low activity compared to the species specific wild type) in

assisting the G M ~ degradation by heterologous Hex A. fusion could identify the mouse/human

domain responsible for the species-specific binding. The mouse/human fusion proteins could bé

made by the PCR three primer method (Tse et al. 1996) and produced in the same manner a s the wild

type activators. The fusion proteins could be than examined for their functionality in enhancing the

degradation of G M ~ in presence of mouse or human Hex A (depending on the results from

preliminary experiments mentioned above). as well as for the R-18 transport function in the

fluorescence dequenching assay which would serve as a control for proper folding of the activator

protein. The obtained data will point to the possible activator residues involved in the Hex A and

hydrophobic binding functions.

REFERENCES

Beilachioma G, Stirling JL, Orlacchio A, Beccari T (1993) Cloning and sequence analysis of a

cDNA clone coding for the mouse GM2 activator protein. Biochem. J. 294:227-230

Fürst W. Sandhoff K (1992) Activator proteins and topology of lysosornal sphingolipid catabolism.

Biochim. Biophys. Acta 1 126: 1- 16

Fürst W. Schubert J, Machleidt W. Meyer HE, Sandhoff K (1990) The complete amino-acid

sequences of human ganglioside Gw activator protein and cerebroside sulfate activator

protein. Eur. J. Biochem. l92:709-7 14

Tse R. Wu YJ, Vavougios G, Hou Y. Hinek A, Mahuran DJ (1996) Identification of Functional

Domains wilhin the a and p Subunits of P-Hexosaminidase A Through the Expression of a-p

Fusion Pro teins. B iochemistry 35: 10894- 10903

Yarnanaka S. Johnson ON, Lyu MS. Kozak CA, Proia RL (1994) The mouse gene encoding the

GM2 activator protein (Gm2a): cDNA sequence, expression. and chromosome mapping.

Genomics X:6O 1-604

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