Identification and Characterization of Asporin
Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1–32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains anN-glycosylation site at Asn281 with a heterogeneous oligosaccharide structure and a potentialO-glycosylation site at Ser54. The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin. Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1–32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains anN-glycosylation site at Asn281 with a heterogeneous oligosaccharide structure and a potentialO-glycosylation site at Ser54. The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin. Cartilage matrix consists of fibrillar networks, primarily of collagen II and highly negatively charged molecules of aggrecan. There are also a number of noncollagenous glycoproteins that apparently contribute to the regulation of tissue assembly and properties. Among them is the family of the leucine-rich repeat (LRR) 1The abbreviations used are: LRRleucine-rich repeatGdnHClguanidinium hydrochlorideHPLChigh-pressure liquid chromatographyMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightPAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reactionproteins, which contains several members found in the extracellular matrix. There are currently 11 known members of this family. These molecules share a common structure with a central stretch of LRRs. This LRR domain is flanked by disulfide bridged loops, with 4 cysteine residues preceding the LRR domain and 2 on its C-terminal side. Apart from chondroadherin, these proteins also contain divergent amino-terminal extensions with features unique for the different proteins. Based on amino acid sequence and gene organization the family can be divided into four distinct groups. leucine-rich repeat guanidinium hydrochloride high-pressure liquid chromatography matrix-assisted laser desorption/ionization time-of-flight polyacrylamide gel electrophoresis polymerase chain reaction Decorin (1Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (415) Google Scholar) and biglycan (2Fisher L.W. Termine J.D. Young M.F. J. Biol. Chem. 1989; 264: 4571-4576Abstract Full Text PDF PubMed Google Scholar) constitute the first group (class I). These proteins have 10 LRRs and carry one and two chondroitin or dermatan sulfate chains, respectively. The glycosaminoglycan chains are linked to serine residues in the amino terminus. The molecules in this group are secreted with a propeptide. The second group (class II) consists of fibromodulin (3Oldberg Å. Antonsson P. Lindblom K. Heinegård D. EMBO J. 1989; 8: 2601-2604Crossref PubMed Scopus (228) Google Scholar), lumican (4Blochberger T.C. Vergnes J.P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google Scholar), keratocan (5Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), PRELP (6Bengtsson E. Neame P.J. Heinegård D. Sommarin Y. J. Biol. Chem. 1995; 270: 25639-25644Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and osteoadherin (7Sommarin Y. Wendel M. Shen Z. Hellman U. Heinegård D. J. Biol. Chem. 1998; 273: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Like the class I proteins they consist of 10 LRRs. With the exception of PRELP, they all carry polylactosamine or keratan sulfate chains linked to the LRR region and sulfated tyrosine residues in the amino-terminal extension. In contrast, the amino terminus of PRELP has a cluster of positively charged amino acid residues that mediates binding to heparan sulfate (8Bengtsson E. Aspberg A. Heinegård D. Sommarin Y. Spillmann D. J. Biol. Chem. 2000; 275: 40695-40702Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Unlike all other family members, osteoadherin contains a COOH-terminal extension (7Sommarin Y. Wendel M. Shen Z. Hellman U. Heinegård D. J. Biol. Chem. 1998; 273: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Epiphycan/PG-Lb/DSPG3 (9Johnson H.J. Rosenberg L. Choi H.U. Garza S. Hook M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Shinomura T. Kimata K. J. Biol. Chem. 1992; 267: 1265-1270Abstract Full Text PDF PubMed Google Scholar, 11Deere M. Johnson J. Garza S. Harrison W.R. Yoon S.J. Elder F.F.B. Kucherlapati R. Hook M. Hecht J.T. Genomics. 