FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019743373> ?p ?o ?g. }

• W2019743373 endingPage "21556" @default.
• W2019743373 startingPage "21545" @default.
• W2019743373 abstract "The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo. The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo. The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage and many other forms of extracellular matrix (for review, see Ref. 1Wagener R. Ehlen H.W. Ko Y.P. Kobbe B. Mann H.H. Sengle G. Paulsson M. FEBS Lett. 2005; 579: 3323-3329Crossref PubMed Scopus (92) Google Scholar). They participate in the formation of fibrillar or filamentous structures (2Chen Q. Johnson D.M. Haudenschild D.R. Tondravi M.M. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1743-1753Crossref PubMed Scopus (55) Google Scholar, 3Chen Q. Zhang Y. Johnson D.M. Goetinck P.F. Mol. Biol. Cell. 1999; 10: 2149-2162Crossref PubMed Scopus (64) Google Scholar, 4Klatt A.R. Nitsche D.P. Kobbe B. Mörgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2000; 275: 3999-4006Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 6Piecha D. Muratoglu S. Mörgelin M. Hauser N. Studer D. Kiss I. Paulsson M. Deák F. J. Biol. Chem. 1999; 274: 13353-13361Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 7Winterbottom N. Tondravi M.M. Harrington T.L. Klier F.G. Vertel B.M. Goetinck P.F. Dev. Dyn. 1992; 193: 266-276Crossref PubMed Scopus (91) Google Scholar) and mediate interactions between collagen-containing fibrils (8Budde B. Blumbach K. Ylöstalo J. Zaucke F. Ehlen H.W. Wagener R. Ala-Kokko L. Paulsson M. Bruckner P. Grässel S. Mol. Cell. Biol. 2005; 25: 10465-10478Crossref PubMed Scopus (109) Google Scholar, 9Wiberg C. Klatt A.R. Wagener R. Paulsson M. Bateman J.F. Heinegård D. Mörgelin M. J. Biol. Chem. 2003; 278: 37698-37704Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and other matrix constituents like aggrecan (10Hauser N. Paulsson M. Heinegârd D. Mörgelin M. J. Biol. Chem. 1996; 271: 32247-32252Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), small leucine-rich proteoglycans (9Wiberg C. Klatt A.R. Wagener R. Paulsson M. Bateman J.F. Heinegård D. Mörgelin M. J. Biol. Chem. 2003; 278: 37698-37704Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), or COMP (11Mann H.H. Ozbek S. Engel J. Paulsson M. Wagener R. J. Biol. Chem. 2004; 279: 25294-25298Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Matrilins form homo- and hetero-oligomers by their C-terminal coiled-coil domain. In addition, the subunits contain epidermal growth factor-like and von Willebrand factor A (VWA) 2The abbreviations used are: VWAvon Willebrand factor AADAMa disintegrin and metalloproteinaseADAMTSa disintegrin and metalloproteinase with thrombospondin-1 motifPBSphosphate-buffered salinePP2Aprotein phosphatase 2ATBSTris-buffered salinesiRNAsmall interfering RNAMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight. 2The abbreviations used are: VWAvon Willebrand factor AADAMa disintegrin and metalloproteinaseADAMTSa disintegrin and metalloproteinase with thrombospondin-1 motifPBSphosphate-buffered salinePP2Aprotein phosphatase 2ATBSTris-buffered salinesiRNAsmall interfering RNAMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight.-like domains, where the latter are presumably the major ligand binding domains (11Mann H.H. Ozbek S. Engel J. Paulsson M. Wagener R. J. Biol. Chem. 2004; 279: 25294-25298Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Mutations in matrilin-3 in humans cause different forms of chondrodysplasia (12Chapman K.L. Mortier G.R. Chapman K. Loughlin J. Grant M.E. Briggs M.D. Nat. Genet. 