Cancer Associated Carbohydrate Antigens Expressed On Mucin-type Glycoproteins

采用免疫亲和层析法从结直肠癌患者腹水中分离到一种携带唾液酸lea抗原的新型糖蛋白（SL-GP）。SL-GP表现出典型的粘蛋白型氨基酸组成，其中Ser、Thr Pro加起来占总氨基酸残基的50%以上。SL-GP中含有大量的碳水化合物（约80%）。携带sialyl-LeA抗原的o -聚糖的数量约占o -糖苷链总数的9%。SL-GP可与IL-1处理的HUVECs结合，抗e -selectin和抗sialyl- lea单克隆抗体可抑制其结合。在SL-GP、SL-GP制备的低聚糖和人乳Sialyl-LeA六糖存在的情况下，研究了结直肠癌细胞LS180与HUVECs的结合。SL-GP的抑制效果最好，而等量的SL-GP低聚糖和牛奶唾液酸- lea六糖的抑制作用较弱。这些结果构成了一个直接的证据，证明在多肽链上一个独特的sialyl-LeA抗原排列，可能是一个簇，是e -选择素结合所必需的。为了检测结扎引起的e -选择素的修饰，HUVECs被代谢标记为^<32> P-phosphate。e -选择素丝氨酸残基磷酸化被证实，与SL-GP的连接提高了磷酸化，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，我们进行了脉冲追踪实验。在SL-GP存在的情况下对e-选择素进行脉冲标记，表明连接SL-GP诱导了e-选择素的降解，预连接SL-GP后标记显示e-选择素的合成增加。这种合成可能反映了对预结扎降解的e-选择素的补偿，导致e-选择素的表达延长。

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.