Functional Analysis of the Combined Role of the O-Linked Branching Enzyme Core 2 β1-6-N-Glucosaminyltransferase and Dimerization of P-selectin Glycoprotein Ligand-1 in Rolling on P-selectin

Mcrae J Smith,Bryan R.E Smith,Michael B Lawrence,Karen R Snapp

doi:10.1074/jbc.m402731200

Abstract

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.

Highlights

Previous studies have shown that P-selectin glycoprotein ligand-1 (PSGL-1) must be prop- glycoprotein ligand-1 (PSGL-1)1 tethers the leukocyte to the erly modified by specific glycosyltransferases including ␣1,3-fucosyltransferase-VII, core 2 ␤1– 6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more ␣2,3-sialytransferases, and a tyrosulfotransferase
The site density of PRIgG used in tether measurements, specificity, rolling cell accumulation, velocity, dissociation constant, or adhesive event lifetime measurements varied from 10 to 200 sites/␮m2 with higher site densities required for interactions involving K562-C320A due to the extremely low formation rate of tether bonds compared with PSGL-1 expressing cell lines
Generation and Characterization of Transfectants Expressing PSGL-1, C320A, and C2GlcNAcT-I—Both the BJAB/FucTVII and K562/FucT-VII cell lines lack the gene for PSGL-1 but express all glycosyltransferases required for appropriate enzymatic modification of PSGL-1

Summary

EXPERIMENTAL PROCEDURES

Generation of Stable Transfectants in K562 and BJAB Cells—The generation of both K562 and BJAB cells stably expressing ␣1,3-fucosyltransferase-VII (FucT-VII) has been described [21, 22]. The site density of PRIgG used in tether measurements, specificity, rolling cell accumulation, velocity, dissociation constant, or adhesive event lifetime measurements varied from 10 to 200 sites/␮m2 with higher site densities required for interactions involving K562-C320A due to the extremely low formation rate of tether bonds compared with PSGL-1 expressing cell lines. The tether bond lifetime (duration of an adhesive event at low site densities of P-selectin) was determined by counting high speed camera image frames starting with a drop in velocity of at least 25% relative to the hydrodynamic velocity of a cell near the wall and ending with an increase in velocity of 12.5 ␮m/s over three frames (24 ms). This resulted in an average lever arm angle (␪) of 60.7°, a lever arm length of 4.3 ␮m, a microvillus tether arm length of 0.8 ␮m, a shear force of 232 piconewtons/dyn/cm, and a force on the tether of 475 piconewtons/dyn/cm that was used to scale the force on the tether for the K562 and BJAB transfectants [26]

RESULTS

DISCUSSION

Mean fluorescence