Isolation of unhydroxylated type I procollagen folding of the protein in vitro

We present a concept for reducing formation of fibrotic deposits by inhibiting self-assembly of collagen molecules into fibrils, a main component of fibrotic lesions. Employing monoclonal antibodies that bind to the telopeptide region of a collagen molecule, we found that blocking telopeptide-mediated collagen/collagen interactions reduces the amount of collagen fibrils accumulated in vitro and in keloid-like organotypic constructs. We conclude that inhibiting extracellular steps of the fibrotic process provides a novel approach to limit fibrosis in a number of tissues and organs.Collagen I is the most abundant structural protein of connective tissues such as skin, bone, and tendon. This protein is first synthesized as a precursor molecule, procollagen, that is characterized by the presence of a rod-like central triple-helical domain flanked by short linear telopeptides and globular N-terminal and C-terminal propeptides (1). Single procollagen molecules are the building blocks for the biologically and mechanically relevant collagen fibrils. Formation of collagen fibrils is initiated by enzymatic cleavage of the N-terminal and the C-terminal propeptides. The N-terminal propeptides are cleaved by a group of enzymes that includes a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-2, -3, and -14, whereas the C-terminal propeptides are cleaved by the metalloprotease bone morphogenetic protein 1 (BMP-1) 2 and by the other members of a closely related family of mammalian tolloid-like metalloproteases (2-4). Such a removal of procollagen propeptides exposes telopeptides, which by engaging in site-specific intermolecular interactions drive collagen self-assembly.In native tissues a precise balance between the processes of biosynthesis and degradation maintains the physiological homeostasis of tissue collagens. At the same time, accelerated biosynthesis is required for proper wound healing, whereas excessive accumulation of collagen is the hallmark of a number of localized fibrotic diseases, such as keloids and hypertrophic scars, and systemic fibrosis, such as systemic scleroderma.Localized fibrotic reactions are quite common and frequently develop as a consequence of surgical procedures. For instance, after surgery of the abdomen, the formation of excessive scar tissue around abdominal organs, such as the intestines, can interfere with the functionality of such organs and may cause severe pain and even death. Another situation where excessive scar formation presents a major complication is in the eye after glaucoma surgery performed to create a pressuremaintenance valve. Frequently, however, excessive scar formation closes this pressure-reducing valve, thereby forcing the intraocular pressure to rise (5). Moreover, excessive scarring of the vocal folds may severely alter their ability to vibrate, thereby causing a number of voice disorders (6).At present, several biological processes critical for development of fibrotic lesions are considered potential targets for inhibitors of fibrosis. These inhibit...

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“…Purification of Human Procollagen I-Procollagen I was purified from cell culture media of normal human dermal fibroblasts, as described (12,13).…”

Section: Methodsmentioning

confidence: 99%

Collagen Fibril Formation

Chung

Steplewski

Chung

et al. 2008

Journal of Biological Chemistry

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“…The effluent was adjusted to 0.4 M NaCl and concentrated by pressure ultrafiltration using an Amicon S1Y100 spiral wound membrane cartridge (Amicon, Inc., Beverly, MA). Purification of type I procollagen was performed by DEAE-cellulose chromatography (14,15). Fractions containing type I procollagen were pooled, dialyzed twice against 500 volumes of 0.4 M NaCl, 0.02% NaN 3 , 0.1 M Tris, pH 7.4, storage buffer, and concentrated by pressure ultrafiltration with Amicon Centriprep 100 filters.…”

Section: Methodsmentioning

confidence: 99%

“…Fractions containing type I procollagen were pooled, dialyzed twice against 500 volumes of 0.4 M NaCl, 0.02% NaN 3 , 0.1 M Tris, pH 7.4, storage buffer, and concentrated by pressure ultrafiltration with Amicon Centriprep 100 filters. The final type I procollagen concentrations were determined by quantitative densitometric analysis using a Molecular Dynamics PDSI 486 laser densitometer and hydroxyproline assay, assuming 10.11% hydroxyproline by weight (15). Enriched culture medium harvested from 30 ϫ 150-mm culture plates (ϳ6 million cells/plate) yielded approximately 1 mg of purified type I procollagen.…”

mentioning

confidence: 99%

Cleavage of Type I Procollagen by Human Mast Cell Chymase Initiates Collagen Fibril Formation and Generates a Unique Carboxyl-terminal Propeptide

Kofford

Schwartz

Schechter

et al. 1997

Journal of Biological Chemistry

181

116

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The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 g/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-␣1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibrilforming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.Mast cells are abundant in connective tissues of skin, lung, and intestine (1, 2). Increased numbers (or increased activity) of mast cells are associated with fibrotic disorders such as scleroderma, pulmonary fibrosis, and Crohn's disease (3-5). The precise role for mast cells in connective tissue biology and fibrosis is not known. One potential mechanism for mast cell involvement in these processes is through the production of unique proteolytic enzymes that may act on matrix proteins. Mast cell enzymes include the neutral proteases tryptase, chymase, cathepsin G, and a zinc-dependent carboxypeptidase (6). Although chymase and tryptase cleave fibronectin (7-9) and chymase cleaves vitronectin (10), no direct effect of these enzymes on procollagen or collagen has been demonstrated.Because procollagen (the precursor of collagen) requires proteolytic cleavage in the extracellular space prior to normal collagen fibril formation, we have speculated that newly secreted procollagen may be a substrate for mast cell proteolytic enzymes. The smooth muscle cells of the intestinal muscularis are a major contributor of collagen to the extracellular matrix in normal and fibrotic human intestine (11). Therefore, in vitro studies were performed to test the above hypothesis utilizing procollagen secreted by human intestinal smooth muscle cells. MATERIALS AND METHODSPurification of Procollagen-Human intestinal smooth muscle cells (12) were grown to confluence in Dulbecco's modified Eagle's medium containing 200 units/ml penicillin, 0.2 mg/ml streptomycin, 50 units/ml Nystatin, and 10% fetal bovine serum. Cell monolayers were then rinsed wit...

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“…Mechanically unstable type II collagen is known to cause dyschondroplasia in craniofacial and limb skeletons (Metsäranta et al, 1992). Examining the effect of α, α'-dipyridyl, which reduces the mechanical stability of collagen (Fiedler-Nagy et al, 1981), on embryogenesis, we found that severe bending of cartilaginous skeletons is also induced by this drug. From the similarity of phenotype, we infer that disulfiram induces bent cartilages by affecting the mechanical stability of collagen, as does α, α'-dipyridyl.…”

Section: Effects Of Disulfiram On Cartilagesmentioning

confidence: 85%

“…To address which process in the development of cartilaginous skeletons is affected by disulfiram, the present study first examined the effects of disulfiram on Hoxd-4 and shh expression in flounder embryos. Then, we compared the phenotype of anomalies induced by α, α'-dipyridyl, which is known to reduces the mechanical stability of collagen (Fiedler-Nagy et al, 1981), with those by disulfiram.…”

Section: Introductionmentioning

confidence: 99%