Expression, Purification, and Characterization of gp160e, the Soluble, Trimeric Ectodomain of the Simian Immunodeficiency Virus Envelope Glycoprotein, gp160
2000; Elsevier BV; Volume: 275; Issue: 45 Linguagem: Inglês
10.1074/jbc.m004905200
ISSN1083-351X
AutoresBing Chen, Genfa Zhou, Mikyung Kim, Yasmin Chishti, Rebecca E. Hussey, Barry K. Ely, J.J. Skehel, Ellis L. Reinherz, Stephen C. Harrison, Don C. Wiley,
Tópico(s)HIV/AIDS Research and Interventions
ResumoThe envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares ∼25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments. The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares ∼25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments. simian immunodeficiency virus human immunodeficiency virus hemagglutinin monoclonal antibody polymerase chain reaction base pair kilobase pair polyacrylamide gel electrophoresis phosphate-buffered saline ethylene glycol bis(succinimidylsuccinate) Chinese hamster ovary endoglycosidase H The SIV1 and HIV envelope glycoproteins, known as gp160, bind to cell surface receptors, effect cell entry by fusion of viral and cellular membranes, and as major surface antigens, induce neutralizing antibodies in the host. gp160 is synthesized as a single chain precursor, which is cleaved after oligomerization by furin, or a similar enzyme, into the two chains gp120 and gp41. Cleavage occurs during transport to the plasma membrane (1Allan J.S. Coligan J.E. Barin F. McLane M.F. Sodroski J.G. Rosen C.A. Haseltine W.A. Lee T.H. Essex M. 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We report the development of expression systems using insect and mammalian cells for production of soluble, oligomeric ectodomain, gp160e, from the SIV envelope glycoprotein. Purified oligomers are shown to bind CD4 and Fab fragments of neutralizing monoclonal antibodies. Experiments show that the molecule is trimeric. These observations should facilitate biochemical and structural characterization of the viral glycoprotein in its native, oligomeric form. SIV gp160e from the SIV strain Mac32H pJ5 (43Rud E.W. Yon J.R. Larder B.A. Clarke B.E. Cook N. Cranage M.P. Brown F. Chanock R.M. Ginsberg H.S. Lerner R.A. Vaccines 92: Modern Approaches to New Vaccines Including Prevention of AIDS. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 229-235Google Scholar) was expressed in CHO-Lec3.2.8.1 cells using the expression construct pSIV-M (44Rhodes A.D. Spitali M. Hutchinson G. Rud E.W. Stephens P.E. J. Gen. Virol. 1994; 75: 207-213Crossref PubMed Scopus (13) Google Scholar) following a procedure described previously (45Liu J. Tse A.G. Chang H.C. Liu J. Wang J. Hussey R.E. Chishti Y. Rheinhold B. Spoerl R. Nathenson S.G. Sacchettini J.C. Reinherz E.L. J. Biol. Chem. 1996; 271: 33639-33646Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Briefly, 20 μg of SalI-linearized pSIV-M DNA was transfected into the Lec3.2.8.1 cells by calcium phosphate precipitation (Stratagene). Transformants were selected in GMEM medium (Life Technologies, Inc.) containing 25 μm methionine sulfoximine and assayed for secretion of soluble SIV gp160e by enzyme-linked immunosorbent assays. Immulon plates (Dynatech Laboratories Inc.) were coated with anti-SIV ENV monoclonal antibody KK19 (46Kent K.A. Gritz L. Stallard G. Cranage M.P. Collignon C. Thiriart C. Corcoran T. Silvera P. Stott E.J. Aids. 1991; 5: 829-836Crossref PubMed Scopus (69) Google Scholar) by incubating a 4 μg/ml solution overnight at 4 °C; the plates were then blocked with 1% bovine serum albumin in PBS at room temperature for 2 h. Cell culture supernatant (50 μl) from each transformed clone was added to the plates and incubated overnight at 4 °C. The plates were washed with 0.1% bovine serum albumin in PBS and incubated with 0.2 μg/ml biotinylated mAb KK41 (47Kent K.A. Rud E. Corcoran T. Powell C. Thiriart C. Collignon C. Stott E.J. AIDS Res. Hum. Retroviruses. 