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Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.

ABSTRACT: Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.

SUBMITTER: Halila R

PROVIDER: S-EPMC1147237 | biostudies-other | 1986 Oct

REPOSITORIES: biostudies-other

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Purification and characterization of the N-terminal propeptide of human type III procollagen.

Project description:The N-terminal propeptide of type III procollagen was purified from human ascitic fluid by using (NH4)2SO4 precipitation, DEAE-Sephacel chromatography at pH 8.6, Sephacryl S-300 chromatography and another DEAE-Sephacel chromatography at pH 4.5. The Mr of the human peptide was about 42 000, which corresponds in size to the propeptide released by the specific N-proteinase during the extracellular processing of collagen. Bacterial-collagenase digestion of the human peptide produced three fragments, which could be separated on a Bio-Gel P-10 column. The human propeptide and its collagenase-derived fragments, an N-terminal non-collagenous domain Col 1, a C-terminal non-helical domain Col 2 and a collagenous domain Col 3, resembled those derived from the N-terminal segment of bovine type III procollagen in their amino acid composition. The human peptide was found to contain sulphate, which may explain its extremely low isoelectric point (3.1). Antibodies against the human N-terminal propeptide reacted similarly with both the purified human peptide and a corresponding segment of bovine type III procollagen. The human propeptide could be used in developing radioimmunoassays for monitoring fibrotic processes.

| S-EPMC1152851 | biostudies-other

Structural Basis for the Acceleration of Procollagen Processing by Procollagen C-Proteinase Enhancer-1.

Project description:Procollagen C-proteinase enhancer-1 (PCPE-1) is a secreted protein that specifically accelerates proteolytic release of the C-propeptides from fibrillar procollagens, a crucial step in fibril assembly. As such, it is a potential therapeutic target to improve tissue repair and prevent fibrosis, a major cause of mortality worldwide. Here we present the crystal structure of the active CUB1CUB2 fragment of PCPE-1 bound to the C-propeptide trimer of procollagen III (CPIII). This shows that the two CUB domains bind to two different chains of CPIII and that the N-terminal region of one CPIII chain, close to the proteolytic cleavage site, lies in the cleft between CUB1 and CUB2. This suggests that enhancing activity involves unraveling of this chain from the rest of the trimer, thus facilitating the action of the proteinase involved. Support for this hypothesis comes from site-directed mutagenesis, enzyme assays, binding studies, and molecular modeling.

| S-EPMC6372009 | biostudies-literature

Procollagen C-proteinase enhancer stimulates procollagen processing by binding to the C-propeptide region only.

Project description:Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner. Here, we performed a detailed quantitative study of the binding of PCPE-1 and of its minimal active fragment (CUB1-CUB2) to three regions of the procollagen III molecule: the triple helix, the C-telopeptide, and the C-propeptide. Contrary to results described elsewhere, we found the PCPE-1-binding sites to be located exclusively in the C-propeptide region. In addition, binding and enhancing activities were found to be independent of the glycosylation state of the C-propeptide. These data exclude previously proposed mechanisms for the action of PCPEs and also suggest new mechanisms to explain how these proteins can stimulate BMP-1/tolloid-like proteinases by up to 20-fold.

| S-EPMC3234718 | biostudies-literature

Data comparing the kinetics of procollagen type I processing by bone morphogenetic protein 1 (BMP-1) with and without procollagen C-proteinase enhancer 1 (PCPE-1).

Project description:This article provides kinetic constants for C-terminal processing of procollagen type I by bone morphogenetic protein 1 (BMP-1; the major procollagen C-proteinase), a reaction stimulated by the connective tissue glycoprotein procollagen C-proteinase enhancer 1 (PCPE-1). Reported are Km , Vmax , Kcat and Kcat /Km (catalytic coefficient) values for BMP-1 alone, BMP-1 with intact PCPE-1, BMP-1 with the CUB (Complement C1r/C1s, Uegf, BMP-1) domains fragment of PCPE-1 as well as its NTR (netrin-like) domain.

| S-EPMC5109235 | biostudies-literature

Preparation and characterization of SARS in-house reference antiserum.

Project description:A reference antiserum for SARS is in urgent need in the development of SARS vaccine and other serological test of SARS research. Convalescent serum was collected from clinical confirmed patient. ELISA, Western-blotting and neutralization assay detected specific antibody against SARS coronavirus. This antiserum was prepared as in-house reference antiserum, freeze-dried and sealed in ampoules. The potency of this reference antiserum is defined to be 52.7 U after extensive calibration. Further, collaborative studies for the evaluation of this serum are needed in order to satisfy the requirements for international reference antiserum.

| S-EPMC7115399 | biostudies-literature

Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing.

Project description:The extracellular matrix (ECM) proteins fibronectin (FN) and type I collagen (collagen I) are codistributed in many tissues, and collagens have been shown to depend on an FN matrix for fibrillogenesis. Microscopic analysis of a fibroblast ECM showed colocalization of procollagen I with FN fibrils, and proteolytic cleavage of procollagen to initiate fibril formation was significantly reduced with inhibition of FN matrix assembly. We examined the role of FN matrix in procollagen processing by the C-propeptide proteinase bone morphogenetic protein 1 (BMP-1). We found that BMP-1 binds to a cell-assembled ECM in a dose-dependent manner and that, like procollagen, BMP-1 colocalizes with FN fibrils in the matrix microenvironment. Binding studies with FN fragments identified a binding site in FN's primary heparin-binding domain. In solution, BMP-1-FN interactions and BMP-1 cleavage of procollagen I were both enhanced by the presence of heparin, suggesting a role for heparin in complex formation during proteolysis. Indeed, addition of heparin enhanced the rate of procollagen cleavage by matrix-bound BMP-1. Our results show that matrix localization of this proteinase facilitates the initiation of collagen assembly and suggest a model in which FN matrix and associated heparan sulfate act as a scaffold to organize enzyme and substrate for procollagen processing.

| S-EPMC6743462 | biostudies-literature

Procollagen type III N-terminal endopeptidase in fibroblast culture.

