Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1

Abstract

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent M r > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent M r 230,000) and CD47 (apparent M r > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser 64 and Ser 79 . Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser 64 .Cell surface proteoglycans play critical roles in cell-matrix interactions. They act as co-receptors for some pathogens and growth and motility factors and regulate the functions of other cell surface signaling receptors (reviewed in Refs. 1-4). Chondroitin sulfate or heparan sulfate chains are attached through a conserved core oligosaccharide sequence to specific Ser residues on the core proteins (5). The most prevalent cell surface heparan sulfate proteoglycans (HSPG) 5 are the syndecans and glypicans, but a number of additional cell surface proteins can be specifically modified with heparan sulfate glycosaminoglycans (GAG) in certain cells and tissues (6). CD4 ϩ T cells express highly sulfated heparan sulfate chains because of their high expression of the N-deacetylase/N-sulfotransferase NDST2 and the 3-O-sulfotransferases 3-OST3 (7). T cell HSPGs have been identified as receptors for histones (8) and cyclophilin B (9, 10) and serve as co-receptors for heparinbinding chemokines such as regulated on activation normal T cell expressed and secreted (RANTES) (11).T cell HSPGs are also co-receptors for retroviral infection. An unidentified cell surface HSPG expressed by the H9 T cell line interacts with the V3 region of the HIV1 envelope gp120-gp41, and heparitinase treatment inhibited binding and entry of the virus into H9 cells (12). The fusion peptide domain of gp41 also interacts specifically with an HSPG on T cells (13). A subsequent study concluded that syndecan functions in trans to promote HIV infection of T cells (14). Similar HSPG binding was demonstrated for the hu...

CD47 Signaling Regulates the Immunosuppressive Activity of VEGF in T Cells

“…1C). Both antibodies detected the 50-55 kDa N-glycosylated and >200 kDa proteoglycan isoforms of CD47 and a previously described 36 kDa proteolytic fragment of CD47 (28, 35) in the immunoprecipitates from WT Jurkat cells that were absent in the corresponding immunoprecipitates from JinB8 cells. The CD47 antibody B6H12 detected several nonspecific bands in VEGFR2 immunoprecipitates from JinB8 cells that were not detected by the CD47 antibody R529.…”

“…TSP1 signaling via CD47 inhibits TCR signaling, including induction of the early activation marker CD69 (28, 33, 42). Consistent with the previous report that VEGF inhibits T cell activation (19), VEGF treatment resulted in a 4-fold reduction in CD69 mRNA expression induced by activation of WT Jurkat T cells in the presence of anti-CD3+CD28 for 24 h (Fig.…”

Regulation of Tumor Angiogenesis and Choroidal Neovascularization by Endogenous Angioinhibitors

“…TSP-1 induces the apoptotic mechanism through CD36 and integrin associated protein (IAP)/Src-family protein kinases/Caspase-3/p38 MAPK [88]. In addition, TSP-1 can also bind to different integrins, CD47 and heparan sulfated proteoglycans [89]. Regulation of tumor angiogenesis and growth by TSP-1 are well documented in various studies.…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…In addition to the undefined protein-protein interaction, binding of TSP1 to CD47 requires post-translational modification of CD47 with a heparan sulfate glycosaminoglycan at Ser 64 (110). Because glycosylation can be cell-type specific, CD47 may vary in its ability to interact with TSP1 to control NO signaling.…”