Identification and Characterization of Asporin
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m010932200
ISSN1083-351X
AutoresPilar Lorenzo, Anders Aspberg, Patrik Önnerfjord, Michael T. Bayliss, Peter J. Neame, Dick Heinegård,
Tópico(s)Cell Adhesion Molecules Research
ResumoAsporin, 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 C.F. Polomeno R.C. Gal A. Drack A.V. Musarella M.A. Jacobson S.G. Young R.S. Weleber R.G. Nat. Genet. 2000; 26: 319-323Crossref PubMed Scopus (282) Google Scholar, 20Pusch C.M. Zeitz C. Brandau O. Pesch K. Achatz H. Feil S. Scharfe C. Maurer J. Jacobi F.K. Pinckers A. Andreasson S. Hardcastle A. Wissinger B. Berger W. Meindl A. Nat. Genet. 2000; 26: 324-327Crossref PubMed Scopus (208) Google Scholar). As is evident from the summary above, the subdivision of LRR proteins into classes based on sequence does not reflect the functions of the molecules. For example, decorin, biglycan, and epiphycan are chondroitin or dermatan sulfate proteoglycans, and may as such be more functionally related than, e.g. the different class II LRR proteins. A major functional property that is shared between most of the class I, II, and IV LRR proteins is a capacity to bind to collagen via the LRR domain. This is a high affinity binding withK d in the nanomolar range. The different NH2-terminal extensions offer a variety of opportunities for interactions with other matrix constituents, including other fibers of collagen, thereby providing cross-linking and stabilization of the fibrillar network. Several of these molecules appear to have roles in modulating the assembly of collagen fibrils as is indicated by experiments in vitro (21Vogel K.G. Trotter J.A. Coll. Relat. Res. 1987; 7: 105-114Crossref PubMed Scopus (278) Google Scholar, 22Vogel K.G. Paulsson M. Heinegård D. Biochem. J. 1984; 223: 587-597Crossref PubMed Scopus (704) Google Scholar, 23Hedbom E. Heinegård D. J. Biol. Chem. 1989; 264: 6898-6905Abstract Full Text PDF PubMed Google Scholar, 24Hedbom E. Heinegård D. J. Biol. Chem. 1993; 268: 27307-27312Abstract Full Text PDF PubMed Google Scholar) as well as by gene inactivation studies (25Danielson K.G. Baribault H. Holmes D.F. Graham H. Kadler K.E. Iozzo R.V. J. Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1183) Google Scholar, 26Chakravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. Cell Biol. 1998; 141: 1277-1286Crossref PubMed Scopus (578) Google Scholar, 27Svensson L. Aszódi A. Reinholt F.P. Fässler R. Heinegård D. Oldberg Å. J. Biol. Chem. 1999; 274: 9636-9647Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Invariably, these studies show altered collagen fiber dimensions when the abundance of the LRR protein is changed. The present work started with a study of altered biosynthesis of proteins in early human osteoarthritis. We found a number of proteins to be up-regulated, one being a component with an apparent size of 39 kDa. This component appeared structurally related to fibromodulin since it cofractionated in a variety of separation procedures. We now define the primary structure including a putative polymorphism, oligosaccharide side chain substituents, and tissue expression of the protein. It represents a novel member of the LRR protein family belonging to the decorin/biglycan group (class I). The protein is named asporin based on the presence of a polyaspartate stretch in the amino-terminal region and the similarity with decorin. Normal human knee cartilage (18.5 g of tissue wet weight, donor age 32 to 50 years) and menisci (3.5 g of tissue wet weight) were obtained at surgery. The tissues were dissected clean, sliced into fine pieces, and disrupted using a high speed homogenizer (Polytron, Kinematica GmbH) in 12 volumes (v/w) of 4m GdnHCl, 0.05 m sodium acetate, pH 5.8, containing protease inhibitors (5 mm benzamidine hydrochloride, 0.1 m 6-aminohexanoic acid). After extraction for 24 h at 4 °C the remaining insoluble material was removed by centrifugation at 20,000 × g at 4 °C for 30 min. Proteins in the cartilage extract were separated from proteoglycans by CsCl density gradient centrifugation with a starting density of 1.5 g/ml under dissociative conditions in 4m GdnHCl as described elsewhere (28Heinegård D. Sommarin Y. Methods Enzymol. 