Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells.
Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells.
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中国仓鼠卵巢细胞表达的重组可溶性人 CD4 的碳水化合物结构。
DOI：
10.1021/bi00223a015
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
vanHalbeek,H
中科院分区：
文献类型：
--
作者：
Spellman,MW;Leonard,CK;Basa,LJ;Gelineo,I;vanHalbeek,H
人类免疫缺陷病毒（HIV）对T淋巴细胞和巨噬细胞的感染是通过HIV包膜糖蛋白与细胞表面受体糖蛋白CD 4的结合介导的。一种可溶性重组CD 4分子（rCD 4），通过在中国仓鼠卵巢（CHO）细胞中表达截短的CD 4基因产生[Smith et al.（1987）Science 238，1704-1707]，作为获得性免疫缺陷综合征（AIDS）治疗中的潜在治疗剂处于临床试验中。在本研究中，可溶性rCD 4的Asn连接的寡糖的结构已被阐明。rCD 4分子有两个潜在的N-糖基化位点，Asn-271和Asn-300。通过反相HPLC纯化含有任一位点的胰蛋白酶糖肽，并酶促释放其寡糖。通过甲基化分析、高pH阴离子交换色谱、快原子轰击质谱和500 MHz NMR光谱确定寡糖的结构。Asn-271被发现携带双触角7V-乙酰乳糖胺型（“复合”）寡糖，其中8%是唾液酸，55%是单唾液酸，37%是二唾液酸。这些结构中约18%含有与还原性GlcNAc残基连接的岩藻糖n（1-*-6）。两种不同的混合结构被发现占34%的寡糖连接到天冬酰胺-300。其余与Asn-300连接的寡糖为双末端N-乙酰乳糖胺型，其中10%为唾液酸，61%为单唾液酸，29%为二唾液酸。在Asn-300处发现约9%的杂合结构和40%的N-乙酰乳糖胺结构含有连接到最内部GlcNAc残基的岩藻糖a（1-* 6）。淋巴细胞分为两大类，它们由细胞表面糖蛋白CD 41和CD 8区分（Reinherz等人，1980; Fitch，1985）。糖蛋白CD 4和CD 8在MHC抗原的识别中起作用。一般来说，CD 4阳性T细胞与免疫系统中表面带有II类MHC抗原的细胞相互作用（Swain，1983）。CD 4分子也被证明在HIV感染CD 4阳性T细胞中起关键作用。HIV的包膜糖蛋白gp 120以高亲和力与CD 4结合（McDougal等人，1986），并且这种结合被认为介导病毒进入CD 4阳性细胞（Maddon等人，1986年）。已经显示，针对CD 4的抗体能够在体外阻断HIV感染和合胞体形成（Dalgleish等人，1984; Klatztnann等人，1984; McDougal等人，1985年）。
Infection of T-lymphocytes and macrophages by human immunodeficiency virus (HIV) is mediated by the binding of the HIV envelope glycoprotein to the cell-surface receptor glycoprotein CD4. A soluble, recombinant CD4 molecule (rCD4), produced by expression of a truncated CD4 gene in Chinese hámster ovafy (CHO) cells [Smith et al.(1987) Science 238, 1704-1707], is in clinical trials as a potential therapeutic agent in the treatment of acquired immunodeficiency syndrome (AIDS). In the present study, the structures of the Asn-linked oligosaccharides of soluble rCD4 have been elucidated. The rCD4 molecule has two potential sites for N-glycosylation, Asn-271 and Asn-300. Tryptic glycopeptides containing either of the sites were purified by reversed-phase HPLC, and their oligosaccharides were released enzymatically. The structures of the oligosaccharides were determined by methylation analysis, high-pH anion-exchange chromatography, fast-atom bombardment mass spectrometry, and NMR spectroscopy at 500 MHz. Asn-271 was found to carry diantennary 7V-acetyllactosamine-type (“complex”) oligosaccharides, of which 8% were asíalo, 55% were monosialyl, and 37% were disialyl. Approximately 18% of these structures contained fucose «(1—*-6) linked to the reducing GlcNAc residue. Two different hybrid structures were found to account for 34% of the oligosaccharides attached to Asn-300. The remainder of the oligosaccharides attáched to Asn-300 were diántennary N-acetyllactosamine-type, of which 10% were asíalo, 61% were monósialyl, and 29% were disiályl. Approximately 9% of the hybrid structures and 40% of the N-acetyllactosamine structures at Asn-300 were found to contain fucose a (l-* 6) linked to the innermost GlcNAc residue.^-Lymphocytes are divided into two major classes, which are distinguished by the Cell-surface glycoproteins CD41 and CD8 (Reinherz et al., 1980; Fitch, 1985). the glycoproteins CD4 and CD8 play a role in the recognition of MHC antigens. In general, CD4-positive T-cells interactwith cells of the immune system that bear class II MHC antigens on their surfaces (Swain, 1983). The CD4 molecule has also been shown to play a key role in thb infection of CD4-positive T-cells by HIV. The envelope glycoprotein of HIV, gpl 20, binds with high affinity to CD4 (McDougal et al., 1986), and this binding is believed to mediate entry of the virus into CD4-positive cells (Maddon et al., 1986). Antibodies to CD4 have been shown to be capable of blocking HIV infection and syncytium formation in vitro (Dalgleish et al., 1984; Klatztnann et al., 1984; McDougal et al., 1985).