2001; 28: 393-396Crossref PubMed Scopus (156) Google Scholar, 13Mostert A.K. Dijkstra P.F. Jansen B.R. van Horn J.R. de Graaf B. Heutink P. Lindhout D. Am. J. Med. Genet. A. 2003; 120: 490-497Crossref Scopus (36) Google Scholar, 14Borochowitz Z.U. Scheffer D. Adir V. Dagoneau N. Munnich A. Cormier-Daire V. J. Med. Genet. 2004; 41: 366-372Crossref PubMed Scopus (60) Google Scholar) and are also linked to the development of hand osteoarthritis (15Stefánsson S.E. Jónsson H. Ingvarsson T. Manolescu I. Jónsson H.H. Olafsdóttir G. Pálsdóttir E. Stefánsdóttir G. Sveinbjörnsdóttir G. Frigge M.L. Kong A. Gulcher J.R. Stefánsson K. Am. J. Hum. Genet. 2003; 72: 1448-1459Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and intervertebral disc degeneration (16Min J.L. Meulenbelt I. Riyazi N. Kloppenburg M. Houwing-Duistermaat J.J. Seymour A.B. van Duijn C.M. Slagboom P.E. Ann. Rheum. Dis. 2006; 65: 1060-1066Crossref PubMed Scopus (49) Google Scholar). von Willebrand factor A a disintegrin and metalloproteinase a disintegrin and metalloproteinase with thrombospondin-1 motif phosphate-buffered saline protein phosphatase 2A Tris-buffered saline small interfering RNA matrix-assisted laser desorption ionization time-of-flight. von Willebrand factor A a disintegrin and metalloproteinase a disintegrin and metalloproteinase with thrombospondin-1 motif phosphate-buffered saline protein phosphatase 2A Tris-buffered saline small interfering RNA matrix-assisted laser desorption ionization time-of-flight. Proteolytic processing of extracellular matrix proteins plays both physiological and pathophysiological roles. Proteolysis is a major post-translational modification used to modify the function of proteins. Tissue homeostasis requires a well balanced synthesis and degradation of extracellular matrix proteins, specifically mediated by protease families like matrix metalloproteinases (17Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3211) Google Scholar), ADAMs (18Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (914) Google Scholar), or ADAMTSs (19Porter S. Clark I.M. Kevorkian L. Edwards D.R. Biochem. J. 2005; 386: 15-27Crossref PubMed Scopus (612) Google Scholar). The development of degenerative diseases is often accompanied by an activation of such proteases. In addition, the cleavage sometimes releases protein fragments that have completely new functions (20O’Reilly M.S. Holmgren L. Chen C. Folkman J. Nat. Med. 1996; 2: 689-692Crossref PubMed Scopus (1150) Google Scholar, 21Bergers G. Javaherian K. Lo K.M. Folkman J. Hanahan D. Science. 1999; 284: 808-812Crossref PubMed Scopus (876) Google Scholar). Determination of which extracellular proteases cleave which substrates is crucial to understand the physiological function of both (22Overall C.M. Blobel C.P. Nat. Rev. Mol. Cell Biol. 2007; 8: 245-257Crossref PubMed Scopus (284) Google Scholar). Physiological cleavage has been described for most members of the matrilin family (4Klatt A.R. Nitsche D.P. Kobbe B. Mörgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2000; 275: 3999-4006Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 6Piecha D. Muratoglu S. Mörgelin M. Hauser N. Studer D. Kiss I. Paulsson M. Deák F. J. Biol. Chem. 1999; 274: 13353-13361Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), but was not yet extensively studied. The adapter function of the matrilins may be modulated by physiological proteolysis that causes the loss of single subunits and thereby decreases the binding avidity (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Interestingly, an earlier identified cleavage site in the hinge region of matrilin-4, N-terminal of the coiled-coil, is conserved throughout the matrilin family (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and it was recently shown that matrilin-3 is cleaved by ADAMTS4 in vitro at this site (23Hills R. Mazzarella R. Fok K. Liu