1987; 144: 319-372Crossref PubMed Scopus (138) Google Scholar). The gradient tube was divided into 4 equal fractions using a Beckman tube slicer, and the top fraction (D4) was used for subsequent purification. The D4 fraction was then concentrated by ultrafiltration (PM-10 membrane, Amicon), followed by diaflow against 4 m GdnHCl, 20 mm Tris-HCl, pH 8, and applied to a Superose 6 column (2.2 × 100 cm) in 2.5-ml aliquots. Fractions of 2.5 ml were collected, monitored for protein content by measuring their absorbance at 280 nm, and analyzed by SDS-PAGE after ethanol precipitation, as previously described (29Paulsson M. Sommarin Y. Heinegård D. Biochem. J. 1983; 212: 659-667Crossref PubMed Scopus (24) Google Scholar). The proteins from the extract were separated into two peaks, a larger containing proteins of high molecular weight (fractions 25 to 45) and a smaller containing the smaller proteins. The latter fractions (46 to 65) were pooled and concentrated by ultrafiltration followed by diaflow against 7 m urea, 20 mm Tris-HCl, pH 8. The pooled material was then loaded onto a 30-ml bed volume column of DEAE-cellulose (1.6 × 15 cm, DE52, Whatman) equilibrated in the urea buffer. After sample loading, the column was washed with 5 bed volumes of the equilibration buffer, and eluted with a 800-ml linear gradient (27 bed volumes) of 0 to 1 m NaCl in the equilibration buffer at a flow rate of 20 ml/h. Fractions of 10 ml were collected, monitored for protein content by measuring their absorbance at 280 nm, and analyzed by SDS-PAGE. The fractions containing asporin were pooled, concentrated by ultrafiltration followed by diaflow against 7 m urea, 10 mm HCOOH, pH 4.0, and chromatographed on a 20-ml bed volume of Q-Sepharose Fast Flow (1.6 × 8.5 cm, Amersham Pharmacia Biotech) anion exchange column equilibrated in urea buffer. The column was washed with 5 bed volumes and the bound proteins were step eluted at a flow rate of 20 ml/h with the equilibration buffer containing 1m NaCl. Fractions of 2 ml were collected, monitored for protein content by measuring their absorbance at 280 nm, and analyzed by SDS-PAGE. The fractions containing asporin were pooled and equilibrated by diaflow to 7 m urea, 10 mm HCOOH, pH 4.0, and applied to a Mono Q HR 5/5 column (Amersham Pharmacia Biotech). The bound proteins were eluted with a 15-ml linear gradient (15 bed volumes) from 0 to 1 m NaCl at a flow rate of 30 ml/h. Fractions of 2 ml were collected, monitored for protein, and analyzed by SDS-PAGE. Initial characterization of the meniscus extract showed a low content of high molecular weight proteoglycan. Therefore, the sample was taken directly to chromatography, omitting cesium chloride gradient centrifugation. Forty milliliters of the meniscus extract were equilibrated to 7 m urea, 20 mm Tris-HCl, pH 8.0, by diaflow and directly chromatographed over Q-Sepharose Fast Flow followed by chromatography over Mono Q, as described above except that the pH was kept at 8.0. After SDS-PAGE analysis, the fractions from the Mono Q chromatography containing asporin were pooled and concentrated by ultrafiltration, followed by diaflow against 4 m GdnHCl, 50 mmsodium acetate, pH 5.8. This material was further chromatographed on two serially coupled columns of Superose 6 and Superdex 200 (Amersham Pharmacia Biotech) equilibrated and eluted at 0.2 ml/min with 4m GdnHCl, 50 mm sodium acetate, pH 5.8. Fractions of 0.5 ml were collected, monitored for protein content by measuring their absorbance at 280 nm. Protein patterns were analyzed by SDS-PAGE. Proteolytic digestion with Lys-C (Roche Molecular Biochemicals) was performed at enzyme to substrate ratios of 1:50 according to the manufacturer's instructions. Peptides were separated by reversed phase HPLC on a Vydac C18 column (2.1 × 30 mm), eluted with a gradient of acetonitrile (0–70% over 45 min) in 0.1% trifluoroacetic acid at a flow rate of 0.2 ml/min. The effluent was monitored at 220 nm. Peptides were sequenced on an Applied Biosystems 477A automated sequencer with on-line analysis of phenylthiohydantoin-derivatives on an Applied Biosystems 120A microbore HPLC. All the molecular