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• W3014328688 abstract "Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo. This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y−Neo. Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y−Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS. Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo. This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y−Neo. Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y−Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS. The heparan sulfate proteoglycan perlecan is localized to the pericellular matrix (PCM) that surrounds chondrocytes in mature hyaline cartilage. Perlecan is a multifunctional and highly conserved protein known to affect the compressive modulus of the PCM (1Wilusz R.E. DeFrate L.E. Guilak F. A biomechanical role for perlecan in the pericellular matrix of articular cartilage.Matrix Biol. 2012; 31: 320-327Crossref PubMed Scopus (56) Google Scholar, 2Guilak F. Alexopoulos L.G. Upton M.L. Youn I. Choi J.B. Cao L. Setton L.A. Haider M.A. The pericellular matrix as a transducer of biomechanical and biochemical signals in articular cartilage.Ann. N.Y. Acad. Sci. 2006; 1068: 498-512Crossref PubMed Scopus (226) Google Scholar) and sequester growth factors (3French M.M. Smith S.E. Akanbi K. Sanford T. Hecht J. Farach-Carson M.C. Carson D.D. Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.J. Cell Biol. 1999; 145: 1103-1115Crossref PubMed Scopus (133) Google Scholar, 4Vincent T.L. McLean C.J. Full L.E. Peston D. Saklatvala J. FGF-2 is bound to perlecan in the pericellular matrix of articular cartilage, where it acts as a chondrocyte mechanotransducer.Osteoarthr. Cartil. 2007; 15: 752-763Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 5Farach-Carson M.C. Warren C.R. Harrington D.A. Carson D.D. Border patrol: Insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.Matrix Biol. 2014; 34: 64-79Crossref PubMed Scopus (93) Google Scholar), indicating that it plays a role in both biomechanical and cell signaling. Mutations of the perlecan encoding gene, Hspg2, in humans result in defective endochondral ossification and skeletogenesis. Dissegmental dysplasia, Silverman-Handmaker type, is a lethal autosomal recessive disorder that presents with severe chondrodysplasia and dysmorphic facial features because of a null mutation in Hspg2 (6Arikawa-Hirasawa E. Wilcox W.R. Le A.H. Silverman N. Govindraj P. Hassell J.R. Yamada Y. Dyssegmental dysplasia, Silverman-Handmaker type, is caused by functional null mutations of the perlecan gene.Nat. Genet. 2001; 27: 431-434Crossref PubMed Scopus (185) Google Scholar). Other mutations in Hspg2 cause the non-lethal Schwartz-Jampel syndrome (SJS), which results in a decreased abundance of functional perlecan and leads to chondrodysplasia, myotonia, and early onset osteoarthritis (7Nicole S. Davoine C.S. Topaloglu H. Cattolico L. Barral D. Beighton P. Hamida D.B. Hammouda H. Cruaud C. White P.S. Samson D. Urtizberea J.A. Lehmann-Horn F. Weissenbach J. Hentati F. Fontaine B. Perlecan, the major proteoglycan of basement membranes, is altered in patients with Schwartz-Jampel syndrome (chondrodystrophic myotonia).Nat. Genet. 2000; 26: 480Crossref PubMed Scopus (210) Google Scholar, 8Rodgers K.D. Sasaki T. Aszodi A. Jacenko O. Reduced perlecan in mice results in chondrodysplasia resembling Schwartz–Jampel syndrome.Hum. Mol. Genet. 