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(Journal of Leukocyte Biology. 2002;72:470-477.)
© 2002 by Society for Leukocyte Biology

Binding of function-blocking mAbs to mouse and human P-selectin glycoprotein ligand-1 peptides with and without tyrosine sulfation

Aravinda Thatte^*, Scott Ficarro, Karen R. Snapp, Martin K. Wild, Dietmar Vestweber, Donald F. Hunt^,|| and Klaus F. Ley^*,#

Departments of
^* Biomedical Engineering,
Chemistry,
^# Cardiovascular Research Center, and
^|| Pathology, University of Virginia, Charlottesville;
Department of Microbiology and Immunology, Northwestern University, Evanston, Illinois; and
Institute of Cell Biology, ZMBE, University of Münster and Max-Planck-Institute of Physiological and Clinical Research, Germany

Correspondence: Klaus Ley, M.D., Department of Biomedical Engineering, Cardiovascular Research Center, MR5 Building, Room 1013, University of Virginia, Box 801394, Charlottesville, VA 22908-1394. E-mail: klausley{at}virginia.edu

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.

Key Words: arylsulfatase • selectin • mass spectrometry • leukocyte adhesion

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