THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biocbemiatry and Molecular Biology, Inc. Vol. 268, No. 18, Issue of June 25, pp. 13242-13247,1993

Printed in U.S.A.

Characterization of Neutral Glycosphingolipids in Human Cataractous Lens*

(Received for publication, October 6, 1992, and in revised form, February 5, 1993)

Manabu Ogiso$§, Atsushi IrieV, Hideo Kubon, Michiji Komoto[[, Toshiyuki Matsuno**, Yuji Koide**, and Motonori Hoshin From the Departments of $Physiology and ~~Ophthulmology, Toho University School of Medicine, Tokyo 143, Japan, the TDepartment of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 227, Japan, and **Research Laboratory, Zenyaku Kogyo Co. Ltd., Tokyo 178, Japan

Neutral glycosphingolipids were purified from hu- man senile cataractous lenses by a combination of sol- vent extraction, Folch’s partition, acetylation, and col- umn chromatography using DEAE-Sephadex and Ia- trobeads. Six major glycosphingolipids (A-F) from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis, secondary ion-mass spectrometry, glycosi- dase digestion, and chromium trioxide oxidation. Their structures suggested that they were closely related in their metabolism: their sugar chains were in sequence and their ceramide moieties were similarly composed, namely C16:O and C24:l constituted most of the fatty acids, and long-chain base components were mostly C18-dihydrosphingosine with a small portion of C18- sphingosine. The sugar chains implied two pathways branching from lactosylceramide: one to globotriao- sylceramide and the other to lactotriaosylceramide, which leads to the production of Le’ glycolipid via neolacto type 2 core chain.

The vertebrate lens is composed of a monolayer of epithelial cells and multiple layers of fiber cells, which accumulate into the lens nucleus throughout life (1). Therefore, the lens is rich in plasma membranes and is a suitable tissue for their purification. It has been suggested that glycosphingolipids (GSLs)’ localized in the plasma membrane surface are in- volved in the differentiation and maturation of epithelial cells to fiber cells (2). Substantial changes in lens GSLs may cause disordered function of the plasma membrane. A few attempts

* This study was supported by Grant-in-aid 03255218 for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan and by the Kowa Life Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 To whom correspondence should be addressed Dept. of Physi- ology, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohta-ku, Tokyo 143, Japan. Tel.: 03-3762-4151 (ext. 2345); Fax:

The abbreviations used are: GSL, glycosphingolipid; Le’, Lewis’; CMH, monohexosylceramide; CDH, dihexosylceramide; CTH, trih- exosylceramide; CTeH, tetrahexosylceramide; CPH, pentahexosyl- ceramide; HP-TLC, high performance thin layer chromatography; HPLC, high performance liquid chromatography; GLC, gas-liquid chromatography; Fuc, fucose; GC-MS, gas chromatography-mass spectroscopy; SIMS, secondary ion-mass spectrometry. Glycolipids are abbreviated according to the recommendations of the IUPAC- IUB Commission on Biochemical Nomenclature (37), but the suffix OseCer is omitted. Ganglio-series gangliosides are abbreviated ac- cording to Svennerholm (38).

03-3761-0546.

have been made to analyze lens neutral GSLs and ganglio- sides, but the data that have been obtained are extremely limited and fragmentary (2-11).

We have found an age-dependent, cataract-related increase in the ganglioside content of human senile cataractous lenses (12). Monkey non-cataractous lenses showed a similar age- dependent increase in the content of gangliosides (13). We recently isolated and identified a Le’ glycolipid, Gal@l-4(Fucal-3)GlcNAc~l-3Galpl-4Glcpl-lceramide (II13FucnLc4), from human cataractous lenses (14). This find- ing prompted us to examine the possibility that the Le” glycolipid accumulates in relation to the initiation and devel- opment of cataracts by a mechanism such as the following. As lens cells accumulate the Le’ glycolipid, cell-to-cell binding mediated through Le’-Le’ interactions would be strengthened, as suggested in embryonic adhesion events mediated by stage- specific embryonic antigen-1 (15-17). The binding might gradually become too strong to maintain the normal functions of lens plasma membranes, eventually leading to cataracts.

