FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2012315214> ?p ?o ?g. }

• W2012315214 endingPage "28880" @default.
• W2012315214 startingPage "28869" @default.
• W2012315214 abstract "Background:The function of MAGP2 was studied by inactivating its gene (Mfap5−/−) in mice.Results: Mfap5−/− mice have a neutrophil deficiency and other phenotypes that are different from MAGP1- and fibrillin-deficient animals.Conclusion: MAGP2 has functional roles in hematopoiesis and in vessel wall maintenance.Significance: Characterization of MAGP2 function is crucial to the identification and treatment of microfibril-related disease.Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5−/− mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2−/−;Mfap5−/−) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGFβ1, TGFβ2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling. The function of MAGP2 was studied by inactivating its gene (Mfap5−/−) in mice. Results: Mfap5−/− mice have a neutrophil deficiency and other phenotypes that are different from MAGP1- and fibrillin-deficient animals. Conclusion: MAGP2 has functional roles in hematopoiesis and in vessel wall maintenance. Significance: Characterization of MAGP2 function is crucial to the identification and treatment of microfibril-related disease. Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5−/− mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2−/−;Mfap5−/−) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGFβ1, TGFβ2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling. The ECM 2The abbreviations used are: ECMextracellular matrixMAGPmicrofibril-associated glycoproteinBMPbone morphogenic proteinDKOMAGP1/2 double knockout2KOMAGP2 knockout1KOMAGP1 knockoutμCTmicrocomputed tomographyBLIbiolayer interferometryNeoneomycin resistance cassettegDNAgenomic DNA. is a complex biopolymer that provides strength to tissues and plays instructive roles in organogenesis and tissue homeostasis (1.Mecham R.P. The Extracellular Matrix. An Overview. Springer-Verlag, Berlin2011Crossref Google Scholar). An abundant component of the ECM is the microfibril, which imparts limited elasticity to tissues, acts as a template for elastin deposition, and is a regulator of signaling events. Microfibrils are 10- to 12-nm fibers built upon a backbone of fibrillin and decorated with function-modifying accessory proteins that include growth factor complexes, fibulins, emilins, and MAGPs (2.Jensen S.A. Robertson I.B. Handford P.A. Dissecting the fibrillin microfibril. Structural insights into organization and function.Structure. 2012; 20: 215-225Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 3.Kielty C.M. Elastic fibres in health and disease.Expert Rev. Mol. Med. 2006; 8: 1-23Crossref PubMed Scopus (212) Google Scholar). Mutations in the genes for microfibril components have varied systemic effects that are not always apparent with in vitro experimentation (3.Kielty C.M. Elastic fibres in health and disease.Expert Rev. Mol. Med. 2006; 8: 1-23Crossref PubMed Scopus (212) Google Scholar, 4.Ramirez F. Sakai L.Y. Biogenesis and function of fibrillin assemblies.Cell Tissue Res. 2010; 339: 71-82Crossref PubMed Scopus (148) Google Scholar, 5.Yanagisawa H. Davis E.C. Unraveling the mechanism of elastic fiber assembly. The roles of short fibulins.Int. J. Biochem. Cell Biol. 2010; 42: 1084-1093Crossref PubMed Scopus (107) Google Scholar). The complexity of microfibril structure necessitates in vivo manipulation of proteins for an accurate understanding of microfibril function and identification of microfibril-related disease. extracellular matrix microfibril-associated glycoprotein bone morphogenic protein MAGP1/2 double knockout MAGP2 knockout MAGP1 knockout microcomputed tomography biolayer interferometry neomycin resistance cassette genomic DNA. MAGP1 (the protein product of the microfibrillar-associated protein 2 (Mfap2) gene) and MAGP2 (the protein product of the Mfap5 gene and also known as MP-25) are a two-member family of