Noncollagenous Peptides Research Articles

Valorization of sturgeon notochord to obtain antioxidant non-collagenous peptides from the purification process of chondroitin sulfate

Notochord is a cartilage-related by-product of sturgeon processing. To increase the utilization of notochord, we have developed an efficient and eco-friendly extraction process that could recycle non-collagenous peptides during chondroitin sulfate (CS) extraction. Alkaline treatment and ultrafiltration were the two critical steps in the overall extraction process. The results of SDS-PAGE and amino acid composition analysis demonstrated that peptides in low molecular fraction were mainly derived from non-collagenous protein. The non-collagenous peptides exhibited high antioxidant activity. The structural analysis (UV, FTIR, 1H NMR and SAX-HPLC) of CS in high molecular fraction proved high purity CS was obtained using new extraction process. The yield of non-collagenous peptides and CS were 16.8% and 14.5%, respectively. Including undenatured type II collagen, the total utilization rate of notochord was increased to 75.6%. It truly realized the maximum utilization of sturgeon notochord. This extraction process can be used as a reference for other cartilage-related by-products.

Antioxidant and fibroblast-activating activities of the by-product of skate chondroitin extractive production

Owing to the increasing popularity of chondroitin sulfate (CS) for joint pain treatment, the CS-production industry has been producing an increasing amount of waste, which includes type II collagen, non-collagenous proteins, and residual CS. To effectively utilize these resources, we intended to develop new products from the by-product of skate chondroitin sulfate production (BP-sCS). In this study, we examined the antioxidant and fibroblast-activating properties of BP-sCS, intending to apply it for a wound-healing promoter. BP-sCS exhibited ABTS and DPPH radical scavenging activities, protected L929 fibroblasts from H2O2- or AAPH-induced oxidative stress, and scavenged intracellular reactive oxygen species. Moreover, BP-sCS promoted L929 fibroblast proliferation/metabolism and stimulated collagen deposition into the extracellular matrix. In addition, BP-sCS counteracted AAPH-induced oxidative stress damage that inhibited fibroblast migration. These effect were attributed to the cooperation among the molecules of BP-sCS, namely, type II collagen peptides, non-collagenous peptides, and CS polysaccharides. Our findings indicate that BP-sCS has the potential as a novel wound-healing promoter. This study is the first step toward the realization of a sustainable CS-production industry by waste utilization in healthcare products.

Girding the loins? Direct evidence of the use of a medieval English parchment birthing girdle from biomolecular analysis.

In this paper, we describe palaeoproteomic evidence obtained from a stained medieval birth girdle using a previously developed dry non-invasive sampling technique. The parchment birth girdle studied (Wellcome Collection Western MS. 632) was made in England in the late fifteenth century and was thought to be used by pregnant women while giving birth. We were able to extract both human and non-human peptides from the manuscript, including evidence for the use of honey, cereals, ovicaprine milk and legumes. In addition, a large number of human peptides were detected on the birth roll, many of which are found in cervico-vaginal fluid. This suggests that the birth roll was actively used during childbirth. This study is, to our knowledge, the first to extract and analyse non-collagenous peptides from a birth girdle using this sampling method and demonstrates the potential of this type of analysis for stained manuscripts, providing direct biomolecular evidence for active use.

Osteocalcin, Vascular Calcification, and Atherosclerosis: A Systematic Review and Meta-analysis.