2007; 16: 515-528Crossref PubMed Scopus (58) Google Scholar). Although rare, these disorders are difficult to treat, and the therapeutic focus is primarily symptom relief (9Nessler M. Puchala J. Kwiatkowski S. Kobylarz K. Mojsa I. Chrapusta-Klimeczek A. Multidisciplinary approach to the treatment of a patient with chondrodystrophic myotonia (Schwartz-Jampel vel Aberfeld Syndrome).Ann. Plast. Surg. 2011; 67: 315-319Crossref PubMed Scopus (6) Google Scholar). Murine models generated to elucidate the function of perlecan during development have provided insight into disease progression and mechanisms underlying perlecan deficiency. Global knockout of Hspg2 in mice is predominantly embryonic lethal, caused by myocardial basement membrane failure under mechanical stress (10Costell M. Gustafsson E. Aszódi A. Mörgelin M. Bloch W. Hunziker E. Addicks K. Timpl R. Fassler R. Perlecan maintains the integrity of cartilage and some basement membranes.J. Cell Biol. 1999; 147: 1109-1122Crossref PubMed Scopus (514) Google Scholar). A model with the same point mutation as a form of SJS, Hspg2C1532Y−Neo, produced mutant mice with similar phenotypes as found in humans, including growth plate defects and myotonia (8Rodgers K.D. Sasaki T. Aszodi A. Jacenko O. Reduced perlecan in mice results in chondrodysplasia resembling Schwartz–Jampel syndrome.Hum. Mol. Genet. 2007; 16: 515-528Crossref PubMed Scopus (58) Google Scholar, 11Stum M. Girard E. Bangratz M. Bernard V. Herbin M. Vignaud A. Ferry A. Davoine C.S. Echaniz-Laguna A. René F. Marcel C. Molgó J. Fontaine B. Krejci E. Nicole S. Evidence of a dosage effect and a physiological endplate acetylcholinesterase deficiency in the first mouse models mimicking Schwartz–Jampel syndrome neuromyotonia.Hum. Mol. Genet. 2008; 17: 3166-3179Crossref PubMed Scopus (36) Google Scholar). A recent in vitro study, which used a fragment of perlecan with a common point mutation found in SJS, found that extracellular signal-regulated kinase (ERK) signaling was disrupted (12Martinez J.R. Grindel B.J. Hubka K.M. Dodge G.R. Farach-Carson M.C. Perlecan/HSPG2: Signaling role of domain IV in chondrocyte clustering with implications for Schwartz-Jampel Syndrome.J. Cell. Biochem. 2019; 120: 2138-2150Crossref Scopus (7) Google Scholar). ERK is known to regulate the differentiation of cartilage to bone during development (13Samsa W.E. Zhou X. Zhou G. Signaling pathways regulating cartilage growth plate formation and activity.Semin. Cell Dev. Biol. 2017; 62: 3-15Crossref PubMed Scopus (32) Google Scholar), and the presence of intact perlecan may prevent endochondral ossification (12Martinez J.R. Grindel B.J. Hubka K.M. Dodge G.R. Farach-Carson M.C. Perlecan/HSPG2: Signaling role of domain IV in chondrocyte clustering with implications for Schwartz-Jampel Syndrome.J. Cell. Biochem. 2019; 120: 2138-2150Crossref Scopus (7) Google Scholar). The absence of functional perlecan in SJS could lead to the activation of ERK signaling and explain why bone differentiation is accelerated. However, because of the multifaceted role of perlecan in regulating signaling within cells through both interactions with the core protein and the sequestration of various growth factors (4Vincent T.L. McLean C.J. Full L.E. Peston D. Saklatvala J. FGF-2 is bound to perlecan in the pericellular matrix of articular cartilage, where it acts as a chondrocyte mechanotransducer.Osteoarthr. Cartil. 2007; 15: 752-763Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 14Gubbiotti M.A. Neill T. Iozzo R.V. A current view of perlecan in physiology and pathology: A mosaic of functions.Matrix Biol. 2017; 57–58: 285-298Crossref PubMed Scopus (102) Google Scholar), there are likely many additional mechanisms that contribute to the SJS phenotype. Although studies often focus on the specific genetic mechanisms behind the disorder, a comprehensive profiling of the developing ECM may provide additional insight into how perlecan deficiency affects musculoskeletal functionality. When the initial skeletal template is specified in the embryo, perlecan and other ECM within the cartilage are less organized (15Xu X. Li Z. Leng Y. Neu C.P. Calve S. Knockdown of the pericellular matrix molecule perlecan lowers in situ cell and matrix stiffness in developing cartilage.Dev. Biol. 2016; 418: 242-247Crossref PubMed Scopus (18) Google Scholar, 16Cescon M. Gattazzo F. Chen P. Bonaldo P. Collagen VI at a glance.J. Cell Sci. 2015; 128: 3525-3531Crossref PubMed Scopus (121) Google Scholar). As chondrogenesis progresses, perlecan and other PCM components (e.g. type VI collagen, nidogens) become restricted to the periphery of the chondrocytes, taking on the organization characteristic of adult cartilage ECM. At the same time, chondrocytes increase synthesis of the bulk matrix, which is comprised of type II collagen fibers, glycosaminoglycans (GAGs) and proteoglycans, decreasing the overall volume fraction of cells (17Han L. Grodzinsky A.J. Ortiz C. Nanomechanics of the cartilage extracellular matrix.Annu. Rev. Mater. Res. 2011; 41: 133-168Crossref PubMed Scopus (113) Google Scholar, 18Lycke R.J. Walls M.K. Calve S. Computational modeling of developing cartilage using experimentally derived geometries and compressive moduli.J. Biomech. Eng. 2019; 141: 81002-81008Crossref Scopus (1) Google Scholar). Chondrocytes either maintain the chondrogenic phenotype on the articulating surface to form mature hyaline cartilage or transition to bone within the rest of the skeletal template by first undergoing hypertrophy then endochondral ossification (19Long F. Linsenmayer T.F. Regulation of growth region cartilage proliferation and differentiation by perichondrium.Development. 1998; 125: 1067-1073PubMed Google Scholar). In perlecan-deficient mice, endochondral ossification and PCM formation are disrupted (1Wilusz R.E. DeFrate L.E. Guilak F. A biomechanical role for perlecan in the pericellular matrix of articular cartilage.Matrix Biol. 2012; 31: 320-327Crossref PubMed Scopus (56) Google Scholar, 20Ishijima M. Suzuki N. Hozumi K. Matsunobu T. Kosaki K. Kaneko H Hassell J.R. Arikawa-Hirasawa E. Yamada Y. Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation.Matrix Biol. 2012; 31: 234-245Crossref PubMed Scopus (48) Google Scholar). Furthermore, we previously demonstrated that the compressive modulus of both cells and ECM is significantly decreased in the Hspg2C1532Y−Neo mouse model (15Xu X. Li Z. Leng Y. Neu C.P. Calve S. Knockdown of the pericellular matrix molecule perlecan lowers in situ cell and matrix stiffness in developing cartilage.Dev. Biol. 2016; 418: 242-247Crossref PubMed Scopus (18) Google Scholar). Based on these observations, we hypothesized that perlecan knockdown would negatively affect the pericellular incorporation of perlecan binding partners, as well as dysregulate molecules that meditate proper ossification, leading to significant changes in ECM composition. To test this hypothesis, we first visualized the distribution of key cartilaginous ECM proteins and GAGs in developing cartilage using immunohistochemistry, then assessed the proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We focused our analysis on the ECM proteins, termed the matrisome (21Naba A. Clauser K.R. Hoersch S. Liu H. Carr S .A. Hynes R.O. The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.Mol. Cell. Proteomics. 2012; 11Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar), to better elucidate and compare the composition of healthy and perlecan-deficient cartilage in embryonic and neonatal mice. We found that perlecan knockdown resulted in expected (e.g. decrease in PCM components) and unexpected (e.g. increase in bulk matrix) trends in ECM abundance. The ultrastructure of the ECM was not significantly different; however, more protein was secreted by Hspg2C1532Y−Neo cartilage in vitro, suggesting that the ability of newly synthesized ECM to incorporate into the matrix was impaired. Ultimately, perlecan knockdown prevents proper PCM formation and leads to ectopic ossification near the articulating surface (22Arikawa-Hirasawa E. Watanabe H. Takami H. Hassell J.R. Yamada Y. Perlecan is essential for cartilage and cephalic development.Nat. Genet. 