Characterization of a soluble form of human CD4. Peptide analyses confirm the expected amino acid sequence, identify glycosylation sites and demonstrate the presence of three disulfide bonds.
CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen-presenting cells bearing the class II major histocompatibility (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells. This 368-amino-acid glycoprotein contains two potential sites of N-linked glycosylation (Asn-271 and Asn-300) and six cysteine residues. Amino-terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P.J. et al. (1985) Cell 42, 93-104]. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed-phase HPLC after digestion of S-carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin-digested rCD4 confirmed that the carboxy-terminus of the protein was Pro-368. Enzymatic digestion of non-reduced rCD4 generated disulfide-bonded fragments that demonstrated the presence of disulfide bonds between Cys-16 and Cys-84, Cys-130 and Cys-159, and between Cys-303 and Cys-345. The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N-glycosidase F demonstrated that both potential N-glycosylation sites are utilized. The tryptic map of rCD4 treated with endo-beta-N-acetylglucosamine H demonstrated that only complex-type oligosaccharides are attached to Asn-271, while Asn-300 has high-mannose or hybrid structures attached in addition to complex-type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn-300 or Asn-271 while no galactosamine was observed. This suggests that rCD4 contains no O-linked oligosaccharides.
Harris
R J
RJ
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco 94080.
Chamow
S M
SM
Gregory
T J
TJ
Spellman
M W
MW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D015704:CD4 Antigens / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis; D004220:Disulfides / Q000032:analysis*; D005260:Female; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D014357:Trypsin
D000596:Amino Acids; D015704:CD4 Antigens; D002241:Carbohydrates; D004220:Disulfides; D006023:Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D014357:Trypsin