The low abundance fibrillar collagen type V is incorporated into and regulates the diameters of type I collagen fibrils. Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays key roles in regulating formation of vertebrate extracellular matrix; it cleaves the C-propeptides of the major fibrillar procollagens I-III and processes precursors to produce the mature forms of the cross-linking enzyme prolysyl oxidase, the proteoglycan biglycan, and the basement membrane protein laminin 5. Here we have successfully produced recombinant pro-␣1(V) 2 pro-␣2(V) heterotrimers, and we have used these to characterize biosynthetic processing of the most prevalent in vivo form of type V procollagen. In addition, we have compared the processing of endogenous pro-␣1(V) chains by wild type mouse embryo fibroblasts and by fibroblasts derived from embryos doubly homozygous null for the Bmp-1 gene and for a gene encoding the closely related metalloprotease mammalian Tolloid-like 1. Together, results presented herein indicate that within pro-␣1(V) 2 pro-␣2(V) heterotrimers, pro-␣1(V) N-propeptides and pro-␣2(V) C-propeptides are processed by BMP-1-like enzymes, and pro-␣1(V) Cpropeptides are processed by furin-like proprotein convertases in vivo.The major fibrillar collagen types I-III are synthesized as procollagens with N-propeptides 1 and C-propeptides that are cleaved to produce mature triple helical monomers capable of forming fibrils (1-3). In particular, failure to remove C-propeptides seems incompatible with fibrillogenesis (4). The major procollagen C-propeptides are cleaved by the metalloprotease bone morphogenetic protein 1 (BMP-1) and by the closely related metalloproteases mammalian Tolloid-like (mTLD) and mammalian Tolloid-like 1 (mTLL-1) (5-7). 2 These enzymes also process the prodomains of various other precursor proteins involved in formation of the extracellular matrix (8 -10) and cleave Chordin (7, 11), an extracellular antagonist of signaling by certain transforming growth factor--like BMPs such as BMP-4 (12). Thus, BMP-1 and related proteases may orchestrate deposition of matrix components with BMP signaling in morphogenetic events.The minor fibrillar collagen type V is broadly distributed in tissues as an ␣1(V) 2 ␣2(V) heterotrimer (13, 14) that is incorporated into type I collagen fibrils and acts to regulate the diameters of the resulting heterotypic fibrils (15)(16)(17)(18)(19). A lower abundance ␣1(V)␣2(V)␣3(V) form of type V collagen has been isolated from placenta (20, 21), whereas a rare ␣1(V) 3 homotrimer has been reported in a limited number of cell types and tissues (22-24). Type XI collagen, a minor fibrillar collagen of cartilage with the chain composition ␣1(XI)␣2(XI)␣3(XI) (25), appears to interact with the major cartilage collagen type II in a manner analogous to the interaction of types V and I in other tissues (26,27). Findings of type XI chains in noncartilaginous tissues (28), type V chains in cartilage (28), and heterotrimers comprising both type V and XI chains (30, 31) now sug...
The transforming growth factors-beta1 and beta2 (TGF-beta) stimulate synthesis of extracellular matrix proteins in vitro and appear upregulated in fibrotic conditions, in scar formation, and in wound healing. The extracellular matrix in turn might also act as a scavenger or repository for TGF-beta. We therefore studied the in situ distribution of latent TGF binding protein-1 (LTBP-1) and latent TGF-beta1 on extracellular matrix elements of normal human skin and skin regenerating from cultured keratinocyte autografts. We localized both LTBP-1 and latent TGF-beta1 to fibrillin-containing (elastic) microfibrils. Both LTBP-1 and latent TGF-beta1 were already present during the earliest stages of the de novo formation of the microfibrillar apparatus, i.e., on fusiform, randomly oriented microfibrils that later coalesced to form the typical candelabra-like structures in the papillary dermis. We show herewith that LTBP-1 exerts a dual role as a component of fibrillin-microfibrils of the skin and in targeting latent TGF-beta1 to the cutaneous microfibrillar apparatus. Thus, this major connective tissue structure does not only serve as a force bearing element and scaffold for elastin deposition in the dermis, but also as an important repository for latent TGF-beta in the skin.
Latent TGF-beta binding proteins (LTBPs) are components of the extracellular matrix (ECM). They belong to the fibrillin/LTBP-superfamily, and are high molecular weight glycoproteins characterized by EGF-like repeats and 8-Cys repeats. Most LTBPs associate with the small latent forms of TGF-beta. Their roles include to facilitate the secretion of latent TGF-beta and to target it to the ECM. In order to identify new matrix-binding domains of LTBP-1 and to characterize their association with the extracellular matrix, we have produced (in a mammalian expression system) partly overlapping recombinant fragments of its shorter form, LTBP-1S, and analyzed the binding of the purified fusion proteins to extracellular matrices of cultured human dermal and lung fibroblasts. Recombinant fragments from three different regions of the N- and C-termini showed affinity to the matrix. These interacting regions contain either the first (hybrid), second or fourth 8-Cys domains of the LTBP-1S molecule. They bound independently to the matrix. Each of them had an ability to inhibit the association of native exogenous LTBP-1 with fibroblast extracellular matrix. The interactions of the LTBP-1 fragments with the extracellular matrix resisted treatment with sodium deoxycholate, suggesting strong, possibly covalent binding. The binding occurred in a time- and dose-dependent fashion. The N-terminal fragments bound more readily to the matrices. With all fragments the binding took place both with intact fibroblast matrices and with matrices isolated by sodium deoxycholate. When using CHO cell layers, which form sparse matrices, only the N-terminal fragment of LTBP-1 was efficiently incorporated. The association of the binding fragments with isolated matrices was enhanced by soluble, cell-derived factors. The current data suggest that LTBP-1 contains three different domains with an ability to associate with the extracellular matrix.
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