DNA AND CELL BIOLOGY Volume 11, Number 6, 1992 Mary Ann Liebert, Inc., Publishers Pp. 489-496

Expression of Human 60-kD Heat Shock Protein (HSP60 or PI) in Escherichia coli and the Development and Characterization of Corresponding Monoclonal Antibodies BHAG SINGH and RADHEY S. GUPTA

ABSTRACT

protein, which is the homolog of the 60- to 65-kD heat shock "common" antigenic protein of nupathogenic organisms (synonyms: HSP60, GroEL homolog, or chaperonin), has been expressed to level in Escherichia coli cells. A large number of well-characterized deletions of this protein spanning high the entire sequence have been constructed and expressed. Methods to purify recombinant human HSP60 protein and its deletions from E. coli have been worked out. In addition, monoclonal antibodies to the human HSP60 protein have been raised and partially characterized. The availability of these materials should greatly aid in understanding the role of this highly conserved and immunologically important protein in Human PI merous

autoimmune diseases and in cell structure and function.

INTRODUCTION PI (referred Thein this paper)65-kD constitutes mycobacteria major antiimmune which for be60-

to

antigen

of

genic protein,

to as

or

HSP60

a

accounts response to T-cells responses in immunized/

tween 20-30% of B- and

infected animals (see Kaufmann, 1990; Young, 1990; Young et al, 1990). In the past few years, it has become clear that this protein corresponds to the previously described bacterial "common antigen" and that it is a member of the heat shock family of proteins (viz. GroEL, HSP60 family) (Lindquist and Craig, 1988; Shinnick et al, 1988; Thole et al, 1988). Recently the gene for the related protein has been cloned and sequenced from a number of different species including Mycobacterium tuberculosis, M. leprae, M. bovis BCG, Escherichia coli, Coxiella burnetii, Ricketssia tsutsugamushi, Cyanobacteria sp., Saccharomyces cerevisiae, Chinese hamster, rat, mouse, and human cells (Mehra et al, 1986; Shinnick, 1987; Hemmingsen et al, 1988; Vodkin and Williams, 1988; Jindal et al, 1989; Picketts et al, 1989; Reading et al, 1989; Miller et al, 1990; Morrison et al, 1990; Sampson et al, 1990; Stover et al, 1990; Venner and Gupta, 1990a,b; Webb et al, 1990). Sequence comparison studies have revealed that the primary sequence of this protein is highly conserved during evolution. For example, the human homolog of HSP60

Department

of

(referred to as PI in our work) shows about 47-50% identity and an additional 20-25% conserved substitutions to its various prokaryotic counterparts (viz. from M. tuberculosis, M. leprae, C. burnetii, E. coli, and R. tsutsugamushi) (Dudani and Gupta, 1989; Jindal et al, 1989). In view of the immunodominant nature of the prokaryotic HSP60, the observed high degree of homology between the bacterial antigen and its human counterpart raises a number of important questions regarding the immune response to this antigen and its possible involvement in the pathogenesis of autoimmune diseases (see Kaufmann, 1990; van Eden, 1990; Young, 1990; Young et al, 1990; Cohen, 1991). There is, in fact, considerable evidence indicating that immune response to the mycobacterial HSP60 may be involved in the etiology of the autoimmune disease rheumatoid arthritis (RA) in humans, and a related condition, adjuvant arthritis (AA) in rats (see van Eden, 1990; Cohen, 1991). Immune response to the MB HSP60 has also been implicated in the development of autoimmune diabetes in human and animal models (see Jones et al, 1990; Cohen, 1991; Elias et al, 1991). Besides being an immunodominant antigen, the HSP60 family of proteins in various systems has been shown to perform a "molecular chaperone" role in the proper folding of newly synthesized polypeptide chains and, in some cases, their assembly into oligomeric protein complexes

Biochemistry, McMaster University, Hamilton, Canada 489

L8N 3Z5.

