L-selectin from human, but not from mouse neutrophils binds directly to E-selectin.
L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin-Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin-Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin-Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin-Ig fusion protein was not observed. In agreement with the biochemical data, the anti-L-selectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin-Ig but not on P-selectin-Ig. No such inhibitory effect was seen with the anti-mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin-binding carbohydrate moieties are attached to different protein scaffolds in different species.
Zöllner
O
O
Institute of Cell Biology, ZMBE, University of Münster, Germany.
Lenter
M C
MC
Blanks
J E
JE
Borges
E
E
Steegmaier
M
M
Zerwes
H G
HG
Vestweber
D
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001854:Bone Marrow Cells; D016466:CHO Cells; D006224:Cricetinae; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D019041:L-Selectin / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D051379:Mice; D009504:Neutrophils / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism
D000911:Antibodies, Monoclonal; D019040:E-Selectin; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012794:Sialic Acids; D019041:L-Selectin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase