HIV-1 envelope protein gp120 expression by secretion in E. coli: assessment of CD4 binding and use in epitope mapping

Journal of VirologicalMethods,29 (1990) 105-l 14 Elsevier

105

VIRMET 01039

Short Communication

HIV-l envelope protein gp120 expression by secretion in E. coli: assessment of CD4 binding and use in epitope mapping Yuko Morikawa’, John P. Moore’ and Ian M, Jones’ NERC Instituteof Virologyand EnvironmentalMicrobiology, Oxford, and ZInstituteof Cancer Research, Chester-BeattyLaboratories, Royal Cancer Hospital, London, U.K. (Accepted

29 March 1990)

Summary A non-glycosylated form of the HIV- 1 envelope protein gp 120 and four truncated derivatives have been expressed as non-fused secreted products in the periplasmic space of E. coli. We show that the full length molecule, whilst folded and soluble, fails to bind to CD4 consistant with other work that suggests an essential role for carbohydrate in gp120 function. In addition, when used in conjunction with the truncated derivatives, rapid epitope mapping of antigpl20 monoclonal antibodies is achieved using both Western-blot and ELISA formats. Epitope mapping; HIV-l; Envelope protein gp120

Interaction of the HIV envelope protein gp120 with its. receptor on lymphocytes, CD4, is the primary event leading to infection of the host cell (Dalgleish et al., 1984; Klatzman et al., 1984). Accordingly, there is a great deal of interest in the structural requirements of both CD4 and gp120 necessary for their successful interaction. For CD4, the gp120 binding site has been precisely mapped to a region in the amino terminal domain of the protein (the VI domain) spanning amino acids 41-52 (Peterson and Seed, 1988; Arthos et al., 1989). For gp120, however, the requirements for CD4 binding appear to be much more complex. A region in the carboxy terminal domain of the protein (residues 397-439) has been shown to Correspondence to: I.M. Jones, NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, U.K. 0166~0934/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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be essential for CD4 binding (Lasky et al., 1987) but deletions from the amino te~inus of the molecule also prevent binding (Dowbenko et al., 1988; Cordonnier et al., 1989). Moreover, whilst it is clear that carbohydrate on CD4 is not required for binding to gpl20 (Arthos et al., 1989), the exact role of carbohydrate on gp120 in complex formation is less clear. In some reports deglycosylated gp120 was reported not to bind to CD4 (Putney et al., 1986; Matthews et al., 1987; Fennie and Lasky, 1989) whereas in another, carbohydrate removal was shown to reduce CD4 binding substantially but not to abolish it (Fenouillet et al., 1989). However, in a number of these reports it was necessary to include a denaturing detergent (SDS) in order to allow complete deglycosylation in vitro (Matthews et al., 1987) or to enhance solubility (Pumey et al., 1986). Thus, the CD4 binding properties of a soluble gp120 expressed in a non-gly~osylating system and in the absence of detergent have yet to be investigated. To address this question we have expressed gpl20 in E. coti using an expression vector that secretes proteins into the bacterial periplasmic space. We have characterised the expressed product in some detail and report on its CD4 binding characteristics. In addition we describe several truncated derivatives of gpl20 produced in the same system and demonstrate their use in rapid epitope mapping. To express gp120 as a secreted protein in E. coli a blunt-ended Asp718-Hind3 fragment encoding the mature gpl20 molecule of HIV-lnau was cloned into the filled-in Hind3 site of expression vector PIN-III-&PA-Hind3 (Rentier-Delrue et al., 1988). As a result of this manipulation, essentially the whole of the mature form of gp120 (amino acids 42-516 inc.) was grafted in phase onto the signal peptide of the major outer membrane protein (OmpA) of E. cc&. Following induction with IPTG, total cell, intracellular and periplasmic fractions were examined for the presence of antigen using Western blotting with a sheep anti-peptide serum to gp120 (D7324-Aalto BioReagents). The results are shown in Fig. 1. Two antigenically active proteins of 55 kDa and 45 kDa were present in total cell extracts with intracellular fractions enriched in the 55 kDa species whilst the periplasmic fraction contained only the 45 kDa product. The 55 kDa protein had the predicted mobility of a full length OmpA-gp120 molecule whilst the 45 kDa protein matched the predicted MW for a mature secreted form of the molecule lacking the bacterial signal sequence. We conclude, therefore, that gp120 (gp120,,ti) is produced in this system and secreted into the bacterial periplasmic space. A feature of expression in the bacterial periplasm is that the expressed products are soluble, disulphide bonded and often fold to give demonstrable functions activity (e.g. Skerra and Pluckthun, 1988; Better et al., 1988; Ward et al., 1989). To test for folding in the case of gpf20,,ii, we prepared total cell extracts from induced cultures in the presence or absence of reducing agent (5% &mercaptoethanol) and examined the gp120,,ii migration profile by Western blotting. Fig. 2 shows that the banding pattern for g~12O,,~i changed substantially when the extract was not reduced, suggesting that disulphide bond formation has occured in this system. Thus, a soluble, non-glycosylated gpl20 molecule is produced in this vector system that is suitable for testing for its ability to bind to CD4. The CD4 binding assay is described in detail elsewhere (Moore, 1990) but relies on a twin site ELISA format using antibodies to gpl20 and CD4 to detect complex formation between gp120