490

SINGH AND GUPTA

(Hemmingsen et al, 1988; Goloubinoff et al, 1989; Ellis, (Promega) to yield the plasmid PGA 8. In Hu HSP60 1990). To facilitate investigations on the role of Hu HSP60 cDNA the first 78 nucleotides following the initiation in immune/autoimmune responses, as well as in cell struc- codon encode mitochondrial targeting presequence. Thereture and function, we describe the expression of Hu fore, deletion of portion of the 5' end sequence from HSP60 in a prokaryotic expression vector. A large number HSP60 cDNA was carried out. The plasmid PGA 8 was of well-defined deletions of this protein have been con- opened with Apa I and Xho I and digested with exonuclestructed, expressed, and purified. In addition, monoclonal ase III for different time periods to delete sequence from antibodies against human HSP60 have been raised and the 5' end of HSP60. After ligation of Nco I linkers (a mixpartially characterized. The availability of these materials ture of 8-mer and 10-mer; Pharmacia Biochemicals) the should prove very useful in understanding the role of this cDNA inserts were released with Nco I and Hind III and protein in immune response, as well as in cell structure and subcloned in the prokaryotic expression vector PKK 233-2 function. (Pharmacia), digested with the above enzymes. To make additional deletions in which portions of the Hu HSP60 sequience from the middle were lacking, a clone PKK 13A, MATERIALS AND METHODS which expressed nearly full-length Hu HSP60 protein (from ami no acid 31 to 547), was digested with Nsi I (at Construction of deletions of Hu HSP60 cDNA nucleotide 1,342). The sequences on both sides of the Nsi I and their expression site were deleted by treatment with exonuclease Bal-31, The clone XC5, which contains the complete cDNA for and the deleted clones were examined for expression. To Hu HSP60 (from nucleotides -45 to 2,197, assuming the delete only a small segment from the carboxy-terminal position of initiating ATG as nucleotides 1 to 3), has been end, the plasmid PKK 13A was digested with Hpa I and described earlier (Jindal et al, 1989). From this phage clone Hind III (the latter in the polyclonal region of the plasmid) (Fig. 1A), a portion of the cDNA flanked by Hha I and and ligated. All constructs studied were partially seDra I (i.e., from nucleotides -24 to 2,176) was excised quenced using oligonucleotide primers specific for either the and subcloned at the Sma I site of the plasmid pGem 7z(f) flanking regions of the plasmid or various regions of the

Hha 1

Nsi 1

(-21)

(1342)

I

Dra 1

Hpa 1 (HAT)

(2175)

_~__5J—

5' —BEC 1000

500

•45 1

1500

-A-A-A-A-3' 2000

1720

2183

B Mature Hu Hsp60

PKK

r

547

t-

30(8)2-

PKK13A

PKK13B

547 547

31

547

65

PKK13C PKK13D

Bal-14 Bal-2 Bal-27

Bal-29

Hpa-1

547

169

A22a Bal-0

547

130(7)

547

ß-gal 211 31

393

366

31

31

547

434 484

547

344

31

335

31

288

100

200

300

400

500

537, 550

Amino acid

FIG. 1. A. Map of human HSP60 cDNA and location of restriction sites used for construction of deletions. B. Description of some of the HSP60 fragments that have been successfully expressed in E. coli. PKK 13B, PKK 13C, and PKK 13D are specific proteolytic fragments derived fromn PKK 13A. The numbers refer to the amino acid sequence in mature HSP60 (PI) protein.

HUMAN HSP60 EXPRESSION AND MONOCLONAL ANTIBODIES Hu HSP60 sequence, to ascertain the letion in each of the clones.

Induction and purification

A strong antibody response was observed in most of the mice within 2 weeks. However, two additional injections in incomplete Freund's adjuvant were given on days 15 and 22. Three days after the last injection, spleens from the immunized animals were removed and the suspension of spleen cells was fused in the presence of 50% polyethylene glycol with 1 x 10' mouse myeloma line P3NSI/1-Ag4/1 as described (Harlow and Lane, 1988). The suspension of fused cells in selective medium (growth medium supplemented with 5.8 x 10"6 M azaserine and 1 x 10~* M hypoxanthine) was aliquoted into 96-well microtiter plates (about 1 x 104 cells/well) and incubated at 37°C in a 95% air-5% C02 incubator. After 7-14 days, the culture supernatants of the wells where clonal growth was observed were tested for the presence of Hu HSP60 antibody by enzyme-linked immunoadsorbent assay (ELISA) using recombinant Hu HSP60 protein bound to 96-well plates as the antigen. The clones showing positive reactions were subcloned, expanded, and used for ascites induction (Harlow and Lane, 1988). The IgG isotype of the monoclonal antibody was determined by a

precise extent of de- complete adjuvant.

