title: Type IIA procollagen amino propeptide is localized in human embryonic tissues.
abstract: 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 NH2 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 NH2 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 NH2 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 NH2 propeptide was observed in the extracellular matrix. The presence of Type IIA procollagen NH2 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 NH2 propeptide was localized in the developing trachea, a cartilage that does not undergo endochondral bone formation. Type IIA NH2 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 NH2 propeptide antiserum indicates, for the first time, that this propeptide is present in the tissue. Co-localization of NH2 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 NH2 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.
authors.affiliation: Department of Orthopaedics, University of Washington, Seattle, USA.
authors.forename: A
authors.initials: A
authors.lastname: Oganesian
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: L J
authors.initials: LJ
authors.lastname: Sandell
mesh_terms: D015153:Blotting, Western; D002356:Cartilage / Q000737:chemistry* / Q000196:embryology*; D003094:Collagen / Q000235:genetics; D004622:Embryo, Mammalian / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D018613:Microscopy, Confocal; D010446:Peptide Fragments / Q000032:analysis*; D011347:Procollagen / Q000032:analysis* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D014018:Tissue Distribution
chemical_list: D010446:Peptide Fragments; D011347:Procollagen; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C109195:procollagen type IIA amino-terminal peptide; D003094:Collagen