Carr S A, Hemling M E, Folena-Wasserman G, Sweet R W, Anumula K, Barr J R, Huddleston M J, Taylor P
Department of Physical and Structural Chemistry, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406.
J Biol Chem. 1989 Dec 15;264(35):21286-95.
The primary structure of a soluble form of the CD4 receptor (sCD4) expressed in Chinese hamster ovary cells has been confirmed by mass spectrometric peptide mapping and and tandem mass spectrometry. These studies corroborated 95% of the 369-amino acid-long sequence and established the fidelity of translation of the NH2 and COOH terminal including the absence of "ragged ends." The arrangement of the three disulfide bonds in recombinant sCD4 was also established by mass spectrometry and comparative high performance liquid chromatography mapping and shown to be identical to that expected from previous studies of intrachain disulfide bonding in T4 antigens derived from sheep and mouse. No other arrangements of disulfides were detected. Carbohydrate mapping by mass spectrometry was used to establish that both potential Asn-linked glycosylation sites in sCD4 (Asn271 and Asn300) have oligosaccharides attached. Structural characterization by mass spectrometry and methylation analysis of the heterogeneous family of oligosaccharides at each of the specific attachment sites indicates that the major components of both families of oligosaccharides have the following biantennary structures: (Formula, see text) where m + n = 0-2, and x = 0,1. Minor carbohydrate components having three N-acetylneuraminic acid (NeuAc) groups and an additional hexose-hexosamine unit were detected by high performance anion-exchange chromatography.
通过质谱肽图谱和串联质谱法已证实了在中国仓鼠卵巢细胞中表达的可溶性CD4受体（sCD4）的一级结构。这些研究证实了369个氨基酸长序列中的95%，并确定了NH2和COOH末端翻译的准确性，包括不存在“参差不齐的末端”。重组sCD4中三个二硫键的排列也通过质谱法以及比较高效液相色谱图谱得以确定，并显示与先前对源自绵羊和小鼠的T4抗原中链内二硫键结合的研究预期一致。未检测到二硫键的其他排列方式。通过质谱法进行的碳水化合物图谱分析确定了sCD4中两个潜在的Asn连接糖基化位点（Asn271和Asn300）均连接有寡糖。通过质谱法和甲基化分析对每个特定连接位点处寡糖的异质家族进行结构表征表明，两个寡糖家族的主要成分具有以下双天线结构：（公式，见原文）其中m + n = 0 - 2，且x = 0,1。通过高效阴离子交换色谱法检测到具有三个N - 乙酰神经氨酸（NeuAc）基团和一个额外的己糖 - 己糖胺单元的次要碳水化合物成分。
