The human Mr 90,000 heat shock protein and the Escherichia coli lon protein share an antigenic determinant

Comp. Biochem. Physiol. Vol. 87B, No. 4, pp. 961-967, 1987

0305-0491/87 $3.00+0.00 © 1987 PergamonJournals Ltd

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THE H U M A N Mr 90,000 HEAT SHOCK PROTEIN A N D THE ESCHERICHIA COLI LON PROTEIN SHARE A N ANTIGENIC D E T E R M I N A N T D. S. LATCHMAN*§,W. L. CHANt, C. E. L. LEAVER*, R. PATEL*, P. OLIVER:~ and N. B. LA THANGUEfll tICRF Tumour Immunology Unit, *Department of Zoology, University College London, Gower Street, London WC1E 6BT, UK (Tel: 01-387-7050); and :~Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK (Received 7 July 1986)

Abstract--l. A monoclonal antibody (TG7A) reacts with a Mr 90,000 mammalian protein, accumulating during virus infection and heat shock. 2. This protein is encoded by a member of the Mr 90,000 heat shock gene family present in a range of organisms form yeast to man. 3. The antibody also recognises a Mr 94,000 protein in E. coli which similarly accumulates in virus infection and heat shock. 4. This protein has been identified as the Lon protease of E. coli. 5. The shared epitope and similar stress inducibility of the two proteins suggests that a functional and/or evolutionary relationship exists between them.

INTRODUCTION In a wide range of organisms from bacteria to mammals exposure to heat shock results in the vigorous expression of a small number of genes, encoding the heat shock proteins and the repression of previously active genes (Ashburner and Bonner, 1979). The range of organisms exhibiting this response (Kelley and Schlesinger, 1978; Loomis and Wheeler, 1980; McAllister and Finkelstein, 1980) is paralleled by the evolutionary conservation of the heat shock protein genes. Originally detected in Drosophila, these genes have now been found in all eukaryotic organisms (Kelley and Schlesinger, 1982) and in the case of hsp70, a bacterial homologue has been detected by DNA hybridisation (Bardwell and Craig, 1984). This evolutionary conservation suggests that the heat shock response is of important survival value. The observation that very many other stimuli, such as amino acid analogues (Hightower, 1980) and viral infection (Nevins, 1982) cause the heat shock proteins to accumulate has led to the idea that the induction of these proteins represents a protective response to stress (Ashburner, 1982). Little data is available however on the functional role these proteins play in the stress response and such findings as have been reported relate almost exclusively to the major heat shock protein hsp70 (Lewis and Pelham, 1985; Ungewickell, 1985). We have recently characterized a monoclonal antibody (TG7A) which reacts with an Mr 90,000 cellular protein that accumulates during lytic infection with §To whom correspondence should be addressed. ][Current address: CRC Eukaryotic Molecular Genetics Research Group, Department of Biochemistry,Imperial College, London SW7 2AZ, UK.

Herpes simplex virus type-2 and also during heat shock (La Thangue and Latchman, submitted). In agreement with the heat shock induction of this protein it has been shown to be encoded by a mammalian member of the hsp90 gene family previously described by others in a range of organisms from yeast to man (Holmgren et al., 1981; Welch and Feramisco, 1982; Farrelly and Finkelstein, 1984). Here we show that this antibody recognises the Lon protein of E. coli (Chung and Goldberg, 1981), an Mr 90,000 protein induced by heat shock and viral infection. We suggest that the presence of a common epitope on the stress-induced hsp90 and Lon proteins is indicative of a functional and/or evolutionary relationship between the two proteins and that given the known role of the Lon protein in degrading abnormal or foreign proteins (Charette et al., 1981; Chung and Goldberg, 1981), the hsp90 protein may have a similar role in the eukaryotic stress response. MATERIALS AND M E T H O D S

Bacteria Escherichia coli strain C600: (hsdR - hsdM ÷ SupE the leu thi LacYl tonA21) and YI090 (A lacU169 proA ÷A Ion araD139 strA SupF) (Huynh et al., 1985) were grown in L

Broth at the temp. indicated for each experiment. To create an isogenic pair of Lon- and Lon + strains, E. coli strain Y1090 (F-) was crossed with HFr Cavalli (Lac+ Lon +) by conjugal mating at 37°C for 10 min. Lac + recombinants were selected on appropriate media. After single colony purification, individual recombinants were tested for the Lon phenotype by exposure to u.v. light (258 nm, Hanovia lamp). A L o n + and a Lon- strain identified by this means were grown at 32°C and transferred to 42°C for 20 min prior to harvesting and immunoblotting. For viral infection, E. coil B/r was grown at 37°C and then infected with bacteriophage T7 at a multiplicity of infection of 10. Equal aliquots of the culture were removed at various times after infection, harvested and immunoblotted.