of HSP60

Escherichia coli JM105 harboring either the control or recombinant plasmids was induced in early to mid-log phase with 1 mA/ IPTG for varying durations. For most constructs, 2-3 hr of induction was found to be optimum. Portions of the induced cultures were pelleted and after lysis in a buffer (containing 2% Nonidet P-40, 5% mercaptoethanol and 10 mM Tris HC1 pH 7.4), the proteins were separated on 10% NaDodS04-polyacrylamide gels. The gels were usually run in duplicate. Of these, one was stained with Coomassie blue and the other blotted on nitrocellulose and treated with a rabbit polyclonal antibody specific for mammalian HSP60 protein (antibody preadsorbed with E. coli extracts to remove any cross-reacting to identify the protein bands corresponding to Hu HSP60 protein. For large-scale preparation of recombinant Hu HSP60 protein, the cell pellet from IPTG-induced cells was washed once with Tris-buffer saline (TBS: 0.9% NaCl, 10 mMTris HC1 pH 7.4) containing 0.5 mM freshly prepared phenylmethyl sulfonylfluoride (PMSF), and then resuspended in the same buffer (about 10 ml buffer per gram wet weight of cells). The cell suspension was sonicated for four 30-sec pulses to break the cells and then centrifuged for 30 min at 12,000 rpm in a Sorvall (SS-34 rotor) centrifuge. The supernatant which contained very little recombinant HSP60 protein was discarded and the pellet was resuspended in the original volume of TBS + PMSF containing 8 M urea. The suspension was gently rocked for 1 hr at 37°C and then centrifuged again for 30 min at 12,000 rpm. The supernatant that contained most of the recombinant HSP60 was carefully removed and passed through a Sephadex G-25 column equilibrated with TBS + PMSF to remove urea. All of the Hu HSP60 eluted in a soluble form after the void volume. Further purification of recombinant Hu HSP60 was carried out by electroelution of the appropriate bands from NaDodS04-PAGE gels.

antibodies)

DNA and protein

491

1

23456

789

94Kd>

67Kd> 43Kd>

'S

30Kd> 20Kd>

%

|

14Kd> I

23456789

sequencing

The nucleotide sequence of various plasmid constructs and deletions in the region of interest was determined by the dideoxy chain-termination method using a Sequenase kit (United States Biochemical). Microsequencing of recombinant proteins electroblotted onto polyvinylidene difluoride membrane was performed by Dr. M. Blum (Department of Biochemistry, University of Toronto, Toronto, Canada). Oligonucleotide sequencing primers were synthesized by the Central Facility of the Institute for Molecular Biology, McMaster University (Hamilton,

Canada).

Preparation of monoclonal antibodies Hu HSP60 protein

to

BALB/c mice were immunized with 150 ni °f purified recombinant human HSP60 protein PKK 13A in Freund's

B FIG. 2. NaDodS04-PAGE analysis of recombinant E. coli clones expressing human HSP60. A. Coomassie blue-stained gel. B. Western blot of a parallel gel treated with a rabbit polyclonal mammalian HSP60 antibody. Lane 1, Control E. coli cells harboring plasmid lacking the cDNA insert; lanes 2-9, independent recombinant clones expressing human HSP60. The start points of HSP60 sequence in different clones are as indicated. Lane 2, PKK 3, nucleotide 133; lanes 3 and 4, PKK 6 and 7, nucleotide -16; lane 5, PKK 8, nucleotide 1; lane 6, PKK 11, nucleotide 143; lane 8, PKK 13A, nucleotide 169; lanes 7 and 9, PKK 12 and 15, not characterized.

492 mouse

SINGH AND GUPTA

isotyping kit obtained

from Bio/Can

Scientific,

Inc.

