Identification and characterization of L-selectin ligands in porcine lymphoid tissues.
A human L-selectin-ZZ fusion protein was used to screen porcine inguinal lymph nodes for the presence of monoclonal antibody (mAb) MECA 79-negative ligands. Fractionation of lymph node-conditioned medium by anion-exchange chromatography revealed two distinct L-selectin-binding fractions, one containing a MECA 79 non-reactive species and the second containing two MECA 79 reactive species of approximately 84 000 and 210 000 molecular weight. The MECA 79 non-reactive species exhibited Ca2+- and lectin-dependent binding to L-selectin-ZZ in a solid-phase capture enzyme-linked immunosorbent assay (ELISA), and this was specifically disrupted by the addition of EDTA, mannose-6-phosphate and the blocking anti-L-selectin mAb, DREG-56. Enzymatic characterization of the ligand by trypsin, O-sialoglycoprotease endopeptidase, heparinases I and III, or chondroitinase ABC lyase digestion indicated that L-selectin binding was predominantly dependent upon a chondroitin sulphate-modified glycoprotein determinant. Although Coomassie Blue staining of sodium dodecyl sulphate (SDS) polyacrylamide gels did not reveal a detectable protein species, carbohydrate-specific staining using GlycoTrack revealed a single, heavily glycosylated protein of high molecular weight (> 220 000). These studies have revealed the existence of a MECA 79 non-reactive, chondroitin sulphate glycosaminoglycan-modified ligand, termed csgp>220, which is secreted by peripheral lymph nodes and may play a role in leucocyte trafficking to the lymph node.
Khan
Adil I
AI
BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, DuCane Road, London W12 0NN, UK.
Haskard
Dorian O
DO
Malhotra
Rajneesh
R
Landis
R Clive
RC
D000818:Animals; D002852:Chromatography, Ion Exchange; D019041:L-Selectin / Q000032:analysis*; D008024:Ligands; D008221:Lymphoid Tissue / Q000276:immunology*; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins; D013552:Swine
D008024:Ligands; D011993:Recombinant Fusion Proteins; D019041:L-Selectin