1999; 23: 354-358Crossref PubMed Scopus (424) Google Scholar). These defects in ECM assembly because of perlecan deficiency result in the abnormal cartilage formation and osteoarthritis characteristic of SJS. All chemicals and reagents were acquired from Sigma-Aldrich (St. Louis, MO) unless otherwise specified Mice heterozygous for Hspg2C1532Y−Neo (Neo/+) on a DBA background were provided by Dr. Sophie Nicole (Inserm, France) (11Stum M. Girard E. Bangratz M. Bernard V. Herbin M. Vignaud A. Ferry A. Davoine C.S. Echaniz-Laguna A. René F. Marcel C. Molgó J. Fontaine B. Krejci E. Nicole S. Evidence of a dosage effect and a physiological endplate acetylcholinesterase deficiency in the first mouse models mimicking Schwartz–Jampel syndrome neuromyotonia.Hum. Mol. Genet. 2008; 17: 3166-3179Crossref PubMed Scopus (36) Google Scholar) and time mated to generate embryonic day (E)16.5 and postnatal day (P)3 mice. All murine experiments were approved by the Purdue Animal Care and Use Committee (PACUC; protocol 1310000973). Cartilage was dissected from the distal humerus of homozygous (Neo/Neo) and Neo/+ knockdown mice and wildtype (+/+) littermates (Fig. 1A). Care was taken to remove ligaments, bone, and ossified cartilage in the epiphyseal plate. Dissected cartilage was rinsed in phosphate-buffered saline (PBS), then immediately processed. Samples were either snap-frozen for proteomic and biochemical assays and stored at −80 °C until used or fixed for transmission electron microscopy (TEM). Tail snips were used to confirm genotypes as previously described (15Xu X. Li Z. Leng Y. Neu C.P. Calve S. Knockdown of the pericellular matrix molecule perlecan lowers in situ cell and matrix stiffness in developing cartilage.Dev. Biol. 2016; 418: 242-247Crossref PubMed Scopus (18) Google Scholar). Forelimbs were embedded in Optimal Cutting Temperature compound (OCT, Sakura Finetek, St, Torrance, CA), frozen in dry ice-cooled isopentane, and stored at −80 °C until sectioned. OCT embedded tissues were sectioned at 17 μm and stored at −20 °C. Before staining, sections were equilibrated to room temperature (RT), rehydrated with 1× PBS for 10 min, fixed with 4% paraformaldehyde (PFA) for 5 min, and rinsed with PBS. Then, sections were permeabilized with 0.1% Triton-X100 (Amresco, Cleveland, OH) in PBS and rinsed with 1× PBS. Background staining was reduced by using the Mouse on Mouse (MOM) staining kit (BMK-2022, Vector Labs, Burlingame, CA). Sections were incubated with MOM IG blocking buffer for 1 h and washed 3 × 2 min with PBS. Sections were primed with MOM protein diluent for 5 min before incubating with primary antibodies overnight and washed 3 × 2 min with PBS. Primary antibodies against COL10A1 (1:50, X53 14-9771-80, Invitrogen, Carlsbad, CA), NID2 (1:50, sc-377424, Santa Cruz Biotechnology, Santa Cruz, CA), HSPG2 (1:50, A7L6 sc-33707, Santa Cruz Biotechnology), type I collagen (1:100, AB765P, Millipore, Burlington, MA) and COL2A1 (1:100, MAB8887, Millipore) were diluted in MOM protein diluent. Biotinylated HABP (385911, Millipore) was diluted 1:150 in MOM protein diluent. Sections were primed again with MOM protein diluent for 5 min before staining with the appropriate secondary detection reagents [Goat anti-mouse IgG1 633 (1:500, Thermo Fisher Scientific, Rockford, IL), Goat anti-mouse IgG1 546 (1:500, Thermo Fisher Scientific), Donkey anti-Rat IgG 488 (1:500, Thermo Fisher Scientific), Donkey Anti-Rabbit 647 (1:500, Thermo Fisher Scientific), Goat Anti-Mouse 546 (1:500, Thermo Fisher Scientific), phalloidin 555 (1:200, Thermo Fisher Scientific), Streptavidin 488 (1:500, Thermo Fisher Scientific), 4′,6-diamidino-2-phenylindole (DAPI, 1:500, Roche, Fishers, IN)] in MOM protein diluent for 30 min in the dark. Incubations occurred at RT unless otherwise stated. Sections were mounted with Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA) and imaged at 10× and 20× using a DMI6000 inverted microscope (Leica, Buffalo Grove, IL). The same acquisition parameters were used for all samples and images were processed using FIJI (23Rueden C.T. Schindelin J. Hiner M.C. DeZonia B.E. Walter A.E. Arena E.T. Eliceiri K.W. ImageJ2: ImageJ for the next generation of scientific image data.BMC Bioinformatics. 