Cancer Associated Carbohydrate Antigens Expressed On Mucin-type Glycoproteins

用免疫亲和层析法从1例结直肠癌患者腹水中分离到新的携带唾液酸糖蛋白(SL-GP)。SL-GP呈典型的粘蛋白型氨基酸组成，Ser、Thr-Pro占氨基酸残基总量的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-糖链约占O-糖苷链总数的9%。SL-gp能与IL-1刺激的人脐静脉内皮细胞结合，其结合可被抗E-选择素和抗唾液酸-Lea单抗抑制。在SL-GP、由SL-GP制备的寡糖和人乳唾液酸-Lea六糖存在下，测定了结直肠癌细胞LS180与人脐静脉内皮细胞的结合。SL-GP抑制作用最强，等量的SL-GP寡糖和乳唾液六糖对其抑制作用较弱。这些结果直接证明了唾液酸化Lea抗原在多肽链上的独特排列，可能是一个簇，对于E-选择素的结合是必不可少的。为了研究连接引起的E-选择素的修饰，我们用^&lt；32&gt；P-磷酸代谢标记HUVEC。E-选择素丝氨酸残基发生了磷酸化，与SL-GP结合后，E-选择素的磷酸化程度增加，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，进行了脉冲追逐实验。在SL-gp存在下对E-选择素的脉冲标记表明，SL-gp可诱导E-选择素的降解，而与SL-gp预连接后的标记显示E-选择素的合成增加。这种合成可能反映了对E-选择素在结扎前降解，导致E-选择素长时间表达的补偿。

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.