Type IIA Procollagen Amino Propeptide Is Localized in Human Embryonic Tissues

Abstract

SUMMARY Type II procollagen is synthesized in two forms generated by the alternative splicing of its precursor mRNA. The alternatively spliced domain, exon 2, encodes the 69-amino-acid cysteine-rich region of the NH 2 propeptide. Studies of mRNA expression have shown that the longer form, designated Type IIA procollagen, is synthesized by chondroprogenitor cells and various noncartilaginous tissues. The shorter form, Type IIB procollagen, is synthesized by differentiated chondrocytes. As the initial step in our investigations of the function of the Type IIA procollagen, the protein domain corresponding to exon 2 was created as a recombinant fusion protein and used to raise antibodies in rabbits. The resulting antiserum was specific for Type IIA procollagen NH 2 propeptide as shown by ELISA, Western blotting, and immunofluorescent co-localization with the triple-helical domain of Type II collagen. Type IIA procollagen was identified in tissue culture medium of 54-day human fetal ribs. Confocal microscopy was used to localize the Type IIA NH 2 propeptide in Day 50 and 53 human embryos. In the digital rays of the developing hand, where only Type IIA procollagen mRNA was detected, Type IIA procollagen NH 2 propeptide was observed in the extracellular matrix. The presence of Type IIA procollagen NH 2 propeptide was observed in the cartilage of the developing long bones of the lower arm and vertebral bodies even though these tissues synthesize Type IIB mRNA at this developmental stage. Type IIA procollagen NH 2 propeptide was localized in the developing trachea, a cartilage that does not undergo endochondral bone formation. Type IIA NH 2 propeptide was also localized in noncartilaginous tissues known to synthesize Type IIA mRNA, such as the intervertebral area, perichondrium, notochordal sheath, and neuroepithelium of the otic vesicle. In most tissues, co-localization with antiserum against the triple-helical domain of Type II collagen was observed. Positive immunoreactivity with the Type IIA NH 2 propeptide antiserum indicates, for the first time, that this propeptide is present in the tissue. Co-localization of NH 2 propeptide antibodies with the triple-helical domain of the collagen molecule suggests that Type IIA procollagen is intact in the extracellular matrix of these tissues. Taken together, these results strongly suggest that around cells that synthesize Type IIA procollagen mRNA, Type IIA procollagen NH 2 propeptide is secreted and deposited into the extracellular matrix. In light of these results, we predict that Type IIA procollagen plays a role in differentiation of tissues that augments its purely architectural function.

Can serum biomarker assays measure the progression of cartilage degeneration in osteoarthritis?

“…They also show that the amino propeptide of an apparently minor splice variant of type II procollagen, type IIA collagen, is detectable in serum even in healthy adults. This propeptide's appearance (both as protein and as messenger RNA) in embryonic development (where it has been localized in the intervertebral disc) (24), its apparent disappearance in maturity, and its reappearances in articular cartilage degeneration in OA (13) have been described previously. Lui and colleagues reported a lack of its expression in hypertrophic chondrocytes (25).…”

Uncoupling of type II collagen synthesis and degradation predicts progression of joint damage in patients with knee osteoarthritis

“…This ELISA is based on a rabbit polyclonal antiserum raised against the recombinant human glutathione S transferase (GST)-exon 2 fusion protein (20) and uses recombinant human GST-exon 2 as a standard. The specificity of the antibody against PIIANP was previously demonstrated by Western blot analyses against the recombinant exon 2 protein (prior to and after cleavage with thrombin) and against type IIA procollagen isolated from the culture medium of human fetal ribs (20).…”

“…On the one hand, type IIB procollagen is expressed at high levels in well-differentiated chondrocytes, forming the framework of normal adult cartilage. On the other hand, type IIA procollagen is temporally expressed in prechondrogenic condensing limb mesenchyme, sclerotome, and early cartilage (17)(18)(19)(20) and can be reexpressed later in development at the onset of cartilage hypertrophy (21). In addition, it has recently been shown that type IIA procollagen is re-expressed by adult articular chondrocytes of affected human OA cartilage (21,22).…”

Trimerization of the Amino Propeptide of Type IIA Procollagen Using a 14-Amino Acid Sequence Derived from the Coiled-Coil Neck Domain of Surfactant Protein D

“…Detection and Purification of IIA/SP-D Fusion Protein-Media from transiently transfected CHO cells were screened for the presence of the fusion protein by an enzyme-linked immunoassay using rabbit antihuman exon 2 (IIA) antibody (26), chicken IgY anti-human Exon3-8 antibody (27), or rabbit anti-rat SP-D antibody (27). Immunopositive proteins labeled with rabbit-horseradish peroxidase secondary antibodies were detected by enhanced chemiluminescence using SuperSignal chemiluminescent substrate (Pierce).…”