Characterization of a murine type IIB procollagen-specific antibody

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.

Keywords: Antibody; Cartilage; Chondrocyte; Type II collagen; Type IIA procollagen; Type IIB procollagen.

Figure 1

Type II procollagen protein isoforms and the antibodies that detect them. A schematic showing the primary structures of type IIA procollagen (A) and its splice variant, type IIB procollagen (B) is shown. The N-propeptide is coded by exons 1-8 which are sequentially spliced to each other to form the N-propeptide of the type IIA procollagen. (B) In type IIB procollagen, the exon 2-derived 69 amino acid sequence is removed due to the splicing of exon 1 to exon 3. (C) The amino acid sequence of the exon 1-3 protein junction in the type IIB procollagen is shown. Sequences specific to exon 1 and exon 3 are shown underlined. The arginine (R) forms at the new junction with sequences derived from both exons due to the nature of the splicing. This peptide sequence, with a cysteine added to the amino terminus for KLH conjugation, was used to raise antibodies in rabbits against the type IIB-specific splice variant. Below each protein isoform are given the names of the antibodies that detect the different structural domains of the type II procollagens. The IIA antibody generated against exon 2 is specific to the type IIA procollagen isoform (Oganesian et al., 1997), while the IIF (the fibrillar antibody against the type II collagen triple helical domain (THD)) and the anti-IIE3-8 (against protein sequences coded by the exons 3-8) (Fukui et al., 2002) antibodies detect both the type IIA and IIB procollagen isoforms. The unique junction formed due to splicing of the exon 1 to exon 3 provides the only unique structural determinant, which is a potential epitope for specific type IIB procollagen detection.

Figure 2

Analysis of the IIBN antibody in western blots. The chondroepiphyseal cartilage was harvested from the limbs of E17.5 wild type (+/+), the Col2a1^+ex2 homozygous knock-in mouse (ki/ki), and the Col2a1^+ex2 heterozygous (ki/+) mouse. Proteins were extracted by heating the ground cartilage with SDS loading buffer containing DTT, and a 8% SDS-PAGE followed by western blot analysis using the antibodies indicated was performed. The asterisks in panel C point to non-specific bands, which allowed for loading controls between the lanes. (THD: triple helical domain). See Figure 1 for a description of the antibodies.

Figure 3

The IIBN antibody is specific to type IIB procollagen. The specificity of IIBN antibody was validated by western blot analysis of cartilage proteins extracted from P14 mice. The chondroepiphyseal cartilage was harvested from the limbs of P14 wild type (+/+) and Col2a1^+ex2 knock-in mouse (ki/ki), proteins extracted by heating the ground cartilage with SDS loading buffer containing DTT, and a 8% SDS-PAGE followed by western blot analysis was performed using the antibodies indicated. The asterisks in panel C point to non-specific bands.

Figure 4

Analyses of collagen distributions using IIF, IIA, and IIBN antibodies by immunofluorescence. (A, B) Double-labeled immunofluorescence analyses with IIF and IIA antibodies in E17.5 wild type (+/+) femur shown either as a composite of IIF plus IIA signals (A) or with signals from IIA only (B). Images are shown for the proliferating zone in the distal growth plate. (C, D) Double-labeled immunofluorescence analyses in the growth plates of the distal femur with IIF and IIBN antibodies in E17.5 wild type (C) and ki/ki (D) mice. Type II collagen distribution from the resting zone to the hypertrophic zone is shown. Higher magnification images with composite signals from IIF plus IIBN, or signals from IIBN only, are shown for the resting zone (E, F) and the proliferative zone (G, H) in the wild type femur. Higher magnification images of the hypertrophic zone in wild type (I, J) and ki/ki mice (K, L) with composite signals from IIF plus IIBN (I, K), or signals from IIBN only (J, L), are shown. Arrows in panels I and J indicate regions where signals from IIF and IIBN colocalize. Insets in panels I and J show a higher magnification of this co-localization indicated by the arrow marked with an asterisk. Notice the yellowish colocalization signals in the inset in panel I surrounded by the red signals from IIF; the corresponding inset in panel J shows the same image but with signals from IIBN (in green) only. Colors represent antibody localizations as follows: green = IIA antibody detecting type II collagen exon 2 coded sequences (A, B), or IIBN antibody detecting the exon 1-3 protein junction of type IIB procollagen (C-L); red = IIF antibody detecting the Col II triple helical domain; yellow = colocalization of IIF and IIA or IIF and IIBN; blue = DAPI-stained nuclei. Bar (A, B, E-L): 10 μm; (C, D): 50 μm; (insets): 5 μm.

Figure 5

Immunohistochemistry with IIBN antibody on E15.5 wild type femur to analyze specificity to cartilage regions. Panel B shows a higher magnification of the hypertrophic zone outlined in A, demonstrating the positive reactivity of IIBN to type IIB procollagen (some designated by arrows) in the hypertrophic zone. Positive staining for IIBN is seen as brown against a counterstain of tartrazine (yellow). Bar (A): 250 μm; (B): 50 μm.