As a first step in examining this possibility, we purified several neutral GSLs from human senile cataractous lenses and proposed a synthetic pathway of these GSLs from their sugar chain sequences.

EXPERIMENTAL PROCEDURES

Materials-Human cataractous lenses were obtained from the op- erating room of Toho University Hospital following intra- or extra- capsular cataract extraction and kept at -70 “C until use. a-Galac- tosidase from green coffee beans, @-galactosidase from jack beans, and @-N-acetylglucosaminidase from jack beans were purchased from Sigma. DEAE-Sephadex A-25 was obtained from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). Iatrobeads 6RS-8060 and 6RSP-6010, and GSL standards were from Iatron (Tokyo, Japan). Polyclonal anti-aSialOGMn and anti-asialoGMl antisera were the kind gifts of Honen Corp. (Tokyo, Japan). The fatty acid methyl ester standards were products of Supelco Inc. (Bellefonte, PA), and the partially methylated alditol acetate standards were from Bio-Carb (Lund, Sweden).

Extraction and Purification of Neutral GSLs from Human Lenses- Neutral GSLs from 250 lens tissues removed by intracapsular extrac- tion were separated and partially purified by solvent extraction, DEAE-Sephadex A-25 column chromatography, and Iatrobeads 6RS- 8060 column chromatography as described previously (14). After acetylation of separated GSL fractions, they were further purified by HPLC on an Iatrobeads column (RSP-8010,4 mm inner diameter X 50 cm) as follows (18). The samples were dissolved in a small amount of chloroform/methanol (2:1, v/v) and injected. The column, previ- ously equilibrated with hexane, was eluted at a flow rate of 1 ml/min with a combined linear gradient between hexane (A) and isopropyl alcohol/hexane/water (43:56:1, v/v/v) (B). The ratio of B was in- creased from 0 to 50% for the first 20 min, and then to 100% for the next 40 min. Acetylated GSLs were monitored at 206 nm and checked by TLC using the solvent system of 1,2-dichloroethane/acetone/water (60:40:1, v/v/v) and orcinol spray. CMH was recovered from the

13242

Le” Glycolipid in Humun Lens 13243

fractions eluted between 20 and 27 min, CDH from those between 30 and 35 min, CTH from those between 37 and 41 min and 45 and 50 min, CTeH from those between 51 and 55 min, and CPH from those between 44 and 48 min. The pooled fractions were deacetylated in sodium methoxide in methanol. CMH, CDH, two CTHs in the order of elution, CTeH, and CPH are designated A, B, C, D, E, and F, respectively, in this report. As described later, CTeH was actually contaminated with CTH.

Carbohydrate and Fatty Acid Analyses-The purified GSLs were methanolyzed in 1 N methanolic HCI at 80 “C for 16 h. Methylgly- cosides and fatty acid methyl esters were analyzed by GLC (Shimadzu GC-7A) equipped with a fused silica capillary column (0.24 mm inner diameter X 25 m) of ULBON HR-101 (Chromato Packings Center, Tokyo) or GC-MS (Shimadzu QP-2OOOA) with the same column as described previously (14).

Methylation Analysis-The GSLs, permethylated according to the method of Hakomori (19), were acetolyzed, hydrolyzed, reduced with NaBH,, and acetylated (20). The partially methylated alditol acetates were analyzed by GLC and GC-MS as described elsewhere (21).

Chromium Trioxide Oxidation-The acetylated GSLs, together with acetylated mannitol as an internal standard, were oxidized with chromium trioxide in acetic acid, and analyzed as alditol acetates by GLC (21,221.

Long-chain Base Analysis-The GSLs were hydrolyzed in 1 N aqueous methanolic HCI at 70 “C for 18 h according to the method of Gaver and Sweeley (23). The resulting free bases were N-acetyl, 0-trimethylsilylated and were analyzed by GLC and GC-MS as described elsewhere (21).