1996; 38: 399-404Crossref PubMed Scopus (22) Google Scholar), mimecan/osteoglycin (12Madisen L. Neubauer M. Plowman G. Rosen D. Segarini P. Dasch J. Thompson A. Ziman J. Bentz H. Purchio A.F. DNA Cell Biol. 1990; 9: 303-309Crossref PubMed Scopus (75) Google Scholar, 13Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and opticin/oculoglycan (14Reardon A.J. Le Goff M. Briggs M.D. McLeod D. Sheehan J.K. Thornton D.J. Bishop P.N. J. Biol. Chem. 2000; 275: 2123-2129Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Friedman J.S. Ducharme R. Raymond V. Walter M.A. Invest. Ophthalmol. Vis. Sci. 2000; 41: 2059-2066PubMed Google Scholar, 16Hobby P. Wyatt M.K. Gan W. Bernstein S. Tomarev S. Slingsby C. Wistow G. Mol. Vis. 2000; 6: 72-78PubMed Google Scholar) form the third group (class III). These are smaller molecules with only 6 LRRs and all contain sulfated tyrosine residues in the amino-terminal extension. In addition, epiphycan carries chondroitin sulfate, other O-linked oligosaccharides, and a cluster of glutamate residues in this region (9Johnson H.J. Rosenberg L. Choi H.U. Garza S. Hook M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The amino-terminal extension of opticin carriesO-linked oligosaccharides (14Reardon A.J. Le Goff M. Briggs M.D. McLeod D. Sheehan J.K. Thornton D.J. Bishop P.N. J. Biol. Chem. 2000; 275: 2123-2129Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and contains a heparin-binding consensus sequence (17Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Chondroadherin (18Neame P.J. Sommarin Y. Boynton R.E. Heinegård D. J. Biol. Chem. 1994; 269: 21547-21554Abstract Full Text PDF PubMed Google Scholar) forms the fourth branch on the extracellular matrix LRR protein family tree (class IV). This protein contains 10 LRRs, but lacks both amino- and COOH-terminal extensions outside the cysteine motifs. Nyctalopin, a recently published glycosylphosphatidylinositol-anchored LRR protein may also be a member of this subfamily (19Bech-Hansen N.T. Naylor M.J. Maybaum T.A. Sparkes R.L. Koop B. Birch D.G. Bergen A.A. Prinsen A. M.A. Young 2000; PubMed Scopus Google Scholar, C. K. H. S. C. J. A. S. A. B. W. A. 2000; PubMed Scopus Google Scholar). is from the the of LRR proteins into on sequence not the of the biglycan, and epiphycan are chondroitin or dermatan sulfate and may be related the different class II LRR proteins. A that is of the class and LRR proteins is a to to collagen the LRR This is a binding in the The different extensions a variety of for with other matrix other of and of the fibrillar of these molecules to have in the assembly of collagen is by in PubMed Scopus Google Scholar, M. Heinegård D. J. PubMed Scopus Google Scholar, E. Heinegård D. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, E. Heinegård D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) by gene H. H. J. Cell Biol. 1997; PubMed Scopus Google Scholar, S. T. C. H. J. Cell Biol. 1998; PubMed Scopus Google Scholar, L. A. R. Heinegård D. Å. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these collagen the of the LRR protein is The with a of of proteins in human found a number of proteins to be one a with of This related to fibromodulin in a variety of the structure a putative oligosaccharide chain and tissue of the It a novel member of the LRR protein family to the group (class I). The protein is asporin on the of a stretch in the amino-terminal region and the similarity with decorin. human cartilage of tissue to and of tissue at The tissues into and a in of for at 4 the was by at at 4 for in the cartilage from by with a of in D. Sommarin Y. PubMed Scopus Google Scholar). The was divided into 4 a and the was used for The was by by 4 and to a 6 in of for protein by at and by M. Sommarin Y. Heinegård D. J. PubMed Scopus Google Scholar). The proteins from the into two a proteins of to and a smaller the smaller proteins. The to and by by The was a of in the the was with of the and with a of to in the at a of of 10 for protein by at and by The asporin by by 10 and on a of in The was with and the proteins at a of with the of 2 for protein by at and by The asporin and by to 10 and to a The proteins with a from to at a of of 2 for and by of the a of proteoglycan. the was to of the to by and by chromatography that the was at the from the chromatography asporin and by by 4 This was on two of 6 and and at with of for protein by at by with was at to of to the by on a with a of in acid at a of The was at on with of on the gel and DNA to J. E. T. A Scholar). The amino acid from asporin used to the with the W. W. D.J. J. Mol. Biol. 1990; PubMed Scopus Google Scholar). The identified from this and The resulting sequence was used for and osteoarthritic articular cartilage was at in and in liquid and mRNA purified P. Neame P. Sommarin Y. Heinegård D. