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Procollagen type III N-terminal endopeptidase in fibroblast culture.

ABSTRACT: 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.

SUBMITTER: Nusgens BV

PROVIDER: S-EPMC1162270 | biostudies-other | 1980 Dec

REPOSITORIES: biostudies-other

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Circulating Procollagen Type III N-Terminal Peptide and Physical Function in Adults from the Long Life Family Study.

Project description:BackgroundCirculating levels of procollagen type III N-terminal peptide (P3NP) may reflect increased fibrosis of skeletal muscle and other tissues with aging. Herein, we tested if P3NP was associated with baseline and 7-year change in physical function.MethodParticipants (n = 400) were from the Long Life Family Study, a study of exceptional familial longevity. Plasma P3NP concentration was measured using a sandwich enzyme-linked immunosorbent assay (inter-assay coefficient of variation <5.5%). At baseline and 7-year follow-up visits, physical function was measured using the Short Physical Performance Battery (SPPB score 0-12), which consists of gait speed, balance, and chair-rise tests. Grip strength was measured using a handheld dynamometer. The association between log-transformed P3NP and physical function was examined using generalized estimating equations adjusted for familial relatedness, age, sex, height, weight, lifestyle characteristics, liver function, kidney function, lung function, and chronic disease prevalence.ResultsParticipants were aged 73.1 ± 15.2 years (range: 39-104), 54% female, had body mass index of 26.6 ± 4.3 kg/m2, and gait speeds of 1.0 ± 0.3 m/s. One standard deviation higher log-transformed P3NP was related to worse baseline SPPB score (β = -0.9points), gait speed (β = -0.05m/s), chair-rises per-second (β = -0.46chair-rises/10 seconds), and grip strength (β = -2.0kg; all p < .001). Higher P3NP was also associated with greater declines in gait speed (β = -1.41, p < .001) and transitioning to being unable to perform chair-rises (β = 0.41, p < .001) after 7 years.ConclusionPlasma P3NP may be a strong, novel biomarker of current and future physical function. Future research is needed to extend our findings to other cohorts and determine mechanisms underlying these associations.

| S-EPMC8355442 | biostudies-literature

Procollagen type III amino-terminal propeptide and insulin-like growth factor I as biomarkers of growth hormone administration.

Project description:The acceptance in 2012 by the World Anti-Doping Agency (WADA) of the biomarker test for human growth hormone (hGH) based on procollagen type III amino-terminal propeptide (P-III-NP) and insulin-like growth factor I (IGF-I) was perhaps the first time that such a method has been used for forensic purposes. Developing a biomarker test to anti-doping standards, where the strict liability principle applies, is discussed. An alternative WADA-accepted approach is based on the measurement of different hGH isoforms, a method that suffers from the very short half-life of hGH limiting the detection period. Modification or withdrawal of the immunoassays, on which the biomarker measurements largely depend, has necessitated revalidation of the assays, remeasurement of samples and adjustment of the decision limits above which an athlete will be assumed to have administered hGH. When a liquid chromatography coupled mass spectrometry (LC-MS) method became a reality for the measurement of IGF-I, more consistency of results was assured. Measurement of P-III-NP is still dependent on immunoassays although work is underway to develop an LC-MS method. The promised long-term detection time for the biomarker assay does not appear to have been realised in practice, and this is perhaps partly the result of decision limits being set too high. Nevertheless, more robust assays are needed before a further adjustment of the decision limit is warranted. In the meantime, WADA is considering using P-III-NP and IGF-I as components of a biomarker passport system recording data from an individual athlete, rather than the population. Using this approach, smaller perturbations in the growth hormone (GH) score would mandate an investigation and possible action for hGH administration.

| S-EPMC9545871 | biostudies-literature

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

Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

Project description:The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.

| S-EPMC1153521 | biostudies-other

Giantin is required for intracellular N-terminal processing of type I procollagen.

Project description:Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

| S-EPMC8103548 | biostudies-literature

Procollagen III N-terminal propeptide and desmosine are released by matrix destruction in pulmonary tuberculosis.

Project description:BackgroundTuberculosis is transmitted by patients with pulmonary disease. Matrix metalloproteinases (MMPs) drive lung destruction in tuberculosis but the resulting matrix degradation products (MDPs) have not been studied. We investigate the hypothesis that MMP activity generates matrix turnover products as correlates of lung pathology.MethodsInduced sputum and plasma were collected prospectively from human immunodeficiency virus (HIV) positive and negative patients with pulmonary tuberculosis and controls. Concentrations of MDPs and MMPs were analyzed by ELISA and Luminex array in 2 patient cohorts.ResultsProcollagen III N-terminal propeptide (PIIINP) was 3.8-fold higher in induced sputum of HIV-uninfected tuberculosis patients compared to controls and desmosine, released during elastin degradation, was 2.4-fold higher. PIIINP was elevated in plasma of tuberculosis patients. Plasma PIIINP correlated with induced sputum MMP-1 concentrations and radiological scores, demonstrating that circulating MDPs reflect lung destruction. In a second patient cohort of mixed HIV seroprevalence, plasma PIIINP concentration was increased 3.0-fold above controls (P < .001). Plasma matrix metalloproteinase-8 concentrations were also higher in tuberculosis patients (P = .001). Receiver operating characteristic analysis utilizing these 2 variables demonstrated an area under the curve of 0.832 (P < .001).ConclusionsIn pulmonary tuberculosis, MMP-driven immunopathology generates matrix degradation products.

| S-EPMC3805234 | biostudies-literature

Muscle quality, physical performance, and comorbidity are predicted by circulating procollagen type III N-terminal peptide (P3NP): the InCHIANTI follow-up study.