Immunofluorescence and

Western

blotting

For immunofluorescence

staining of human diploid ficoverslips were fixed by treatment with cold methanol (-20°C) and then incubated with the monoclonal HSP60 antibody (either 1:20 dilution of the supernatant from hybridoma cells or 1:500 dilution of the ascites fluid) for 30 min at 37°C in a moist chamber. After rinsing the coverslips several times with TBS, they were treated similarly with a fluorescein-conjugated goat anti-mouse antibody. The stained cells were examined by epifluorescent illumination and photographed as described (Gupta and Dudani, 1987). Western blot analysis of proteins was carried out as described previously (Dudani and Gupta, 1989). broblasts, cells growing

on

RESULTS

Expression of Hu HSP60 proteins and its deletion in E. coli

The cDNA for Hu HSP60 contains a 45-bp upstream sea 78-bp mitochondrial targeting sequence which is not present in the mature protein (Fig. 1A). Therefore, for expression of this cDNA in E. coli sequential deletions from the 5' end were made and subcloned at the Nco I site of the prokaryotic expression vector PKK 233-2. In this plasmid, the ATG codon, which is contained within the Nco I site, is preceded by the lacZ ribosome binding site and a regulated and highly expressed trp-lac fusion promoter (Amann and Brosius, 1985). Several of the clones obtained from different time points were induced with IPTG (1 mM for 2 hr) and the total cellular proteins were analyzed by NaDodS04-PAGE and Western blotting using an antibody specific for mammalian HSP60 protein. It was observed that a large number of clones from the early time points after exo III digestion expressed new protein bands in the MT range of 40- to 65-kD, which cross-reacted with the PI (viz. mammalian HSP60) antibody in Western blots (Fig. 2). In contrast, in control cells containing the non-recombinant plasmid, no PI antibody cross-reactivity was observed (Fig. 2, lane 1). The recombinant HSP60 protein from a large number of deletions constructed above have been purified by gel filtration column followed by electroelution of specific bands from NaDodS04-PAGE gels. The NaDodS04-PAGE profile of various recombinant Hu HSP60 protein preparations that have been purified is shown in Fig. 3. Western blot analysis show that all of these deletions react with a polyclonal antibody to mammalian HSP60 protein (results not quence and

shown).

The lower MT bands in induced recombinant cells, which cross-reacted with the PI antibody, presumably resulted from proteolytic cleavage of the high Mr protein. This inference was supported by the observation that upon deletion of the 5' end segment from HSP60 cDNA, while the MT of the upper band varied with the size of the cDNA in-

FIG. 3. NaDodS04-PAGE analysis of some purified and well-characterized deletions of HSP60 proteins. Lane 1, PKK 30 (8); lane 2, PKK 13A; lane 3, PKK 13B (the lower band is due to proteolysis); lane 4, Hpa-l; lane 5, PKK 13C; lane 6, PKK 13D; lane 7, clone X22a fusion protein; lane 8, Bal-0 deletion; lane 9, Bal-14 deletion; lane 10, Bal-2 deletion; lane 11, Bal-29 deletion; lane 12, Bal-27 deletion. Further description of these deletions is provided in Fig. 1.

(see Fig. 2, lanes 2-5), the positions of the lower bands did not change. This observation indicates that the lower Mr bands that cross-react with PI antibody result from proteolytic cleavage at specific sites distal from the aminoterminal ends. This proteolytic cleavage occurred despite the presence of 0.5 mM PMSF in all extracts and buffers. The start point of the cDNA insert in a large number of the clones that expressed recombinant Hu HSP60 protein was determined by DNA sequencing using PKK 233-2 sequencing primer. In the clones expressing recombinant protein, the start point of the insert ranged from nucleotide -16 to 169 in the Hu HSP60 cDNA (Table 1). For these clones a good correlation was observed between the size of the cDNA insert and the Mr of the polypeptide chain expressed. In contrast to these clones, in other clones in which deletions have extended beyond nucleotide 170, no expression of Hu HSP60 was observed in any experiments even though they contained cDNA inserts in the correct reading frames (results not shown). To make deletions which extended beyond nucleotide 170, a clone PKK 13A, which expressed a high level of Hu HSP60, was cut at an internal Nsi I site (in the Hu HSP60 coding region; see Fig. 1A) and Bal-31 digestion was carried out for varying times. A number of the clones thus obtained expressed lower Mr bands (20-50 kD) in NaDodS04-PAGE which reacted with PI antibody. The precise extent of deletion in these Bal clones was determined by DNA sequencing using internal oligonucleotide primers specific for Hu HSP60 cDNA. In all of the clones examined the correct reading frame was restored following sert

