Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin.
Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.
Smith
McRae J
MJ
Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22903, USA.
Smith
Bryan R E
BR
Lawrence
Michael B
MB
Snapp
Karen R
KR
D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D000939:Epitopes; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D007091:Image Processing, Computer-Assisted; D020014:K562 Cells; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D018715:Microscopy, Video; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000502:physiology*; D019007:P-Selectin / Q000737:chemistry*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D000939:Epitopes; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase