A unique tool to selectively detect the chondrogenic IIB form of human type II procollagen protein

作者：Aubert Foucher Elisabeth; Mayer Nathalie; Pasdeloup Marielle; Pagnon Aurelie; Hartmann Daniel; Mallein Gerin Frederic^*

来源：Matrix Biology, 2014, 34: 80-88.

DOI：10.1016/j.matbio.2013.09.001

• Type II procollagen
• Alternative splicing
• Chondrocytes
• Cartilage
• New antibodies

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 BB) 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 11 collagen protein. Antibodies directed against the cysteine-rich extra domain found in procollagens ILA and IID are already available but antibodies detecting only the chondrogenic BB 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 ILA/D and with the fibrillar procollagens I, Ill 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 LIND and IIB during normal development and in pathological situations.

• 出版日期2014-2