biological procedures, including agarose gel electrophoresis, restriction enzyme digestion, ligation, bacterial transformation, and DNA sequencing, were performed according to standard methods (30Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The amino acid sequences obtained from endoproteinase Lys-C-digested asporin were used to search the GenBankTM data base with the TBLASTN 2.1 program (31Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70747) Google Scholar). The EST sequences identified from this search were aligned and assembled. The resulting full-length sequence was used for designing primers h39k-S (5′-CTTCTACACTAAGACACC-3′) and h39k-AS (5′-AAATGGACATTACCAATTAC-3′). Human osteoarthritic articular cartilage was obtained at surgery after total hip replacement, kept in phosphate-buffered saline during dissection, shaved and frozen in liquid nitrogen. Total RNA and mRNA were purified as described previously (32Lorenzo P. Neame P. Sommarin Y. Heinegård D. J. Biol. Chem. 1998; 273: 23469-23475Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). First strand cDNA was primed with oligo-(dT)15 and reverse transcribed with Superscript II reverse transcriptase (Life Technologies). After digestion of the mRNA with RNase H, the asporin cDNA was obtained using the polymerase chain reaction (PCR) with primers h39k-S and h39k-AS and Pfu DNA polymerase. After an initial denaturation step at 95 °C for 1 min, the DNA was amplified for 30 cycles of 45 s at 95 °C, 45 s at 54 °C, and 2 min 40 s at 72 °C. The resulting 1.2-kilobase product was isolated from an agarose gel, purified using the QiaQuick kit (Qiagen), and ligated into the pCR-Script Amp SK(+) vector (Stratagene). The PCR product and several of the resulting pCR-Script clones were sequenced using the BigDye kit (ABI) and run on a ABI 310 DNA sequencer. In addition to primers T3, T7, h39k-S, and h39k-AS two internal primers were used: h39k-IntS (5′-ATGAAAATAAAGTTAAGAAAATAC-3′) and h39k-Int AS (5′-AGGGTTTGCACTCATTTC-3′). The resulting sequence tracings were assembled using the SeqMan II module of the LaserGene 99 software (DNAstar Inc). A first draft full-length mouse asporin sequence was assembled from sequences obtained through a BLASTN search of the mouse EST section of GenBankTM with the human asporin sequence. Using this draft sequence the primers m39k-S (5′-ACTTGTACACAGGCCAGC-3′), m39k-AS (5′-TTTTATATTTAATGGATGTCATG-3′), m39k-IntS (5′-GACCTTCAAAATAATAAAATC-3′), and m39k-IntAS (5′-TGGTATATTAGCAAAAGTTC-3′) were designed. Mouse aorta first strand cDNA was prepared and asporin cDNA amplified from this by reverse transcriptase PCR using primers m39k-S and m39k-AS, as described above. The PCR product was cloned into pCR-Script and sequenced using all four m39k primers, as well as T3 and T7 primers. The human and mouse asporin cDNA sequences were deposited in GenBankTM with the accession numbers AF316824 and AF316825, respectively. For Northern blot analysis 10 μg of total RNA isolated from human osteoarthritic articular cartilage were electrophoresed on 1% formaldehyde-agarose gel, and transferred to a nitrocellulose filter (NitroPure, Micron Separation). Membranes of Multiple Tissue Northern blot and Human RNA Master Blot were from CLONTECH. The membranes were hybridized with a 463-base pair cDNA fragment (nucleotides 382–845 of the human sequence, Fig. 3) labeled with [α-32P]dCTP by using the Random Primed DNA labeling kit (Roche Molecular Biochemicals). Hybridization and washing of the membranes were according to the manufacturer's instructions. The membranes were allowed to expose x-ray film (Biomax MS, Kodak) or analyzed by the Bas2000 phosphoimaging system (Fuji). Samples to be digested were precipitated with ethanol, resuspended in 0.1 m Tris-HCl, pH 6.8, containing 0.1% SDS, and incubated in a boiling water bath for 3 min. Then an equal volume of 0.125 m Tris-HCl, pH 6.8, was added, plus 5 μl of 0.5% Nonidet P-40, 1 μg of trypsin inhibitor (from chicken egg white type II-0, Sigma), and 1 unit ofN-glycosidase F (Roche Molecular Biochemicals). An aliquot of the mixtures before and after digestion was diluted with sample buffer (2% SDS, 0.125 m Tris-HCl, pH 6.8, 