Prokopishyn N L, Puzon-McLaughlin W, Takada Y, Laferté S
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
J Cell Biochem. 1999 Feb 1;72(2):189-209.
Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.
肿瘤发生过程中的碳水化合物表位是人类结肠直肠癌的一个常见特征，但其生物学意义仍不清楚。我们之前已经表明，识别2型血型A和B寡糖上一个决定簇的单克隆抗体（MAb）3A7，可检测到偶氮甲烷诱导的大鼠结肠肿瘤和一些人类结肠癌细胞系中的肿瘤发生变化。（Laferté S等人，[1995] J. Cell. Biochem. 57:101 - 119）。在本研究中，我们着手纯化gp140，它是人类结肠癌细胞表达的3A7表位的主要糖蛋白载体，这是阐明3A7表位及其主要糖蛋白载体对结肠癌进展贡献的第一步。为此，从HT29细胞中纯化出gp140，并用于制备多肽特异性单克隆抗体。五种单克隆抗体（7A8、7B11、8C7、8H7和11D4）免疫沉淀出一种140 kDa的3A7免疫反应性糖蛋白复合物，随后通过部分蛋白质测序鉴定为α3β1整合素。对表达不同人类整合素链的中国仓鼠卵巢（CHO）细胞进行流式细胞术分析表明，单克隆抗体7A8和7B11检测α3整合素亚基，而单克隆抗体8C7和8H7检测β1整合素亚基。不与任何CHO转染子结合的单克隆抗体11D4检测到2型血型A决定簇。对一组从血型A、AB或B个体获得的人类结肠癌细胞系进行流式细胞术分析，结果显示3A7表位的细胞表面表达与α3整合素亚基之间存在直接相关性，这表明α3β1整合素是结肠癌细胞中3A7表位的首选靶点。使用凝集素和糖苷酶进一步研究α3β1整合素的碳水化合物结构，我们证明它是一种含有N - 连接和O - 连接寡糖的唾液酸糖蛋白。此外，α3和β1整合素亚基均表达β1 - 6分支的N - 连接寡糖和短的多聚N - 乙酰乳糖胺单位（Galβ1 - 4GlcNAc - R；n≤3），这些聚糖先前与癌症转移有关。因此，人类结肠癌细胞表达的α3β1整合素是肿瘤发生过程中碳水化合物表位的主要载体，其存在可能调节肿瘤细胞的黏附、迁移和/或侵袭。