M. Nemirovskiy O. Leone J. Zack M.D. Arner E.C. Viswanathan M. Abujoub A. Muruganandam A. Sexton D.J. Bassill G.J. Sato A.K. Malfait A.M. Tortorella M.D. J. Biol. Chem. 2007; 282: 11101-11109Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Here we studied matrilin processing in some detail and identified another member of the ADAMTS family, ADAMTS5, as being able to cleave matrilin-3 and -4. Such cleavage is likely to alter the cohesion of the extracellular matrix. All recombinant matrilin proteins were expressed in the human embryonic kidney cell line 293EBNA (Invitrogen). Expression and affinity purification of wild type full-length matrilin-3 and -4 with an N-terminal BM40 signal peptide and a C-terminal StrepII tag was described earlier (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Mann H.H. Ozbek S. Engel J. Paulsson M. Wagener R. J. Biol. Chem. 2004; 279: 25294-25298Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The cDNAs encoding murine wild type full-length matrilin-1 and matrilin-2 were amplified by PCR using primers that inserted a SpeI restriction site at the 5′-end and a NotI site at the 3′-end, respectively (5′-matn1spe GCC CAC TAG TCC CCC AGC CCA GAG and 3′-matn1not CAA TGC GGC CGC GAT GAT TCT GTT CTC CAG G; 5′-matn2spe GCC CAC TAG TTA GAG AGC GTC CCC AAG CC and 3′-matn2not CAA TGC GGC CGC TCT GTA TTT TAG GCG ATT TTC). After digestion with SpeI and NotI, the amplified cDNA fragments were inserted into the expression vector pCEP-Pu-StrepII tag (C-terminal) in-frame with the sequence of the signal peptide of BM40 (24Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (201) Google Scholar). The expression construct for the short matrilin-4 splice variant lacking the VWA1 domain (wt M4 ΔA1) was cloned in the same manner into a modified version of pCEP-Pu carrying a C-terminal His8 tag (pCEP-PuV27, kindly provided by Manuel Koch, Cologne) using 5′-SpeI and 3′-BamHI sites in the primers: m4dA1fw, GCC CAC TAG TAA AGG ACC TGT GTG CTG AGT TGG, and m4dA1rev, CAA TGG ATC CCT TTC GGC TAG CCA GCT GG. The matrilin-4 571EE → AA (M4AA) and 571EE → QQ (M4QQ) mutations were introduced into a matrilin-4 full-length cDNA clone in pBluescript KS using the TransformerTM Site-directed Mutagenesis Kit (Clontech) according to the manufacturer’s protocol. Mutagenesis primers were CAG CAT TTG CCC AGC GGC GGG CAT TGG C for M4AA, GCA TTT GCC CAC AGC AGG GCA TTG GC for M4QQ, and GTG ACT GGT GAG GCC TCA ACC AAG TC to switch a ScaI to a StuI restriction site in the vector sequence for easier selection of mutant clones. Matrilin-2 910EE → AA and matrilin-3 429EE → QQ mutant constructs were generated by PCR amplification of two fragments of each cDNA, which overlapped in the region in which the mutations and a new unique restriction site (NheI for M2AA and EcoRV for M3QQ) were introduced. As for the wild type constructs, SpeI and NotI sites were introduced via the primers at the 5′- or 3′-ends, respectively. Primer pairs were 5′-matn2spe (see above) and matn2AAas (GCA TTG GTC CTG GCT AGC TGC CAA AGG GTT TCC TG); 5′-matn3spe (GCC CAC TAG TCC GTT TGG CCC GCG CGA GC) and matn3QQas (GAG GCT TCG GGC TTG TTG GAT ATC TGA ACA TGT C) for the 5′-fragments and matn2AAs (CAG GAA ACC CTT TGG CAG CTA GCC AGG ACC AAT GC) and 3′-matn2not (see above); matn3QQs (CAT GTT CAG ATA TCC AAC AAG CCC GAA GCC TC) and 3′-matn3not (CAA TGC GGC CGC ACG ATG TAC TTG TCC ATA TTC) for the 3′-fragments. The amplified 5′- and 3′-fragments were digested with the appropriate restriction enzymes, ligated, cloned into pBluescript KS for easy screening, and finally cloned into the expression vector pCEP-Pu-Strepll tag. The chimeric matrilin-4 constructs “M1 hinge” and “mut M1 hinge” (Fig. 3) were generated by the same strategy introducing unique