493

HUMAN HSP60 EXPRESSION AND MONOCLONAL ANTIBODIES

Table 1. Partial Nucleotide Sequence of Some Constructs That Express Hu HSP60 Protein

Clone

kDa

Hu HSP60 sequence

Vector sequence *

PKK 6 PKK

30(22)

ATGGC ATGGCT

PKK 15

ATGGCT

PKK

ATGGCT

30(8)

PKK61

ATGGCT

PKK 11

ATGGC

PKK 13

ATGGCT

-16 CCGC CGC CCC GCA 1 ATG CTT CGG TTA 43 TCC AGG GTA CTG 88 GTA AAA TTT GGT 112 GCC TTA ATG CTT 144 CGAT GCT GTG GCC 169 CCA AAG GGA AGA

* indicates the beginning of Hu HSP60 sequence in different clones. Sequences on the left of the asterisk including the initiation codon are derived from the Nco I linkers. The numbers refer to the nucleotides in the Hu HSP60 sequence.

FIG. 4. NaDodS04-PAGE analysis of proteins extracted from PKK 13A cells by 8 M urea, after passing through a G-25 column. The bands marked A, B, C, and D show reactivity with PI antibody in Western blots and correspond to the proteins PKK 13A, PKK 13B, PKK 13C, and PKK

13D, respectively.

the deletions. The extent of sequence deletion in various Bal-31 clones that express truncated Hu HSP60, and which have been fully characterized, is shown in Fig. IB. One interesting feature of PI and related proteins is the presence of a Gly-Gly-Met repeat at the carboxy-terminal end. The functional role of the Gly-Gly-Met repeat is at present not clear, but it could be responsible for the unusually high antigenicity of this family of proteins. For the purpose of investigating the possible role of this carboxyterminal repeat, a deletion of PKK 13A was made in which the carboxy-terminal 34 amino acids were specifically removed by digesting the DNA with Hpa I and Hind III. The clones lacking this region showed similar level of expression of truncated protein as the parental strain PKK 13A (results not shown).

Purification of recombinant protein In the clones that expressed Hu HSP60 protein, much of it was found associated with the 12,000 x g paniculate fraction (see Materials and Methods), indicating that it was present within inclusion bodies. Treatment of the 12,000 X g pellet with 8 M urea extracted most of the recombinant protein from this fraction. Besides Hu HSP60 protein, only a few other proteins were released in the 8 M urea fraction. The urea was removed by passing the 8 M urea fraction through a G-25 column. Gel electrophoretic analysis of the eluate from PKK 13A cells showed that it consisted of five to six well-defined bands (Fig. 4) of which the bands marked A, B, C, and D reacted with the PI anti-

body (results

not

shown).

To determine if these lower Mr bands result from proteolytic cleavage at specific site, two of the lower Afr bands from PKK 13A, designated PKK 13B and PKK 13D, were

electroblotted on PVDF membranes, and the excised bands were subjected to protein microsequencing. The amino acid yields during repetitive cycles indicated that the amino-terminal ends of PKK 13B and PKK 13D were homogeneous, confirming that they resulted from proteolytic cleavage at specific internal sites. The partial amino-terminal amino acid sequence data on these peptides (PKK 13B; DIGAFLVQDVANNT; PKK 13D, K G V I T V K DGKTLNDE) matched exactly with the Hu HSP60 protein sequence beginning with residues 65 and 169, respectively. Preliminary results indicate that the protein sequence in the band PKK 13C begins with the amino acid residue 130 in the Hu HSP60 protein. The sequence data on these fragments thus show that proteolytic cleavage in each case occurred after a basic residue such as lysine or arginine. Although a large number of Lys or Arg residues are present in this protein, only a selected few sites are susceptible to the proteolysis under the conditions employed. The well-defined ends of PKK 13B, PKK 13C, and PKK 13D combined with their large yield have provided us with additional well-characterized deletions for various studies.

