|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1995 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1994 : ¥1,200,000 (Direct Cost : ¥1,200,000)
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.