1992; 8: 1147-1151Crossref PubMed Scopus (89) Google Scholar) for 2 h at room temperature. After washing again, the plates were developed with horseradish peroxidase-conjugated streptavidin (Sigma). The highest expressing cells were chosen for rescreening with the same assay. The expression of SIV gp160e was also confirmed by immunoprecipitation. Large scale production of SIV gp160e was carried out following the protocol previously described (45Liu J. Tse A.G. Chang H.C. Liu J. Wang J. Hussey R.E. Chishti Y. Rheinhold B. Spoerl R. Nathenson S.G. Sacchettini J.C. Reinherz E.L. J. Biol. Chem. 1996; 271: 33639-33646Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) in 175-cm2 tissue culture flasks or 850-cm2 roller bottles (Corning). The protein expressed from pSIV-M in Lec3.2.8.1 cells was found to have two N termini (see Fig. 1). To produce SIV gp160e variants with the V1, V2, and V3 (gp160eΔ(V1V2V3)) or just the V1 and V2 (gp160eΔ(V1V2)) segments deleted and a corrected N terminus in the Lec3.2.8.1 cells, a shorter version of the tissue plasminogen activator leader sequence was fused with the N terminus of SIV gp160. pSIV-M was amplified by PCR with primers BC-16 (5′-TAGTCTCATTGACCATGTCT-3′) and BC-45 (5′-GCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGAGCTAGCACTCAATATGTCACAGTC-3′). The resulting 424-bp PCR product was gel-purified and reamplified with BC-16 and BC-44 (5′-GCTCTAGAAGGGACGCTGTGAAGCAATCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGT-3′) to give a 477-bp fragment, which was subsequently digested withXbaI and NsiI and gel-purified. This fragment was ligated with a 1.4-kb NsiI–EcoRI fragment from pFBSIVΔV1V2 (see below) or a 1.4-kb NsiI–EcoRI fragment from pFBSIVΔV1V2V3 (see below) and then cloned into pEE14 (48Kingston R.E. Kaufman R.J. Bebbington C.R. Rolfe M.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.D. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 2nd Ed. John Wiley & Sons, Inc., New York1992: 55-62Google Scholar) digested with XbaI and EcoRI to generate pNES9ΔV1V2 and pNES1ΔV1V2V3, respectively. Expression of pNES9ΔV1V2 and pNES1ΔV1V2V3 followed the same procedure except that transformants were were screened with different mAbs, as mAb KK9 (46Kent K.A. Gritz L. Stallard G. Cranage M.P. Collignon C. Thiriart C. Corcoran T. Silvera P. Stott E.J. Aids. 1991; 5: 829-836Crossref PubMed Scopus (69) Google Scholar) and biotinylated mAb KK41 used for pNES9ΔV1V2, mAb 2C3 2M. Kim, B. Chen, R. E. Hussey, Y. Chishti, D. Montefiori, J. A. Hoxie, D. C. Wiley, S. C. Harrison and E. L. Reinherz, manuscript in preparation. and biotinylated mAb KK41 for pNES9ΔV1V2 by enzyme-linked immunosorbent assays.Figure 1Constructs for expression of the ectodomain, gp160e, of the SIV envelope glycoprotein in mammalian and insect cells. Schematic representations for the expression constructs, pSIV-M (44Rhodes A.D. Spitali M. Hutchinson G. Rud E.W. Stephens P.E. J. Gen. Virol. 1994; 75: 207-213Crossref PubMed Scopus (13) Google Scholar), pNES1ΔV1V2V3, pFBSIV-His1, and pFBSIVΔV1V2V3-His1, are shown. In all cases, the posttranslational cleavage sites (residues 512 and 523) between gp120 and gp41 have been mutated as shown to prevent cleavage and dissociation of gp120. pNES1ΔV1V2V3 has a shorter tissue plasminogen activator leader sequence than pSIV-M, while both pFBSIV-His1, and pFBSIVΔV1V2V3-His1 use the honeybee melittin (HM) secretion signal. The cleavage sites after the leader sequences were confirmed by N-terminal sequencing of the expressed products and are indicated by arrows beneath the