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). was with and with II of the mRNA with the asporin was the polymerase chain reaction with and and DNA at for the DNA was for of at at and 2 at The resulting was isolated from purified the and into the The and several of the resulting the and on a DNA In to and two and The resulting sequence the II of the A first mouse asporin sequence was from a of the mouse of with the human asporin this sequence the and first was and asporin from this by and The was into and all four and The human and mouse asporin in with the and respectively. 10 of isolated from human osteoarthritic articular cartilage on and to a of and from The with a of the human with by the DNA and of the to the The to or by the to be with in and in a for of was of of and of the and was with and at for 4 and on the polyacrylamide by with on and in the gel in a and and 10 and at and at respectively. and at of or at in The was by the of 10 of which also the of the a at purified from the acid used to the sequence coverage. and that the to a smaller the was used M. C. H. M. H. E. Chem. 2000; PubMed Scopus Google Scholar). The mass was of protein from the gel H. S. A. B. 1997; PubMed Scopus Google Scholar) by to the acid the matrix. was used for a matrix-assisted laser desorption/ionization time-of-flight mass The was used in the with and of 26 the and for of of protein the was with of The used to the and oligosaccharide and structure W. Chem. 2000; PubMed Scopus Google Scholar) and C. E. PubMed Scopus Google Scholar), respectively. of human articular cartilage with 4 by the matrix proteins from the of the in the of the by gel on 6 in two one proteins and the other with proteins of The proteins in this by asporin was in the also These on a at asporin and fibromodulin they from other proteins in the to asporin from fibromodulin on a at with a the two proteins with fibromodulin the to asporin and fibromodulin by gel with in the and chromatography A to the collagen of fibromodulin E. Heinegård D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) to the protein with collagen the that also asporin was with the collagen not The of a of human by electrophoresis a of not The was on a in was in a identified by These and by gel on two of and 6 in a with a of the of the protein from by and two but by of the of the amino at was to both with a of the protein was not and isolated that with the that of these was from to but the of these are in the sequence of the identified by are the and the The the amino acid of the in a the identified by are the and the The the amino acid of the is sequence in a putative extracellular matrix The deduced sequence contains several leucine-rich and the two COOH-terminal cysteine residues of the extracellular matrix protein family. was used the in a number of other of these a that a and the amino-terminal of the extracellular matrix LRR proteins. The of the novel LRR protein was from human osteoarthritic cartilage, to the and of the consensus The mouse was identified of the mouse with the human sequence and from mouse on the sequences. The human and mouse asporin are in The amino acid of the two proteins are The four amino-terminal the of decorin and biglycan Mol. Biol. 1997; PubMed Scopus Google Scholar), which asporin a member of the class I branch of the LRR proteins. decorin and biglycan, asporin contains a highly putative sequence acid residues The putative site to the site in biglycan Y. A.D. P.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of the LRRs of asporin to decorin and biglycan a in amino acid sequence repeat of a tree of the extracellular matrix LRR proteins confirmed that asporin to the I the decorin and biglycan branch Unlike decorin and biglycan, asporin contains consensus glycosaminoglycan in its amino terminus. There one consensus site for and in the human and mouse asporin In contrast to all identified extracellular matrix LRR proteins, asporin has a stretch of aspartic acid residues in its amino-terminal region. found that the number of consecutive aspartic acid residues is of the human asporin the sequence the first residues in the human sequence revealed that at this several with of residues M. Johnson J. Garza S. Harrison W.R. Yoon S.J. Elder F.F.B. Kucherlapati R. Hook M. Hecht J.T. Genomics. 