Abstract
Lectin-carbohydrate recognition between the selectins and their ligands are among the earliest events in leukocyte recirculation, leukocyte recruitment into inflamed areas, and abnormal egress of leukocytes in diseases. Previously, we have described a dimeric sialoglycoprotein from myeloid cells with subunits of molecular mass = 120 kDa, which is selectively recognized by P-selectin (Moore, K.L., Stults, N.L., Diaz, S., Smith, D.F., Cummings, R.D., Varki, A., and McEver, R.P. (1992) J. Cell Biol. 188, 445-456). Here, we demonstrate that this P-selectin ligand carries alpha 2-3-linked sialic acids and the sialyl-Lewisx (SLex) tetrasaccharide motif. This glycoprotein contains < 1% of the total membrane-bound sialic acids and a very small fraction of the total SLex on neutrophil membranes. In spite of a relative resistance to sialidase digestion, the predominant form of sialic acid on the ligand is N-acetylneuraminic acid. Selective periodate oxidation of the side chain of sialic acids does not affect P-selectin binding and allows the introduction of tritium label into the truncated sialic acids. beta-Elimination with alkaline borohydride releases labeled O-linked oligosaccharides both from the labeled neutrophil ligand and from the ligand purified from HL-60 cells metabolically labeled with [3H]glucosamine. The ligand from both neutrophils and HL-60 cells is also susceptible to cleavage by the enzyme O-sialoglycoprotease from Pasteurella hemolytica. Analysis of the specificity of this enzyme suggests that the P-selectin ligand carries large numbers of closely spaced sialylated O-linked oligosaccharides. O-Sialoglycoprotease abolishes both direct binding of P-selectin to HL-60 cells and the adhesion of HL-60 cells to immobilized P-selectin, without significantly decreasing overall cell surface SLex expression. This indicates that the 120-kDa ligand may be the major determinant of P-selectin:myeloid cell interaction in vivo. Finally, based on the current and previous data, we hypothesize that the high affinity recognition site(s) of this P-selectin ligand may be derived from a "clustered saccharide patch" of sialylated fucosylated O-linked oligosaccharide sequences.
Highlights
Lectin-carbohydrate recognition between the selec- the current anpdrevious data, we hypothesize that the tins and their ligandasre among the earliest events in high affinity recognition site(s)of this P-selectin ligand leukocyte recirculation, leukocyte recruitment into in-may be derived froma “clustered saccharide patcho”f flamed areas, and abnormal egress of leukocytes in sialylated fucosylated 0-linked oligosaccharide sediseases
P-selectin binding andallows the introduction of trit- assume that it plays a major role in recognition of such cells ium label into the truncated sialicacids. &Elimination with alkaline borohydride releases labeled 0-linked oligosaccharides both from the labeled neutrophil ligand and from the ligand purified from HL-60 cells metabolically labeled with [SH]glucosamine
Our previous work has suggested a role for a specific cellsurface glycoprotein ligand in thehigh affinity recognition of neutrophils and HL-60cells by P-selectin (26).In thisstudy, we have looked more closely at the nature of the sialic acids and theoligosaccharides involved in this binding
Summary
(Received for publication, November 10, 1992, and in revised form, January 21, 1993). Periodate OnidutionlTritiated Borohydride Reduction-The WGA eluate or purified P-selectin ligand from neutrophil membranes was incubated with freshly prepared 2 mM periodate in phosphate-buffered saline, pH 6.5, at 4 "C for 30 min in the dark. Ureafaciens sialidase in 100pl of 100 mM sodium acetate, pH5.5, containing 0.5% Triton X-100 for 14-16 h a t 37 "C under a toluene atmosphere, Released sialic acids were purified by dialysis, followed by sequential ion exchange chomatography on Bio-Rad AG50 1x8 (hydrogen form) and Bio-Rad AG3x4A (formate form) as previously described (40, 41). HPLC Analysis of Underivatized Sialic Acids-The P-selectin ligand was metabolically labeled with [3H]glucosaminein HL-60 cells as described above and affinity-purified. This purified ligand was fractionated by SDS-PAGE under reducing conditions and the 120kDa ligand was excised and digested with Pronase.
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