small microfibril-associated proteins ∼31 kDa and 25 kDa in size, respectively (6.Gibson M.A. Hughes J.L. Fanning J.C. Cleary E.G. The major antigen of elastin-associated microfibrils is a 31-kDa glycoprotein.J. Biol. Chem. 1986; 261: 11429-11436Abstract Full Text PDF PubMed Google Scholar, 7.Gibson M.A. Finnis M.L. Kumaratilake J.S. Cleary E.G. Microfibril-associated glycoprotein-2 (MAGP-2) is specifically associated with fibrillin-containing microfibrils but exhibits more restricted patterns of tissue localization and developmental expression than its structural relative MAGP-1.J. Histochem. Cytochem. 1998; 46: 871-886Crossref PubMed Scopus (79) Google Scholar). Both proteins are found only in vertebrates, and phylogenetic studies suggest that MAGP2 arose from MAGP1 through gene duplication early in vertebrate evolution (8.Segade F. Functional evolution of the microfibril-associated glycoproteins.Gene. 2009; 439: 43-54Crossref PubMed Scopus (17) Google Scholar). The two MAGPs share a functional C-terminal matrix-binding domain that is characterized by conserved cysteine residues (9.Segade F. Trask B.C. Broekelmann T.J. Pierce R.A. Mecham R.P. Identification of a matrix-binding domain in MAGP1 and MAGP2 and intracellular localization of alternative splice forms.J. Biol. Chem. 2002; 277: 11050-11057Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 10.Hatzinikolas G. Gibson M.A. The exon structure of the human MAGP-2 gene.J. Biol. Chem. 1998; 273: 29309-29314Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). MAGP2 has a conserved proprotein convertase cleavage site within this domain that makes MAGP2 a substrate for multiple proprotein convertase family members (11.Donovan L.J. Cha S.E. Yale A.R. Dreikorn S. Miyamoto A. Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2.Matrix Biol. 2013; 32: 117-122Crossref PubMed Scopus (7) Google Scholar). Also unique to MAGP2 is an RGD integrin-binding motif located at the N terminus. Amino acid sequences in the N-terminal regions of MAGP1 and MAGP2 are dissimilar, but both are enriched in acidic amino acids. MAGP1 contains two consensus sequences for O- but not N-glycosylation (12.Trask B.C. Broekelmann T. Ritty T.M. Trask T.M. Tisdale C. Mecham R.P. Post-translational modifications of microfibril associated glycoprotein-1 (MAGP-1).Biochemistry. 2001; 40: 4372-4380Crossref PubMed Scopus (28) Google Scholar), whereas MAGP2 contains five conserved, predicted O-linked glycosylation sequences. Functional studies with MAGP1 show that the acidic N terminus contains a growth factor interaction motif capable of binding active TGFβ and bone morphogenic protein (BMP) (13.Weinbaum J.S. Broekelmann T.J. Pierce R.A. Werneck C.C. Segade F. Craft C.S. Knutsen R.H. Mecham R.P. Deficiency in microfibril-associated glycoprotein-1 leads to complex phenotypes in multiple organ systems.J. Biol. Chem. 2008; 283: 25533-25543Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). MAGP1 is associated with all microfibrils except those directly adjacent to the plasma membrane of aortic endothelial cells and those at the junction of the zonule of the eye (14.Davis E.C. Immunolocalization of microfibril and microfibril-associated proteins in the subendothelial matrix of the developing mouse aorta.J. Cell Sci. 1994; 107: 727-736Crossref PubMed Google Scholar, 15.Henderson M. Polewski R. Fanning J.C. Gibson M.A. Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure of fibrillin-containing microfibrils as visualized by the rotary shadowing technique.J. Histochem. Cytochem. 1996; 44: 1389-1397Crossref PubMed Google Scholar, 16.Kumaratilake J.S. Gibson M.A. Fanning J.C. Cleary E.G. The tissue distribution of microfibrils reacting with a monospecific antibody to MAGP, the major glycoprotein antigen of elastin-associated microfibrils.Eur. J. Cell Biol. 1989; 50: 117-127PubMed Google Scholar). Although MAGP2 has been localized to both elastin-associated microfibrils and elastin-free microfibrils in a number of tissues, the protein exhibits patterns of tissue localization and developmental expression that are more restricted than those of MAGP1 (7.Gibson M.A. Finnis M.L. Kumaratilake J.S. Cleary E.G. Microfibril-associated glycoprotein-2 (MAGP-2) is specifically associated with fibrillin-containing microfibrils but exhibits more restricted patterns of tissue localization and developmental expression than its structural relative MAGP-1.J. Histochem. Cytochem. 