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Mammalian cells

TG7A Hybridoma was grown in RPMI 1640 containing 10% foetal calf serum (La Thangue et al., 1984). All experiments were performed with culture supernatants. Filter screening

Bacteria were plated out and incubated at 32°C. After 6 hr growth the plates were overlayed with nitrocellulose filters and kept at various temperatures for 3 hr. After this time filters were removed and probed with the monoclonal TG7A followed by a second layer of ~25I-labelled rabbit anti-mouse antibody as described by Huynh et al. (1985). Western blotting

After equalizing amounts of bacteria by taking the optical density, samples were harvested, solubilized in SDS sample buffer and run on an 8% polyacrylamide gel. The gel was blotted and probed with antibody TG7A as previously described (La Thangue et al., 1984).

RESULTS

In initial attempts to isolate the gene(s) encoding the mammalian proteins defined by the T G 7 A antibody, we screened a c D N A library prepared in the

bacteriophage expression vector ~ gtll (Young and Davis, 1983). This screening was unsuccessful because of high binding of the antibody to the entire bacterial lawn rather than to a few specific plaques expressing the mammalian gene product as a fusion protein (Fig. 1A-C). This high, non-localizing binding was specific to the T G 7 A antibody, being absent with antibodies of similar isotype (IgG2a) when the same polyclonal rabbit anti-mouse immunoglobulin was used as a second layer (Fig. 1D). This result suggested that the T G 7 A antibody recognized a protein synthesized constitutively by the bacteria and therefore distributed throughout the plate. Such a reaction of an antibody prepared against one protein with another protein could be indicative of a relationship between the two proteins or may be due to a fortuitous similarity in otherwise unrelated proteins (Lane and Koprowski, 1982). In order to assess the nature of the cross-reaction, we investigated whether the bacterial protein had other characteristics similar to those of the mammalian protein. A preliminary experiment in which the bacterial plates were incubated at different temperatures sug-

Fig. 1. Filter screen of E. coli strain C600 with antibody TG7A. Bacteria were plated out and incubated at 32°C. After 6 hr growth the plates were overlayed with nitrocellulose filters and either kept at 32°C (A) or transferred to 37°C (B) or 42°C (C). After a further 3 hr incubation the filters were removed and reacted with the TG7A monoclonal. Filter D was treated identically to B but reacted with another monoclonal antibody (T156, La Thangue et al., 1984).

Heat shock protein and E. coli Lon protease gested that this was indeed the case, since a higher level of protein was detected at elevated temperatures, an analogous situation to the heat shock induction of the mammalian protein (see Fig. 1A-C). To investigate this further we performed immunoblots using bacterial proteins prepared from liquid cultures incubated at different temperatures. Figure 2 shows that the predominant protein recognized by the TG7A antibody in E. coli strain C600 is similar in size to that recognized in mammalian cells (M r 94,000 compared to M r 90,000, see tracks 3 and 4). Like the mammalian protein, the level of the bacterial protein is elevated by heat shock either in transiently (track 5) or continuously (track 6) shocked cultures. The mammalian M r 90,000 protein is also induced by infection with Herpes simplex virus type-2 (Fig. 2, track 3). To see if the bacterial protein was similarly induced by viral infection of E. coli we used bacteriophage T7. Infection of E. coli with this virus like HSV-2 infection of mammalian cells results in the repression of most host gene expression and eventual cell lysis (Chamberlin et al., 1970). However, as shown in Fig. 3, such infection results in the accumulation of the Mr 94,000 protein recognized by TG7A

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exactly paralleling the accumulation of hsp90 during HSV-2 infection of mammalian cells. In order to identify the gene encoding the Mr 94,000 E. coli protein we studied mutant strains of E. coli with a view to identifying one which lacked this protein. Figure 2 (tracks 7-9) shows the results with one such strain (YI090) which has a deletion removing the Ion gene, which encodes a protease of Mr94,000 which is induced by heat shock and is responsible for the degradation of abnormal or foreign proteins (Baker et al., 1984; Goff et al., 1984; Phillips et al., 1984). In L o n - strains, a low level of a constitutively synthesized Mr94,000 protein is recognized by the antibody in cells grown at low temperature but upon heat shock this protein is repressed, in complete contrast to the behaviour of Lon + strains. The heat inducible Mr 94,000 protein recognized by the antibody in Lon ÷ strains thus appears to be the product of the Ion gene. In order to confirm the identity of the Mr 94,000 protein we introduced the Ion gene into the L o n strain by conjugation and isolated an isogenic pair of strains differing only in the Ion gene. The Lon ÷ member of this pair exhibits a heat inducible Mr 94,000 protein reacting with the antibody which is