Project description:A peptidase activity capable of excising in a single fragment the N-terminal extension of the precursor of collagen type III (p-N-collagen type III) was observed in calf tendon fibroblast culture medium. A new procedure was developed for detecting this peptidase (p-N-collagen type III peptidase). It is based on the use of 14C-labelled p-N-collagen type III obtained by carboxymethylation of the half-cystine residues with iodo-[14C]acetamide. The released labelled N-terminal extension is soluble in 27% (v/v) ethanol, whereas the uncleaved substrate and the collagen are precipitated under these conditions. The endopeptidase nature of p-N-collagen type III peptidase is supported by the similarity in molecular weight of the product of cleavage of p-N-collagen III by the enzyme to those obtained by cleavage with bacterial collagenase. An apparent Km of 0.3 X 10(-6)M was established. The pH optimum of p-N-collagen type III peptidase is similar to that of p-N-collagen type I peptidase, i.e. about 7.5. Both peptidases are inhibited by dithiothreitol and by Cu2+ and Zn2+, but not by other bivalent ions. p-N-collagen type III peptidase does not cleave p-N-collagen I or p-N-gelatin I. Partial purification of p-N-collagen type III peptidase from fibroblast culture medium was performed by sieve chromatography on Ultrogel AcA-34 to yield two peaks of activity, of mol.wts. 170000 and 100000. Part of the activity was retained on affinity chromatography on concanavalin A--Sepharose. Studied as a function of the age of the culture, p-N-collagen type III peptidase activity produced by tendon fibroblasts parallels that of p-N-collagen type I peptidase and collagen synthesis.

| S-EPMC1162270 | biostudies-other

Structure of cDNA clones coding for the entire prepro alpha 1 (III) chain of human type III procollagen. Differences in protein structure from type I procollagen and conservation of codon preferences.

Project description:Two overlapping cDNA clones that cover the complete length of the mRNA for human type III procollagen were characterized. The data provided about 2500 base pairs of sequence not previously defined for human type III procollagen. Two tripeptide sequences of -Gly-Xaa-Yaa- were identified that were not detected previously by amino acid sequencing of human type III collagen. The two additional tripeptide units, together with three previously detected, establish that the alpha 1 (III) chain is 15 amino acids longer than either the alpha 1 (I) or alpha 2 (I) chains of type I collagen. The additional tripeptide units made hydropathy plots of the N-terminal and C-terminal regions of type III collagen distinctly different from those of type I collagen. The data also demonstrated that human type III procollagen has the same third base preference in codons for glycine, proline and alanine that was previously found with human and chick type I procollagen. In addition, comparison of two cDNA clones from the same individual revealed a variation in structure in that the codon for amino acid 880 of the alpha 1 (III) chain was -CTT- for leucine in one clone and -TTT- for phenylalanine in the other.

| S-EPMC1138697 | biostudies-other

Preparation and application of rat myostatin antiserum.

Project description:To prepare and identify a polyclonal antibody against rat myostatin and investigate myostatin expression in the rat atrophic gastrocnemius muscle after tibial nerve crush.The purified fusion protein was used as antigen to immunize rabbits for the preparation of polyclonal antibody. The polyclonal antibody of the protein was measured by enzyme linked immunosorbent assay (ELISA), western-blot and immunochemistry. Myostatin protein expression levels in normal and atrophic gastrocnemius muscle were detected by western-blot and immunochemistry assays.The GST-myostatin had a purity of 96% and possessed high titer and specificity. The level of myostatin in gastrocnemius muscle significantly increased one week after tibial nerve crush, reached the peak on day 14, and then returned to normal level on day 28.We have successfully made antiserum of rat myostatin and found that the expression level of myostatin protein in the gastrocnemius after tibial nerve crush-induced atrophy was time-dependent. This study provides an experimental basis to clarify the possible role of myostatin during skeletal muscle atrophy.

| S-EPMC5552581 | biostudies-other

Effect of dextran on synthesis, secretion and deposition of type III procollagen in cultured human fibroblasts.

Project description:When subconfluent cultures of primary human skin fibroblasts are incubated for 20 h in the presence of 5% neutral dextran, the newly synthesized procollagens are shifted from the medium into the pericellular matrix fraction. This is accompanied by an overall decrease by 30-50% in the secretion rates of these proteins, as indicated by the incorporation of tritiated proline into collagenase-sensitive macromolecules and by radioimmunoassays for the propeptide regions of type I and III procollagens. Inhibition of tyrosine sulphation in newly formed type III procollagen by NaClO3 does not change the distribution of this protein between the medium and matrix fractions either in the presence or in the absence of the polymer. Processing of type III procollagen at its N-terminus is incomplete in this situation, irrespective of whether the protein is present mainly in the medium or in the pericellular matrix. The concentrations of the mRNAs for the pro alpha 1(I)- and pro alpha 1(III)-chains of procollagens and for actin were monitored for up to 20 h; in the dextran-treated cultures these are not different from the corresponding concentrations in control cultures, indicating that the rapid down-regulation of the synthesis of type I and III procollagens in response to the enhanced pericellular matrix deposition induced by dextran is not due to transcriptional mechanisms.

| S-EPMC1151545 | biostudies-other

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