Harris R J, Chamow S M, Gregory T J, Spellman M W
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco 94080.
Eur J Biochem. 1990 Mar 10;188(2):291-300. doi: 10.1111/j.1432-1033.1990.tb15402.x.
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.
CD4是一种糖蛋白，在免疫系统的多种细胞表面表达，被认为参与这些细胞与携带II类主要组织相容性（MHC）抗原的抗原呈递细胞之间的相互作用。CD4还通过与病毒糖蛋白gp120结合而作为人类免疫缺陷病毒（HIV）的受体。重组可溶性CD4（rCD4）是人类CD4的截短形式，由转染的中国仓鼠卵巢细胞分泌。这种368个氨基酸的糖蛋白含有两个潜在的N-连接糖基化位点（Asn-271和Asn-300）和六个半胱氨酸残基。氨基末端序列分析表明，该序列从最初根据cDNA分析预测的多肽的第三个残基开始[马登，P.J.等人（1985年）《细胞》42卷，93 - 104页]。通过用胰蛋白酶消化S-羧甲基化的rCD4后，对经反相HPLC纯化的肽进行分析，证实了其余的一级序列。对胰蛋白酶消化的rCD4进行无水胰蛋白酶亲和层析，证实该蛋白的羧基末端是Pro-368。对未还原的rCD4进行酶消化产生了二硫键连接的片段，表明在Cys-16和Cys-84、Cys-130和Cys-159以及Cys-303和Cys-345之间存在二硫键。发现rCD4碳水化合物结构的组成单糖为岩藻糖、甘露糖、半乳糖、N-乙酰葡糖胺和N-乙酰神经氨酸。用肽：N-糖苷酶F处理后rCD4的胰蛋白酶图谱特征表明，两个潜在的N-糖基化位点均被利用。用内切β-N-乙酰葡糖胺H处理rCD4的胰蛋白酶图谱表明，只有复合型寡糖连接到Asn-271，而除了复合型寡糖外，Asn-300还连接有高甘露糖或杂合结构。仅在含有Asn-300或Asn-271的糖肽中观察到葡糖胺，而未观察到半乳糖胺。这表明rCD4不含有O-连接寡糖。