NdeI restriction sites in the mutated regions and 5′-SpeI and 3′-BamHI sites at the respective ends. pCEP-PuV27 was used as expression vector. Primer pairs were m4dA1fw (see above) and m4dA1m1as (GGC TTT CGC ATT CGC AGG GGT CCT CCT CTG GGC ATA TGC TGC CTT TGA GA); m4dA1fw (see above) and m4dA1m1QQas (GGC TTT CGC ATT CGC AGG GGT CCT GCT GTG GGC ATA TGC TGC CTT TGA GA) for the 5′-fragments; m4dA1m1s (TCT CAA AGG CAG CAT ATG CCC AGA GGA GGA CCC CTG CGA ATG CGA AAG CC), m4dA1rev (see above), and m4dA1m1QQs (TCT CAA AGG CAG CAT ATG CCC ACA GCA GGA CCC CTG CGA ATG CGA AAG CC) for the 3′-fragments. Each of the expression constructs was transfected into 293EBNA cells with FuGENE 6 (Roche), the cell lines were selected with puromycin (1 μg/ml) and cultured under serum-free conditions prior to harvest of conditioned cell culture supernatants. C-terminal StrepII-tagged M2, M2AA, M3, M3QQ, M4, M4AA, and M4QQ were affinity purified from conditioned supernatants using Streptactin-Sepharose Affinity Resin (IBA, Göttingen) as described (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). For the detection of intracellular matrilin-4, transfected 293EBNA cells were cultured for 2 days under serum-free conditions. Cells were then washed twice with PBS and digested with 0.1% trypsin for 15 min at 37 °C to harvest cells and degrade extracellular and cell surface proteins. Digestion was stopped by addition of 10 volumes of prechilled PBS followed by five washing steps in PBS at 4 °C. The cell pellet was ground under liquid nitrogen, homogenized at 4 °C in 100 mm Tris, pH 8.0, 1 tablet/50 ml of “Complete protease inhibitor mixture” (Roche), and cell debris was removed by centrifugation. The supernatant was subjected to SDS-PAGE. Tissues from C57/Bl6 mice were dissected and proteins sequentially extracted at 4 °C with 10 volumes (ml/g wet tissue) of 150 mm NaCl, 50 mm Tris, pH 7.4 (TBS), for 4 h and TBS containing 10 mm EDTA for 16 h. All extraction buffers contained 2 mm phenylmethylsulfonyl fluoride and 2 mm N-ethylmaleimide. To generate a specific cleavage neo-epitope antibody against processed matrilin-4, the peptide matn4pC (H2N-572GIGAGTELRSPC-CONH2) was synthesized and used to immunize two rabbits and two guinea pigs (Pineda Antibody Service, Berlin). The antisera were tested by immunoblot and one rabbit serum, which showed the desired reactivity toward processed matrilin-4, was affinity purified on the matn4pC peptide coupled to CNBr-Sepharose. Antibodies were eluted in 0.1 m glycine, pH 2.5, and neutralized with 1 m Tris, pH 8.8. To assess the specificity, the purified antibody was in some cases incubated with the peptide (1 μg/ml diluted antibody) for 16 h at 4 °C prior to detection by immunoblot or immunofluorescence microscopy. SDS-PAGE was performed as described by Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). For immunoblots the proteins were transferred to nitrocellulose and incubated with the appropriate affinity-purified rabbit antibody diluted in TBS containing 5% low fat milk powder. Bound antibodies were detected by luminescence using peroxidase-conjugated swine anti-rabbit IgG (Dako), 3-aminophthalhydrazide (1.25 mm), p-coumaric acid (225 mm), and 0.01% H2O2. Primary antibodies against the following antigens were used: matrilin-1 (26Hauser N. Paulsson M. J. Biol. Chem. 1994; 269: 25747-25753Abstract Full Text PDF PubMed Google Scholar), matrilin-2 (6Piecha D. Muratoglu S. Mörgelin M. Hauser N. Studer D. Kiss I. Paulsson M. Deák F. J. Biol. Chem. 1999; 274: 13353-13361Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), matrilin-3 (4Klatt A.R. Nitsche D.P. Kobbe B. Mörgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2000; 275: 3999-4006Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), matrilin-4 (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), StrepII-tag (IBA, Göttingen), protein-disulfide isomerase (Stressgen), Golgi matrix protein GM130 (BD Biosciences), protein phosphatase 2A (Santa Cruz), and matn4pC (see above). For N-terminal sequencing, proteins were reduced with 5 mm dithiothreitol and alkylated with ⅓ volume of 6 m acrylamide, subjected to SDS-PAGE, and electroblotted to a polyvinylidene difluoride membrane (Immobilon PSQ, Millipore). Protein bands were cut out and their N-terminal amino acid sequences determined in a Procise Protein Sequencer (Applied Biosystems). Peptide mass fingerprinting (27Sengle G. Kobbe B. Morgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2003; 278: 50240-50249Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) and MALDI-TOF mass spectrometry (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 27Sengle G. Kobbe B. Morgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2003; 278: 50240-50249Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) were performed as described. For mass determination of the processed matrilin-3 coiled-coil domains, the affinity purified proteins were reduced in 8 m urea, 10 mm dithiothreitol at 80 °C for 20 min, alkylated with 20 mm iodoacetamide for 1 h in the dark, and desalted on C18 reversed-phase columns (Millipore) with a stepwise elution with 20, 40, 60, and 80% acetonitrile in 0.1% trifluoroacetic acid. Subsequent MALDI-TOF analysis was conducted on a dihydroxybenzoic acid matrix. Transfected 293EBNA cells were grown to confluence, washed three times with PBS, and cultured for 24 h under serum-free conditions to remove residual serum proteins. Subsequently the cells were cultured in serum-free media supplemented with 0.1, 1, 10, and 30 μm GM 6001, 0.1, 1, and 10 μg/ml aprotinin, E- 64, E-64d, and pepstatin A, 1, 10, and 100 μm amastatin, leupeptin, and decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone, 10, 100, and 1000 μm 1,10-o-phenanthroline, and 20, 200, and 2000 μm 4-(2-aminoethyl)benzenesulfonyl fluoride or only the solvents. The media were changed after 12 and 28 h and the conditioned supernatants harvested at each medium change and after 52 h. For pulse-chase analysis of matrilin-4 processing, transfected 293EBNA cells were grown to confluence, washed in PBS, and cultured in Dulbecco’s modified Eagle’s medium without l-methionine and l-cysteine. Radiolabeling was performed for 90 min by addition of 50 μCi/ml NEG722 Easytag Express Protein Labeling Mix (PerkinElmer Life Sciences) to the medium (pulse). After two washes with PBS, cells were cultured in serum-free medium and cells and conditioned supernatants were harvested after 30, 60, 90, and 120 min, 6 and 24 h. Cells were lysed in TBS containing 1% Nonidet P-40 and 35S-labeled matrilin-4 was affinity precipitated from cell lysates and conditioned supernatants with 20 μl of Streptactin-Sepharose beads (IBA, Göttingen) per sample for 3 h on a rocking platform. The beads were recovered by centrifugation (1000 × g, 5 min), washed three times in 100 mm Tris, pH 8.0, and eluted in 30 μl of elution buffer (0.1 m Tris, 2.5 mm desthiobiotin). Eluted proteins were separated by SDS-PAGE, the gel dried and exposed to a storage phosphorscreen (GE Healthcare) and detected with a PhosphorImager (GE Healthcare). The autoradiograph was analyzed with ImageQuant 5.1 software (GE Healthcare). Transfected 293EBNA cells expressing recombinant matrilin-4 were grown to confluence, incubated for 24 h in serum-free medium, washed three times in PBS, and recovered from the cell culture dishes with a cell scraper. After three additional washing steps cells were recovered in homogenization buffer (0.25 m sucrose, 10 mm triethanolamine acetate, pH 