Premium
Identification and characterization of L‐selectin ligands in porcine lymphoid tissues
Author(s) -
Khan Adil I.,
Haskard Dorian O.,
Malhotra Rajneesh,
Landis R. Clive
Publication year - 2002
Publication title -
immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.297
H-Index - 133
eISSN - 1365-2567
pISSN - 0019-2805
DOI - 10.1046/j.1365-2567.2002.01393.x
Subject(s) - chemistry , microbiology and biotechnology , biochemistry , monoclonal antibody , lectin , chondroitin sulfate , glycoprotein , affinity chromatography , staining , glycosaminoglycan , biology , antibody , enzyme , immunology , genetics
Summary 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 ≈ 84 000 and 210 000 molecular weight. The MECA 79 non‐reactive species exhibited Ca 2+ ‐ 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.