sequences. The sequences where the V1, V2, and V3 segments were deleted and replaced with a GAG linker (in italics) are shown for pNES1ΔV1V2V3 and pFBSIVΔV1V2V3-His1. The sequences where the transmembrane segment of gp41 was truncated are also shown for all constructs. Leader sequences are shown in normal type, and SIV gp160 sequences included in the expression constructs are inboldface type and underlined; sequences not included are printed with astrike-through.View Large Image Figure ViewerDownload Hi-res image Download (PPT) pFBSIV-His1 was constructed to express SIV gp160e in insect cells using the Bac-to-Bac expression system (Life Technologies, Inc.). The pSIV-M was amplified by PCR using primers BC-1 (5′-CGCGGATCCGACTCAATATGTCACAGTCTTTTAT-3′) and BC-2 (5′-GGCCGAATTCTATATCCAAGAAGCAAGG-3′) to produce a DNA fragment encompassing the complete SIV gp160e coding sequence. The PCR product was digested with BamHI and EcoRI, gel-purified, and cloned into pMelBac (Invitrogen) to generate an intermediate construct pBacSIV-5, which was sequenced using primers BC-1, BC-2, BC-6 (5′-CACCAACGGCAGCATCAA-3′), BC-7 (5′-AGATGTAATGACACAAAT-3′), BC-8 (5′-AAATTGGAAGGATGCAAT-3′), BC-9 (5′-GAGACCTCACGTGTAACT-3′), BC-10 (5′-GTGCAGCAACAGCAACAG-3′), and BC-16 to verify the entire sequence of SIV gp160e open reading frame. pBacSIV-5 was then amplified using primers BC-14 (5′-TTTATGGTCGTATACATTTCTTACATCTATGCGACTCAATATGTCACAGTC-3′) and BC-16. The resulting PCR product was reamplified by BC-13 (5′-CGCGGATCCATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATT-3′) and BC-16 to produce a 447-bp fragment, which was digested withBamHI and NsiI, gel-purified, and designated as fragment 1. This fragment allows fusion of the N terminus of SIV gp160e and the honeybee melittin secretion signal sequence. pBacSIV-5 was digested with NsiI and NcoI and gel-purified to give a 1.5-kb fragment, designated as fragment 2, which encompassed a major portion of SIV gp160e open reading frame. To introduce a histidine tag at the C terminus of SIV gp160e, a 757-bp PCR product was amplified using pBacSIV-5 as template and BC-9 and BC-24 (5′-CCGGAATTCTCAATGATGATGATGATGATGAGTGCGACCTTCGATTTGTATATACTTTATCCAAGA-3′) as primers and digested with NcoI andEcoRI, gel-purified, and designated as fragment 3. Fragments 1, 2, and 3 were ligated together and cloned into pFastBac-1 (Life Technologies, Inc.), digested with BamHI andEcoRI, to give the expression construct pFBSIV-His1. Constructs were also made to express SIV gp160e variants with deletions of segments V1, V2, and V3; of V1 and V2 only; or of V3 only. pFBSIV-His1 was amplified by PCR with primers BC-2 and BC-32 (5′-CCATTATGCATTGGAGCAGGTCACTGTAACACTTCTATTAT-3′), which replaces V1 and V2 segments with a short linker GAG. The PCR product was digested with NsiI and NcoI, gel-purified, and inserted into pFBSIV-His1, which was digested with NsiI and NcoI to generate pFBSIVΔV1V2-His1. pFBSIVΔV3-His3 was constructed by overlapping PCR. pBacSIV-5 was amplified with primers BC-1 and BC-43 (5′-ACACCAACCTGCTCCTCTACATTTCATTGTTAGATT-3′) and primers BC-18 (5′-TTGTATATACTTTATCCAAGAAGCAAG-3′) and BC-42 (5′-TGTAGAGGAGCAGGTTGGTGTTGGTTTGGAGGAAAT-3′) to produce two overlapped 892-bp and 1.0-kb fragments, respectively. These two fragments were gel-purified, mixed, and reamplified with primers BC-1 and BC-18 to give a 1.9-kb fragment in which the V3 segment was replaced with GAG linker. This fragment was then digested withNsiI and NcoI and cloned into pFBSIV-His1, which was also digested with NsiI and NcoI to generate pFBSIVΔV3-His3. To construct pFBSIVΔV1V2V3-His1, pFBSIVΔV3-His3 was used as a template to produce a 1.4-kb PCR fragment with primers BC-2 and BC-32 to replace V1 and V2 segments with a GAG. The PCR