1998; 46: 871-886Crossref PubMed Scopus (79) Google Scholar, 17.Visel A. Thaller C. Eichele G. GenePaint.org. An atlas of gene expression patterns in the mouse embryo.Nucleic Acids Res. 2004; 32: D552-D556Crossref PubMed Google Scholar). The association of MAGP1 and MAGP2 with other microfibrillar proteins is covalent, requiring reducing agents for their extraction (18.Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. Further characterization of proteins associated with elastic fiber microfibrils including the molecular cloning of MAGP-2 (MP25).J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Genetic deletion of extracellular MAGP1 in mice results in an array of phenotypes, including a bleeding diathesis, obesity, and osteopenia (13.Weinbaum J.S. Broekelmann T.J. Pierce R.A. Werneck C.C. Segade F. Craft C.S. Knutsen R.H. Mecham R.P. Deficiency in microfibril-associated glycoprotein-1 leads to complex phenotypes in multiple organ systems.J. Biol. Chem. 2008; 283: 25533-25543Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19.Craft C.S. Broekelmann T.J. Zou W. Chappel J.C. Teitelbaum S.L. Mecham R.P. Oophorectomy-induced bone loss is attenuated in MAGP1-deficient mice.J. Cell. Biochem. 2012; 113: 93-99Crossref PubMed Scopus (25) Google Scholar, 20.Craft C.S. Zou W. Watkins M. Grimston S. Brodt M.D. Broekelmann T.J. Weinbaum J.S. Teitelbaum S.L. Pierce R.A. Civitelli R. Silva M.J. Mecham R.P. Microfibril-associated glycoprotein-1, an extracellular matrix regulator of bone remodeling.J. Biol. Chem. 2010; 285: 23858-23867Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). MAGP1-deficient animals also have a pronounced deficiency in circulating and tissue monocytes. Here we report the generation and initial characterization of mice with null alleles of the MAGP2 gene (Mfap5) and both MAGP1 and MAGP2 (Mfap2−/−;Mfap5−/−) (DKO) genes. We find that Mfap5−/− (2KO) phenotypes are non-overlapping with mice lacking MAGP1 (1KO). However, the absence of both MAGPs causes changes in large vessel architecture. Biochemical studies show that MAGP2 protein binds active TGFβ1, TGFβ2, and BMP2. Taken together, these data show that MAGP2 has unique, MAGP1-independent functions in hematopoiesis and that MAGPs have redundant functions in large vessels. Homologous recombination was used to insert a gene-targeting cassette containing the coding sequence for neomycin resistance into the exon 9 region of the Mfap5 gene in murine ES cells. G418-resistant ES cell colonies were screened by Southern blot analysis, and ES cell clones, positive for recombination, were injected into C57Bl/6 blastocysts. Chimeric mice were bred for germ line transmission. Genotype analysis was performed on genomic DNA isolated from tail tissue using primers listed in supplemental Table 1. Generation of mice deficient in both MAGP1 and MAGP2 was achieved by breeding MAGP2 mutant mice with previously generated animals deficient in MAGP1 (13.Weinbaum J.S. Broekelmann T.J. Pierce R.A. Werneck C.C. Segade F. Craft C.S. Knutsen R.H. Mecham R.P. Deficiency in microfibril-associated glycoprotein-1 leads to complex phenotypes in multiple organ systems.J. Biol. Chem. 2008; 283: 25533-25543Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Mice used for these studies were maintained on a mixed-strain background, and wild-type controls were generated from the same parental stock as MAGP-deficient animals. Animals were housed in a pathogen-free facility, and the Washington University Animal Studies Committee approved all procedures. Genomic DNA isolated from adult mouse tail tissue was used with primers listed in supplemental Table 1 to confirm the location and orientation of the targeting construct within the Mfap5 gene locus. For all sequencing reactions, bands were extracted from agarose gel, and PCR products were isolated using a QIAquick gel extraction kit according to the protocol of the manufacturer (Qiagen Inc., Valencia, CA). Amplicons were then ligated into the pGEM-T vector using the rapid DNA ligation kit according to the protocol of the manufacturer (Roche Diagnostics, Mannheim, Germany) and introduced into DH5α Escherichia coli (Promega Corp., Madison, WI). The growth medium was inoculated with ampicillin-resistant, color-selected colonies, and plasmid products were isolated using QIAprep Spin Miniprep kits according to the instructions of the manufacturer (Qiagen). The Protein and Nucleic Acid Chemistry Laboratory (Washington University, Saint Louis, MO) performed DNA sequencing. All DNA alignments were performed using DNASTAR Lasergene 9 software. Total RNA was isolated from adult mouse lung, kidney, and heart using TRIzol reagent, and reverse transcription cDNA amplification was performed using 1 μg of RNA and SuperScript III first-strand synthesis system with oligo(dT) according to the specifications of the manufacturer (Invitrogen). Complement DNA was amplified using primers listed in supplemental Table 1. Ethidium bromide gels for nonsense-mediated decay studies were imaged with the ChemiDoc MP system and Image Lab version 4.0 software using identical exposure and filter settings (Bio-Rad). For sequencing, PCR band excision and sequencing were performed according to methods detailed previously. RNA probe generation and in situ hybridization for Mfap5, Mfap2, and collagen1α1 (Col1a1) was performed as described previously using mRNA isolated from adult mouse heart and primers listed in supplemental Table 1 (21.Ehrman L.A. Yutzey K.E. Lack of regulation in the heart forming region of avian embryos.Dev. Biol. 1999; 207: 163-175Crossref PubMed Scopus (68) Google Scholar, 22.Lincoln J. Alfieri C.M. Yutzey K.E. BMP and FGF regulatory pathways control cell lineage diversification of heart valve precursor cells.Dev. Biol. 2006; 292: 292-302Crossref PubMed Scopus (153) Google Scholar, 23.Shelton E.L. Yutzey K.E. Tbx20 regulation of endocardial cushion cell proliferation and extracellular matrix gene expression.Dev. Biol. 2007; 302: 376-388Crossref PubMed Scopus (95) Google Scholar). RNA probes were 806 bp in length for Mfap5, 802 bp for Mfap2, and 846 bp for Col1a1. Rabbit polyclonal antibodies against mouse MAGP2 (24.Lemaire R. Farina G. Kissin E. Shipley J.M. Bona C. Korn J.H. Lafyatis R. Mutant fibrillin 1 from tight skin mice increases extracellular matrix incorporation of microfibril-associated glycoprotein 2 and type I collagen.Arthritis Rheum. 2004; 50: 915-926Crossref PubMed Scopus (42) Google Scholar) and MAGP1 (13.Weinbaum J.S. Broekelmann T.J. Pierce R.A. Werneck C.C. Segade F. Craft C.S. Knutsen R.H. Mecham R.P. Deficiency in microfibril-associated glycoprotein-1 leads to complex phenotypes in multiple organ systems.J. Biol. Chem. 2008; 283: 25533-25543Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) have been described previously. For Western blot analysis of serially extracted mouse tissue lysates, adult mouse lung was homogenized in 990 μl of PBS (pH7.4) and 10 μl of protease inhibitor mixture (catalog no. P8340, Sigma Aldrich, Saint Louis, MO) and extracted 24 h at 4 °C. The samples were pelleted by centrifugation at 16,000 × g or 20 min at 4 °C. The liquid portion of the sample was removed for Western blotting, and the pellet was resuspended in 1 m NaCl and protease inhibitors and extracted for 24 h. The process was repeated using 8 m urea, PBS, and 20 mm DTT (seen in Fig. 5C,D), and 8 m urea + DTT. Western blotting was performed using standard procedures. Samples were run on 7.5% or 12% SDS-PAGE gels and transferred to nitrocellulose membranes. Ponceau S stain was applied for 5 min and removed