0.002% bromphenol blue, and 20% glycerol), boiled at 100 °C for 4 min and electrophoresed on the gradient polyacrylamide gel. Proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Serva). Coomassie-stained bands on SDS-PAGE gels were excised and washed extensively using 40% acetonitrile in 25 mm NH4HCO3, pH 7.8. After washing, the gel pieces were dried in a SpeedVac and subsequently reduced and alkylated using 10 mmdithiothreitol and 55 mm iodoacetamide at 56 °C (30 min) and at 20 °C (30 min), respectively. Samples were then washed and dried before digestion overnight at 37 °C using 10–20 μl of sequencing grade endoproteinases such as trypsin (Promega) or Glu-C (Roche Molecular Biochemicals) at 25 ng/μl in 25 mmNH4HCO3, pH 7.8. The digestion was terminated by the addition of 10 μl of 2% trifluoroacetic acid, which also extracted the peptides out of the gel. After a minimum 1-h extraction at room temperature, peptides were purified from buffer using miniaturized C-18 reversed phase tips (ZiptipsTM, Millipore). Purified peptides were eluted directly onto the sample target using acetonitrile, 0.1% trifluoroacetic acid (1:1). Various matrices were used to increase the sequence coverage. When using water-soluble matrices such as 2,4,6-thihydroxyacetophenone and 2,5-dihydroxybenzoic acid, an AnchorchipTM target (Bruker Daltonik GmbH, Bremen, Germany), that confines the sample to a smaller area increasing the sensitivity was used (33Schuerenberg M. Luebbert C. Eickhoff H. Kalkum M. Lehrach H. Nordhoff E. Anal. Chem. 2000; 72: 3436-3442Crossref PubMed Scopus (300) Google Scholar). The intact mass was obtained after elution of intact protein from the gel (34Ehring H. Strömberg S. Tjernberg A. Norén B. Rapid Commun. Mass Spectrom. 1997; 11: 1867-1873Crossref PubMed Scopus (43) Google Scholar) followed by direct application to the AnchorchipTM target using ferulic acid as the matrix. Carbonic anhydrase was used for external calibration. Mass spectrometric studies were performed using a Bruker Scout 384 Reflex III matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. The instrument was used in the positive ion mode with delayed extraction and an acceleration voltage of 26 kV. Peptide samples were mainly analyzed using the reflector detector and 50–150 single-shot spectra were accumulated for improved signal-to-noise ratio. Spectra were internally calibrated using autolysis fragments of trypsin. For analysis of intact protein the linear detector was used, with an acceleration voltage of 20 kV. The software used to identify the obtained peptide masses and N-linked oligosaccharide composition and structure were ProFound (35Zhang W. Chait B.T. Anal. Chem. 2000; 72: 2482-2489Crossref PubMed Scopus (551) Google Scholar) and GlycoMod (36Cooper C. Gasteiger E. Packer N. Proteomics. 2001; 1: 340-349Crossref PubMed Scopus (363) Google Scholar), respectively. Extraction of human articular cartilage with 4 m GdnHCl followed by cesium chloride gradient centrifugation separated the matrix proteins from the bulk of the large proteoglycans in the cartilage. Fractionation of the extract by gel filtration on Superose 6 resulted in two pools, one containing large proteins and the other with proteins of lower molecular masses (≤67 kDa). The proteins in this latter pool were fractionated by DEAE ion exchange chromatography, where asporin was observed in the fractions also containing fibromodulin. These fractions were then chromatographed on a Q-Sepharose column at low pH. Although asporin and fibromodulin still coeluted, they were separated from other proteins in the pool. We then tried to separate asporin from fibromodulin on a Mono Q column using elution at low pH with a linear NaCl gradient. Again the two proteins eluted together with fibromodulin as the predominant component (Fig.1). Further attempts to separate asporin and fibromodulin by Superose 12 gel filtration with 1% SDS in the buffer, heparin-Sepharose chromatography, anti-fibromodulin antibody affinity chromatography, and C-18 reverse phase chromatography were unsuccessful. A final attempt to use the collagen affinity of fibromodulin (24Hedbom E. Heinegård D. J. Biol. Chem. 1993; 268: 27307-27312Abstract Full