Monoclonal antibodies to Hu HSP60 protein The recombinant Hu HSP60 protein was injected into several BALB/c mice with the aim of developing monoclonal antibodies (MAbs). Surprisingly, despite the very

SINGH AND GUPTA

494

of sequence similarity between human and HSP60 proteins (the two differ in only 13 amino acids, most of which are conservative substitutions; see Venner and Gupta, 1990a), a strong antibody response was observed in most (11 out of 12) of the mice within 2 weeks of injection. By fusion of the spleen cells from these mice to the mouse myeloma line P3 NSI/I, we have been successful in raising several stable hybridoma cell lines that produce MAbs to Hu HSP60 protein. One of these hybridoma clones, MAb 11-13 has been investigated in detail. The IgG secreted by it has been identified as IgG2a based on its reactivity with a mouse MAb isotyping kit (Line Immunoassay; Bio/Can Scientific). In Western blots of total cellular proteins from various species (viz. human, Chinese hamster, snake, dolphin, and mosquito) this antibody reacts specifically with a protein of MT s 60 to 62 kD that corresponds to HSP60 or PI protein (Fig. 5A). In contrast,

high degree mouse

FIG. 5. Cross-reactivity of Mab clone II-13 towards other species and with various deletions of HSP60 protein. A. Western blot of total cellular proteins from different species. Approximately equivalent amounts of proteins from different species ( = 20 ni) were electrophoresed in 10% NaDodS04-PAGE and blotted onto nitrocellulose. The blots were treated sequentially with 1:500 dilution of MAb 11-13 and with a 1:3,000 diluted alkaline phosphatase-conjugated goat antimouse antibody and then developed. The original of cells/cell lines from different species has been described earlier (Gupta and Dudani, 1987). Lane 1, E. coli; lane 2, yeast; lane 3, mosquito; lane 4, rainbow trout; lane 5, snake; lane 6, dolphin; lane 7, CHO cells; lane 8, human diploid fibroblasts. B. Western blot showing reactivity of Mab 11-13 with different deletions of HSP60 protein: lane 1, Bal-29; lane 2, Bal-14; lane 3, Bal-0; lane 4, X 22a; lane 5, PKK 13D; lane 6, PKK 13C; lane 7, PKK 13B; lane 8, PKK 13A. Description of different deletions is given in Fig. 1.

no significant cross-reaction was observed with the proteins from either E. coli or yeast cells. To map the antigenic epitope which is recognized by this antibody, its cross-reactivity to various deletions was examined. Based on its observed reactivity (Fig. 5B), the antigenic epitope recognized by it has been tentatively mapped to between amino acid residues 288 and 366 in the human HSP60 protein. In immunofiuorescence experiments, this antibody showed specific staining of worm- or string-shaped mitochondria in human fibroblasts (Fig. 6) which is the expected result, based on the mitochondrial localization of mammalian HSP60 protein (Gupta and Duani, 1987; Pick-

ettsera/., 1989).

DISCUSSION This paper reports the successful expression of human HSP60 cDNA in E. coli cells and production of hybridoma cell lines secreting antibodies to it. Under optimal induction conditions, human HSP60 comprise 10-15% of the total cellular proteins in the recombinant cells. Besides the fulllength protein, we have also successfully expressed several deletions of HSP60 in which portions of the sequence from either amino-terminal, middle, or carboxy-terminal end are lacking. Interestingly, in deletions in which the sequence from the 5' end was deleted more than 170 nucleotides, no expression in E. coli was observed. This suggests that the sequence in this region may be important in determining either the stability of the protein in E. coli cells or its expression. The availability of recombinant protein was instrumental in our developing hybridoma cell lines that secrete monoclonal antibodies to it. Further, deletions of HSP60 have enabled us to map the antigenic epitope which is recognized by one of these monoclonal antibody clones (11-13) to between amino acids 288 and 366 in the human HSP60 sequence. The species cross-reactivity of this monoclonal antibody indicates that the antigenic epitope

FIG. 6. Immunofluorescent staining of human diploid fibroblast with monoclonal antibody 11-13. The string- or worm-shaped structures seen here correspond to mitochondria, as established earlier (Gupta and Dudani, 1987).