Osteocalcin (OC) is an intriguing hormone, concomitantly being the most abundant non-collagenous peptide found in the mineralized matrix of bone, and expanding the endocrine function of the skeleton with far-reaching extra-osseous effects. A new line of enquiry between OC and vascular calcification has emerged in response to observations that the mechanism of vascular calcification resembles that of bone mineralisation. To date, studies have reported mixed results. This systematic review and meta-analysis aimed to identify any association between OC and vascular calcification and atherosclerosis. Databases were searched for original, peer reviewed human studies. A total of 1,453 articles were retrieved, of which 46 met the eligibility criteria. Overall 26 positive, 17 negative, and 29 neutral relationships were reported for assessments between OC (either concentration in blood, presence of OC-positive cells, or histological staining for OC) and extent of calcification or atherosclerosis. Studies that measured OC-positive cells or histological staining for OC reported positive relationships (11 studies). A higher percentage of Asian studies found a negative relationship (36%) in contrast to European studies (6%). Studies examining carboxylated and undercarboxylated forms of OC in the blood failed to report consistent results. The meta-analysis found no significant difference between OC concentration in the blood between patients with "atherosclerosis" and control (p = 0.13, n = 1,197). No definitive association was determined between OC and vascular calcification or atherosclerosis; however, the presence of OC-positive cells and histological staining had a consistent positive correlation with calcification or atherosclerosis. The review highlighted several themes, which may influence OC within differing populations leading to inconclusive results. Large, longitudinal studies are required to further current understanding of the clinical relevance of OC in vascular calcification and atherosclerosis.

Characterization of an intronless collagen gene family in the marine sponge Microciona prolifera.

Two independent clones from the genomic DNA of a marine sponge Microciona prolifera were isolated by hybridization to the Caenorhabditis elegans Col-1 gene and one clone was obtained from genomic DNA by PCR. They contain open reading frames (MpCol1, MpCol2, MpCol3, MpCol4) capable of coding for a family of collagens different from those previously found in sponges. Southern blotting of genomic DNA suggested the presence of several other homologous genes. cDNA clones covering most of the triple-helical coding domain and the 3' untranslated region of MpCol1 were isolated by specific primers and reverse PCR. Two cDNA clones end in the middle of an AATAAA sequence 170 bp downstream from the translation stop codon of MpCol1. The putative NH2-terminal noncollagenous peptide is composed of only seven amino acid residues. The 1074-bp triple-helical coding region is not interrupted by intervening sequences. It codes for a polypeptide of 120 Gly-Xaa-Yaa triplets with only one short interruption near the COOH terminus. A putative N-glycosylation sequence (Asn-Gly-Ser), three Arg-Gly-Asp triplets known as cell recognition peptides, frequent Lys residues in the Yaa position (which are templates for hydroxylation), several Lys-Gly-Asn/Xaa-Arg peptides known as the lysyl oxidase recognition site, and long stretches without imino acids could be found within the triple-helical domain. The short COOH-terminal noncollagenous domain closely resembles that of nematode cuticular collagens and vertebrate nonfibrillar collagens. Our results strongly support the idea that the diversity of collagen genes and gene families found in higher organisms already existed in sponge.

Human cDNA clones transcribed from an unusually high-molecular-weight RNA encode a new collagen chain

Human collagen (COL) cDNA clones were isolated from a library representing transcripts synthesized by an established rhabdomyosarcoma (RH) cell line. The 0.6-kb insert of the first isolate encodes a discontinuous collagenous sequence not homologous to type I-XVI COL chains. Sequencing of a second clone with a 4-kb insert revealed an open reading frame (ORF) of 2154 nucleotides. The deduced amino acid (aa) sequence begins with an 186-aa noncollagenous region containing seven cysteines (Cys). Several of the Cys and surrounding aa residues can be aligned with those in type XVI, XII and IX COL. Due to the presence of two long interruptions, the 524-aa collagenous region is separated into three subdomains that each have smaller interruptions of 1-6 aa. The protein terminates with an 8-aa noncollagenous peptide including an unusual single Cys which would be expected to form an interchain disulfide bond. Results of Northern blot hybridization suggest that the new COL chain may be uncommonly large since the clone identified a low-abundance RNA at least 12.4kb in size. The gene coding for RH COL is located on human chromosome 6. It is now important to elucidate the role of this unusual COL in the infrastructure of extracellular matrix.

Immunochemical studies of the Alport antigen

The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, alpha 3(IV) NC1, and alpha 4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from alpha 1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of alpha 5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-alpha 5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and alpha 5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of alpha 5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of alpha 3(IV) NC1 and alpha 4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.