Glycosidase Digestion-The GSLs were incubated in 200 pl of 0.1 M citrate buffer, pH 5.0, containing 250 pg of sodium taurodeoxycho- late (Sigma) and 20 milliunits of several glycosidase preparations, a t 37 “C for 16 h (14). The hydrolyzed GSLs were recovered from Folch’s lower phase (24) and analyzed by TLC on precoated HP-TLC plates (Merck 5641, Merck, Darmstadt, Germany) using chloroform/meth- anol/water (65:254, v/v/v) as the solvent.

Negative Ion Secondary Ion-Mass Spectrometry (SIMS)-The GSLs were analyzed in a matrix of triethanolamine by negative ion SIMS using a Hitachi mass spectrometer M-80B. The conditions were as follows: Xenon beam, 3 kV; filament emission current, 3.8 mA; accelerating voltage, 8 kV.

Imrnunostaining of Lens Neutral GSLS on Thin-layer Plates-An aliquots of neutral GSLs, which were partially purified as described above, was developed on silica gel plastic TLC sheets (Polygram Si1 G, Macherey-Nagel, Duren, Germany) in chloroform/methanol/water (65:25:4, v/v/v). TLC-immunostaining using anti-asialoGM2 and anti- asieloGM, antisera was performed as described previously (13, 14). Immunodetection was done by horseradish peroxidase-conjugated goat anti-rabbit I& antiserum. Asialo& and asialo& standards were obtained by acidic hydrolysis of GMa and G M l purified from human cataractous lenses.’

Compositional Changes in CTH of Individual Lenses-The neutral GSL fraction was extracted from individual lenses of 16- to 80-year- old subjects and partially purified by the following steps: chromatog- raphy on a DEAE-Sephadex column and an Iatrobeads 6RS-8060 column, mild alkaline hydrolysis, and Folch’s partition (14). An aliquot of GSLs from each lens was separated on an Empore TLC silica sheet (Analytichem International, Harbor City, CA) in a solvent system of chloroform/methanol/water (65:254, v/v/v). The band corresponding to CTH was cut off from the sheet after iodine vapor exposure and extracted in chloroform/methanol (2:1, v/v) overnight. After acetylation, CTH was developed on the HP-TLC plate in a solvent system of 1,2-dichloroethane/acetone/water (6040:1, v/v/v) and detected by orcinol spray.

RESULTS

Purification of GSLs from Human Cataractow Lenses-The neutral GSLs in human lens tissues were separated by map- ping on the column of Iatrobeads (Fig. 1). Five groups (1-5) of GSLs were distinguished between CMH and CPH on the basis of TLC mobilities, and pooled separately. Contaminants, presumably phospholipids, in CTeH and CPH (Fig. 1, arrow- heads) were removed by mild alkaline hydrolysis and Folch’s partition. After HPLC separation of acetylated derivatives on

M. Ogiso, T. Okinaga, M. Ohta, M. Komoto, and M. Hoshi, unpublished data.

-cMn

‘CQH

- c w -Glob

1 20 Lo 60 80 FRACTION NUMBER

FIG. 1. A typical elution pattern of neutral CSLs from hu- man cataractous lens tissues on an Iatrobeads column. The GSLs were eluted from the column (1.7 X 80 cm) with a linear gradient of chloroform/methanol/water from 85:15:0.5 to 20805 (v/ v/v). An aliquot of the fraction was developed on an HP-TLC plate in a solvent system of chloroform/methanol/water (65:25:4. v/v/v) and detected with orcinol reagent. Arrowha& indicate non-GSL contaminants. I , CMH; 2, CDH; 3, CTH; 4. CTeH; 5, CPH; Glob, globotetraosylceramide, Gb4.

””-”

A B C D E F s t FIG. 2. TLC pattern of GSLs purified from human lens tis-

sues. An aliquot of purified GSLs: CMH (A). CDH (R) . CTH (C), CTH (D), CTeH (E). and CPH (F), was developed on an HP-TLC plate and detected with orcinol reagent, as shown in Fig. 1.

