Interestingly, 3 positions in this region are perfectly conserved in all stud ied species. The transmem 1A. Among the conserved positions, Ser 346, Arg 347, Lys 348 and Ser 358 are associated with moesin binding to your cytoplasmic domain of human PSGL one. In all sequences, the C terminal region is ended by 11 almost completely conserved residues. Human L and P selectin interact with human, rat, bovine, pig or equine CHO PSGL one cells CHO cells co expressing human FucT VII and C2GnT I and human, bovine, pig, rat or equine PSGL 1 had been pre pared. The five transfectants expressed very similar levels of sLex and CLA. PSGL one expression was detected utilizing a mAb reacting with PSGL 1 C terminal 6 ? His tag. The anti human PSGL one mAbs PL1, KPL1 and PL2 did not react with bovine, pig, rat or equine PSGL one.
Flow cytometric evaluation of human P or L selectin binding to your different CHO PSGL one trans fectants showed that P and L selectin bind similarly to human, bovine, pig, rat or equine PSGL 1 expressed by transfected CHO cells. inhibitor SB 431542 As the reactivity of mouse PSGL 1 with human selectins was previously described. we did not repeat these analyses. Human L. P and E selectin bind heterogeneously to human, bovine, pig or rat neutrophils PSGL one expressed by CHO transfectants differ within their gly cosylation pattern from mammalian neutrophil PSGL one. In CHO transfectants, the different mammalian PSGL one are glycosylated by FucT VII and C2GnT I of human origin, while in mammalian neutrophils PSGL 1 is glycosylated by their very own glycosyltransferases.
Because the glycosylation pattern may possibly affect PSGL 1 interactions with L or P selec tin, we examined the reactivity of human selectins with mammalian neutrophils. L and Clinofibrate P selectin chi mera strongly reacted with human and bovine PSGL one, even though a weaker reaction was observed with pig and rat. The L and P selectin carbohydrate ligands sLex and CLA, identified by CSLEX one and HECA 452 mAbs respectively, have been strongly expressed by human neutrophils and in addition, surprisingly, by equine neutrophils. By contrast, despite substantial selectin binding, sLex and CLA were undetectable on bovine, pig and rat neutrophils. As selectin binding is dependent on cell surface expression of fucosylated ligands, we examined FucT VII mRNA expression by RT PCR amplification of total RNA from bovine, pig, rat and equine neutrophils. FucT VII mRNA transcripts have been detected in all investigated species.
Thus, as previously established for mouse leukocytes. the lack of reactivity of mAbs CSLEX 1 and HECA 452 with most mammalian PSGL one is likely due to the powerful specificity of these mAbs for human oligosaccharides. Furthermore, the observation that mAbs CSLEX 1 and HECA 452 strongly react with equine neutrophils suggests that human and equine neutrophils exhibit widespread carbohydrate structures, which are not detectable in mouse, rat, pig or bovine.