ABSTRACT

α-Enolase is an important glycolytic enzyme in the cytoplasm of prokaryotic and eukaryotic cells

and is considered a multifunctional protein. α-Enolase is expressed on several cell surface, where

it acts as a plasminogen receptor, hydrolyzing the inactive plasminogen into active proteolytic

plasmin. In addition to glycolytic enzyme and plasminogen receptor functions, α-

Enolaseperforms other cellular functions and found at varying subcellular localizations that are

distinct from its well-established function in glycolysis. Therapeutically α-enolase is an essential

enzyme as many streptococcal species such as Streptococcus mutansuses plasminogen binding

site of α- enolase to spread infective endocarditis. In this study functional identity of recombinant

α-enolase has been confirmed by its ability to catalyze glycolytic reactions and its enzyme

kinetics.α-Enolase is localized on the surface of hematopoietic cells such as monocytes, T cells

and B cells, neuronal cells, and endothelial cells as a strong plasminogen receptor, modulating

pericellularfibrinolytic activity.

1

Table of content

TOPIC PAGE NO.

ABSTRACT 1

INTRODUCTION 3

METHOD 6

DISCUSSION 14

REFERENCES 15

2

INTRODUCTION

Enolase (2-phospho-D-glycerate hydrolyase, EC4.2.1.11) is a key glycolytic enzyme [1] in the

cytotoplasma of both prokaryotes and eukaryotes and is also considered as multifunctional

protein. It is also one of the most abundantly expressed cytosolic enzyme in many organisms

discovered in 1934 by Lohman and Mayerhof. Itcatalyzes the dehydration of 2-phospho D-

glycerate (2PGA) to phosphoenolpyruvate (PEP) in glycolysis as shown in figure 2, and the

reverse reaction in gluconeogenesis.Before binding with substrate enolase form complex with

divalent metal cations and hence it is characterize as metalloenzyme. It shows highest activity

when bound to metallic cation Mg+2. Enolaseexist in three isoformsin mammals and birds:α

enolase encoded by Eno1 gene found in all human tissues, β enolaseencoded by Eno3 gene found

only in muscle tissues and γ enolase encoded by Eno2 gene found only in neuron and

neuroendocrine tissues [2] .the three isoforms of enolase share high structural and kinetic

similarities [3,4]. Enolase exist in a dimeric form either homodimer or heterodimer composed of

two subunits arranged in antiparallel fashion as shown in figure 1 [4,5].

3

1st α subunit of α enolasehomodimer

2nd α subunit of α enolasehomodimer

Figure 1: PDB 2PSN structural model of α enolase. It’s a homodimer with two structural units

facing each other in antiparallel fashion

4

Figure 2: Metabolic chain reactions of glycolysis, the central pathway for the catabolism of

carbohydrates that takes place in the cytoplasm of almost all prokaryotic and eukaryotic cells.

The insert shows different enolaseisoenzymes in vertebrates.

α-Enolase mRNA translation is under developmental control which is highly unregulated during

cellular growth [6,7]. Recent studies have shown that in addition to its innate glycolytic function

α-enolase play key role in several biological and pathophysiological processes. α-Enolase has an

alternative stop codon by using it α-enolase m-RNA can be translated into an additional 37 kDa

protein which lacks the first 96 amino acids residues and called as c-myc promoter binding

protein 1 (MBP- 1) localized in nucleus and negatively regulates transcription of the

protooncogene [8].

α-Enolaseis a multifunctional protein as is found on the surface of hematopoietic cells such as

monocytes, T cells and B cells, neuronal cells, and endothelial cells as a strong plasminogen

receptor, modulating pericellularfibrinolytic activity. It is also described neurotrophic factor [9],

a heat-shock protein (HSP48) [10], and a hypoxic stress protein [11], it’s a part of the crystallin

lens of vertebrates [12], binds to fragments of F-actin and tubulin [13], and has been detected

associated to centrosomes in HeLa cells [14].α-Enolase also binds with other adjacent glycolytic

enzyme such aspyruvate kinase, phosphoglyceratemutase and andolase (which is associated with

cytoskeletal protein) with high affinity [15] (figure 3).

Sterptococcus mutans is a gram positive bacterium and a causative agent of oral cavity and

infectious endocarditis. S.mutans cells are found to associate with blood component and then

eventually colonizes cardiovascular endothelium causing deadly infectious endocarditis. they

associate with fibronectin and degrade by activating plasminogen, hence spread through blood in

body of host organisum.