product was digested with NsiI and NcoI, gel-purified, and inserted into pFBSIV-His1, which was digested with NsiI andNcoI to generate pFBSIVΔV1V2V3-His1. pFBSIVΔV1V2 and pFBSIVΔV1V2V3 were also made to express gp160e variants without histidine tags. pBacSIV-5 was used as a template for PCR with primers BC-2 and BC-10. The PCR product was then digested with NcoI and EcoRI, gel-purified, and inserted into NcoI- and EcoRI-digested pFBSIVΔV3-His3 and pFBSIVΔV1V2V3-His1, respectively, to yield pFBSIVΔV1V2 and pFBSIVΔV1V2V3. Both restriction digestion and DNA sequencing verified all of the expression constructs. Recombinant baculovirus was generated according to the manufacturer's protocol and amplified in Sf9 insect cells in TNM-FH medium (Sigma). Expression of SIV gp160e was confirmed by a Western blot using sheep antisera against SIV envelope glycoprotein. The optimal amount of virus and postinfection harvest time was determined by small scale tests in 100-ml spinner flasks. Infected Trichoplusia ni (Hi-5) cells were found to secrete significantly more SIV gp160e than Sf9 cells, judging by Western blot. For large scale protein production, 10 liters of T. ni (Hi-5) cells (2 × 106 cells/ml) in Ex-Cell 405 medium (JRH Biosciences) were infected at a multiplicity of infection of 2.5. 3 days after infection, the supernatant was harvested by centrifugation and concentrated to 1 liter in a tangential flow filtration system, ProFlux M 12 (Millipore Corp.). The CHO Lec3.2.8.1 supernatants containing secreted SIV gp160e were harvested by centrifugation or by filtration through a Corning filter (0.22 mm). SIV gp160e was purified by an immunoaffinity chromatography using a mAb 17A11 3J. A. Hoxie,unpublished results. affinity column (5-ml bed volume), where the monoclonal antibody 17A11 was cross-linked at 5 mg/ml to GammaBind Plus Sepharose (Amersham Pharmacia Biotech) with dimethyl pimelimidate (Pierce). The supernatants were passed through the column with a flow rate of about 0.5 ml/min. After extensive washing with PBS, the protein was then eluted with 100 mmglycine (pH 3.0), followed by immediate neutralization with 2m Tris-HCl (pH 8.0). The fractions were analyzed by SDS-PAGE. The fractions containing SIV gp160e were pooled, concentrated, and further purified by gel filtration chromatography on Superdex 200 or Superose 6 (Amersham Pharmacia Biotech) with a buffer containing 25 mm Tris-HCl (pH 8.0) and 150 mmNaCl. SIV gp160eΔ(V1V2) and gp160eΔ(V1V2V3) proteins were purified following the same procedure. SIV gp160e expressed from insect cells was purified by metal chelate affinity chromatography with ProBond resin (Invitrogen). Concentrated insect cell culture supernatants were immediately changed into 1× column buffer (25 mm sodium phosphate (pH 8.0), 250 mm NaCl) in a ProFlux M 12 flow filtration system to remove small molecules in the medium that interfere with the binding of His-tagged SIV gp160e to the nickel column. After centrifugation at 5000 rpm in a JA-14 rotor (Beckman) for 15 min to remove insoluble materials, imidazole was added to the final concentration of 15 mm to reduce nonspecific binding to the resin. Batch binding was then performed at 4 °C for 3 h. After the column was packed (about 5-ml bed volume), the beads were washed by 100 ml of 1× column buffer containing 15 mm imidazole, followed by further washing with 50 ml of 40 mm imidazole in 1× column buffer. The protein was eluted with 300 mm imidazole in 1× column buffer. The fractions were analyzed by SDS-PAGE. The fractions containing SIV gp160e were pooled, concentrated, and further purified by gel filtration chromatography on Superdex 200 or Superose 6 (Amersham Pharmacia Biotech) with a buffer containing 25 mmTris-HCl and 150 mm