an Iatrobeads 6RSP-8010 column, six GSLe (A-F) were pu- rified as demonstrated by HP-TLC (Fig. 2). CSLs (A-F) purified from 250 lens tissues weighed approximately 0.25, 0.90, 0.70,0.20, 0.25, and 0.25 mg, respectively. CU: Analysis of Sugar Moieties of Lens GSh-The sugar

composition of the purified GSLs (A-F) was determined by GLC analysis of the 0-trimethylsilyl derivatives of methylgly- cosides (hexosamines were analyzed after N-acetylation) (Fig. 3). The relative ratios of the sugars, estimated from their areas on gas-liquid chromatograms, are summarized in Table I. Fucose was detected only in CPH (F), and the peak area ratio of Fuc to Glc was 0.49. Trace yet meaningful amounts of the derivatives of GalNAc were observed in CTH (D) and CTeH (E). On the basis of their sugar composition, CMH (A), CTH (D), and CTeH (E) were presumed to be mixtures of at least two closely related GSLs.

Methylation Analysis of Lens CSLs-Partially O-methyl- ated alditol acetates derived from permethylated GSLs were analyzed by GLC (Fig. 4). The presence of terminal Glc and Gal residues was revealed in CMH (A), as predicted from i t s sugar composition (Fig. 3A and Table I). Methylation analysis of CDH (B) and CTH (C) showed the presence of terminal Gal and 4-0-substituted Glc, and terminal Gal, 4-0-substi- tuted Gal and 4-0-substituted Glc, respectively. CTH (D) showed the presence of a mixture having two different ter- minal amino sugars, GlcNAc and GalNAc, in addition to 3- 0-substituted Gal and 4-0-substituted Glc. CTeH (E) showed the presence of terminal Gal and GalNAc, 4-0-substituted GlcNAc, 3-0-substituted Gal, 4-0-substituted Clc, and 4-0- substituted Gal. CPH (F) corresponds to the pattern of the Le’ glycolipid, Galp1-4(Fucal-3)ClcNAcBl-3Gal~1-4GlcB1- lceramide (II13FucnLc4), as described elsewhere (14). In Fig. 4 (D and E ) , the peak with a retention time of about 5 min

13244 Le" Glycolipid in Human Lens

D

C I F I

i 0 12 i6 ' i i r i is RETENTION TIME (mid

FIG. 3. Gas-liquid chromatograms of 0-trimethylsilyl de- rivatives of methylglycmides from lens GSLs (A-F). The sugar derivatives were analyzed by Shimadzu gas chromatograph GC-7A on a fused silica capillary column of ULBON HR-101 (0.24 mm X 25 m, Chromato Packings Center, Tokyo, Japan) at a temperature programmed at 2 "C/min from 170 to 230 'C. I , Gal; 2, Glc; 3, GalNAc; 4, GlcNAc; 5, Fuc.

TABLE I Carbohydmte mtios of neutral GSLS isolated from human cataractous

lenses Peak area ratios are expressed relative to peak areas of glucose.

Glycolipid Fuc Gal Glc GalNAc GlcNAc

CMH (A) 0.60 1.00 CDH (B) 0.67 1.00 CTH (C) 1.68 1.00 CTH (D) 0.86 1.00 0.11 1.45 CTeH (E) 1.38 1.00 0.95 1.23 CPH (F) 0.49 1.85 1.00 0.99

was not terminal Fuc, because the mass fragmentation pat- terns differed from those of terminal Fuc. Instead, a series of fragments with a CHZ-unit (m/z 14) difference was observed, suggesting that this peak represents a contaminant with a hydrocarbon chain (data not shown). The appearance of ter- minal GalNAc residues in CTH (D) and CTeH (E) coincided with the results of the sugar composition analysis (Fig. 3).

Anomeric Configuration of Glycosidic Bonds and Sugar C h i n Sequence of Lens GSLs-Chromium trioxide oxidation of neutral GSLs (22) suggested that only Fuc in CPH (F) and one of the Gal residues in CTH (C) were linked a-glycosidi- cally (their yields were 60.1 and 76.6%, respectively). While the yield of Fuc was lower than expected, a-fucosidase suc- cessfully released Fuc residue from CPH (F) (14). More than 90% of the other Glc and Gal residues except for CTH (C) were destroyed after oxidation, suggesting &linkages. All GalNAc and GlcNAc residues disappeared after oxidation.