Fig. 2. Western blot of mammalian and bacterial samples probed with antibody TG7A. Samples track 1, mock infected baby hamster kidney (BHK) cells; track 2, BHK cells infected with HSV-1; track 3, BHK cells infected with HSV-2. Tracks 4-6, E. coli strain C600 grown at 32°C (track 4), grown at 32°C followed by 20 min at 42°C just prior to harvesting (track 5) and grown at 42°C (track 6). Tracks 7-9, E. coli strain Y1090 grown at 32°C (track 7), grown at 32°C and then for 20 min at 42°C (track 8) and grown at 42°C (track 9). Arrows indicate the positions and sizes of mol. wt markers.

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Fig. 3. Western blot of E. coli strain B/r proteins at various times after infection with bacteriophage T7, probed with antibody TG7A. Track I, uninfected cells; tracks 2-5, infected cells at time 5 min (track 2), 20 min (track 3), 40 min (track 4) and 60 min (track 5) after infection. Complete lysis of the culture occurred shortly after the 60 min time point was taken. Arrows indicate the positions and sizes of mol. wt markers. absent from the Lon- strain (Fig. 4) confirming that this protein is the product of the Ion gene. The low level of constitutively expressed protein detected by the antibody in L o n - strains (see Fig. 2, track 7) parallels the finding that nonsense mutants in the Ion gene still synthesize an M r 94,000 protein with ATP-dependent protease activity which however, has a different isoelectric point and is less stable than the protein found in Lon ÷ strains (Chung and Goldberg, 1981). Such a finding of a protein closely related to Lon but repressed by heat shock parallels the situation in eukaryotes where genes closely related to the heat shock genes but repressed by heat shock have been described (Ingolia and Craig, 1982; Lowe and Moran, 1984). It is noteworthy that L o n - strains (as well as E. coli strain B/r, which has a low level of the Ion gene product--see Phillips et al., 1984) also express two additional proteins not seen in the Lon ÷ strain, one of which, an M~ 46,000 protein is induced both by heat shock and virus infection (see Figs 2 and 3). These two proteins are present at very low levels in the Lon + strain C600 and can be detected only by very long exposures of Western blots. A survey of a wide variety of other strains of E. coli suggested that this difference between the strains used in the experi-

ments shown in Fig. 2 is not due to mutational differences at loci other than Ion but is rather a consequence of the Ion mutation itself (data not shown). The fact that L o n - strains over-express two other proteins recognized by the antibody, one of which is heat inducible, suggests that these proteins may in some way substitute for the Lon protein in the heat shock response of L o n - bacteria. In agreement with this idea we were unable to detect any differences between the growth rates of our isogenic strains of Lon- and Lon ÷ bacteria at different temperatures (see Fig. 5) or in the max. temp. at which growth could occur (45°C in each case).

DISCUSSION

We have shown previously (La Thangue and Latchman submitted), that the M r 90,000 mammalian protein recognized by TG7A is encoded by a member of the previously defined heat shock gene family found in mammalian (Welch and Feramisco, 1982), Drosophila (Holmgren et al., 1981) and yeast (Farrely and Finkelstein, 1984) cells. The work presented here demonstrates that this antibody also recognizes the

Heat shock protein and E. coli Lon protease

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Fig. 4. Western blot ofisogenic Lon- and Lon + strains probed with antibody TG7A. Bacteria were grown at 37°C, prior to harvesting. Track 1, Lon ÷ isolate; track 2, Lon isolate. Arrows indicate the sizes and positions of mol. wt markers.

Lon protein of E. coli. Such a reaction of an antibody prepared against a protein of one species with a protein of another species may be due to a fortuitous sharing of epitopes or may indicate a functional and/or evolutionary relationship between the two proteins (Lane and Koprowski, 1982). In the case of the TG7A antibody the finding that the mammalian and bacterial proteins are both induced in response to heat shock and viral infection suggests that this cross reaction is not fortuitous and that the domain defined by the TG7A antibody may play a functional role in the stress response of a wide range of organisms. The presence of this domain on both the mammalian hsp90 protein and the E. coli Lon protein may be an example of convergent evolution between two

proteins of similar function. Alternatively in view of the remarkable evolutionary conservation of the heat shock proteins in a range of eukaryotic organisms (Kelley and Schlesinger, 1982), it is possible that the Lon protein is evolutionarily related to the eukaryotic hsp90 proteins as is the case for the E. coli dnak gene and the hsp70 proteins of eukaryotes (Bardwell and Craig, 1984). In this regard it is of interest that although antibodies prepared against eukaryotic heat shock proteins of one species react with similar proteins in other eukaryotes (Kelley and Schlesinger, 1982), none (whether against the Mr 70,000 or the Mr 90,000 protein) has been shown to react with a comparable prokaryotic protein. If the Lon protein and hsp90 are indeed homologous, the observation