7.4, 2 mm phenylmethylsulfonyl fluoride, 2 mm N-ethylmaleimide, Complete protease inhibitor mixture (Roche)) and homogenized with 15 strokes in a Dounce homogenizer. Samples were centrifuged at 1000 × g and 4 °C for 10 min. Postnuclear supernatants were separated for 20 h at 38,000 × g in a L7-55 ultracentrifuge (Beckman) equipped with the SW41TI rotor using a 10–40% sucrose gradient, which was poured on a 1-ml cushion of 40% sucrose. Fractions (1 ml) were recovered, proteins were precipitated with ethanol and analyzed by immunoblot. Primary murine chondrocytes were isolated from ribcages of newborn C57/Bl6 mice and cultured as described earlier (8Budde B. Blumbach K. Ylöstalo J. Zaucke F. Ehlen H.W. Wagener R. Ala-Kokko L. Paulsson M. Bruckner P. Grässel S. Mol. Cell. Biol. 2005; 25: 10465-10478Crossref PubMed Scopus (109) Google Scholar). Cultured cells or frozen tissue sections (7 μm) were fixed for 5 min in 95% ethanol, 5% acetic acid at −20 °C, washed three times in TBS, and blocked for 1 h in blocking solution (2% fetal bovine serum in TBS, 0.1% Triton X-100). Frozen sections were in addition digested for 30 min at 37 °C with hyaluronidase (500 units/ml in 100 mm NaH2PO4, 100 mm sodium acetate, pH 5.0) and washed three times in TBS, 0.1% Triton X-100 prior to blocking. Cells or sections were incubated for 3 h with primary antibodies (anti-matrilin-4 (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), anti-matn4pC, or peptide inhibited anti-matn4pC) and 1 h with Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen) in blocking solution. Truncated human recombinant ADAMTS1, ADAMTS4, and ADAMTS5 (28Will H. Dettloff M. Bendzkô P. Sveshnikov P. J. Biomol. Tech. 2005; 16: 459-472PubMed Google Scholar) were from Invitek GmbH, Berlin. ADAMTS1 consisted of amino acids Phe253–Asn617, ADAMTS4 of Phe213–Ala579, and ADAMTS5 of Ser262–Gly625. All contained a C-terminal His6 tag. The aggrecanase activity of the recombinant ADAMTSs was determined by cleavage of recombinant aggrecan interglobular domain. Recombinant murine matrilin-3 and -4 were expressed in 293EBNA cells and purified as described above. Matrilin-1/-3 hetero-oligomers were extracted from fetal bovine rib cartilage and purified as described before (29Mann H.H. Sengle G. Gebauer J.M. Eble J.A. Paulsson M. Wagener R. Matrix Biol. 2007; 26: 167-174Crossref PubMed Scopus (18) Google Scholar). The matrilins were reconstituted in 150 mm NaCl, 5 mm CaCl2, 50 mm Tris, pH 7.5, at concentrations of 40 μg/ml. Samples (2 μg) were incubated with ADAMTS4 (0.2 μg), ADAMTS1 (0.4 μg), or ADAMTS5 (0.4 μg) in a total volume of 50 μl at 37 °C. The incubation was stopped by addition of 1 ml of ice-cold ethanol, the proteins were precipitated overnight and after centrifugation the pellet was resolved in 10 μl of water. After addition of 10 μl of sample buffer the samples were submitted to SDS-PAGE. cDNAs encoding human full-length ADAMTS4 and ADAMTS5 (Invitek GmbH) were cloned into the expression vector pCEP4 (Invitrogen). Each of the expression constructs was transfected into 293EBNA cells expressing recombinant matrilin-4 with LipofectamineTM 2000 (Invitrogen), the cell lines were selected with hygromycin (200 μg/ml) and cultured under serum-free conditions prior to harvest of conditioned cell culture supernatants. Cells transfected with the empty pCEP4 vector were used as control. The cells were washed with PBS and digested with 0.05% trypsin for 20 min at 37 °C to degrade extracellular and cell surface proteins. The harvested cells were counted and identical numbers were lysed in 150 mm NaCl, 5 mm EDTA, 1 mm sodium orthovanadate, 1% SDS, 50 mm HEPES, 1% Triton X-100, 0.5% Nonidet-P40, 10 mm sodium fluoride, 10% glycerol, pH 6.8, and Complete protease