2017; 18: 529Crossref PubMed Scopus (2134) Google Scholar, 24Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. Tinevez J.Y. White D.J. Hartenstein V. Eliceiri K. Tomancak P. Cardona A. Fiji: an open-source platform for biological-image analysis.Nat. Methods. 2012; 9: 676Crossref PubMed Scopus (21316) Google Scholar). Glycosaminoglycans (GAGs) were extracted from distal humeral cartilage using a guanidine extraction buffer. Briefly, snap-frozen cartilage from right and left humeri was pooled (∼1.5 and 2 mg wet-weight tissue from E16.5 and P3 cartilage, respectively) and pulverized using a tissue homogenizer (Ace Glass, 8325–08, Vineland, NJ) sitting in a liquid nitrogen bath. Pulverized samples were placed on ice for 15 min to allow the temperature to equilibrate. Samples were resuspended in 1 ml guanidine extraction buffer (4 m guanidine HCl, 50 mm sodium acetate, 100 mm N-ethylmaleimide, pH 5.8) by douncing twenty times with the homogenizer, transferred into 1.7 ml MaxyClear microtubes (Axygen, Union City, CA). Sulfated-GAGs were assayed following (25Hoemann C.D. Molecular and biochemical assays of cartilage components.Methods Mol. Med. 2004; 101: 127-156PubMed Google Scholar). Briefly, 250 μl of DMB solution (40 mm DMB, 40 mm NaCl, 40 mm Glycine, pH 3.00, 20 μm filtered) was added to 10 μl samples and 10 μl 1× PBS with 1 mm ethylenediaminetetraacetic acid (PBE). Chondroitin 6-sulfate (shark cartilage, C4384) in PBE was used as a standard, adding 250 μl of DMB solution to 10 μl standards and 10 μl guanidine extraction buffer. Optical density was measured at 530 and 590 nm, where the 590-baseline peak was subtracted from the 530 nm signal to increase assay sensitivity. PFA fixed cryosections were stained with Harris' hematoxylin (Electron Microscopy Sciences) containing 4% glacial acetic acid (v/v) for 5 min, rinsed under tap water, and washed with PBS 2 × 10 min. Sections were stained with safranin O solution (0.1% safranin O w/v in H2O, 0.22 μm pore vacuum filtered, Polysciences Inc., Warrington, PA) for 5 min, rinsed under tap water, and washed with PBS 3 × 10 min. Sections were mounted with Permount mounting medium (Electron Microscopy Sciences) and imaged at 10× and 20× using a DMI6000 inverted microscope. Protein was extracted from snap-frozen cartilage following (26Hsueh M.F. Khabut A. Kjellström S. Önnerfjord P. Kraus V.B. Elucidating the molecular composition of cartilage by proteomics.J. Proteome Res. 2016; 15: 374-388Crossref PubMed Scopus (34) Google Scholar). Samples resuspended in guanidine extraction buffer were incubated 24 h on a rocking platform at 4 °C and then reduced with 4 mm dithiothreitol for 30 min on an orbital shaker at 56 °C and alkylated with 16 mm iodoacetamide for 1 h in the dark at RT. Extracts were ethanol precipitated (9:1 ethanol/extract) overnight on a rocking platform at 4 °C, followed by centrifugation at 13,750 × g for 30 min at 4 °C. Pellets were washed with cold ethanol for 4 h at −20 °C and dried under vacuum. Extracts were resuspended in 500 μl digestion buffer (100 mm Tris base, 2 mm calcium chloride, 10% acetonitrile v/v, pH 8.0), transferred to tissue homogenizers on ice, dounced twenty times, and returned to 1.7 ml microtubes. Trypsin digestion was performed with 2.5 μg MS grade trypsin (Thermo Fisher Scientific) per mg wet-weight tissue for 16 h on an orbital shaker at 37 °C. Peptides were acidified with 1% trifluoroacetic acid (v/v) and ultra-filtrated using Ultra-micro C-18 SpinColumns (The Nest Group, Southboro, MA) according to the manufacturer's instructions. The eluent was dried using a CentriVap vacuum concentrator (Labconoco, Kansas City, MO) at 45 °C and resuspended in 10 μl running buffer (3% acetonitrile, 0.1% formic acid v/v). Peptide concentration was measured by 205 nm peptide absorbance using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) according to