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-FiveTM insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH2 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. 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-FiveTM insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH2 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. Metalloproteases responsible for extracellular (ECM) 1The abbreviations used are: ECMextracellular matrixADAMTSLadisintegrin-like andmetalloprotease domain withthrombospondin type I motifs likeADAMTSadisintegrin-like andmetalloprotease domain withthrombospondin type I motifsADAMadisintegrin-like andmetalloproteaseMSmass spectrometryESTexpressed sequence tagLC-ESMSliquid chromatography-electrospray mass spectrometryMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMMPmatrix metalloproteaseORFopen reading framePBSphosphate-buffered salineRACErapid amplification of cDNA endsTSPthrombospondinTSthrombospondin type I domainHexNAcN-acetylhexosamineNeuAcN-acetylneuraminic acid 1The abbreviations used are: ECMextracellular matrixADAMTSLadisintegrin-like andmetalloprotease domain withthrombospondin type I motifs likeADAMTSadisintegrin-like andmetalloprotease domain withthrombospondin type I motifsADAMadisintegrin-like andmetalloproteaseMSmass spectrometryESTexpressed sequence tagLC-ESMSliquid chromatography-electrospray mass spectrometryMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMMPmatrix metalloproteaseORFopen reading framePBSphosphate-buffered salineRACErapid amplification of cDNA endsTSPthrombospondinTSthrombospondin type I domainHexNAcN-acetylhexosamineNeuAcN-acetylneuraminic acid turnover have a modular structure. Matrix metalloproteinases (MMPs) (1.Murphy G. Knauper V. Matrix Biol. 1997; 15: 511-518Crossref PubMed Scopus (258) Google Scholar), adisintegrin-like andmetalloprotease (ADAMs) (2.Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), and proteases of the ADAMTS family (3.Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 4.Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) are composed of characteristic domains arranged in a precise order that is the hallmark of each family. These enzymes are structurally and functionally bipartite consisting of an enzymatic domain attached to nonenzymatic or ancillary domains. The ancillary domains localize these proteases to substrates, the cell surface, or to the ECM. The ancillary domains of the gelatinases MMP-2 and MMP-9 are among the best studied of the substrate-binding domains. The fibronectin type II domains of the gelatinases are involved in binding to gelatin and some collagens as well as to fibronectin and heparin as in the case of MMP-2 (5.Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 6.Steffensen B. Bigg H.F. Overall C.M. J. Biol. Chem. 1998; 273: 20622-20628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The gelatin-binding domain of MMP-2 binds the matricellular proteins thrombospondin-1 (TSP1) and TSP2 (7.Bein K. Simons M. J. Biol. Chem. 2000; 275: 32167-32173Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Although neither is a substrate for MMP-2, the interaction may mediate the clearance of MMP-2 and affect cell-adhesive properties (8.Bornstein P. Armstrong L.C. Hankenson K.D. Kyriakides T.R. Yang Z. Matrix Biol. 2000; 19: 557-568Crossref PubMed Scopus (133) Google Scholar). The MMP-2 hemopexin domain interacts with the carboxyl terminus of the tissue inhibitor of metalloproteases-2, facilitating pro-MMP-2 activation by membrane-type MMPs (1.Murphy G. Knauper V. Matrix Biol. 1997; 15: 511-518Crossref PubMed Scopus (258) Google Scholar, 5.Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 6.Steffensen B. Bigg H.F. Overall C.M. J. Biol. Chem. 1998; 273: 20622-20628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9.Overall C.M. King A.E. Sam D.K. Ong A.D. Lau T.T. Wallon U.M. De Clerck Y.A. Atherstone J. J. Biol. Chem. 1999; 274: 4421-4429Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The MMP-2 hemopexin domain also interacts with a chemokine called monocyte chemoattractant protein-3, which allows its processing by the catalytic domain (10.McQuibban G.A. Gong J.H. Tam E.M. McCulloch C.A. Clark-Lewis I. Overall C.M. Science. 2000; 289: 1202-1206Crossref PubMed Scopus (631) Google Scholar). The disintegrin domains of ADAMs such as ADAM-15 are implicated in cell-cell adhesion (2.Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 11.Zhang X.P. Kamata T. Yokoyama K. Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1998; 273: 7345-7350Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 12.Zolkiewska A. Exp. Cell Res. 1999; 252: 423-431Crossref PubMed Scopus (50) Google Scholar), and the ancillary domains of ADAMTS-1 are required for its binding to the ECM (13.Kuno K. Matsushima K. J. Biol. Chem. 1998; 273: 13912-13917Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In some ADAMs, the zinc-binding active site is nonfunctional, suggesting that they do not function as proteases at all but may instead have a primary role in adhesion via their ancillary domains (2.Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). extracellular matrix adisintegrin-like andmetalloprotease domain withthrombospondin type I motifs like adisintegrin-like andmetalloprotease domain withthrombospondin type I motifs adisintegrin-like andmetalloprotease mass spectrometry expressed sequence tag liquid chromatography-electrospray mass spectrometry matrix-assisted laser desorption ionization time-of-flight matrix metalloprotease open reading frame phosphate-buffered saline rapid amplification of cDNA ends thrombospondin thrombospondin type I domain N-acetylhexosamine N-acetylneuraminic acid extracellular matrix adisintegrin-like andmetalloprotease domain withthrombospondin type I motifs like adisintegrin-like andmetalloprotease domain withthrombospondin type I motifs adisintegrin-like andmetalloprotease mass spectrometry expressed sequence tag liquid chromatography-electrospray mass spectrometry matrix-assisted laser desorption ionization time-of-flight matrix metalloprotease open reading frame phosphate-buffered saline rapid amplification of cDNA ends thrombospondin thrombospondin type I domain N-acetylhexosamine N-acetylneuraminic acid With this background, it is conceptually possible that gene products containing only the ancillary domains of ADAMTS may have specific functions in cell-cell or cell-matrix interactions or may regulate ADAMTS proteases. We have identified an ADAMTS-like (ADAMTSL) molecule named punctin, 2Approved gene symbols ADAMTSL1 and Adamtsl1 indicate human and mouse orthologs, respectively. The corresponding protein product of these genes, ADAMTSL-1, is designated by the trivial name punctin because of its punctate distribution beneath transfected cells. 2Approved gene symbols ADAMTSL1 and Adamtsl1 indicate human and mouse orthologs, respectively. The corresponding protein product of these genes, ADAMTSL-1, is designated by the trivial name punctin because of its punctate distribution beneath transfected cells. which is the product of a gene distinct from any in the ADAMTS family and is composed of ADAMTS ancillary domains alone. We have purified and characterized recombinant punctin produced in insect cells, visualized it by electron microscopy, and demonstrated that it is a glycoprotein and a component of the ECM. Using BLAST programs from the National Center for Biotechnology Information, we scanned the data base of ESTs using the protein sequences of ADAMTS proteases previously cloned by us (4.Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 14.Georgiadis K.E. Hirohata S. Seldin M.F. Apte S.S. Genomics. 1999; 62: 312-315Crossref PubMed Scopus (28) Google Scholar) and identified a human EST (GenBankTM accession number AA482392 encoded by IMAGE The EST a with a to the carboxyl of ADAMTS members but with in or other protein and data Using based on the sequences at the and ends of the IMAGE and human skeletal cDNA as the we and the cDNA at and ends by as previously (4.Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 14.Georgiadis K.E. Hirohata S. Seldin M.F. Apte S.S. Genomics. 1999; 62: 312-315Crossref PubMed Scopus (28) Google Scholar). tissue from adult human and mouse to a punctin a cDNA from the of the punctin by for the of we a of from an that has characterized for the mouse Seldin M.F. Scholar). on the by from the mouse data base and the and from the and with and with the Adamtsl1 cDNA with accession to for was determined by analysis. order was determined by all and among all that determined to a in the of the gene the for the human punctin cDNA sequence was used for BLAST of the human The in the domain of punctin was using purified by liquid and molecular was by was at the NH2 terminus for to was in and used for with the and at for an in in was measured by using by of from COS-1 cells transfected with punctin cDNA Although from and the best was with by chromatography of using the High-FiveTM cells on tissue and at in insect cell as The punctin was from with and and the corresponding in The insect cell punctin with a and was transfected High-FiveTM cells using and was and for that and in containing Punctin by was by of conditioned using antibody and antibody protein cells in in insect cell or containing heparin in and was at with was binding containing was using of and a on an protein liquid