107 kD

1

2

3

116-

58-

37-

Fig. 1. Detection of gPl2Omti in various cell fractions. Cells were grown to an ODm of 0.5 at 37’C, induced by the addition of IPTG to 1 mM and grown for a further 1 h at 37°C. Bacteria were harvested and either lysed directly or fractionated to cellular and periplasmic fractions (Bigggins and k&die, 1983). The equivalent of 0.05 ml of induced culture was subjected to 12% SDS-PAGE and transferred to Immobilon (Millipote) by standard procedures (Brunette, 1983). gp120 antigens were detected with D7324 followed by an anti-sheep Ig. alkaline phosphatase conjugate. Track 1, total lysate; track 2, cellular fraction: track 3, periplasmic fraction, kDa indicates molecular weight standards run in parallel the sires of which are shown (in kilodaltons).

and soluble CD4 (sCD4) in solution. The assay is extremely sensitive, detecting binding of gp120 at concentrations as low as 10 r&ml. To test for CD4 binding by gp12Oc,ii,periplasmic extracts of induced cultures were prepared as described (Higgins and Hardie, 1983), quantitated by ELBA (Moore and Jarrett, 1988) and assayed in parallel with similar amounts of mammalian gpl20 (gp120m- as a positive controi), The results are shown in Fig. 3. The gp120mm binds to sCD4 over a rarige of concentrations within the assay with the limit of detection in the region of l-10 @ml. However, even at concentrations lOO-fold higher, gPilto,,li, failed to show any detectable binding to sCD4. It is concluded from this that, gPl2Ocoribinding to sCD4 is at least 100-fold less efficient than gp120mamm.The =120&i molecule that we have expressed starts at amino acid 42 of the full length molecule and it could be argued that deletion of the amino terminus abrogates CD4 binding. However, this is unlikely to be the case as deletions as far as amino acid 46 of gp120 are reported not to affect CD4 binding substantially (Cordon&r et al., 1989). We conclude, therefore, that bacterial expression of gpI20 in the periplasm of E. cdi results in a molecule that is antigenic, soluble and appatently folded yet

108

kD

1

M

2

116-

49-

37-

Fig. 2. gP12Oc,li induced as described in the legend in Fig. 1 was lysed in 3% SDS with or without 5% P-mercapto-ethanol @Me) and Western blotted as described in the legend to Fig. 1. Track 1, no me; track 2, + ,C?Me;track M shows a set of prestained markers (Sigma SDS-7B) the sizes of which are shown in kilodaltons (kDa).

is functionally inactive in binding CD4. This result is consistant with recent data suggesting an essential role for at least some of the carbohydrate on gp120 in CD4 binding (Fennie and Lasky, 1989) although it remains possible that subtle aspects of gp120 conformation rather than the absence of carbohydrate are the primary cause of failure to bind to CD4. The successful expression of the full length gp120 molecule in the periplasmic expression system prompted us to construct a further four truncated derivatives of gpl20,ti for use in epitope mapping of monoclonal antibodies raised against recombinant gp120. Table 1 details the manipulation carried out and the predicted resulting products. To test for expression of each deleted gp120, cultures were induced and analysed by Western blotting using a polyvalent antiserum raised against gp 120,,, (S216, from the MRC Aids reagent repository). In addition, to evaluate their use in direct epitope mapping, Western blotting was also done using a commercial antigp120 peptide serum (D7324), the epitope for which has not previously been published. The results are shown in Fig. 4. Western blotting with a polyvalent serum (S216) identified several gp120 proteins in each induced extract, the largest of which in each case had a molecular weight corresponding to that predicted by the truncations detailed in Table 1. The major antigens produced by each construct appeared as a doublet representing proteins with and without the

1.0

/ 1

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0

0

0.5

0

/

/

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--

&---05f+-.-~_, ~~ 1

0

10

nglml

100

1000

gp120

Fig. 3. Binding of gp120mam,,,(0) or gPl2Oati (0) to soluble CD4 in solution. gp120-sCD4 complexes formed during a 2 h incubation period were captured to a solid phase using D7324 (antigpl20) and detected using 0KT4 (antiCD4) followed by an anti-mouse Ig. alkaline phosphatase conjugate. Alkaline phosphatase activity (i.e, complex formation) was quantitated using a commercial enzyme substrate (AMPAK, Novo BioLabs, Cambridge) by measuring absorbance at 495 nm.

