Dimerization of P-selectin glycoprotein ligand-1 (PSGL-1) required for optimal recognition of P-selectin

Abstract

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with alpha1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.

Figure 1

Dimerization of PSGL-1 is mediated by cysteine 320. (A) SDS-PAGE analysis of KPL1 immunoprecipitates made from cell surface biotinylated K562 cells stably transfected with either wild-type PSGL-1 (lane 1) or C320A (lane 2). KPL1 immunoprecipitates were electrophoresed under nonreducing conditions, transferred to nitrocellulose and probed with avidin-HRPO. (B) Native gel electrophoresis of wild-type PSGL-1 (lane 1) or C320A (lane 2). Cell lysates were analyzed by native PAGE on 5% polyacrylamide gels, transferred to nitrocellulose and probed with KPL1. Under both denaturing and native conditions, K562 cells transfected with PSGL-1 express primarily the 240-kD form of the molecule, whereas cells transfected with C320A express only the monomeric form of PSGL-1. (C) Cross-linking analysis of PSGL-1 and C320A dimerization. Cells were incubated with 3-100 μM BS³ cross-linker and analyzed under reducing conditions by SDS-PAGE followed by Western blotting with KPL1.

Figure 1

Dimerization of PSGL-1 is mediated by cysteine 320. (A) SDS-PAGE analysis of KPL1 immunoprecipitates made from cell surface biotinylated K562 cells stably transfected with either wild-type PSGL-1 (lane 1) or C320A (lane 2). KPL1 immunoprecipitates were electrophoresed under nonreducing conditions, transferred to nitrocellulose and probed with avidin-HRPO. (B) Native gel electrophoresis of wild-type PSGL-1 (lane 1) or C320A (lane 2). Cell lysates were analyzed by native PAGE on 5% polyacrylamide gels, transferred to nitrocellulose and probed with KPL1. Under both denaturing and native conditions, K562 cells transfected with PSGL-1 express primarily the 240-kD form of the molecule, whereas cells transfected with C320A express only the monomeric form of PSGL-1. (C) Cross-linking analysis of PSGL-1 and C320A dimerization. Cells were incubated with 3-100 μM BS³ cross-linker and analyzed under reducing conditions by SDS-PAGE followed by Western blotting with KPL1.

Figure 2

Expression of PSGL-1, C320A, and FucT-VII–dependent epitopes on stably transfected K562 cells. Expression of either wild-type PSGL-1 or C320A was determined by staining with KPL1, and FucT-VII enzyme activity was determined by HECA-452 staining. Note that equal or higher levels of C320A and FucT-VII are found on K562/FucT-VII/C320A transfectants compared with K562/FucT-VII/PSGL-1 cells. HL60 cells are included as controls.

Figure 3

RT-PCR analysis of FucT-VII and C2GnT mRNA expression in K562 transfectants. RNA was isolated, and RT-PCR was performed as described in Materials & Methods. “+” and “−” refer to the presence or absence, respectively, of RT in the RT reaction. Pgk1 is a housekeeping enzyme included as an internal control. Indistinguishable levels of C2GnT mRNA are seen in K562/FucT-VII/PSGL-1 and K562/FucT-VII/C320A cells, whereas slightly higher levels of FucT-VII mRNA are seen in K562/FucT-VII/C320A cells, consistent with higher staining by HECA-452 (Fig. 2).

Figure 4

Binding of soluble P-selectin to K562 transfectants. Binding of soluble recombinant P-selectin fusion proteins was assessed by flow cytometry. Data are given as the mean channel fluorescence of ∼10,000 light scatter gated events. K562/FucT-VII/ C320A cells bound P-selectin poorly in comparison to K562/ FucT-VII/PSGL-1.

Figure 5

Binding of K562/FucT-VII/PSGL-1 and K562/FucT-VII/C320A to COS cells transfected with P-selectin. Binding of cells to COS cells expressing high levels of P-selectin was assessed under low shear conditions as described in Materials and Methods. Untransfected K562 cells, which express neither PSGL-1 nor FucT-VII, and HL60 cells are included as negative and positive controls, respectively.

Figure 6

Rolling of K562/FucT-VII/PSGL-1 or K562/FucT-VII/ C320A cells on P-selectin under defined shear stress. The number of adhesive interactions of cells rolling on CHO cells expressing P-selectin were quantitated at a shear stress of 1.0 dynes/cm². U937 cells are included for comparison and as a positive control. The number of events for the C320A cells was virtually identical to that of cells transfected with FucT-VII cDNA alone (data not shown).

Figure 7

Cell surface distribution of PSGL-1 and C320A on transfected K562 cells. (A) Low power photomicrograph (3,000×) of K562/FucT-VII/PSGL-1 cells. Note the prominent and varied microvilli and ruffles on these cells. (B) High power (8,000×) of K562/FucT-VII/PSGL-1 cells. (C) High power (8,000×) of K562/FucT-VII/C320A cells. Both cell types exhibited a uniform distribution of PSGL-1 on both microvilli and the cell body.

Figure 7

Cell surface distribution of PSGL-1 and C320A on transfected K562 cells. (A) Low power photomicrograph (3,000×) of K562/FucT-VII/PSGL-1 cells. Note the prominent and varied microvilli and ruffles on these cells. (B) High power (8,000×) of K562/FucT-VII/PSGL-1 cells. (C) High power (8,000×) of K562/FucT-VII/C320A cells. Both cell types exhibited a uniform distribution of PSGL-1 on both microvilli and the cell body.

Figure 7

Cell surface distribution of PSGL-1 and C320A on transfected K562 cells. (A) Low power photomicrograph (3,000×) of K562/FucT-VII/PSGL-1 cells. Note the prominent and varied microvilli and ruffles on these cells. (B) High power (8,000×) of K562/FucT-VII/PSGL-1 cells. (C) High power (8,000×) of K562/FucT-VII/C320A cells. Both cell types exhibited a uniform distribution of PSGL-1 on both microvilli and the cell body.