chromatography the was with of binding of in binding was used to from the was with four of in binding containing was by and of and by as containing punctin was determined using the and by acid using an in Science. Scholar). The sequence of recombinant punctin was determined by Edman Recombinant punctin was on to and with and to Edman on an in the Biotechnology of the for recombinant punctin was on and for carbohydrate using a glycoprotein staining In this glycoprotein molecular consisting of of and proteins used as staining was also enzymatic of punctin with F. of as well as punctin was with a using as a N-linked in punctin, stably transfected insect cells in the presence or of tunicamycin of protein from of and cells by with antibody at the of tunicamycin The molecular mass of punctin was measured by and by was with a using acid as the matrix and as a protein 1997; PubMed Scopus Google Scholar). of punctin and are ± at was with a mass 1997; PubMed Scopus Google Scholar, Z. Y. N. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). was used as the at and was from a of punctin, a of was used with mass a of and at an of and liquid chromatography was at a of 5 on a using an liquid chromatography and acid with of the to the mass of punctin are as the ± glycopeptide punctin was from a 6 with with in and with of at in from the with containing in a in of and by using with the mass as for protein based on and N-acetylneuraminic acid 1997; PubMed Scopus Google Scholar). at and in a at a at mass at a and in the Rotary shadowing was as previously PubMed Scopus (78) Google Scholar). of punctin at was with of and using an The was in a and using a electron at to the a The was with and using a electron at site and a site used to a of IMAGE and it corresponding in IMAGE corresponding to the of the punctin cDNA to a and this used to and the sequence for the of in which the was with a tag or a tag and was with using the sequence as a The cloned the and for with a tag or a tag and respectively. COS-1 cells number on tissue in with and of and cells and transfected with or His-tagged punctin using 6 as and cells for an in or medium. a cells transfected with the The was and with for at of cells was by of was to the and the ECM was of cell and ECM under reducing the of on and to was using antibody antibody or antibody on the used for binding was using the antibody by using from COS-1 cells on in and transfected as in or medium. The was The cells on with containing and and for on with of containing antibody or with four for each with in in on for with and with at cells with for at with and blocked with in at with the antibody to in containing The COS-1 cells transfected with the or COS-1 cells with the or transfected cells with from the in which the punctin and the cells with or as with the that the with at In the of or punctin as cells with in for to staining with antibody to in with for the of punctin or antibody to focal adhesion in with antibody for the of antibody was used for the of punctin, and antibody was used for the of or focal adhesion We identified a novel EST (GenBankTM accession number from human and with to ADAMTS proteases. The of the corresponding IMAGE a an a a and by a short and The and sequence by as well as by EST (GenBankTM accession number The region encoded in IMAGE a signal by a of the sequences by a signal of the The signal was by a a sequence at at to M. J. Cell Biol. PubMed Scopus Google Scholar) was The sequence by was by cloned human and mouse ESTs accession for human EST and for mouse The of the cDNA was by amplification of the punctin from human skeletal cDNA as well as by of the arranged on human chromosome The punctin protein contains and has the typical domain of the ancillary of an ADAMTS The secreted of punctin is with a molecular mass of Da and a calculated of the ADAMTS each domain in punctin has an number of that each domain may have disulfide bonds such bonds are in and that punctin of a of and domains. Punctin contains other domains from previously in the ADAMTS family. The punctin sequence contains for N-linked Y. G. PubMed Scopus Google Scholar) at is any acid and also contains a of and The punctin sequence is to human the ADAMTS enzymes to punctin is to human The punctin domains have a of to other ADAMTS-like proteins and ADAMTS proteases to and The as by of of acid sequences identified by BLAST of the first domain of punctin to domains from are as human human and mouse human human human human domains in the ADAMTS punctin domains do not sequences in that have as heparin or binding P. PubMed Scopus Google Scholar). do not binding sequences such as is the acid and is any acid P. PubMed Scopus Google Scholar). The of