1996; 38: 399-404Crossref PubMed Scopus (22) Google Scholar, L. Neubauer M. Plowman G. Rosen D. Segarini P. Dasch J. Thompson A. Ziman J. Bentz H. Purchio A.F. DNA Cell Biol. 1990; 9: 303-309Crossref PubMed Scopus (75) Google Scholar, 13Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, A.J. Le Goff M. Briggs M.D. McLeod D. Sheehan J.K. Thornton D.J. Bishop P.N. J. Biol. Chem. 2000; 275: 2123-2129Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Friedman J.S. Ducharme R. Raymond V. Walter M.A. Invest. Ophthalmol. Vis. Sci. 2000; 41: 2059-2066PubMed Google Scholar) identified in the human In addition, the sequence of human asporin for the used in the human asporin was from tissue from several that this a found in the stretch of the mouse which and tree of extracellular matrix LRR proteins. The tree was with W. the of the extracellular matrix LRR protein A of with and for the and mouse respectively. The human asporin gene is on chromosome 9q31.1–32, the This also contains the for the LRR proteins and mimecan/osteoglycin A asporin was from sequence and a number of to the and of the sequence of a from chromosome that the asporin sequence number The first is also in the in the asporin gene 26 kilobases and consists of 8 the the and the the the in decorin and biglycan the are in the in decorin and biglycan It is exons are in the of the is the in decorin. Like biglycan consensus TATA is found of the first of the asporin A number of binding identified of the asporin in the human asporin sequence is from number exons number are in the of this The of of the sequence is in sequence in and in to of the first or the second of the The sequence is from number exons number are in the of this The of of the in a sequence is in sequence in and in to of the first or the second of the that the asporin gene for a of kilobases a human tissue found that the of was in the by the the was in the other articular cartilage is not on the was not to asporin in cartilage with that in other the of human osteoarthritic cartilage a that 10 of cartilage of was with 2 of from the other and that the the of asporin may be higher in articular cartilage in the other tissues A for the and of asporin was by of a mRNA which and tissues The asporin with a of human tissues with the levels in and levels found in heart, and in the and asporin was in and There was a of in the central in and A asporin was in mass spectrometry was used to the protein with the with the from the novel The identified of asporin are in The identified 84% of the protein The only in the sequence is the amino-terminal the consecutive This is to the of this which and mass spectrometry from asporin identified by mass acid with a mass of identified only are in in a with a mass of identified only are in The protein has one site at of asporin with confirmed that the protein contains oligosaccharides by the in its on This in the of the Asn281 by mass spectrometry this was not in protein A of mass was which can be by of to a H. T. G. C. 2000; PubMed Scopus Google Scholar). be both in in A mass of was of the oligosaccharide and putative of the linked to Asn281 in asporin from cartilage and are in the of the The of oligosaccharides at Asn281 was confirmed This has a different resulting in mass This mass with the and putative of on human mass at in human asporin was with mass and structure not in a mass at in human asporin was with mass and structure not The amino-terminal of the protein contains a potentialO-glycosylation site at which may be with oligosaccharide not the time-of-flight mass two with and that to the amino-terminal linked oligosaccharide The mass the two which be of linked oligosaccharide This is by the of in these the of a in the amino-terminal sequence that have consecutive residues in the a of and is for the with and respectively. to that the protein contains the sequence is to its this not be from the partially purified asporin the of the protein from the gel the of by mass of was for the protein from the The mass was to in the by the of with the that of the gel with This in with a higher The mass of the protein with consecutive residues in the amino-terminal is by a mass for oligosaccharide of and for O-linked oligosaccharide of to the the mass that the amino-terminal is in the is a member of the LRR protein family closely related to decorin and biglycan. The four amino-terminal the of the class I LRR proteins. decorin and biglycan asporin contains a putative with a site to the sequence for the to Y. A.D. P.