NaCl. SIV gp160eΔ(V1V2) and gp160eΔ(V1V2V3) proteins were purified following the same procedure. N-terminal analyses of purified proteins were carried out by the HHMI biopolymer facility. For chemical cross-linking experiments, SIV gp160e protein was dialyzed extensively against PBS. In 20-μl reactions, SIV gp160e (1 mg/ml) was incubated with ethylene glycol bis(succinimidylsuccinate) (EGS; Pierce) at concentrations of 0.06, 0.18, 0.55, 1.67, and 5 mm respectively, on ice for 30 min. The reactions were then quenched by adding 5 μl of 100 mmTris-glycine (pH 7.0) and incubated at room temperature for 45 min. The cross-linked products were analyzed by SDS-PAGE. Cross-linked phosphorylase b (Sigma) was used as an SDS-PAGE molecular weight standard. Analytical ultracentrifugation was performed on a Beckman XL-A analytical ultracentrifuge at 4 °C. Experiments were performed at concentrations of 1.6 μm, 3.3 μm, and 6.5 μm protein and centrifuged at a rotor speed of 6000 rpm. Data were fitted to a single-species model. The protein partial specific volume and solvent density were calculated according to Laue et al. (49Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge1992: 90-125Google Scholar). Briefly, all glycans in gp160e from CHO-Lec3.2.8.1 cells were found to be (GlcNAc)2(Man)5 4M. Kim and E. L. Reinherz, unpublished results. by mass spectroscopy using previously described methods (45Liu J. Tse A.G. Chang H.C. Liu J. Wang J. Hussey R.E. Chishti Y. Rheinhold B. Spoerl R. Nathenson S.G. Sacchettini J.C. Reinherz E.L. J. Biol. Chem. 1996; 271: 33639-33646Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and assuming that all 25 potential glycosylation sites are occupied, the partial specific volume of gp160e was calculated to be 0.70 ml/g. Four-domain sCD4 expressed from CHO cells, affinity-purified, and sized by Superdex 75 gel filtration was kindly provided by Dr. Yi Xiong. Monoclonal antibodies were purified from cell supernatants of hybridomas growing in roller bottles using a 5-ml GammaBind Plus Sepharose (Amersham Pharmacia Biotech) affinity column. Fab fragments were produced as described (45Liu J. Tse A.G. Chang H.C. Liu J. Wang J. Hussey R.E. Chishti Y. Rheinhold B. Spoerl R. Nathenson S.G. Sacchettini J.C. Reinherz E.L. J. Biol. Chem. 1996; 271: 33639-33646Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), from the neutralizing monoclonal antibodies KK9 (46Kent K.A. Gritz L. Stallard G. Cranage M.P. Collignon C. Thiriart C. Corcoran T. Silvera P. Stott E.J. Aids. 1991; 5: 829-836Crossref PubMed Scopus (69) Google Scholar), 17A11 (a kind gift of J. Hoxie), and 9G3.2 Purified SIV gp160e was incubated at room temperature for 1 h with CD4 or Fab fragments. The complexes were separated from excess unbound CD4 or Fabs by a gel filtration chromatography on Superose 6 (Amersham Pharmacia Biotech). Molecular weights were calculated based on a standard curve plotted from the elution volumes of known proteins. Peak fractions were verified to contain both gp160e and CD4 or Fabs by SDS-PAGE. CD spectra were recorded at 25 °C using SIV gp160e at a concentration of 0.56 mg/ml in PBS with a circular dichroism spectrometer model 62DS (Aviv). The molar ellipticity [θ] was monitored as the average of 12 scans with 0.5-nm bandwidth and 1.0-nm wavelength increments from 195 to 260 nm. The spectra were corrected with a base line obtained using buffer alone under the same conditions. Endoglycosidase H (Endo H) digestion was carried out using 20 μg of the SIV gp160e expressed in CHO-Lec3.2.8.1 cells and 500 units of Endo H (New England Biolabs) for each reaction at room temperature under native and denaturing conditions with buffers supplied by
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