inhibitor mixture (Roche). siRNAs were obtained from Qiagen (Hs_ADAMTS4_1, ACA GAT GTG GTT GCA TCC TAA; Hs_ADAMTS5_2, CCC GTT AAC TTC ATA GCA AAT). Transfections of siRNA were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection mixtures were prepared by adding 1 μl (50 μm) of siRNA to 250 μl of Opti-MEM (Invitrogen) and 5 μl of Lipofectamine 2000 solution to 250 μl of Opti-MEM. After a 20-min incubation, the transfection solution was poured over cells previously covered with 500 μl of supplement-free culture medium. Mock transfection without siRNA served as a control. The transfection solution was removed after 4 h to reduce cytotoxicity and the medium added. The cells were cultured for 96 h and transferred to serum-free conditions prior to harvest of conditioned cell culture supernatants and the analysis of these by SDS-PAGE. To investigate the proteolytic processing of matrilins we established stably transfected 293EBNA human embryonic kidney cell lines that recombinantly express full-length murine matrilin-1, -2, -3, or -4 fused to an N-terminal BM40 signal peptide and a C-terminal StrepII tag. Conditioned media were analyzed by SDS-PAGE and immunoblot using antibodies directed either against the different matrilins or against the StrepII tag (Fig. 1A). Most recombinant matrilins showed heterogeneous band patterns. As previously demonstrated by MALDI-TOF mass spectrometry and Edman degradation (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), matrilin-4 consisted of unprocessed trimers (t, 219 kDa), 3To allow simple identification of protein components, we introduced a nomenclature where q represents tetramer, t represents trimer, d represents dimer, m represents monomer, and cc represents coiled-coil (5Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These designations are followed by a −cc or a +cc. The −cc indicates that the coiled-coil region is absent from the subunit, and +cc shows that the fragment carries additional coiled-coil regions bound by disulfide bonds and derived from other cleaved subunits. The digit before cc refers to the number of these coiled-coil fragments when more than one is carried. For example, m+2cc means a monomer connected by disulfide bonds to two additional coiled-coil region fragments thereby forming a triple coiled-coil α-helix. For a schematic depiction of this nomenclature, see Fig. 1B. processed dimers (d+cc, 153 kDa) and monomers (m+2cc, 87 kDa) containing an intact coiled-coil as well as cleaved-off monomers (m-cc, 66 kDa; Fig. 1, A and B). The cleavage site lies in the hinge region between the VWA2 domain and the coiled-coil oligomerization domain (Fig. 1, B and C). On the basis" @default.
• W2019743373 created "2016-06-24" @default.
• W2019743373 creator A5014968495 @default.
• W2019743373 creator A5018491631 @default.
• W2019743373 creator A5032800024 @default.
• W2019743373 creator A5034143227 @default.
• W2019743373 creator A5049839584 @default.
• W2019743373 creator A5052431937 @default.
• W2019743373 creator A5057980089 @default.
• W2019743373 date "2009-08-01" @default.
• W2019743373 modified "2023-10-13" @default.
• W2019743373 title "Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms" @default.
• W2019743373 cites W1505603609 @default.
• W2019743373 cites W1526252019 @default.
• W2019743373 cites W1595542501 @default.
• W2019743373 cites W1604074848 @default.
• W2019743373 cites W1755569971 @default.
• W2019743373 cites W1967479602 @default.
• W2019743373 cites W1967683284 @default.
• W2019743373 cites W1984186264 @default.
• W2019743373 cites W1984341575 @default.
• W2019743373 cites W1992660677 @default.
• W2019743373 cites W1996384857 @default.
• W2019743373 cites W2001050259 @default.
• W2019743373 cites W2011227409 @default.
