title: Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses.
abstract: Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.
authors.affiliation: School of Biomedical Sciences, Western Australian Biomedical Research Institute, Curtin University of Technology, GPO Box U1987, Perth, Western Australia 6845, Australia. d.coombe@curtin.edu.au
authors.forename: Deirdre R
authors.initials: DR
authors.lastname: Coombe
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Stevenson
authors.forename: Beverley F
authors.initials: BF
authors.lastname: Kinnear
authors.forename: Neha S
authors.initials: NS
authors.lastname: Gandhi
authors.forename: Ricardo L
authors.initials: RL
authors.lastname: Mancera
authors.forename: Ronald I W
authors.initials: RI
authors.lastname: Osmond
authors.forename: Warren C
authors.initials: WC
authors.lastname: Kett
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D007136:Immunoglobulins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011993:Recombinant Fusion Proteins