Effect of chlorpromazine on pz-peptidase and several other peptidase activities in cloned osteoblastic cells (MC3T3-E1)

The effect of chlorpromazine (CPZ) on the degradation of collagen and non-collagenous peptides in clonal osteoblastic MC3T3-E1 cells was investigated by measuring the activities of PZ-peptidase, collagenase-like peptidase (CL-peptidase), dipeptidyl-aminopeptidase (DAP), leucine aminopeptidase (LAP) (EC 3.4.11.1), and post-proline cleaving enzyme (PPCE) (EC 3.4.21.26). CPZ increased PZ-peptidase and CL-peptidase activities in a dose-related fashion, but it had no effect on LAP and PPCE activities in the cells. CPZ (10 μg/ml) enhanced the specific activities of PZ-peptidase, CL-peptidase, and DAP for 72 hr after the start of CPZ stimulation; in particular, about a 3.3-fold increase of PZ-peptidase activity was observed at 12 hr of culture. Furthermore, other phenothiazine derivatives specifically enhanced the PZ-peptidase, CL-peptidase, and DAP activities as well as CPZ. Since PZ-peptidase, CL-peptidase, and DAP, involved in the degradation of collagen peptides, were induced significantly by CPZ (and/or other phenothiazine derivatives) in comparison with LAP and PPCE, involved in the degradation of non-collagenous peptides, these results show that CPZ specifically stimulated collagen catabolism by inducing the collagen-catabolizing enzymes. In addition, CPZ specifically inhibited collagen synthesis in clonal osteoblasts.

Degradation of glomerular basement membrane in diabetes. II. Proteolytic activity of diabetic and nondiabetic glomeruli.

The proteolytic effects of isolated glomeruli of diabetic rats on glomerular basement membrane of nondiabetic rats was investigated at various times after streptozotocin injection. One week after induction of diabetes, proteolytic activity remained unchanged as compared with nondiabetic controls. However, 4 and 10 weeks after streptozotocin injection, glomerular degradation of collagenous (but not noncollagenous) peptides of basement membranes increased (+24% as compared with control experiments). Using diabetic basement membrane as substrate, degradation of collagenous and noncollagenous peptides caused by diabetic glomeruli was 2.6-fold and 1.7-fold higher than in control experiments with nondiabetic glomeruli. The results indicate that the disturbed degradation of glomerular basement membrane in diabetes is not due to a decreased activity of glomerular proteolytic enzymes. In contrast, it can be concluded that the increased resistance of diabetic basement membrane to proteolytic degradation could be partially compensated by quantitative and qualitative changes of the proteolytic potential of diabetic glomeruli.

Degradation of glomerular basement membrane in diabetes. I. Susceptibility of diabetic and nondiabetic basement membrane to proteolytic degradation of isolated glomeruli.

Degradation of glomerular basement membrane in diabetic and nondiabetic rats was measured by incubating isolated basement membrane with a homogenate of glomeruli obtained from metabolically healthy rats. When diabetic basement membrane was used, there was a marked decrease in the amount of collagen-typical (hydroxyproline, hydroxylysine, glycine) and noncollagen-typical amino acids (proline, lysine, leucine) released in the supernatant of the incubation assay. A negative correlation was found between the amount of collagen-typical amino acids released by diabetic basement membrane and the duration of diabetes. The results indicate that the collagenous and noncollagenous peptides of diabetic basement membrane are less susceptible to proteolytic degradation than those of nondiabetic controls. This may be due to increased nonenzymatic glycosylation of diabetic basement membrane.

Effect of prostaglandin E2 on PZ-peptidase and several other peptidase activities in a clonal osteoblast-like cell line derived from newborn mouse calvaria.