• W2019743373 cites W2012152900 @default.
• W2019743373 cites W2014447402 @default.
• W2019743373 cites W2015809080 @default.
• W2019743373 cites W2017644547 @default.
• W2019743373 cites W2020456020 @default.
• W2019743373 cites W2030146437 @default.
• W2019743373 cites W2030246164 @default.
• W2019743373 cites W2031027318 @default.
• W2019743373 cites W2040263753 @default.
• W2019743373 cites W2043111550 @default.
• W2019743373 cites W2043146701 @default.
• W2019743373 cites W2043297474 @default.
• W2019743373 cites W2050790881 @default.
• W2019743373 cites W2052684852 @default.
• W2019743373 cites W2053628893 @default.
• W2019743373 cites W2054318886 @default.
• W2019743373 cites W2061945526 @default.
• W2019743373 cites W2074587230 @default.
• W2019743373 cites W2078974495 @default.
• W2019743373 cites W2084346169 @default.
• W2019743373 cites W2086908525 @default.
• W2019743373 cites W2089048746 @default.
• W2019743373 cites W2090510511 @default.
• W2019743373 cites W2100837269 @default.
• W2019743373 cites W2109790859 @default.
• W2019743373 cites W2115330717 @default.
• W2019743373 cites W2127923502 @default.
• W2019743373 cites W2128049766 @default.
• W2019743373 cites W2133032095 @default.
• W2019743373 cites W2157827546 @default.
• W2019743373 cites W2287041072 @default.
• W2019743373 doi "https://doi.org/10.1074/jbc.m109.016568" @default.
• W2019743373 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2755879" @default.
• W2019743373 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19531486" @default.
• W2019743373 hasPublicationYear "2009" @default.
• W2019743373 type Work @default.
• W2019743373 sameAs 2019743373 @default.
• W2019743373 citedByCount "20" @default.
• W2019743373 countsByYear W20197433732012 @default.
• W2019743373 countsByYear W20197433732013 @default.
• W2019743373 countsByYear W20197433732014 @default.
• W2019743373 countsByYear W20197433732015 @default.
• W2019743373 countsByYear W20197433732016 @default.
• W2019743373 countsByYear W20197433732018 @default.
• W2019743373 countsByYear W20197433732023 @default.
• W2019743373 crossrefType "journal-article" @default.
• W2019743373 hasAuthorship W2019743373A5014968495 @default.
• W2019743373 hasAuthorship W2019743373A5018491631 @default.
• W2019743373 hasAuthorship W2019743373A5032800024 @default.
• W2019743373 hasAuthorship W2019743373A5034143227 @default.
• W2019743373 hasAuthorship W2019743373A5049839584 @default.
• W2019743373 hasAuthorship W2019743373A5052431937 @default.
• W2019743373 hasAuthorship W2019743373A5057980089 @default.
• W2019743373 hasBestOaLocation W20197433731 @default.
• W2019743373 hasConcept C181199279 @default.
• W2019743373 hasConcept C185592680 @default.
• W2019743373 hasConcept C2781307694 @default.
• W2019743373 hasConcept C55493867 @default.
• W2019743373 hasConcept C86803240 @default.
• W2019743373 hasConceptScore W2019743373C181199279 @default.
• W2019743373 hasConceptScore W2019743373C185592680 @default.
• W2019743373 hasConceptScore W2019743373C2781307694 @default.
• W2019743373 hasConceptScore W2019743373C55493867 @default.
• W2019743373 hasConceptScore W2019743373C86803240 @default.
• W2019743373 hasIssue "32" @default.
• W2019743373 hasLocation W20197433731 @default.
• W2019743373 hasLocation W20197433732 @default.
• W2019743373 hasLocation W20197433733 @default.
• W2019743373 hasLocation W20197433734 @default.
• W2019743373 hasOpenAccess W2019743373 @default.
• W2019743373 hasPrimaryLocation W20197433731 @default.
• W2019743373 hasRelatedWork W1531601525 @default.
• W2019743373 hasRelatedWork W2319480705 @default.

• next