Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120.
High-performance cation-exchange chromatography of recombinant soluble CD4 (rCD4) allowed the resolution of four charge variants. This charge heterogeneity could be eliminated by neuraminidase treatment of rCD4 and therefore can be attributed to different degrees of sialylation of the carbohydrate portion of this glycoprotein. A single acidic variant was observed upon cation-exchange chromatography of neuraminidase-treated rCD4 that had been stored in liquid solution, pH 7.2, at 25 degrees C for 6 months. This acidic variant was isolated by semipreparative cation-exchange chromatography and subjected to tryptic mapping analysis. Tryptic peptides were characterized by fast atom bombardment mass spectrometry (FABMS). The results of this analysis demonstrated that the acidic variant of neuraminidase-treated rCD4 is generated from deamidation at Asn-52. Digestion of the deamidated rCD4 with endoproteinase Asp-N confirmed Asn-52 as the primary site of deamidation. The ability of the deamidated rCD4 variant to bind gp120 was assessed by use of an ELISA-based binding assay. The binding capacity of the deamidated variant was 24% of the binding capacity of unmodified rCD4. The overall structure of the V1 domain in the deamidated variant was not markedly different from that of the native protein as probed with eight conformationally dependent anti-V1 monoclonal antibodies. Therefore, it appears that Asn-52 is directly involved in binding to gp120.
Teshima
G
G
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
Porter
J
J
Yim
K
K
Ling
V
V
Guzzetta
A
A
D000595:Amino Acid Sequence; D001216:Asparagine; D015704:CD4 Antigens / Q000235:genetics / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001216:Asparagine; D009439:Neuraminidase; D010450:Endopeptidases