To examine the sugar chain sequence of GSLs (B-E), about 1-2 pg of each GSL was incubated with various glycosidases in the presence of sodium taurodeoxycholate (Fig. 5). CDH (B) was not affected by a-galactosidase but was completely hydrolyzed by @-galactosidase to CMH. The structure of CDH was determined to be a lactosylceramide, GalS1-4GlcSl- lceramide. The terminal Gal residue of CTH (C) was partly removed by a-galactosidase under the conditions used, but

4 b l2 16 20 24 b 3 12 l 6 2 0 2 b RETENTION TIME (mid

FIG. 4. Gas-liquid chromatograms of partially methylated alditol acetate derivatives from lens GSLs (A-F). Column tem- perature was 160 "C for the initial 16 min and subsequently increased by 4 "C/min to 200 'C. 1. Clcl-; 2. Gall-; 3, -4Glcl-; 4, -Gall-; 5 , - 3Gall-; 6, ClcNAcl-; 7, GalNAcl-, 8, -4GlcNAcl-; 9, Fucl-; IO. - 3,4GlcNAcl-.

Cot4

mCTH Glob

1 2 3 1 2 3 1 2 3 LBA LCA

FIG. 5. Glycosidase digestion of lens GSLs (B-D). Each GSL (1-2 pg) was incubated with 20 milliunits of enzyme preparation and 250 pg of sodium taurodeoxycholate in 0.1 M citrate buffer at 37 'C for 16 h. The hydrolyzed GSLs were separated by Folch's partition, and the lower organic phase was analyzed by HP-TLC with the same solvent system as in Fig. 1. R and C: 1, no enzyme control; 2, Q-

galactosidase; 3, 8-galactosidase. D 1 , no enzyme control; 2, 8-N- acetylglucosaminidase; 3, 8-N-acetylglucosaminidase plus 8-galacto- sidase.

not by /%galactosidase, and further addition of &galactosidase produced CMH (data not shown). Incubation with an in- creased amount of a-galactosidase removed the terminal Gal residue of CTH (C) almost completely (data not shown). The Gala1-4Galj31-4Clc-R structure was also confirmed by TLC- immunostaining using monoclonal anti-Pk antibody (Bio- Carb) as described elsewhere.3 Terminal GlcNAc and GalNAc residues of CTH (D) were completely removed by 8-N-ace- tylglucosaminidase, and the resulting CDH were hydrolyzed to CMH by the addition of &galactosidase. Thus, CTH (D) was probably composed of GlcNAcS1-3GalB1-4Glc~l- lceramide (Lc3) and a trace of GalNAcS1-3GalB1-4Clc~l- lceramide.

Enzymatic hydrolysis of CTeH (E) demonstrated the pres- ence of two compounds with different carbohydrate chains (Fig. 6) as suggested by sugar composition and methylation analyses. /3-Galactosidase hydrolyzed the lower bands of CTeH ( l a n e 3). The resulting CTH was further hydrolyzed by 8-N-acetylglucosaminidase and finally converted to CMH by the addition of &galactosidase (lanes 4 and 5 ) . 8-N- Acetylglucosaminidase treatment alone also produced a small

3M. Ogiso, M. Ohta, A. !ne. M. Hoshi, 1. Niahiyama, and M. Komoto, unpublished data.

Le" Glycolipid in Human Lens 13245

amount of CDH because of contaminating @-galactosidase activity (lanes 6 and 7).

To examine the heterogeneity of CTeH, @-galactosidase- treated CTeH was separated to CTH and CTeH on an HPLC Iatrobeads column after acetylation as described above. Meth- ylation analysis showed the presence of terminal GalNAc, trace terminal Gal, 4-0-substituted GlcNAc, 3-0-substituted Gal, 4-0-substituted Glc, and 4-0-substituted Gal in the re- maining CTeH. In turn, terminal GlcNAc, 3-0-substituted Gal, and 4-0-substituted Glc, but no 4-0-substituted Gal, were found in the CTH released from CTeH (data not shown).

..