The effects of prostaglandin E2(PGE2) on the degradation of collagen and non-collagenous peptides in clonal osteoblastic MC3T3-E1 cells were investigated by using highly sensitive assay methods for PZ-peptidase, collagenase-like peptidase (CL-peptidase), dipeptidyl-aminopeptidase (DAP), leucine aminopeptidase (LAP), and post-proline cleaving enzyme (PPCE). PGE2, at concentrations of 0.1 to 4.0 micrograms/ml, doubled the PZ-peptidase and CL-peptidase activities in the cells on 24 h culturing in a dose-dependent manner. PGE2, at a concentration of 2.0 micrograms/ml, enhanced the specific activities of PZ-peptidase, CL-peptidase, DAP, LAP, and PPCE for 75 h after the start of PGE2 stimulation. The time dependent changes in PZ-peptidase and CL-peptidase activities showed similar patterns, and 3- and 2-fold increases were seen after 48 h, respectively. The protein and DNA contents gradually increased after addition of PGE2. Since the PZ-peptidase and CL-peptidase, involved in degradation of collagen peptides, were significantly induced by PGE2 in comparison with LAP and PPCE, involved in the degradation of non-collagenous peptides, these results show that PGE2 specifically stimulates induction of collagen catabolizing enzymes in clonal osteoblasts.

Synthesis and characterization of cDNA encoding a cartilage-specific short collagen.

Hyaline cartilage contains a unique set of collagenous proteins. Type II collagen is the most abundant, constituting about 85% of the total cartilage collagen. In addition, several minor collagenous components have been described. To study the structure and developmental regulation of chondrocyte-specific collagens, we have constructed a cDNA library from embryonic chicken sternal cartilage mRNA. We report here on the isolation and characterization of a 3200 base-pair-long cDNA that codes for a collagenous polypeptide of unusual structure in that the total length of the molecule is only about half of pro alpha 1(II) collagen chains. The mRNA for this polypeptide is considerably smaller than mRNA encoding the pro alpha chains of interstitial collagens. In addition, the peptide encoded by the cDNA appears to contain at least three domains with triple-helical potential separated by short, noncollagenous peptides. Between the three collagenous domains are several cysteinyl residues.

Assembly and processing of procollagen V (AB) in chick blood vessels and other tissues.

The biosynthesis and processing of type V procollagens was investigated in chick embryo blood vessels labeled with radioactive amino acids. Monomeric, pepsin-sensitive pro alpha 1 V and pro alpha 2 V chains are slowly assembled into triple helically folded molecules. A small proportion of these procollagen V molecules contain interchain disulfide bridges, and the disulfide-linked heterodimer and heterotrimer (pro alpha 1 V)2pro alpha 2 V were found. A relatively fast conversion of procollagen V to p-collagen V is followed by slow change to collagen V. This time course is similar to the processing of procollagen III and is much slower than the rate of appearance of type I collagen in blood vessels. A combination of sedimentation and electrophoretic analyses was used to measure the relative size of the type V chains and to demonstrate attachment of noncollagenous peptides (Mr = 33,000) to p alpha 2 V by disulfide linkage. Similar quantitative pulse-chase studies were made with calvaria and crop. As the same unusual features of assembly and processing of type V chains were seen in muscle, bone, and blood vessels, we conclude that these are characteristic of type V collagen biosynthesis.

Chemical characterization of glomerular and tubular basement membranes of men of different ages

Pairs of glomerular and tubular basement membrane preparations were obtained from kidneys of individuals of premature age up to 80 years. The purity of the preparations was established with light and electron microscopy and by estimating the total phosphorus content. The amino acid and carbohydrate composition was determined and statistically evaluated for 33 to 38 preparations divided over five age groups. Comparison of glomerular and tubular basement membranes from the same kidneys showed that regardless of age glomerular basement membranes contain more 3-hydroxyproline and more of the heteropolysaccharide constituents neuraminic acids and mannose. These differences cannot be ascribed solely to the presence of a minor amount of interstitial collagen. The chemical composition of the two types of basement membranes changes with age. For the first few years after birth the contents of glycine and total imino acids increase and those of collagen-nonspecific amino acids decrease, whereas constituents of the heteropolysaccharide units do not change markedly. These results suggest that proportions of collagenous and noncollagenous peptide moieties gradually change with age in both glomerular and tubular basement membranes. In addition, the extent of hydroxylation of proline and lysine increases significantly with age, reaching an adult level for glomerular basement membranes after 4 to 7 months of age and for tubular basement membranes during late childhood. The contents of glucose and galactose rise with age to an extent comparable with that of hydroxylysine. The age-related changes in basement membrane composition may influence functional properties of these extracellular renal structures.

