Constitutive and accelerated shedding of murine syndecan-1 is mediated by cleavage of its core protein at a specific juxtamembrane site.
Syndecan-1 is a developmentally regulated cell surface heparan sulfate proteoglycan (HSPG). It functions as a coreceptor for a variety of soluble and insoluble ligands and is implicated in several biological processes, including differentiation, cell migration, morphogenesis, and recently feeding behavior. The extracellular domain of syndecan-1 is proteolytically cleaved at a juxtamembrane site by tissue inhibitor of metalloprotease-3 (TIMP-3)-sensitive metalloproteinases in response to a variety of physiological stimulators and stress in a process known as shedding. Shedding converts syndecan-1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. We found that replacing syndecan-1 juxtamembrane amino acid residues A243-S-Q-S-L247 with human CD4 amino acid residues can completely block PMA-induced syndecan-1 ectodomain shedding. Furthermore, using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), we identified the proteolytic cleavage site of syndecan-1 as amino acids A243 and S244, generated by constitutive and PMA-induced shedding from murine NMuMG cells. Finally, we show that basal cleavage of syndecan-1 utilizes the same in vivo site as the in vitro site. Indeed, as predicted, transgenic mice expressing the syndecan-1/CD4 cDNA do not shed the syndecan-1 ectodomain in vivo. These results suggest that the same cleavage site is utilized for basal syndecan-1 ectodomain shedding both in vitro from NMuMG and CHO cells and in vivo.
Wang
Zihua
Z
Division of Developmental and Newborn Biology, Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Götte
Martin
M
Bernfield
Merton
M
Reizes
Ofer
O
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D018076:DNA, Complementary; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D053668:Syndecan-1; D053667:Syndecans; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism
D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D011994:Recombinant Proteins; C508241:SDC1 protein, human; C508242:Sdc1 protein, mouse; D053668:Syndecan-1; D053667:Syndecans; D019717:Tissue Inhibitor of Metalloproteinase-3; D010447:Peptide Hydrolases