HUMAN HSP60 EXPRESSION AND MONOCLONAL ANTIBODIES

should

495

conserved sequence within this reregard that several of the gion. monoclonal antibodies raised against mycobacterial proteins also bind to human and rodent HSP60 protein and are directed against conserved epitopes within this protein family (Anderson et al, 1988; Dudani and Gupta, 1989). As indicated earlier, PI or Hu HSP60 protein is the human homolog of the 60- to 65-kD heat shock protein which constitutes the major antigenic protein of numerous pathogenic organisms. In view of the high degree of sequence conservation for this protein, antigenic mimicry between the host HSP60 protein and that of the invading organisms is suspected to play an important role in the development of several autoimmune diseases. Further, the specific reactivity or stimulation of a large proportion of human and murine T lymphocytes (both with aß and 76 receptors) with HSP60 has led to the suggestion that this interaction plays an important role in immune surveillance and regulation, perhaps by acting as the first line of defense against infection (see Kaufmann, 1990; Young,

chaperone in facilitating protein folding and assembly processes, and in the transport of protein across membranes (Bochkareva et al, 1988; Goloubinoff et al, 1989; Ellis, 1990). Whether the molecular chaperone function of HSP60 is in any way related to its strong antigenicity (despite very high degree of sequence conservation) is unclear at present. It should be of much interest in this regard to investigate the involvement of HSP60 in the antigen presentation process, particularly since a protein related to HSP70 (another highly conserved and strongly antigenic protein) has been shown to be involved in the antigen presentation process (Lakey et al, 1987). Another intriguing aspect of HSP60 function relates to its possible role in in vivo assembly of microtubules. Our studies with mutants,

With the availability of purified recombinant human HSP60 protein, it should now be possible to directly examine the reativity of the T-cell clones with the autologous protein and to determine whether autoantibodies to this protein are present in sera of patients with various autoimmune diseases. In cases where a positive response is observed, further studies with the deletions of HSP60 should enable us to localize the antigenic epitope responsible for the immune reativity. Recently, it has been shown that a human yô T-cell clone isolated based on reactivity to mycobacterial HSP60 as well as synovial fluid T lymphocytes from inflammatory synovitis are stimulated by the recombinant Hu HSP60 (Haregewoin et al, 1991; Pope et al, 1992). These studies demonstrate the usefulness of these reagents in establishing the link between cross-reactivity to human HSP60 and autoimmunity. The monoclonal antibodies to HSP60 should enable us to examine the cellular distribution of this protein in healthy and diseased persons. It is of particular interest to know whether this antigen is localized at the sites where inflammation and/or tissue damage due to autoimmune response is observed. Although HSP60 in eukaryotic cells is mainly localized in mitochondria, its distribution in specialized cells or under conditions of stress, may be altered or different. Our recent studies show that in pancreatic beta cells, HSP60, in addition to being localized in mitochondria, also shows specific association with the insulin core of the mature secretory granules (Brudzynski et al, 1992). In view of the molecular chaperone role of HSP60, it could be involved in the insulin packaging and secretory processes. Likewise, a number of recent reports indicate surface expression of HSP60 in human B- and T-cell lines (see Jarjour et al, 1990). In view of these obsevations, it is of interest to examine the cellular distribution of HSP60 in different specialized cell types and those subjected to different stresses using the monoclonal antibodies specific for it. As indicated above, one of the main cellular functions of the HSP60 family of protein is to act as molecular

ACKNOWLEDGMENTS

correspond

to a

It is of interest in this

which have altered HSP60 and are resistant to antimitotic drugs, suggest that this protein should play an important role in the in vivo assembly of microtubules (see Gupta, 1990). The reagents developed here should greatly aid in the investigation of these and related questions related to HSP60's cellular functions.

1990).

This work was supproted by a research grant from the Medical Research Council of Canada.

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DUDANI, A.K., and GUPTA, R.S. (1989). Immunological characterization of a human homolog of the 65-kilodalton mycobacterial antigen. Infect. Immunol. 57, 2786-2793. ELIAS, D., RESHEF, T., BIRK, O.S., VAN DER ZEE, R., WALKER, M.D., and COHEN, I.R. (1991). Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. Proc. Nati. Acad. Sei. USA 88, 3083-3091. ELLIS, R.J. (1990). The molecular chaperone concept. Sem. Cell Biol. 1, 1-9.

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SINGH AND GUPTA

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reprint requests to: Dr. R.S. Gupta Department of Biochemistry McMaster University Hamilton, Ontario, Canada L8N 3Z5 Received for publication October 17, 1991, and in revised form December 18, 1991.