Carbohydrate structures of recombinant soluble lamp-1 and leukosialin containing sialyl Le(x) terminus.
Recombinant soluble lamp-1 and soluble leukosialin can be produced from CHO cells which express sialyl Le(x) structures after stable transfection of fucosyltransferase-III. It was shown previously that those soluble lamp-1 and leukosialin are potent inhibitors for E-selectin-mediated adhesion of human colonic tumor cells (Sawada, R.; Tsuboi, S.; Fukuda, M. J. Biol. Chem., 1994, 269, 1425). In the present study, we have determined the amount of the sialyl Le(x) structure present in recombinant, soluble lamp-1 and soluble leukosialin. CHO cells were metabolically labeled with [3H]-galactose and recombinant soluble lamp-1 and leukosialin were purified from the spent medium. Glycopeptides containing N-glycans derived from lamp-1 were fractionated by sequential lectin affinity chromatography. Similarly, O-glycans released from leukosialin were fractionated by Bio-Gel P-4 gel filtration. The terminal structures of carbohydrate chains were determined by sequential digestion with specific glycosidases. The results clearly indicate that soluble lamp-1 contains much more sialyl Le(x) structure than soluble leukosialin. Considering that soluble leukosialin and lamp-1 are almost equally effective as inhibitors for E-selectin-mediated adhesion, the results strongly suggest that densely clustered O-glycans are better presenters for E-selectin ligands than N-glycans.
Sueyoshi
S
S
Glycobiology Program, La Jolla Cancer Research Foundation, CA 92037, USA.
Sawada
R
R
Fukuda
M
M
D000818:Animals; D015703:Antigens, CD; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D006020:Glycopeptides / Q000737:chemistry; D051917:Leukosialin; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000235:genetics; D000080506:Sialyl Lewis X Antigen; D012995:Solubility
D015703:Antigens, CD; D006020:Glycopeptides; D051917:Leukosialin; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D000080506:Sialyl Lewis X Antigen