P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils.
Under physiological shear stress, neutrophils roll on P-selectin on activated endothelial cells or platelets through interactions with P-selectin glycoprotein ligand-1 (PSGL-1). Both P-selectin and PSGL-1 are extended molecules. Human P-selectin contains an NH2-terminal lectin domain, an EGF domain, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the length of P-selectin affected its interactions with PSGL-1, we examined the adhesion of neutrophils to CHO cells expressing membrane-anchored P-selectin constructs in which various numbers of CRs were deleted. Under static conditions, neutrophils attached equivalently to wild-type P-selectin and to constructs containing from 2-6 CRs. Under shear stress, neutrophils attached equivalently to wild-type and 6 CR P-selectin and nearly as well to 5 CR P-selectin. However, fewer neutrophils attached to the 4 CR construct, and those that did attach rolled faster and were more readily detached by increasing shear stress. Flowing neutrophils failed to attach to the 3 CR and 2 CR constructs. Neutrophils attached and rolled more efficiently on 4 CR P-selectin expressed on glycosylation-defective Lec8 CHO cells, which have less glycocalyx. We conclude that P-selectin must project its lectin domain well above the membrane to mediate optimal attachment of neutrophils under shear forces. The length of P-selectin may: (a) facilitate interactions with PSGL-1 on flowing neutrophils, and (b) increase the intermembrane distance where specific bonds form, minimizing contacts between the glycocalyces that result in cell-cell repulsion.
Patel
K D
KD
W. K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
Nollert
M U
MU
McEver
R P
RP
D000818:Animals; D016466:CHO Cells / Q000166:cytology; D002448:Cell Adhesion / Q000276:immunology; D002462:Cell Membrane / Q000737:chemistry*; D002465:Cell Movement / Q000276:immunology; D006224:Cricetinae; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000502:physiology; D009504:Neutrophils / Q000166:cytology*; D019007:P-Selectin / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D011134:Polysaccharides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000276:immunology
D006023:Glycoproteins; D019007:P-Selectin; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins