Characterization of a murine type IIB procollagen-specific antibody |
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| Institution: | 1. Department of Orthopaedic Surgery, Washington University School of Medicine, BJCIH, St. Louis, MO 63110, USA;2. Department of Cell Biology and Physiology, Washington University School of Medicine, BJCIH, St. Louis, MO 63110, USA;3. Department of Biomedical Engineering, Washington University School of Medicine, BJCIH, St. Louis, MO 63110, USA;1. Department of Physics and Astronomy, University of Western Ontario, London, Ontario N6A 3K7, Canada;2. Department of Chemical and Biochemical Engineering, University of Western Ontario, London, ON N6A 5B9, Canada;2. Research Associate, Department of Oral Biology, University of Illinois at Chicago, Chicago, IL;3. Assistant Professor, Department of Oral and Maxillofacial Surgery, University of Illinois at Chicago, Chicago, IL;4. Visiting Professor, Department of Orthopaedic Surgery, Rush University, Chicago, IL;5. Professor and Head, Department of Oral and Maxillofacial Surgery, University of Illinois at Chicago, Chicago, IL;1. 3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal;2. ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal;1. Rheumatology, Biomarkers and Research, Nordic Bioscience, Herlev, Denmark;2. Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark;3. Center for Sensory-Motor Interaction (SMI), Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Denmark;4. The D-BOARD European Consortium for Biomarker Discovery, United Kingdom;5. Department of Veterinary Pre-Clinical Sciences, School of Veterinary Medicine, University of Surrey, Guildford GU2 7AL, United Kingdom;6. Faculty of Health and Medical Sciences, Duke of Kent Building, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom;7. Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, Queen''s Medical Centre, Nottingham NG7 2UH, United Kingdom;8. Center of Excellence in Genomic Medicine Research (CEGMR), King Fahd Medical Research Center (KFMRC), Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia;9. Fibrosis Biology, Biomarkers and Research, Nordic Bioscience, Herlev, Denmark |
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| Abstract: | Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1+ ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1+ ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking. |
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