Text PDF PubMed Google Scholar) to precipitate the protein with collagen I, gave the interesting result that also asporin was recovered with the collagen precipitate (data not shown). The analysis of a GdnHCl extract of human meniscus by electrophoresis showed a low content of large proteoglycans (data not shown). The extract was thus directly applied on a Q-Sepharose anion exchange column in 7m urea, 20 mm Tris-HCl, pH 8.0. Asporin was recovered in a few fractions identified by SDS-PAGE. These fractions were pooled and further fractionated by gel filtration on two tandemly arranged columns of Superdex 200 and Superose 6 (Fig.2). Asporin eluted in a few fractions together with a minor proportion of the fibromodulin. After Lys-C digestion of the intact protein excised from an SDS-polyacrylamide gel, peptides were separated by reversed phase HPLC. Peaks were collected and analyzed. Some peaks gave two sequences, but by analysis of the relative yields of the amino acids at each cycle, it was possible to determine both sequences with a high degree of confidence. As the protein was not reduced and carboxymethylated, no peptides were isolated that contained cysteine. TBLASTN searches with the 9 peptide sequences obtained (Table I) showed that six of these were contained within an EST clone (GenBankTM accession numberAK000136). One peptide was derived from fibromodulin. Two peptides were too short to produce BLAST hits, but the sequences of these are present in the AK000136 sequence (Fig. 3).Table IIdentification of peptide sequencesPeptideTBLASTN hitAlignment1None (see peptide 2)KsRRLYLS2KsRRLYLSHNTLsEIP:::::::: :::::AK000136118 KLRRLYLSHNQLSEIP 1653KxTDIExGSLANIPRxREi:::: :::::::: :::AK000136493 KITDIENGSLANIPRVREI 5494KxLPpTLLELxlden:::::::: :: :AK000136376 KGLPPTLLELHLDYN 4205KxVYFQNNQITSIQE:::::::::::::Fibromodulin342 KYVYFQNNQITSIQE 3866KxTDIENGSLANIPR:::::::::::::AK000136493 KITDIENGSLANIPR 5377KxLTSLYGLILNN:::::::::::AK00013634 KGLTSLYGLILNN 728KELQxLGLGNN:::: ::::::AK000136460 KELQRLGLGNN 4929NoneKsLAELGenBank™ entries containing the obtained peptide sequences were identified by TBLASTN searches. Alignments are between the query peptides (on top) and the hit sequences (below). The numbers represent the amino acid numbering of the GenBank™ entries. Open table in a new tab GenBank™ entries containing the obtained peptide sequences were identified by TBLASTN searches. Alignments are between the query peptides (on top) and the hit sequences (below). The numbers represent the amino acid numbering of the GenBank™ entries. AK000136 is an EST sequence deposited in GenBankTM as a putative extracellular matrix protein. The deduced AK00136 sequence contains several leucine-rich repeats and the two COOH-terminal cysteine residues typical of the extracellular matrix LRR-repeat protein family. When AK000136 was used as the query in further BLAST searches, a number of other EST sequences were identified. Assembly of these sequences produced a longer open reading frame that included a signal peptide and the amino-terminal 4-cysteine motif of the extracellular matrix LRR proteins. The cDNA of the novel LRR protein was cloned through reverse transcriptase PCR from human femoral head osteoarthritic cartilage, using primers corresponding to the 5′- and 3′-untranslated regions of the assembled consensus sequence. The mouse homologue was similarly identified through BLAST searches of the mouse EST data base with the human sequence and cloned from mouse aorta cDNA through reverse transcriptase PCR using primers based on the EST sequences. The human and mouse asporin sequences are shown in Fig. 3. The predicted amino acid sequences of the two proteins are 90% identical. The four amino-terminal cysteines show the C-X 3-C-X-C-X 6-C pattern typical of decorin and biglycan (37Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar), which clearly identifies asporin as a member of the class I branch of the LRR proteins. Indeed, like decorin and biglycan, asporin contains a highly conserved putative propeptide sequence (amino acid residues 15–32). The putative propeptide cleavage site conforms to the bone morphogen
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