ABSTRACT
Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.
Subject(s)
CD4 Antigens/chemistry , Gene Products, env/chemistry , Glycoproteins/chemistry , Immunoglobulin Fab Fragments/chemistry , Retroviridae Proteins, Oncogenic/chemistry , Viral Fusion Proteins/chemistry , Amino Acids/chemistry , Binding Sites , Blotting, Western , CD4 Antigens/metabolism , Chromatography , Dimerization , HIV/metabolism , Humans , Kinetics , Ligands , Models, Statistical , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Surface Plasmon Resonance , Time Factors , UltracentrifugationABSTRACT
Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (K(D) = 1-2 nm) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.
Subject(s)
Gene Products, env/isolation & purification , HIV Envelope Protein gp41/isolation & purification , HIV-1 , Membrane Glycoproteins/isolation & purification , Recombinant Proteins/isolation & purification , Retroviridae Proteins/isolation & purification , Simian Immunodeficiency Virus , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , CD4 Antigens/metabolism , CHO Cells , Cricetinae , Gene Products, env/genetics , Gene Products, env/immunology , Glycosylation , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/immunology , HIV-1/chemistry , HIV-1/pathogenicity , Immunoglobulin Fab Fragments/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Molecular Sequence Data , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/immunology , Retroviridae Proteins/genetics , Retroviridae Proteins/immunology , Simian Immunodeficiency Virus/chemistry , Simian Immunodeficiency Virus/pathogenicity , env Gene Products, Human Immunodeficiency VirusABSTRACT
The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 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.
Subject(s)
Gene Products, env/chemistry , Animals , Antibodies, Monoclonal/metabolism , CD4 Antigens/metabolism , CHO Cells , Cell Line , Chromatography, Gel , Circular Dichroism , Cricetinae , Cross-Linking Reagents , Electrophoresis, Polyacrylamide Gel , Gene Deletion , Gene Products, env/genetics , Gene Products, env/isolation & purification , Gene Products, env/metabolism , Glycosylation , Insecta , Models, Genetic , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , UltracentrifugationABSTRACT
Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic alphabeta heterodimeric structure (Ti) non-covalently associated with the monomorphic CD3 signaling components. A crystal structure of an alphabeta TCR-anti-TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cbeta domain, situated beneath the Vbeta domain. This loop, along with the partially exposed ABED beta sheet of Cbeta, and glycans attached to both Cbeta and Calpha domains, forms a cavity of sufficient size to accommodate a single non-glycosylated Ig domain such as the CD3epsilon ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre-TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module.
Subject(s)
Immunoglobulin Fab Fragments/chemistry , Mitogens/immunology , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Amino Acid Sequence , Animals , CD3 Complex/immunology , Crystallography, X-Ray , Humans , Immunoglobulin Fab Fragments/immunology , Mice , Molecular Sequence Data , Protein Conformation , Receptors, Antigen, T-Cell, alpha-beta/immunology , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the "Velcro" leucine zipper sequence to facilitate alpha-beta pairing. Upon secretion in culture media, the VSV-8-specific/H2-Kb-restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2-Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15(s), a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cbeta domain, was thrombin-cleaved and endoglycosidase H-digested, and the two derivatives were termed iN15DeltaH and N15(s)DeltaH, respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 alpha and beta subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cbeta-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15DeltaH and N15(s)DeltaH. Both N15(s)DeltaH-Fab[H57] and iN15DeltaH-Fab[H57] complexes crystallize, with the former diffracting to 2.8-A resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.