TABLE 1 Plasmid designation

Manipulation

gPl2Ocoli

None

42-516 (full length)

AMst

Deletion of sequence 3’ to MsrII site

42-370

ABgl - Bgl

Deletion of sequence between BglII sites and Klenow repair of ends to restore reading frame

42-279 + 472-5 16

ABgl - Sma

Deletion of sequence 3’ to BgfII site

42-279

AStu

Deletion of secmence 3’ to SruI site

42-208

of gp120 gene

gp120 amino acids remaining

bacterial signal peptide. In addition, there are a number of proteolytic breakdown products. When blotted with D7324, however, only two products are highlighted (full length and ABgl-Bgl) allowing a direct assignment of the D7324 epitope to the terminal 44 amino acids of gp120. Furthermore, essentially no breakdown products were visualised by D7324 suggesting that the majority of bacterial proteolysis occurred from the carboxy terminus. An advantage of these truncated products over those already described (Putney et al., 1986; Dowbenko et al., 1988) is that their solubility allows their use in ELISA assays as well as Western blots. This is an attractive feature for mapping epitopes that are sensitive to the denaturation involved during the Western blotting procedure (Bumette, 1981). To evaluate this type of assay for epitope mapping, soluble lysates of each truncated gp120 molecule were prepared from the periplasm

110

B 3

4

5

5049-

27-

/ // // /I-/!_

Fig. 4. Detection of truncated gp120 molecules and epitope mapping of D7324. Cultures of the truncations detailed in Table 1 were induced and total protein extracts Western blotted as described in the legend to Fig. 1. In panel A antigens were identified using S216, a polyvalent antigpl20 serum, whilst in panel B the blot was prepared using D7324. In each panel the tracks are (I), gpl2O,,ti; (2), AMst; (3), ABglSma; (4), ABgl-Bgl; (5), AStu. Molecular weights of protein standards are shown on the left of panel A and are in kilodaltons.

4

a

m

a

Fig. 5. Epitope mapping of mAb 221 using a ELISA assay. Microtitre plates were coated overnight with S216 at l/1000 dilution in 0.1 M sodium bicarbonate pH 9.0 and subsequently with periplasmic extracts prepared from each gp120 truncation mutant. After 1 h at room temperature the antigens were removed and the plate washed and treated with mAb 221 at 1 &ml. Bound antibody was detected using an antimouse alkaline phosphatase conjugate with para-nitrophenol as substrate. mAb binding was quantitated by absorbance at 405 nm. All washes and dilutions were. done in 3% milk powder in water.

of induced E. coli cultures and captured to a solid phase using S216. Following washing, each truncated gp120 protein was probed with monoclonal antibody ADP 22 1 (a gift from Rod Daniels) raised against recombinant HIV envelope protein, and subsequently, with an anti-mouse Ig. alkaline phosphatase conjugate. The results

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are shown in Fig. 5. The epitope for mAb 221 could be mapped directly using this assay procedure and, in this case, was found to be within the terminal 44 amino acids of gp120, the same region of the molecule recognised by D7324. ADP221 and D7324 do not however, bind to the same epitope as they can both bind simultaneously to gp120,in twin site EISA (Moore et al., 1990). Using the same methodology we have also mapped a further monoclonal antibody (CRA1 from Mark Page) to this site suggesting the conserved carboxyl te~inus of gp120 is an immunodominant region of the protein. Similar conclusions have been reached by others (Palker et al., 1987; Bahraoui et al., 1989). Epitope mapping of anti gp120 mAbs has been previously reported using Western blotting combined with a Xgtl 1 @galactosidase-fusion protein) expression library (Dowbenko et al., 1988) but as pgal-fusion proteins are rarely soluble (see Jones and Brownlee 1985; Jones et al., 1986) an ELISA format for co~o~ation~ly sensitive epitopes was not described. Similarly, epftope mapping can be achieved by pepscanning (e.g. Gehsen et al., 1984) but this technology is only successful for epitopes that lie within short linear peptides, In conclusion, we have constructed and tested a number of E. coli strains that express portions of the HIV- 1 envelope protein gp 120. We fail to detect any receptor binding by the largest of these proteins in keeping with the suggested essential role of carbohydrate in gp120 function. ..4lthough sugar appears important however, we have shown elsewhere that the type of carbohydrate on gp120 can change without substantial loss of CD4 binding ability (Morikawa et al., 1990). These observations suggest that sugar residues are not involved in contact with CD4 but that they aid the gp120 molecule gain the correct ~onfo~~on for biological activity. In addition to an examination of CD4 binding by gpl20,li we have demonstrated the use of a set of truncation mutants for rapid epitope mapping of monoclonal antibodies to gp120 by both Western blot and ELISA type assays. Finally, as these proteins appear to be folded and soluble in the absence of detergent, they may have advantages as immunogens over the largely denatured recombinant products that have already been examined (e.g., Putney et al., 1986). Acknowledgements

We thank Tony Wtlkinson (University of York) for the gift of a gp120 clone, Rod Daniels (NIMR, Mill Hill) for ADP 221, Mark Page and Harvey Holmes (NIBSC) for CRA-1 and S2 16 and Paul Stephens (Celltech) for gpl20-,. All reagents described in this work are available from the MRC AIDS reagent repository by contacting Dr H. Holmes, National Institute for Biological Standards and Control, South Mimms, Herts. The work was supported by the Medical Research Council’s AIDS directed program, and J.P.M. is also supported by the Cancer Research Campaign.

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