CD47-independent effects mediated by the TSP-derived 4N1K peptide.
4N1K is a peptide fragment derived from the C-terminal, globular domain of thrombospondin which has been shown to mediate integrin-dependent cell adhesion and promote integrin activation acting via the cell-surface receptor, CD47. However, some studies found that 4N1K could act independently of CD47, putting in question the specificity of 4N1K for CD47. This led us to characterize the cellular and non-cellular effects of 4N1K. We found that 4N1K stimulated a potent increase in binding of a variety of non-specific IgG antibodies to cells in suspension. We also found that these same antibodies, as well as CD47-deficient cells, could bind substrate-immobilized 4N1K significantly better than a control peptide, 4NGG. Furthermore, we found that cells treated with 4N1K at higher concentrations inhibited, while lower concentrations promoted cell adhesion to immobilized fibronectin as an integrin substrate. Importantly, both the stimulatory and the inhibitory activity of 4N1K occurred as efficiently in the CD47-deficient JinB8 cells, as it did in the CD47-expressing parental or in JinB8 cells reconstituted with CD47 expression. Given these results, we suggest that 4N1K interacts non-specifically with epitopes commonly found on the cell surface, and conclude that it is not a suitable peptide for use to study the consequences of CD47 receptor ligation.
Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada; Department of Cell and Developmental Biology, University of British Columbia, Vancouver, British Columbia, Canada; Child and Family Research Institute, BC Children's Hospital, Vancouver, British Columbia, Canada.
Pascal
P
Leclair
Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada; Child and Family Research Institute, BC Children's Hospital, Vancouver, British Columbia, Canada.
Chinten James
CJ
Lim
D051928:CD47 Antigen / Q000378:metabolism*; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D039482:Integrin alpha5 / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism; D019169:Jurkat Cells; D008024:Ligands; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019699:Thrombospondins / Q000378:metabolism*
C120273:4N1K peptide; D051928:CD47 Antigen; C497894:CD47 protein, human; D007074:Immunoglobulin G; D039482:Integrin alpha5; D019012:Integrin beta1; D008024:Ligands; D009842:Oligopeptides; D010446:Peptide Fragments; D019699:Thrombospondins