title: Punctin, a novel ADAMTS-like molecule, ADAMTSL-1, in extracellular matrix.
abstract: Punctin (ADAMTSL-1) is a secreted molecule resembling members of the ADAMTS family of proteases. Punctin lacks the pro-metalloprotease and the disintegrin-like domain typical of this family but contains other ADAMTS domains in precise order including four thrombospondin type I repeats. Punctin is the product of a distinct gene on human chromosome 9p21-22 and mouse chromosome 4 that is expressed in adult skeletal muscle. His-tagged punctin expressed in stably transfected High-Five(TM) insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH(2) terminus is not blocked and has the sequence EEDRD and so forth as determined by Edman degradation, demonstrating signal peptidase processing. Recombinant epitope-tagged punctin has a calculated mass of 59,991 Da but exhibits major molecular species of 61970 +/- 6 Da and 62131 +/- 5 Da as measured by liquid chromatography electrospray mass spectrometry. Punctin is a glycoprotein based on carbohydrate staining and liquid chromatography electrospray mass spectrometry glycopeptide analysis. Glycosylation occurs at a single N-linked site as demonstrated by altered electrophoretic migration of punctin expressed in the presence of tunicamycin A. Punctin contains disulfide bonds based on antibody accessibility and electrophoretic migration under reducing versus nonreducing conditions. Rotary shadowing demonstrates that punctin is hatchet-shaped having a globular region attached to a short stem. In transfected COS-1 cells, punctin is deposited in the cell substratum in a punctate fashion and is excluded from focal contacts. Punctin is the first member of a novel family of ADAMTS-like proteins that may have important functions in the extracellular matrix.
authors.affiliation: Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hirohata
authors.forename: Lauren W
authors.initials: LW
authors.lastname: Wang
authors.forename: Masaru
authors.initials: M
authors.lastname: Miyagi
authors.forename: Lin
authors.initials: L
authors.lastname: Yan
authors.forename: Michael F
authors.initials: MF
authors.lastname: Seldin
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Keene
authors.forename: John W
authors.initials: JW
authors.lastname: Crabb
authors.forename: Suneel S
authors.initials: SS
authors.lastname: Apte
mesh_terms: D000071096:ADAMTS Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D019556:COS Cells; D002853:Chromatography, Liquid; D002874:Chromosome Mapping; D002899:Chromosomes, Human, Pair 9; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019483:Disintegrins / Q000096:biosynthesis / Q000737:chemistry*; D004220:Disulfides; D000939:Epitopes; D005109:Extracellular Matrix / Q000201:enzymology*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection
chemical_list: C454120:ADAMTSL1 protein, human; D018076:DNA, Complementary; D019483:Disintegrins; D004220:Disulfides; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D011994:Recombinant Proteins; D000071096:ADAMTS Proteins; D008666:Metalloendopeptidases