, *coH

""""

1 2 3 4 5 6 7 "E-

FIG. 6. Glycosidase digemtion of CTeH (E). Hydrolysis of CTeH was carried out as described in Fig. 5, and the products were analyzed by HP-TLC. 1, no enzyme control; 2, a-galactosidase; 3, 8- galactosidase; 6, 8-N-acetylglucosaminidase for 24 h; 7, 8-N-acetyl- glucosaminidase for 40 h. After 8-galactosidase was eliminated by Folch's partition, 8-galactosidase-treated CTeH was further hydro- lyzed with 8-N-acetylglucosaminidase ( 4 ) and 8-galactosidase plus 8- N-acetylglucosaminidase (5).

a

*Gg3

"1 *CMH

CI

N N st

mCDH

CfH

'Glob

FIG. 7. Expreseion of asialoGm (Gg3) in neutral CSL frac- tion. a, neutral GSLs prepared from p o o l e d cataractous lenses ( N ) were developed on plastic TLC sheets using the same solvent as in Fig. 1. The sheets were immunostained with anti-asialoGM2 and anti- asialo& antisera. Desialylated derivatives of G M ~ and GMI were used for positive controls (Gg3 and Gg4). b, neutral GSL standards (s t ) were developed in a parallel lane and detected with orcinol reagent.

It was therefore postulated that CTeH (E) is a mixture of Gal@1-4GlcNAc@1-3Ga1@1-4Glc~l-lceramide (nLc4) and GalNAc@1-4Gal@1-4Glc~1-lceramide (asialo&, Gg3), al- though the co-existence of GalNAcp1-4GlcNAc@l-3Gal1!3l- 4Glcp1-lceramide cannot be ruled out. In fact, authentic asialoGM2 (Gg3) had a TLC mobility similar to that of the upper bands of CTeH (E), which were incompletely hydro- lyzed by @-N-acetylglucosaminidase ( d a t a not shown). The presence of asialoGM2 (Gg3) was immunologically confirmed in neutral GSLs, but asialoGM, (Gg4) was not detectable (Fig. 7).

From these data, a hypothetical pathway of GSL synthesis in human lens was proposed (Fig. 8). AsialoGMz (Gg3) may also originate from the desialylation of ganglio-series ganglio- sides, but the details remain unknown. Thus, human lens GSLs consisted mainly of globo-series and neolacto-series GSLs, leading to the synthesis of Gb3 and III"FucnLc4. re- spectively.

Ceramide Composition of Lens GSLs-Fatty acid methyl esters obtained by methanolysis of lens GSLs were examined by GLC (Table 11). Palmitic (C16:O) and nervonic (C24:l) acids were detected as the principal fatty acids in all GSLs. Only CTH (C) contained C14:O and C180 at a ratio of more than 10% of the total fatty acid moiety. No hydroxy fatty acids were seen in A-F by selected ion monitoring at mlz 90 using GC-MS.

The long-chain bases were analyzed by GLC and GC-MS as the N-acetyl derivatives of 1,3-di-O-trimethylsilyl ethers. In all GSLs, C18-dihydrosphingosine was predominant (7s- 85% of total long-chain base) and C18-sphingosine was ap- preciably detected. Other long-chain bases were present in trace amounts or not detectable.

Negative Ion SIMS of Lens Neutral GSb-SIMS of lens GSLs (A-F) showed two dehydrogenated molecular ions (M- H)-, which corresponded to molecular species containing a fatty acid of C16:O or C24:l and C18-dihydrosphingosine (Fig. 9). The mass spectra of all the GSLs contained the same fragment ions of ceramide at m/z 538 and 648 and confirmed their sugar chain sequence8 as those deduced in Fig. 9. The (M-H)- ions corresponding to Hex-HexNAc-Hex-Hex-cer- amide (m/z 1,227 and 1,337) could be seen in CTeH (E), but not those corresponding to HexNAc-HexNAc-Hex-Hex-cer- amide (m/z 1,268 and 1,378).

Changes in CTH Composition of Individual Lenses-We have shown significant changes in the composition of neutral GSLs from subjects between 16 and 80 years of age, particu- larly in the expression of Le' glycolipid, III"FucnLc4 (14). The tendency for an age-related increase in CTH expression was seen. Therefore, synthetic activity directed to Le' glyco- lipid expression was examined at the branching point of lacto-

CYM Con CTH man CPH

FIG. 8. Proposed synthetic pathway of neutral CSLs in human cataractous lens. AsialoCM2 (Cg3) may be synthesized not only from lactosylceramide (dotted l ine) but also from the desialylation of ganglio-series gangliosides.

