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Cancer Associated Carbohydrate Antigens Expressed On Mucin-type Glycoproteins

粘蛋白型糖蛋白上表达的癌症相关碳水化合物抗原

基本信息

批准号：
06672211
负责人：
NAKADA Hiroshi
金额：
$ 1.54万
依托单位：
kyoto Sangyo Uni.
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Novel glycoproteins carrying sialyl-LeA antigens (SL-GP) were isolated from ascites fluid from a patient with colorectal cancer by immunoaffinity chromatography. SL-GP showed a typical mucin type amino acid composition in which Ser, Thr Pro together accounted for greater than 50 % of the total amino acid residues. A large amount of carbohydrate (about 80 %) was present in SL-GP.The number of O-glycans carrying sialyl-LeA antigens comprised about 9 % of the total number of O-glycosidic chains. SL-GP could bind to IL-1 treated HUVECs, and the binding was inhibited by anti-E-selectin and anti-sialyl-LeA monoclonal antibodies. The binding of colorectal cancer cells, LS180, to HUVECs was assayd in the presence of SL-GP,oligosaccharides prepared from SL-GP and human milk Sialyl-LeA hexasaccharide. SL-GP inhibited most effectively, whereas equivalent amounts of the SL-GP oligosaccharides and milk sialyl-LeA hexasaccharide inhibited it slightly. These results constitute direct evidence that a unique arrangement of sialyl-LeA antigens on the polypeptide chain, probably a cluster, is essential for the binding of E-selectin.To examine the modification of E-selectin caused by ligation, HUVECs were metabolically labeled with ^<32> P-phosphate. Phosphorylation at serine residue of E-selectin was demonstrated and ligation with SL-GP elevated the phosphorylation, which might have led to the activation of E-selectin metabolism. To further investigate the effect of SL-GP on the metabolic behavior of E-selectin, pulse-chase experiments were performed. While pulse-labeling of E-selectin in the presence of SL-GP indicated that the degradation of E-selectin was induced by SL-GP ligation, labeling after pre-ligation with SL-GP revealed an increase in the synthesis of E-selectin. The synthesis may reflect compensation for the E-selectin degraded on pre-ligation, resulting in prolonged expression of E-selectin.

用免疫亲和层析法从结直肠癌患者腹水中分离出携带唾液酸LeA抗原的新型糖蛋白（SL-GP）。SL-GP具有典型的粘蛋白型氨基酸组成，其中Ser、Thr Pro共占总氨基酸残基的50%以上。SL-GP中含有大量的碳水化合物（约80%），携带唾液酸LeA抗原的O-聚糖约占O-糖苷链总数的9%。SL-GP可与IL-1处理的HUVECs结合，这种结合可被抗E-选择素和抗sialyl-莱亚单克隆抗体所抑制。在SL-GP、由SL-GP制备的寡糖和人乳Sialyl-LeA六糖存在下，测定结肠直肠癌细胞LS 180与HUVEC的结合。SL-GP抑制最有效，而等量的SL-GP低聚糖和牛奶唾液酸-LeA六糖抑制它略有。这些结果构成了直接的证据，唾液酸-LeA抗原在多肽链上的独特排列，可能是一个簇，是E-选择素结合所必需的<32>。结果表明，E-选择素的丝氨酸残基存在磷酸化，与SL-GP连接后，磷酸化水平升高，这可能导致E-选择素代谢的激活。为了进一步研究SL-GP对E-选择素代谢行为的影响，进行了脉冲追踪实验。而在SL-GP存在下的E-选择素的脉冲标记表明，E-选择素的降解是由SL-GP连接诱导的，用SL-GP预连接后的标记揭示了E-选择素合成的增加。合成可能反映了对预连接时降解的E-选择素的补偿，导致E-选择素的延长表达。