manufacturer's instructions and normalized to 0.2 mg/ml. Trypsin-digested peptides equivalent to 1 μg were analyzed with a Dionex UltiMate 3000 RSLC Nano System coupled to a Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Primary spectra were collected from 400 to 1600 m/z at 120,000 resolution, a maximum injection time of 100 ms, and a dynamic exclusion of 15 s. The top 20 precursors were fragmented using higher-energy C-trap dissociation at a normalized collision energy of 27%. Tandem spectra were acquired in the Orbitrap at a resolution of 15,000 with a maximum injection time of 20 ms. Xcalibur RAW files were processed by MaxQuant (version 1.6.1.0) (27Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367Crossref PubMed Scopus (7486) Google Scholar) to align primary spectra, identify proteins, and calculate relative protein abundance for LFQ. Tandem spectra were searched against the complete Mus musculus reference database (ID 000000589, downloaded from UniProt on 04/06/2018) comprising 53,127 proteins and a contaminants database. MaxQuant search parameters are tabulated in supplemental Table S1. Carbamidomethylation was defined as a fixed modification and variable modifications included oxidation of methionine, deamidation of asparagine, and hydroxylation of proline and lysine (28Naba A. Pearce O.M.T. Del Rosario A. Ma D. Ding H. Rajeeve V. Cutillas P.R. Balkwill F.R. Hynes R.O. Characterization of the extracellular matrix of normal and diseased tissues using proteomics.J. Proteome Res. 2017; 16: 3083-3091Crossref PubMed Scopus (80) Google Scholar). Initial precursor mass tolerances were set to 20 ppm in the first search and 4.5 ppm in the main search, and fragment mass tolerances were set to 20 ppm. Enzyme cleavage was set to trypsin, allowing for cleavage before proline residues and for a maximum of two missed cleavages. Results were manually filtered, removing contaminants and proteins identified with only modified peptides or by reverse database matching, and requiring a minimum of two razor and unique peptides across all sample replicates for identification and quantification. Raw data sets are available in the MassIVE repository (29Ocken A.R. Ku M. Kinzer-Ursem T.L. Calve S. Hspg2C1532YNeo DBA distal humeral cartilage proteomic comparison dataset.MassIVE. 2019; Google Scholar). Gene Ontology (GO) analysis was conducted for each pairwise comparison of interest (+/+: E16.5 versus P3, E16.5: Neo/Neo versus +/+, and P3: Neo/Neo versus +/+). All proteins identified in a sample were used to query g:GOSt, the functional enrichment analysis of the g:Profiler web server (30Reimand J. Arak T. Adler P. Kolberg L. Reisberg S. Peterson H. Vilo J. g:Profiler—a web server for functional interpretation of gene lists. (2016 update).Nucleic Acids Res. 2016; 44: W83-W89Crossref PubMed Scopus (635) Google Scholar). Leading protein gene names were submitted, specifying organism as Mus musculus, statistical domain scope to only annotated genes, and significance thresholding by g:SCS (α = 0.05). Positive identification of biological process terms required a minimum of three protein gene names. Hydroxyproline (Hyp) was assayed from snap-frozen cartilage. Both humeri were pooled, dried at 110 °C for 48 h, and weighed on a microbalance (∼80 and 110 μg dry-weight tissue from E16.5 and P3 cartilage, respectively). Hyp content was assessed using a Hyp assay kit (MAK008) according to the manufacturer's instructions. Water and HCl were added to maintain dry-weight tissue concentration at 4 μg/μl (water or HCl). Hyp and hydroxylysine (Hyl) levels were also analyzed by including as variable modifications in MaxQuant. Analyses of hydroxylation were calculated from the MaxQuant evidence table outputs. Raw intensity (XIC) of each evidence (e) was multiplied by the number of modified residues (mMod,e) for a given modification, either Hyp or Hyl (Mod). These values were summed for each experiment (i). The summation