A unique tool to selectively detect the chondrogenic IIB form of human type II procollagen protein.
Type II collagen, the major fibrillar collagen of cartilage, is synthesized as precursor forms (procollagens) containing N- and C-terminal propeptides. Three splice variants are thought to be translated to produce procollagen II isoforms (IIA/D and IIB) which differ in their amino propeptide parts. The IIA and IID are transient embryonic isoforms that include an additional cysteine-rich domain encoded by exon 2. The IIA and IID transcripts are co-expressed during chondrogenesis then decline and the IIB isoform is the only one expressed and synthesized in fully differentiated chondrocytes. Additionally, procollagens IIA/D can be re-expressed by dedifferentiating chondrocytes and in osteoarthritic cartilage. Therefore, it is an important point to determine which isoform(s) is (are) synthesized in vivo in normal and pathological situations and in vitro, to fully assess the phenotype of cells producing type II collagen protein. Antibodies directed against the cysteine-rich extra domain found in procollagens IIA and IID are already available but antibodies detecting only the chondrogenic IIB form of type II procollagen were missing so far. A synthetic peptide encompassing the junction between exon 1 and exon 3 of the human sequence was used as immunogen to produce rabbit polyclonal antibodies to procollagen IIB. After affinity purification on immobilized peptide their absence of crossreaction with procollagens IIA/D and with the fibrillar procollagens I, III and V was demonstrated by Western blotting. These antibodies were used to reveal at the protein level that the treatment of dedifferentiated human chondrocytes by bone morphogenic protein (BMP)-2 induces the synthesis of the IIB (chondrocytic) isoform of procollagen II. In addition, immunohistochemical staining of bovine cartilage demonstrates the potential of these antibodies in the analysis of the differential spatiotemporal distribution of N-propeptides of procollagens IIA/D and IIB during normal development and in pathological situations.
Université Lyon 1, Univ Lyon, CNRS, UMR 5305-LBTI: Laboratoire de Biologie Tissulaire et Ingénierie thérapeuthique, IBCP, 7 passage du Vercors, F69367 Lyon, France.
Elisabeth
E
Aubert-Foucher
Université Lyon 1, Univ Lyon, CNRS, UMR 5305-LBTI: Laboratoire de Biologie Tissulaire et Ingénierie thérapeuthique, IBCP, 7 passage du Vercors, F69367 Lyon, France.
Nathalie
N
Mayer
Université Lyon 1, Univ Lyon, CNRS, UMR 5305-LBTI: Laboratoire de Biologie Tissulaire et Ingénierie thérapeuthique, IBCP, 7 passage du Vercors, F69367 Lyon, France.
Marielle
M
Pasdeloup
Novotec, 13-15 rue Jacques Monod, F69007 Lyon, France.
Aurélie
A
Pagnon
UCBL 1/ISPB, Faculté de Pharmacie, UMR CNRS 5510/MATEIS, Equipe I2B-"Interactions Biologiques et Biomatériaux", 8 avenue Rockefeller, F69373 Lyon, Cedex 08, France.
Daniel
D
Hartmann
Université Lyon 1, Univ Lyon, CNRS, UMR 5305-LBTI: Laboratoire de Biologie Tissulaire et Ingénierie thérapeuthique, IBCP, 7 passage du Vercors, F69367 Lyon, France. Electronic address: f.mallein-gerin@ibcp.fr.
Frédéric
F
Mallein-Gerin
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002356:Cartilage / Q000254:growth & development / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation / Q000235:genetics*; D019902:Chondrocytes / Q000378:metabolism; D020219:Chondrogenesis / Q000235:genetics*; D024043:Collagen Type II / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005091:Exons; D006801:Humans; D020033:Protein Isoforms / Q000235:genetics*; D012333:RNA, Messenger; D011817:Rabbits
D000906:Antibodies; C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D024043:Collagen Type II; D020033:Protein Isoforms; D012333:RNA, Messenger
Alternative splicing; Cartilage; Chondrocytes; New antibodies; Type II procollagen