Intracellular maturation of the mouse metalloprotease disintegrin MDC15.
Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-alpha. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks pro-domain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro. Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.
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Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
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Blobel
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D051722:ADAM Proteins; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D019556:COS Cells; D003001:Cloning, Molecular; D019483:Disintegrins / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008168:Lung / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
D019483:Disintegrins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D051722:ADAM Proteins; C490902:Adam15 protein, mouse; D008666:Metalloendopeptidases