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). the sequence and of the LRR of asporin are to of decorin and biglycan to other members of the proteins. This is also from the tree of the LRR proteins. the decorin A. Genomics. PubMed Scopus Google Scholar) and biglycan L.W. U. W. W. Termine J.D. Young M.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar) the human asporin gene is divided into 8 The are in the sequence at the to of decorin and biglycan. The exons of the human asporin gene span 26 kilobases on chromosome It is not the asporin gene also contains the decorin gene A. Genomics. PubMed Scopus Google Scholar). Like in the biglycan gene L.W. U. W. W. Termine J.D. Young M.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar), TATA was found in the region of of a number of for in the of the deduced The extracellular matrix LRR protein to be in of and epiphycan (class and to chromosome Asporin, and (class and are found on chromosome a gene a LRR protein amino-terminal has asporin and osteoadherin J. T. Y. Genomics. 1998; PubMed Scopus Google Scholar). PRELP, and opticin (class and to chromosome (class is unique in not of a cluster but found in on chromosome It that several have resulting in the organization of the LRR The biglycan gene may have to chromosome four LRR protein to be one on chromosome and on chromosome The asporin amino-terminal extension is in stretch of aspartate to asporin recently identified in the and the S. A. S. C. J. J. Mol. 2000; PubMed Scopus Google Scholar). These proteins to by the to the class I LRR proteins on the amino-terminal cysteine and the amino acid sequence of the LRRs. of aspartic acid residues in the amino-terminal extensions these proteins of Unlike in human the mouse and the two are by other amino acid the of a number of and in this region in the amino terminus for this negatively charged amino acid In contrast to decorin and biglycan, asporin is not a proteoglycan. It contains consensus for glycosaminoglycan the and the amino-terminal cysteine decorin (1Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (415) Google Scholar) and biglycan (2Fisher L.W. Termine J.D. Young M.F. J. Biol. Chem. 1989; 264: 4571-4576Abstract Full Text PDF PubMed Google Scholar) have one and two respectively. There a consensus sequence for in the human This was confirmed by which in a on and of the by mass The linked oligosaccharide in all the from different tissue or all are at a in the tissue is not The that the protein was from a of tissue from several can of also contribute to the on in to oligosaccharide may on the of The protein contains putative site in the human that to be with linked has in the amino-terminal extension of epiphycan and It to be oligosaccharide in this region may the of this The of the of class I LRR proteins is It has that this sequence the glycosaminoglycan structure of decorin Å. Antonsson P. J. 1996; PubMed Scopus Google Scholar) and biglycan P. D.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The of a in which not contain glycosaminoglycan consensus that the may have other mRNA is expressed in a number of different articular cartilage, the levels found in and uterus, by levels in other tissues with of The of the protein are not in to the protein from fibromodulin in a collagen both proteins to to This be in with of other LRR proteins of the members have to to collagen with in the of aspartic acid residues in asporin are a of the extracellular matrix of and has a acid sequence in the of the protein Å. A. Heinegård D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: PubMed Scopus Google Scholar). This protein to and may have a in J. 1994; PubMed Scopus Google Scholar). asporin also has a in is not In this the of the protein in is of in of the of in this in S. C. D. K. M. L. Scholar). A may be the found in epiphycan (9Johnson H.J. Rosenberg L. Choi H.U. Garza S. Hook M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and in Å. A. Heinegård D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The stretch of the protein has to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. 1994; PubMed Scopus Google Scholar). putative for asporin be to the stretch in with other matrix in with fibromodulin and lumican and also of the glycosaminoglycan chains of decorin and biglycan, the family This for the collagen in the The other two members of the family decorin and biglycan have to A. M. L.M. Heinegård D. Ruoslahti E. J. 1994; PubMed Scopus Google Scholar). also asporin has this to be are to and at the of for with time-of-flight mass