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 en dothelium 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. Ho wever, previous affinity isolation experiments with an E-selectin-Ig f usion protein had failed to detect L-selectin among the isolated E-sel ectin ligands from mouse neutrophils. We show here that L-selectin fro m 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 di rect, since purified L-selectin could be reprecipitated with E-selecti n-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 observ ed, In agreement with the biochemical data, the anti-L-selectin mAb DR EG56 inhibited rolling of human neutrophils on immobilized E-selectin- Ig but not on P-selectin-Ig, No such inhibitory effect was seen with t he anti-mouse L-selectin mAb MEL14 on mouse neutrophils, Rolling of E- selectin transfectants on purified and immobilized human L-selectin wa s inhibited by mAb DREG56. We conclude that L-selectin on human neutro phils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear differenc e between human and mouse L-selectin suggests that E-selectin-binding carbohydrate moieties are attached to different protein scaffolds in d ifferent species.