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OD I

~

Ion-37"C Ion+37"C

Ion-42~C Ion*42~C

Time (hr)

Fig. 5. Growth curves (optical density at 540nm against time) of isogenic Lon- and Lon ÷ bacteria. Open squares, Lon bacteria grown at 37°C; closed squares Lon ÷ bacteria grown at 37°C; open circles Lon- bacteria grown at 42°C; closed circles Lon ÷ bacteria grown at 42°C.

that at least one region of the proteins is exceptionally well conserved (such that one antibody can recognize both the mammalian and bacterial forms), whilst others have diverged, again suggests that this region is of functional importance. Hence whether the relationship of hsp90 and Lon is purely a functional one or is also an evolutionary one, the weight of evidence suggests that antibody TG7A recognizes a common functional domain on the two proteins. This idea is in good agreement with other data from both mammalian and bacterial systems where smaller proteins reactive with the antibodies also exist. Thus in L o n - bacteria two such small proteins are present at much higher levels than in Lon ÷ bacteria and one of these is inducible by heat shock and viral infection. If the TG7A antibody does indeed recognize a functional domain, it is possible that the over-expression of these two proteins and the stress induction of the smaller protein compensate for the absence of the Lon product in the stress responses of Lon- bacteria. Such a compensatory response is consistent with the finding that L o n - bacteria have generally not been found to be more heat sensitive than Lon + strains (see, for example, Phillips et aL, 1984) even when (as in our experiments) isogenic strains differing only at the Ion locus are compared (see Fig. 5). A similar situation occurs in mammalian systems where in addition to M, 90,000 protein, TG7A also recognized Mr 40,000 cellular protein which shares

the epitope recognized by the antibody but is otherwise unrelated to the M r 90,000 protein on the basis of peptide mapping (La Thangue and Latchman, submitted). Although both heat shock and HSV-2 infection induce the M, 90,000 protein, infection with the closely related virus, HSV-I, causes the accumulation of the Mr 40,000 protein (see Fig. 2, track 2). Such a result is difficult to explain except on the basis of different stresses inducing proteins with a common function mediated by a common functional domain. Our finding that the Ion gene product of E. coliand the mammalian M, 90,000 heat shock protein share a common functional domain, suggests the possibility of using the known data about Ion mutants as a means of deducing the role of this functional domain in the eukaryotic stress response. The Lon phenotype was originally described as an increase in sensitivity to u.v. light and increased accumulation of capsular polysaccharides (Howard-Flanders et al., 1964; Huas and Markovitz, 1972). It is now known that the Ion gene encodes an ATP-dependent protease (Chung and Goldberg, 1981; Charette et al., 1981) that is involved in degradation of abnormal or short-lived proteins and it is the loss of this activity which is believed to cause the properties of the phenotype (Mizusawa and Gottesman, 1983). It has recently been shown that introduction of foreign proteins into E. coli results in induction of the Ion gene (Goff and Goldberg, 1985) and this has led to the suggestion that the induction of the gene in heat shock is of a similar nature, being a homeostatic response to the production of denatured protein. Such a response to heat or other stresses is not confined to prokaryotes but is seen also in eukaryotes: treatments of eukaryotic cells with amino acid analogues (Hightower, 1980) or oxidizing agents (Leenders and Berendes, 1972), which denature or damage proteins can cause the heat shock response, as can the introduction into cells of denatured proteins (Ananthan et al., 1986). Indeed the production of such damaged proteins has been postulated to be the ultimate inducer of the stress response in eukaryotes (Goff and Goldberg, 1985). The role of the Mr 90,000 protein in eukaryotic cells by analogy with the Lon protein may be to minimize damage by rapidly digesting abnormal or denatured proteins resulting from stress. Such an idea would predict that the Mr 90,000 mammalian protein would possess protease activity. We are currently using affinity chromatography on a monoclonal antibody column to purify the protein in order to test this hypothesis. Acknowledgements--We thank N. A. Mitchison, M. Raft and P. W. J. Rigby for helpful discussion and D. Roscoe for E. coli B/r and bacteriophage T7. D. S. Latchman thanks P. W. J. Rigby for providing him with laboratory space during the early part of this work. C. E. L. Leaver and W. L. Chan were supported by grants from the Nuttield Foundation and the MRC respectively. This work was partly supported by a grant to D. S. Latchman from the Cancer Research Campaign.

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