using 0.1 m NaOH. All blocking steps were performed with blocking buffer containing 5% casein, 0.1% coldwater fish skin gelatin, and 0.01% Tween 20 in TBS. Primary antibodies were applied overnight at 4 °C at a dilution of 1:10,000 for MAGP2 (in 5% BSA) or 1:1000 for MAGP1 (in 5% BSA). Secondary antibodies were applied at 1:4000 in casein block and incubated for 1 h at room temperature. ECL reagents were used for detection of horseradish peroxidase, and chemiluminescence was read using the ChemiDoc MP system and Image Lab version 4.0 software (GE Healthcare). Immunohistochemistry for paraffin sections was performed using the ultrasensitive ABC peroxidase rabbit IgG staining kit (Fisher Scientific, Pittsburgh, PA) according to the instructions of the manufacturer, with the following modifications for MAGP2 detection. After peroxidase quenching, the sections were washed in 0.1% BSA in TBS. Hyaluronidase digestion solution (1 mg/ml hyaluronidase in 0.1 m sodium acetate buffer (pH5.5) containing 0.85% NaCl) was applied, and the sections were incubated in a humid chamber for 30 min at 37 °C. The sections were washed in BSA/TBS three times for 5 min. A guanidine/DTT solution was applied to the sections for 15 min. The sections were washed with BSA/TBS three times for 5 min before incubation with iodoacetamide solution for 15 min. The sections underwent a final BSA/TBS wash before application of ultra-sensitive staining kit block solution. Primary anti-MAGP2 antibody was diluted 1:8000 in blocking solution and applied to the sections overnight at 4 °C. Secondary antibody solution was made using 2.25 μl/ml of secondary antibody. Horseradish peroxidase reactivity was visualized using the metal enhanced DAB substrate kit (Fisher). Imaging was performed on a Zeiss Axioskop using identical exposure and filter settings. Image processing was performed using Adobe Photoshop CS5 software. Monoclonal antibodies used for flow cytometry were as follows: phycoerythrin-conjugated hamster anti-mouse CD3e (BD Biosciences), FITC rat anti-mouse CD335 (NKp46, eBioscience, San Diego, CA), FITC rat anti-mouse Ly-6G (eBioscience), phycoerythrin rat anti-mouse CD115 (eBioscience), allophycocyanin rat F4/80 (Abcam, Cambridge, MA). Phycoerythrin hamster IgG1, FITC rat IgG2a, FITC rat IgG2b, phycoerythrin rat IgG2a control, and allophycocyanin rat IgG2b isotype controls were utilized according to the suggestions of the manufacturer. Mice were killed by CO2 narcosis, and the chest and abdominal cavity were exposed to reveal the heart, dorsal aorta, and spleen. Whole blood was collected from the dorsal aorta in a heparinized syringe. Red cells were lysed with NH4Cl buffer for 10 min at room temperature and washed twice with FACs buffer (1× PBS and 2% FBS). After blocking with mouse Fc Block (BD Biosciences) for 20 min, 2 × 105 cells were incubated with antigen-specific or isotype control antibodies for 30 min at 4 °C. Cells were washed twice with FACs buffer and fixed with 4% paraformaldehyde for 15 min, washed twice, and stored overnight. Spleens were removed, and splenocytes were isolated after passage through a 70-μm nylon mesh filter. Lysis, blocking, incubation, and fixation were identical to the white blood cell treatment above. Cells were sorted on a FACSCalibur machine (BD Biosciences) with Cell-Quest software (BD Biosciences). The percentage of positive cells for each cell type was determined by gating on isotype control, nonspecific labeling (percentage of positive cells within gate < 5% for control), followed by subtraction of background percentage from antibody-specific results. A minimum of 105 live cell events were recorded for each analysis. Experiments were performed with at least five animals of each genotype (n ≥ 5), and experiments were repeated at least two times with mice 2 and 6 months of age. Microcomputed tomography analysis of left tibias from 6-month-old mice was performed as described previously (19.Craft C.S. Broekelmann T.J. Zou W. Chappel J.C. Teitelbaum S.L. Mecham R.P. Oophorectomy-induced bone loss is attenuated in MAGP1-deficient mice.J. Cell. Biochem. 