Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function.
BACKGROUND
P-selectin glycoprotein ligand-1 (PSGL-1) plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog) and examined mammalian PSGL-1 interactions with human selectins.


RESULTS
A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250-280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human) and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR). Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin. By contrast, pig or rat neutrophils were much less efficiently recruited than human or bovine neutrophils on human selectins. Horse PSGL-1, glycosylated by human or equine glycosyltransferases, did not interact with P-selectin. In all five species, tyrosine sulfation of PSGL-1 was required for selectin binding.


CONCLUSIONS
These observations show that PSGL-1 amino acid sequence of the transmembrane and cytoplasmic domains are well conserved and that, despite a poor conservation of PSGL-1 N-terminus, L- and P-selectin binding sites are evolutionary conserved. Functional assays reveal a critical role for post-translational modifications in regulating mammalian PSGL-1 interactions with selectins.
Baïsse
Bénédicte
B
Service and Central Laboratory of Hematology, Centre Hospitalier Universitaire Vaudois, Bugnon 46, 1011 Lausanne, Switzerland. Benedicte.Baisse@chuv.ch
Galisson
Frédérique
F
Giraud
Sylvain
S
Schapira
Marc
M
Spertini
Olivier
O
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002448:Cell Adhesion; D006224:Cricetinae; D003412:Cricetulus; D019143:Evolution, Molecular; D005434:Flow Cytometry; D045683:Furin / Q000235:genetics; D006736:Horses; D006801:Humans; D016130:Immunophenotyping; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D020556:Neutrophil Infiltration / Q000235:genetics*; D051381:Rats; D013045:Species Specificity; D013552:Swine; D014162:Transfection
D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; C468731:FURIN protein, human; D045683:Furin