Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-beta 1 and BMP-2

Type II procollagen is expressed as two splice forms. One form, type IIB, i s synthesized by chondrocytes and is the major extracellular matrix compone nt of cartilage. The other form, type IIA, contains an additional 69 amino acid cysteine-rich domain in the NH,propeptide and is synthesized by chondr ogenic mesenchyme and perichondrium. We have hypothesized that the addition al protein domain of type IIA procollagen plays a role in chondrogenesis. T he present study was designed to determine the localization of the type IIA NH2-propeptide and its function during chondrogenesis. Immunofluorescence histochemistry using antibodies to three domains of the type IIA procollage n molecule was used to localize the NH2-propeptide, fibrillar domain, and C OOH-propeptides of the type IIA procollagen molecule during chondrogenesis in a developing human long bone (stage XXI). Before chondrogenesis, type II A procollagen was synthesized by chondroprogenitor cells and deposited in t he extracellular matrix. Immunoelectron microscopy revealed type IIA procol lagen fibrils labeled with antibodies to NH2-propeptide at similar to 70 nm interval suggesting that the NH2-propeptide remains attached to the collag en molecule in the extracellular matrix, As differentiation proceeds, the c ells switch synthesis from type IIA to IIB procollagen, and the newly synth esized type IIB collagen displaces the type IIA procollagen into the inter- territorial matrix. To initiate studies on the function of type IIA procoll agen, binding was tested between recombinant NH2-propeptide and various gro wth factors known to be involved in chondrogenesis, A solid phase binding a ssay showed no reaction with bFGF or IGF-1, however, binding was observed w ith TGF-beta 1 and BMP-2, both known to induce endochondral bone formation. BMP-2, but not IGF-1, coimmunoprecipitated with type IIA NH2-propeptide. R ecombinant type IIA NH2-propeptide and type IIA procollagen from media coim munoprecipitated with BMP-2 while recombinant type IIB NH2-propeptide and a ll other forms of type II procollagens and mature collagen did not react wi th BMP-2, Taken together, these results suggest that the NH2-propeptide of type IIA procollagen could function in the extracellular matrix distributio n of bone morphogenetic proteins in chondrogenic tissue.