2012; 113: 93-99Crossref PubMed Scopus (25) Google Scholar), with the exception that the tibias were not frozen. The tibias were embedded in 2% agarose and scanned using a Scanco μCT 40 (Scanco Medical AG, Zurich, Switzerland). For trabecular bone measurements, the growth plate was excluded and 30 × 16-μm sections were contoured. For cortical bone, the sections were analyzed just above the tibiofibular junction, and three sections per tibia were analyzed. Contouring and analysis were performed by the Washington University Musculoskeletal Research Facility using Scanco MicroCT software. Identical contouring and analysis parameters were used for all tibias. The tibias from at least five male mice of each genotype (n ≥ 5) were used for analysis. Six-month-old male mice were weighed and then anesthetized using 1.5% isoflurane, keeping the body temperature constant. A solid-state catheter was inserted into the right common carotid artery, isoflurane was reduced to 0.5%, and blood pressure measurements were recorded (25.Faury G. Pezet M. Knutsen R.H. Boyle W.A. Heximer S.P. McLean S.E. Minkes R.K. Blumer K.J. Kovacs A. Kelly D.P. Li D.Y. Starcher B. Mecham R.P. Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency.J. Clin. Invest. 2003; 112: 1419-1428Crossref PubMed Scopus (207) Google Scholar, 26.Wagenseil J.E. Nerurkar N.L. Knutsen R.H. Okamoto R.J. Li D.Y. Mecham R.P. Effects of elastin haploinsufficiency on the mechanical behavior of mouse arteries.Am. J. Physiol. Heart Circ. Physiol. 2005; 289: H1209-H1217Crossref PubMed Scopus (152) Google Scholar). The hearts were excised and weighed, and the aortas and left carotid arteries were removed for mechanical testing as reported previously (25.Faury G. Pezet M. Knutsen R.H. Boyle W.A. Heximer S.P. McLean S.E. Minkes R.K. Blumer K.J. Kovacs A. Kelly D.P. Li D.Y. Starcher B. Mecham R.P. Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficiency.J. Clin. Invest. 2003; 112: 1419-1428Crossref PubMed Scopus (207) Google Scholar). At least 12 male mice of each genotype (n ≥ 12) were used for analysis. The coding sequence for mouse MAGP2 protein, excluding the N-terminal signal sequence, was cloned into pET23b as a tagless construct. This plasmid was transformed into Rosetta 2(DE3) E. coli cells for protein expression. MAGP2 proteins were purified from inclusion bodies, followed by oxidative refolding as described previously (27.Swiecki M. Scheaffer S.M. Allaire M. Fremont D.H. Colonna M. Brett T.J. Structural and biophysical analysis of BST-2/tetherin ectodomains reveals an evolutionary conserved design to inhibit virus release.J. Biol. Chem. 2011; 286: 2987-2997Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Recombinant His6-tagged MAGP1, used for Western blotting, was expressed and isolated as described previously for this protein (13.Weinbaum J.S. Broekelmann T.J. Pierce R.A. Werneck C.C. Segade F. Craft C.S. Knutsen R.H. Mecham R.P. Deficiency in microfibril-associated glycoprotein-1 leads to complex phenotypes in multiple organ systems.J. Biol. Chem. 2008; 283: 25533-25543Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Native MAGP2 protein was expressed in mammalian cells and purified as follows. Full-length mature-form MAGP2, without the endogenous signal sequence, was cloned into the pHL-Avitag3 vector (28.Aricescu A.R. Lu W. Jones E.Y. A time- and cost-efficient system for high-level protein production in mammalian cells.Acta Crystallogr. D. Biol. Crystallogr. 2006; 62: 1243-1250Crossref PubMed Scopus (532) Google Scholar) containing an optimized signal sequence at the N terminus as well as a BirA biotin ligase recognition sequence and hexahistidine (His6) tag at the C terminus. Protein was expressed by large-scale transient transfection of FreeStyle 293F cells cultured in serum-free Freestyle 293 medium (Life Tech.). Culture supernatants were collected 72 h post-transfection, and protein was purified to homogeneity using nickel affinity chromatography. Purity was assessed by SDS-PAGE. Site-specific biotinylation of the C-terminal tags were carried out using E. coli BirA biotin ligase. All surface plasmon resonance binding was performed on a Reichert SR7000 surface plasmon resonance system. To bind MAGP2 protein to the carboxymethyl dextran (CM-5) chip, the chip was preactivated with a mixture of 200 mm 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride and 50 mm N-hydroxysuccinimide, and then 100 μl MAGP2 was diluted in 5 mm acetate buffer (pH 4.5) and injected with a flow rate of 10 μl/min at a concentration of 20 μm. This resulted in ∼1300 resonance units of MAGP2 protein coupled to the chip. Excess reactive sites on the chip were then quenched with a 70-μl injection of 0.5 m ethanolamine (pH 8). To measure MAGP2 binding to active TGFβ1, carrier-free active human TGFβ1 (R&D Systems, Minneapolis, MN) was diluted in surface plasmon resonance running buffer (10 mm HEPES, 150 mm NaCl, 0.05% Triton X-100 (pH 7.4)) and injected for 100 s at a flow rate of 50 μl/min. The dissociation was monitored for 300 s. The chip was recycled with 0.2 m glycine (pH 2.3) before subsequent injections. The concentrations of TGFβ1 injected were 100, 50, 50, 25, 25, and 12.5 nm. Background subtraction and calculation of the dissociation constant for individual curves was calculated using Scrubber2 software (Center for Biolmolecular Interaction Analysis, University of Utah). The Octet system for Biolayer interferometry (Pall Life Sciences, Ann Arbor, MI) was also used to assess MAGP2 binding to active TGFβ1 as well as TGFβ2 and BMP2. Streptavidin-coated biosensors from ForteBio were used to capture biotinylated MAGP2 onto the surface of the sensor. After reaching base line, sensors were moved to the association step containing 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 nm active mouse TGFβ1, TGFβ2, or BMP2 for 300 s and then dissociated for 300 s. A buffer-only reference was subtracted from all curves. The running buffer consisted of 10 mm HEPES (pH 7.4), 150 mm NaCl, 3.4 mm EDTA, 1% BSA, 0.01% azide, 0.05% Tween, and 0.005% Triton X-100. Affinities were estimated from global kinetic analysis. Statistical significance was determined by Student's t test. Data are reported as a mean ± S.D. Homologous recombination was used to disrupt the MAGP2 gene (Mfap5) by replacing exon 9 with a neomycin resistance cassette (Neo), causing a downstream frameshift. Our attempts to target exon 1 and the upstream promoter sequence were unsuccessful. Exon 9 encodes the matrix-binding domain. If expressed, the mutant protein lacking this domain would be incapable of binding to fibrillin and, hence, would be a functional null. PCR primers flanking each exon from 2–10, as well as within the Neo cassette itself, were used to walk along the targeted Mfap5 gene and confirm localization of the Neo cassette to exon 9. Genomic DNA (gDNA) isolated from WT and Mfap5−/− mice yielded identical amplicons using primers flanking exons 2–8 of the Mfap5 gene (Fig. 1, A and B, and supplemental Table 1). An exon 9 amplico" @default.
• W2012315214 created "2016-06-24" @default.
• W2012315214 creator A5008838841 @default.
• W2012315214 creator A5016948672 @default.
• W2012315214 creator A5023087890 @default.
• W2012315214 creator A5031970005 @default.
• W2012315214 creator A5037029746 @default.
• W2012315214 creator A5037115495 @default.
• W2012315214 creator A5040638170 @default.
• W2012315214 creator A5044351338 @default.
• W2012315214 creator A5054704857 @default.
• W2012315214 creator A5068418680 @default.
• W2012315214 creator A5074263750 @default.
• W2012315214 creator A5088601071 @default.
• W2012315214 date "2013-10-01" @default.
• W2012315214 modified "2023-10-12" @default.
• W2012315214 title "Microfibril-associated Glycoprotein 2 (MAGP2) Loss of Function Has Pleiotropic Effects in Vivo" @default.
• W2012315214 cites W132710040 @default.
• W2012315214 cites W1480702700 @default.