was multiplied by the LFQ normalization coefficient (Ni) to give the normalized LFQ abundance of a modification for a given sample, IMod,LFO,i (Eq. 1). IMod,LFQ,i=∑e,i(mMod,e*XICe)*Ni(1) Next, the number of prolines or lysines modified in each sample was calculated, using the ratio of modified to the total number of residues to calculate relative abundance. First, the number of modified residues (mMod,e) was divided by the total number of residues (nRes,e) for a given modification; either Hyp and proline or Hyl and lysine (Res). This ratio was multiplied by the raw intensity of each evidence. These values were summed for each experiment to yield the relative intensity of modified to all residues, IMod,i (Eq. 2). IMod,i=∑e,i(mMod,enRe⁡s,e*XICe)(2) Similarly, the relative intensity of unmodified to all residues was calculated IUnmod,i (Eq. 3). In this case, one minus the ratio of modified to total residues was multiplied by each evidence raw intensity. This value was summed for each experiment. IUnmod,i=∑e,i((1−mMod,enRe⁡s,e)*XICe)(3) Finally, the relative intensity of modified residues was divided by the total relative intensity of all residues and multiplied by the LFQ normalization coefficient (Ni) to give the LFQ normalized percent of modified residues for a given sample, RMod,LFQ,i (Eq. 4). RMod,LFQ,i=IMod,iIMod,i+IUnmod⁡,i*Ni(4) Freshly dissected cartilage samples were fixed in 2% PFA and 2% glutaraldehyde in 0.1 m cacodylate buffer overnight at 4 °C. Fixed samples were rinsed with cacodylate buffer (3 × 5 min), impregnated with 1% osmium 0.8% ferricyanide for 1 h, rinsed in water (3 × 5 min), stained with 2% aqueous uranyl acetate for 30 min, and rinsed again with water (3 × 5 min). Samples were then ethanol dehydrated (50, 70, 95, 100% sequentially for 1 × 30, 1 × 30, 1 × 30, and 3 × 15 min, respectively) and transitioned to acetonitrile (2 × 15 min) before infiltration with Embed 812 (Electron Microscopy Sciences) and acetonitrile (2:1) for 2 h, (1:2) rotating overnight, and resin for 3 h rotating. Samples were embedded in flat molds and cured overnight at 70 °C in fresh resin. Ultrathin sections were cut at 85 nm with a 45-degree diamond knife (Diatome, Hatfield, PA) on a UC6 ultramicrotome (Leica) and collected on 100 mesh formvar-coated copper grids and post stained in 4% aqueous uranyl acetate and 2% aqueous lead citrate. Sections were imaged on a FEI T12 80kV TEM (FEI Company, Hillsboro, OR) in the Purdue Life Science Microscopy core facility. Fibril area fraction and diameter measurement protocols were adapted from (31Starborg T. Kalson N.S. Lu Y. Mironov A. Cootes T.F. Holmes D.F. Kadler K.E. Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization.Nat. Protoc. 2013; 8: 1433Crossref PubMed Scopus (138) Google Scholar). For fibril area fraction measurements, uniform interstitial matrix regions between chondrocytes of the distal humeral cartilage were imaged at 30,000×. Three regions of interest (ROI) for both Neo/Neo and +/+ samples were segmented from each of n = 4 biological replicates using FIJI. The top layer of cells from the articular surface was excluded so that ROIs included only subsurface ultrastructure to avoid surface irregularities. The mean intensity of the ROI was divided by the maximum intensity range value (256) to calculate the fibril area fraction percentage. For fibril diameter measurements, uniform interstitial matrix regions were imaged at 120,000× magnification. ROIs were thresholded at" @default.
• W3014328688 created "2020-04-10" @default.
• W3014328688 creator A5015630020 @default.
• W3014328688 creator A5033362440 @default.
• W3014328688 creator A5036020518 @default.
• W3014328688 creator A5055742599 @default.
• W3014328688 date "2020-07-01" @default.
• W3014328688 modified "2023-10-15" @default.
• W3014328688 title "Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development" @default.
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