5

Figure 3: Shows multifunctional characteristics of α-enolase

Recombinant DNA or Molecular Cloning

In this process Eno1 gene forms covalent interaction with plasmid vector. Many copies of the

hybrid DNA may be produced by the progeny of the recipient cells; hence, the DNA molecule is

cloned. If theinserted fragment contains a functional gene carrying the code for a specific

protein, many copies of that gene and translated protein may be produced in the host cell. This

process has become important for the large-scale production of proteinsthat are of value in

medicine and basic science.

6

Figure 4: Different steps of molecular cloning

7

Materials and Methods

Preparation of α-enolase antiserum

Antisera against S. mutans α-enolase was prepared by immunizing subcutaneously New

Zealand female rabbits with 200 μg of purified his-tagged α-enolase protein emulsified in

Freund's incomplete adjuvant containing 10% muramyl peptide. Rabbits were boosted two

weeks later with the same amount of his-tagged α-enolase in Freund's incomplete adjuvant and

after two additional weeks were bled. Antiserum to the α-enolase of S. pyogenes (anti-SEN)

was a gift from Vijaykumar Pancholi [10].

Isolation of cellular protein fractions

Extracellular, cytoplasmic, and cell wall protein fractions of S. mutans M51 were prepared

from 1-liter cultures grown at 37 ° C for 48 hours in Todd-Hewitt broth as described by

Kuykindoll and Holt [17]. Total cell lysates of S. mutans M 51 were prepared from 20 ml of

culture grown overnight at 37 ° C in Todd-Hewitt broth. Cells were collected by centrifugation

and resuspended in 10 mM phosphate buffered saline, pH 7.0. The cell suspension was

transferred to a 2 ml bead beater tube and 0.4 mg of 0.1 mm zirconia/silica beads per ml of cell

suspension were added. The tube was placed in a Mini-Beadbeater 3110BX and run in one

minute time periods with cooling on ice between time periods for a total of 3 minutes. The

beads were allowed to settle and the supernatant fluid (total cell extract) was removed.

Source of DNA fragment

Eno1 gene is isolated from Streptococcus mutans.

The Polymerase Chain Reaction Amplifies Specific DNA Sequence

PCR amplifies Eno1 gene , given below is α enolase protein sequence.This gene that encodes

alpha enolase is use to design oligonucleotide PCR primer for sequence amplification. There will

be two primers forward primer (5′- ATGTCA ATT ATT ACT GAT GTT TAC GCT CGC-3′)

that starts from base 1 and ends at base 30 and reverse primer (5′- TTATTT TTT CAA GTT

GTA GAA TGA TTT AAG ACC ACG-3′) began at base 1263 and ends at base 1296 of the

sequence including termination codon.The forward primer began at base 1 and ended at base 30,

8

while the backward primer began at base 1263 and ended at base 1296 of the sequence and

includes a termination codon.The primer pair was used in PCR reactions with denaturing,

annealing, and extension temperatures and times of 94 ° C for 1 minute, 50 ° C for 30 seconds,

and 72 ° C for 3 minutes; respectively. The amplified PCR product generated was approximately

1.3 kb in size, which is the correct size based on the nucleotide sequence. The α-enolase PCR

product was cloned into the TA expression vector pCR T7/NT TOPO-TA. The resulting plasmid

DNA was transformed into E. coli TOP 10F' cells and spread on LB plates supplemented with

100 μg/ml of ampicillin. Plasmid DNA was isolated from several transformants and digested

with restriction enzymes to verify the size of the insert DNA (figure 6 and 7). The plasmid DNA

was then sequenced for determination of the sequence of the insert DNA using Big Dye

terminator reactions.

Figure 6: Plasmid vector for Eno1 gene

9

Figure 6: Ligation of amplified PCR product into Invitrogen's TOPO TA Cloning System

For the fast and efficient cloning of polymerase chain reaction product the recommended clonind

system is Invitrogen's TOPO TA Cloning System (TOPO Cloning). This is a linearized plasmid

3’ overhang consisting of deoxythimidine (T) which form covalent interaction with

topoisomerase I. On the other hand PCR product 3’ overhang consist of deoxyadenine (A)

which is complementary to that of the plasmid 3’ polyT overhang, allowing fast ligation in the

presence of topoisomerase 1. Engineered plasmid is then transformed into competent E. coli

TOP 10F' cells [32].