Project description:Sarcopenia is characterized by skeletal muscle quantitative and qualitative alterations. A marker of collagen turnover, procollagen type III N-terminal peptide (P3NP), seems to be related to those conditions. This study aims to assess the predictive role of P3NP in muscle density and physical performance changes. In the InCHIANTI study, a representative sample from the registry lists of two towns in Tuscany, Italy, was recruited. Baseline data was collected in 1998, and follow-up visits were conducted every 3 years. Out of the 1453 participants enrolled at baseline, this study includes 1052 participants. According to P3NP median levels, population was clustered in two groups; 544 (51.7%) of the 1052 subjects included were classified in the low median levels (LM-P3NP); at the baseline, they were younger, had higher muscle density, and performed better at the Short Physical Performance Battery (SPPB), compared to the high-median group (HM-P3NP).LM-P3NP cases showed a lower risk to develop liver chronic diseases, CHF, myocardial infarction, and osteoarthritis. HM-P3NP levels were associated with a longitudinal reduction of muscle density, and this effect was potentiated by the interaction between P3NP and leptin. Moreover, variation in physical performance was inversely associated with high level of P3NP, and directly associated with high fat mass, and with the interaction between P3NP and muscle density. Our data indicate that P3NP is associated with the aging process, affecting body composition, physical performance, and clinical manifestations of chronic degenerative age-related diseases.

| S-EPMC10828316 | biostudies-literature

Role of ?-isomerized C-terminal telopeptides (?-CTx) and total procollagen type 1 amino-terminal propeptide (tP1NP) as osteosarcoma biomarkers.

Project description:IntroductionFew serum markers are valid and useful for the diagnosis or therapeutic effect monitoring of osteosarcoma. This study aimed to investigate the role of ?-isomerized C-terminal telopeptides (?-CTx) and total procollagen type 1 amino-terminal propeptide (tP1NP) as serological biomarkers for osteosarcoma patients.Materials and methodsA total of 48 patients with osteosarcoma and 55 healthy volunteers were investigated. Serum ?-CTx and tP1NP levels were measured by electrochemiluminescence immunoassay. Data were analyzed by t test with Walth's correction and receiver operating characteristic (ROC) curve analysis.ResultsThe baseline levels of ?-CTx and tP1NP were found to be significantly higher in patients with osteosarcoma than the healthy volunteers. The mean areas under the ROC curves were 0.919 (range, 0.864-0.973) for ?-CTx and 0.866 (range, 0.792-0.939) for tP1NP. The levels of ?-CTx and tP1NP were lower in patients with stable disease after operation than those before operation.ConclusionThese findings support our hypothesis that ?-CTx and tP1NP are promising serum biomarkers for diagnosing or monitoring osteosarcoma.

| S-EPMC4358526 | biostudies-literature

Serum Procollagen Type I N-Terminal Propeptide and Osteocalcin Levels in Korean Children and Adolescents.

Project description:PURPOSE:Bone markers can be useful for the diagnosis and treatment of skeletal diseases in children and adolescents. Owing to high skeletal growth velocity and rapid bone turnover, children and adolescents have higher bone marker levels than adults. Thus, a valid age- and sex-specific reference should be established for pediatric populations living in similar environments. We aimed to assess the associations of procollagen type I N-terminal propeptide (P1NP) and osteocalcin with age and sex in a group of healthy Korean children and adolescents. MATERIALS AND METHODS:The participants (290 boys and 290 girls, age range 0-18 years) were Korean outpatients. Serum P1NP and osteocalcin levels were measured in control materials and patient samples by electrochemiluminescence immunoassay using an automated Cobas e411 analyzer. RESULTS:Significant age-dependent variations in bone marker levels were observed in both sexes (p<0.001). The highest P1NP levels were observed during the first year of life; thereafter, levels decreased until puberty. There was no postnatal peak for osteocalcin; however, its levels remained higher than the adult reference range throughout childhood. Significant differences were observed between boys and girls (p<0.05), especially between the ages of 12 and 17 years. Cobas e411 results for P1NP showed satisfactory precision and linearity. CONCLUSION:We established reference data for P1NP and osteocalcin levels in healthy Korean children and adolescents, as the first and only study of these parameters in pre-adulthood in Korea. Cobas e411-quantified bone markers may be useful for determining bone metabolism indices.

| S-EPMC6881701 | biostudies-literature

COL1A1 C-propeptide mutations cause ER mislocalization of procollagen and impair C-terminal procollagen processing.

Project description:Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ± PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.

| S-EPMC7521940 | biostudies-literature

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