• W2012315214 cites W1530181168 @default.
• W2012315214 cites W1896434730 @default.
• W2012315214 cites W1964922567 @default.
• W2012315214 cites W1965508013 @default.
• W2012315214 cites W1972978465 @default.
• W2012315214 cites W1973457542 @default.
• W2012315214 cites W1981074262 @default.
• W2012315214 cites W1981381366 @default.
• W2012315214 cites W1981471190 @default.
• W2012315214 cites W1984015906 @default.
• W2012315214 cites W1985901090 @default.
• W2012315214 cites W1993010243 @default.
• W2012315214 cites W1993230767 @default.
• W2012315214 cites W1994785305 @default.
• W2012315214 cites W1995073326 @default.
• W2012315214 cites W1996073438 @default.
• W2012315214 cites W2004913444 @default.
• W2012315214 cites W2005804802 @default.
• W2012315214 cites W2024039709 @default.
• W2012315214 cites W2024707653 @default.
• W2012315214 cites W2030017298 @default.
• W2012315214 cites W2035742371 @default.
• W2012315214 cites W2042571711 @default.
• W2012315214 cites W2047254900 @default.
• W2012315214 cites W2055115221 @default.
• W2012315214 cites W2058345218 @default.
• W2012315214 cites W2063650823 @default.
• W2012315214 cites W2065865744 @default.
• W2012315214 cites W2075529332 @default.
• W2012315214 cites W2080057626 @default.
• W2012315214 cites W2084403389 @default.
• W2012315214 cites W2087256455 @default.
• W2012315214 cites W2089282865 @default.
• W2012315214 cites W2093238184 @default.
• W2012315214 cites W2101242266 @default.
• W2012315214 cites W2101870218 @default.
• W2012315214 cites W2105892139 @default.
• W2012315214 cites W2106365753 @default.
• W2012315214 cites W2123464577 @default.
• W2012315214 cites W2128677374 @default.
• W2012315214 cites W2131588425 @default.
• W2012315214 cites W2140256258 @default.
• W2012315214 cites W2140409722 @default.
• W2012315214 cites W2143286808 @default.
• W2012315214 cites W2151448988 @default.
• W2012315214 cites W2151779288 @default.
• W2012315214 cites W2152350731 @default.
• W2012315214 cites W2153316018 @default.
• W2012315214 cites W2168265052 @default.
• W2012315214 cites W2171591131 @default.
• W2012315214 cites W2172270516 @default.
• W2012315214 cites W2304879549 @default.
• W2012315214 cites W4210739731 @default.
• W2012315214 cites W2031289652 @default.
• W2012315214 doi "https://doi.org/10.1074/jbc.m113.497727" @default.
• W2012315214 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3789982" @default.
• W2012315214 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23963447" @default.
• W2012315214 hasPublicationYear "2013" @default.
• W2012315214 type Work @default.
• W2012315214 sameAs 2012315214 @default.
• W2012315214 citedByCount "56" @default.
• W2012315214 countsByYear W20123152142013 @default.
• W2012315214 countsByYear W20123152142014 @default.
• W2012315214 countsByYear W20123152142015 @default.
• W2012315214 countsByYear W20123152142016 @default.
• W2012315214 countsByYear W20123152142017 @default.
• W2012315214 countsByYear W20123152142018 @default.
• W2012315214 countsByYear W20123152142019 @default.
• W2012315214 countsByYear W20123152142020 @default.
• W2012315214 countsByYear W20123152142021 @default.
• W2012315214 countsByYear W20123152142022 @default.
• W2012315214 countsByYear W20123152142023 @default.
• W2012315214 crossrefType "journal-article" @default.
• W2012315214 hasAuthorship W2012315214A5008838841 @default.
• W2012315214 hasAuthorship W2012315214A5016948672 @default.
• W2012315214 hasAuthorship W2012315214A5023087890 @default.
• W2012315214 hasAuthorship W2012315214A5031970005 @default.
• W2012315214 hasAuthorship W2012315214A5037029746 @default.
• W2012315214 hasAuthorship W2012315214A5037115495 @default.

• next