13246 Le" Glycolipid in Human Lens

series and globo-series glycolipids. The CTH fraction from an individual lens was partially purified by preparative TLC and developed on an HP-TLC plate after acetylation (Fig. 10). Two acetylated CTHs were separated on the HP-TLC plate. At least among the specimens we analyzed, there appeared to be an age-dependent shift in CTH expression from the upper to the lower bands. The upper and lower bands were assigned to be Lc3 and Gb3, respectively, by comparison with the acetylated derivatives of purified CTHs. The lower band

TABLE I1 Fatty acid compositions of neutral GSLs isolated from human

cataractous lenses Fatty acids are denoted as chain 1ength:number of double bonds.

Fatty CMH (A) CDH (B) CTH (C) CTH (D) CTeH(E) CPH (F) acid

14:O 1.7 0.6 10.3 2.0 2.3 3.2 14:l 0.5 0.2 3.9 1.0 1.2 2.8 1 6 0 35.1 29.2 13.8 27.4 36.7 23.9 1 6 1 1.9 0.6 7.1 2.0 2.9 3.6 180 7.2 1.7 12.1 3.4 5.2 7.1 181 0.7 1.0 7.7 2.0 3.3 6.2 20:o 1.1 0.7 0.3 0.7 0.7 1.4 20:l 0.4 0.1 0.2 22:o 3.1 4.3 3.6 2.9 4 .O 22:l 4.5 4.1 1.1 5.2 1.9 7.8 23:O 1.3 2.0 0.2 1.8 1.1 1.5 23:l 0.9 249 5.8 7.2 7.2 5.4 9.2 5.6 24:l 30.1 44.0 19.7 40.7 24.6 25.0 Others 6.6 4.5 12.1 5.5 6.9 11.9

(Gb3) was also confirmed by TLC-immunostaining using monoclonal anti-Pk antibody after deacetylation by immers- ing the TLC sheet in ammonia water (data not shown). To determine whether or not CTH expression in human lens actually shows age-dependent changes, we will have to analyze multiple individuals at different ages. However, as discussed below, it is impractical to expect that whole lenses from many individuals will become available for analysis.

DISCUSSION

Our recent study provided evidence that human senile cataractous lens accumulates the Le" glycolipid (II13FucnLc4) in an age-dependent, cataract-related manner (14). Although the presence of fucosylglycolipids and fucogangliosides in human cataractous lenses had been postulated from the analy- sis of sugar composition, their configuration and metabolic pathways had not been elucidated (2, 8). The current study was therefore undertaken to clarify the metabolic relationship of neutral GSLs to the synthesis of the Le" glycolipid in the human cataractous lens.

As summarized in Fig, 8, lens GSLs were mainly composed of globo-series and neolacto-series GSLs. They have a similar ceramide structure of C16:O or C24:l fatty acid and C18- dihydrosphingosine (Fig. 9). The synthetic pathway of lens GSLs is also suggested in Fig. 8. At least three glycosyltrans- ferases are involved in the CTH synthesis of the branching point: /31-3-N-acetylglucosaminyltransferase forms Lc3 (25), al-4-galactosyltransferase forms Gb3 (26), and pl-4-N-ace- tylgalactosaminyltransferase forms asialoGM2 (Gg3) (27, 28).

0

100 500

.- LOO 700 no0 900 IO00 1100 , 200 139" 1.00 1100

C w lcla (any acd ) - Iunl H e x f ~ X f ~ X $ c ~ r 648 (c2. , Iatly a d I s v) lei 620640

.E 0

; ' O 538 101.

l u n l

FIG. 9. Negative ion secondary ion mass spectra and the fragmen- tation diagrams of lens GSLs (A-F). Two molecular ion simals were observed

.- to" I,,. e 1 0 662 972

al a 800 900 I IO0 1100 L

600 700 IOOP I ,on 1.00 I50" .- - in all the GSLs, corresponding to C16:O or C24:l fatty acid and C18-sphinganine

?2

in the ceramide moiety.

