Binding of function-blocking mAbs to mouse and human P-selectin glycoprotein ligand-1 peptides with and without tyrosine sulfation.
P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin-expressing endothelial cells under shear flow. Function-blocking monoclonal antibodies (mAbs) against mouse and human PSGL-1 recognize an anionic segment at the N-terminus of PSGL-1. High affinity interaction of PSGL-1 with P-selectin requires sulfation of tyrosines 46, 48, and 51 (human) or 54 and 56 (mouse). We tested binding of two anti-human (KPL1 and PL1) and two anti-mouse (4RA10 and 2PH1) PSGL-1 mAbs to synthetic peptides of N-terminus of human and mouse PSGL-1 and found binding to be independent of tyrosine sulfation. In peptide-blocking experiments, sulfated and nonsulfated human and mouse peptides competed with antibody binding to PSGL-1 expressed on myeloid cells. Arylsulfatase treatment significantly reduced P-selectin binding but had no effect on antibody binding. Our data show, in three independent assay systems, that function-blocking antibodies to mouse or human PSGL-1 do not require sulfation of N-terminal tyrosines for binding.
Thatte
Aravinda
A
Department of Biomedical Engineering, University of Virginia, Charlottesville 22908-1394, USA.
Ficarro
Scott
S
Snapp
Karen R
KR
Wild
Martin K
MK
Vestweber
Dietmar
D
Hunt
Donald F
DF
Ley
Klaus F
KF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D001667:Binding, Competitive; D018922:HL-60 Cells / Q000187:drug effects; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D010446:Peptide Fragments; C005662:tyrosine O-sulfate; D014443:Tyrosine