10

Figure 7: Transformation of E.coli

11

RESULTS

Alpha-enolase activity and enzyme kinetic properties of recombinant Enolase(rEno)

Similar to the enzyme reaction catalyzed by α enolase recombinant proteinrEno also possessed

the same functional capacity to catalyze the conversion of 2-phosphoglycerate to

phosphoenolpyruvate.

Kinetic properties of rEno was determined by enzyme reaction kinetics using purified his-tagged

protein with various concentrations of 2-phosphoglycerate. Values for Km and Vmax were

calculated from reciprocal plots. This analysis revealed a Km value of 9.5 mM for 2-

phosphoglycerate and a Vmax of 31 mMphosphoenolpyruvate/min/mg for rSmEno. On the other

hand α enolaseKmvalue of1.492 mMfor 2-phosphoglycerate andVmaxof31.25

mMphosphoenolpyruvate/min/mg[16]

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Above double reciprocal curve showes enzyme kinetics of purified recombinant α- enolase. The

curve measures the rate of conversion of 2-phospho D-glycerate (2PGA) to phosphoenolpyruvate

(PEP) in the presence of varying concentration of substrate 2PGA and and 5 μg of purified

rcombinanat enolase. Data was plotted by the method of Michaelis-Menten(inset) and Vmax and

Km were calculated from double reciprocal plots.

Plasminogen binding by α-enolase.

To test the ability of the α-enolase to bind plasminogen, protein in a total cell extract

and an extracellular protein fraction along with purified rSmEno were separated

by SDS-PAGE and transferred to nitrocellulose membranes. The blot was incubated with human

plasminogen and then probed with antibodies against human plasminogen. The

resultsdemonstrated that plasminogen bound to a protein of approximately 50 kDa in the total

cellextract, extracellular protein fraction and with purified rEno that migrated with a mobility

that was very similar to a protein in each of the preparations that reacted with anti-rEno sera.

13

Above blot shows Plasminogen binding to the α-enolase of S. mutans.SDS-polyacrylamide

gelelectrophoresis of a total protein extract and extracellular protein of S. mutans and purified

rSmEno. Lane 1, molecular weight markers.

DISCUSSION

This study has demonstrated that the chemical attributes of α- enolase and recombinant α-

enolase don’t differ considerably. Both α- enolase as well as recombinant α- enolase processes

their innate ability to catalyze dehydration of 2-phospho D-glycerate (2PGA) to

phosphoenolpyruvate (PEP). The enzyme kinetic data reveled both enolase exhibits same Vmax .

The α- enolase of streptococcus specieshave on its C-terminal end two lysine residues that have

been reported to interact with the lysine binding sites of plasminogen [33,34]. Bergmann et al.

[35] have demonstrated that the enolase of S. pneumoniae, designated Eno, has an additional

internal plasminogen binding epitope and that this internal motif is crucial for the interaction of

the Eno protein with human plasminogen. These investigators identified a motif of nine amino

acids, FYDKERKVY (amino acid residues crucial for plasminogen binding are bold and

underlined), that bound human plasminogen. The acidic amino acids, aspartic acid (position 3)

and glutamic acid, and the lysine residues at positions 4 and 7 in the motif were found to be

important for plasminogen binding by the S. pneumoniae enolase. Furthermore, mutant strains of

S.pneumoniae with substitutions in the critical amino acids of the internal plasminogen-binding

motif were substantially less virulent than a wild-type strain of S. pneumoniae. The S.

mutansα-enolase has the sequence, FYDNG**VY, that has five amino acids in common with

the internal plasminogen-binding motif of S. pneumoniae. However of the amino acids required

for plasminogen binding, only the aspartic acid at position 3 is present and two of the amino

acids in the motif are not present at all. A similar sequence was found in the enolase of S.

sobrinus, another caries causing oral streptococcus. This finding indicates that the S. mutans

enolase does not have a functional internal plasminogen-binding site and suggests that enolase

mediated plasminogen binding by S. mutans occurs primarily through interaction of

plasminogen with the C-terminal lysyl residues. Previously, we have shown that plasminogen

activation by S. mutans is almost totally inhibited by lysine or the lysine analog, ε-aminocaproic

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acid [36].

In conclusion, we have characterized the α-enolase of S. mutans and provided evidence that

this typically cytoplasmic protein can translocate the cell wall of the oral bacterium, S.

mutans, and function as a receptor for plasminogen. This finding suggests that this generally

non-invasive oral organism may have virulence potential similar to that of more pathogenic

streptococci

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