(D -

Le' Glycolipid in Human Lens 13247

c L c 3 - A ~ ' w - Gb3-Ac

k

"""-4 "

1 2 3 4 5 6 7 8 910 - " - . * * e *

FIG. 10. Change in CTH composition between age 16 and 80 years. The CTH fraction of individual lenses was isolated by preparative TLC, acetylated, and developed on an HP-TLC plate in a solvent of 1,2-dichloroethane/acetone/water (60:401, v/v/v). Acet- ylated forms of crude CTH (lane 9 ) and purified Gb3 (lane 10) were developed in parallel lanes. I , 16 (male); 2,35 (male); 3,50 (male); 4, 55 (male); 5, 67 (female); 6, 75 (male); 7, 75 (female); 8, 80 years old (female). -; normal, +; presumably non-cataract, +; immature cata- ract, ++; mature cataract.

For the synthesis of neolacto type 2 chain in nLc4, galactose must be transferred from UDP-galactose to Lc3 by @1-4- galactosyltransferase (29). Finally, nLc4 is fucosylated by al- 3-fucosyltransferase to produce Le' glycolipid ( II13FucnLc4) (30). Further studies on the acceptor specificity and biochem- ical properties of al-3-fucosyltransferase would clarify the synthetic mechanism of Le' glycolipid in lens. Additionally, a trace of GalNAc@1-3Gal@1-4Glc@l-lceramide in CTH (D) may be secondarily synthesized by PI-3-N-acetylglucosami- nyltransferase or ~1-4-N-acetylgalactosaminyltransferase, but the details on the biochemical properties of these enzymes remain unknown.

However, the result shown in Fig. 10 does not necessarily support de mu0 synthesis of Le' glycolipid in aged, catarac- tous lenses. Although we predict the tendency for an age- dependent increase in the ratio of Gb3 to Lc3, the data presented herein are too fragmentary to draw any conclusions. At present, it is practically impossible to obtain whole lenses because of the technical progress in cataract surgery, i.e. removal of lens tissues by phacoemulsification and aspiration, and ethical problems regarding the use of Eye Bank lenses. The human lens shows a statistically significant age-depend- ent, cataract-related increase in ganglioside content as lens fibers accumulate with aging (12). On this basis, it is plausible that human lens accumulates Gb3 with aging, as one of the membrane components. It is also noteworthy that Gb3 has a fatty acid composition somewhat different from other GSLs (Table 11), suggesting an age-dependent change in ceramide synthesis.

At present, it is highly likely that the increase in Le' glycolipid is due to the accelerated desialylation of sialylated Le' gangliosides. Generally, it is more difficult to imagine that de mu0 synthesis of Le' glycolipid occurs in fiber cells that lack nuclei and cell organelles, especially in mature fibers accumulated in the lens nucleus (31, 32). In fact, Le' and sialylated Le' epitopes were immunologically detectable in both cortical and nuclear regions of human lenses, and the

molar ratio of Le' to sialylated Le' was about 1:2 to 1:4.' One of the sialylated Le' gangliosides was recently identified as NeuAca2-3Gal~1-4(Fucal -3)ClcNAc~l-~Gal~l -4Glc~l - l ceramide. Therefore, the appearance of asialoGM2 (Gg3) (Fig. 7) may be also attributable to the desialylation of GM2 or partial degradation of ganglio-series gangliosides, rather than to de m u 0 synthesis of Cg3.

A large proportion of membrane CSLs are implicated in ion transport, cell surface markers, cellular interaction, dif- ferentiation, and oncogenesis (33). Some changes in the con- tent, composition, or distribution of lens GSLs may affect the physiological functions required for the maintenance of lens transparency and cell integrity. The specific carbohydrate- carbohydrate interaction between Le'-Le' determinants or between GM3 and asialoGMz (Cg3) glycolipids has been pro- posed as the initial step in the cell recognition process (17. 34-36). It is extremely important to determine whether the expression of Le' glycolipid in human lens is related to cata- ract formation and/or the aging process.

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