Adamtsl1 in an in the gene order ± ± ± and Adamtsl1 at a of on mouse chromosome 4 in the of the gene of the mouse data base not any this The that the ADAMTSL1 is on human chromosome The was by the of the human The punctin is encoded by of to of the in site human in the of the ADAMTSL1 with is to is to and type are to ADAMTSL1 is expressed in human and mouse skeletal with a major of in species species of was also in some human skeletal and was not in or Punctin expressed in High-FiveTM cells with and was secreted the conditioned of as well as Punctin was by antibody and tag as a under reducing conditions. was purified from the using Ni-chromatography The a of purified protein as determined by acid analysis. and of punctin demonstrated of molecular mass and data not suggesting the of and at The of punctin to by disulfide bonds as by rapid migration in under nonreducing under reducing on under nonreducing the protein was not with antibody not suggesting that the was not of disulfide mass of His-tagged punctin by a suggesting that the major molecular species of ± and ± respectively. of the protein the major molecular species to ± 6 and ± which and Da the calculated mass of punctin based on acid of the protein a single which at and so punctin by of conditioned or purified staining of purified punctin demonstrated a single of staining carbohydrate that recombinant punctin is a glycoprotein and mass spectrometry demonstrated molecular species with of recombinant protein with not in a in molecular the of glycoprotein staining was not from cells only a single punctin species as demonstrated by The the of the major punctin molecular species and is to the mass of a molecular species also apparent by which by mass that the mass of a of the protein by using to including molecular species with of ± and ± The mass these to to the mass of N-acetylneuraminic acid these data that punctin is specific have to characterized of the acid sequence in punctin was identified by mass including the that the protein has on the the and calculated of punctin, the recombinant protein contains carbohydrate by of punctin in the of of and on not The molecular species with measured of ± and ± as determined by The of these the terminus as the The an that the is from the carboxyl The of to this suggesting that it was by a Rotary shadowing of purified recombinant punctin demonstrated a hatchet-shaped or molecule in Punctin of a single globular domain of in with a short at of the visualized protein was in with the of and but have not in cells or and on to the of punctin or respectively. these punctin was the cells to their in the substratum on The staining was punctate and was the of the cells and and under The punctin of from suggesting cells or staining on the cell Punctin was not in the substratum in not corresponding to the cells. cells with to of transfected cells on the substratum with a staining as under cells. was in some not with cell In other cell punctin We this to from of and the of with antibody or antibody staining of protein using the and staining with staining cells in the presence or of and using or proteins not staining for or focal adhesion not of focal that punctin staining not to of focal contacts. staining was in in cells, cells transfected with cells a primary or cells with as we protein of the for punctin and for the His-tagged or in the cell and the substratum or ECM of transfected COS-1 cells In cells transfected with or cells not not a from the in which not on not In to the catalytic the ADAMTS-like proteins do not disintegrin-like domains. that the disintegrin-like domain and catalytic domain may a functionally domain in ADAMTS for this from the of other proteins with a to the of we of a such molecule encoded by the gene (GenBankTM accession number that we designated as T. K. N. A. N. Res. 1998; PubMed Scopus Google Scholar). We have cloned a ADAMTS-like (GenBankTM accession B. and S. punctin to a distinct protein family. and from punctin in their and and also have domains and These in in In to and which are is expressed in muscle. secreted ECM such as and also the ancillary domains of the ADAMTS family in the precise order as punctin is to and some ADAMTS proteases it is to mouse is a glycoprotein in the Biol. 1999; PubMed Scopus Google Scholar). has the of including as well as a single and In it contains of a novel inhibitor inhibitor and domains. to the of the Biol. 1999; PubMed Scopus Google Scholar). from may an of M. because the are in their domain and primary is also a protein N. Y. M. J.H. 2000; PubMed Google Scholar). Although these proteins have inhibitor contains such domains N. Y. M. 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