Propeptides of procollagen V (A,B) in chick embryo crop.

The biosynthesis of type V (A,B) collagens was recently found to proceed through the sequential forms pro alpha (A,B), p alpha (A,B), and f alpha (A,B). All these chains are larger than the A,B chains extracted from tissues after pepsin digestion. This report shows that all forms contain substantial peptides which are resistant to bacterial collagenase and concludes that type V collagens differ from the intestitial collagens (types I, II, III) in retaining large noncollagenous peptides in tissues. A peptide becomes transiently attached to the processing intermediate p alpha A by a reducible linkage. The conversion of procollagen V to p-collagen V was inhibited by colchicine and arginine. Previously, the disulfide-linked heterotrimer [(pro alpha B)2 (pro alpha A)] was found and additional procollagens containing only B type molecules were inferred. Further investigations reported here agree with these conclusions and also indicate that some trimeric molecules containing more than one chain related to A may exist.

Characterization of collagenous and non-collagenous peptides of a glycoprotein isolated from alveoli of patients with alveolar proteinosis

A glycoprotein of Mr 62 000 was isolated from lung lavage material of patients with alveolar proteinosis. The glycoprotein was found to contain (per molecule) 72 residues of glycine, 5 residues of hydroxyproline, 3 molecules of sialic acid, 4.9 molecules of mannose, 4.0 molecules of galactose, 0.9 molecule of fucose and 7.0 molecules of N-acetylglucosamine. Limited pepsin digestion of the glycoprotein resulted in six peptides, three of which contained hydroxyproline and nearly 30% glycine, and two of which contained all the carbohydrate present in the glycoprotein of Mr 62 000. The three peptides containing hydroxyproline and with high content of glycine contained a repeating -Gly-X-Y-sequence in the peptide chain. Partial amino acid-sequence analyses on the peptides derived from the digestion of the alveolar glycoprotein with various proteolytic enzymes indicate that this glycoprotein is characterized by the presence of alternating collagenous and non-collagenous regions in the same polypeptide chain.

Isolation of a unique collagenous fraction from limited pepsin digests of human placental tissue. Characterization of one of the constituent polypeptide chains.

Fractionation of the highly soluble collagens released during limited pepsin digestion of whole human placenta has resulted in the isolation of a unique collagenous fraction comprised exclusively of high molecular weight aggregates. On reduction and alkylation, the aggregates dissociated yielding collagen-like polypeptides with an apparent Mr = 40,000 as well as a heterogeneous mixture of much smaller noncollagenous peptides. Ion exchange chromatography of the collagen-like, 40,000-dalton subunits results in recovery of a single relatively acidic component and a mixture of at least two relatively basic components. Both the basic and acidic components contain large amounts of cysteine, hydroxylysine glycosides, and glucosamine, and exhibit compositional features indicative of the presence of collagenous and noncollagenous domains. These unusual features have been verified for the more acidic component through isolation of its major cyanogen bromide cleavage products. Characterization of the cyanogen bromide fragments further indicated that this particular subunit is characterized by alternating collagenous and noncollagenous domains as opposed to a lengthy collagenous domain which is either preceded or followed by noncollagenous sequences. The molecular organization and possible derivation of these unique pepsin-resistant collagenous components is discussed.

Studies on the sulfhydryl groups in type III collagen

The Type III collagen molecule, [α(III)] 3, is comprised of three α(III) chains each of which contains two cysteinyl residues. Free sulfhydryl groups, however, could not be detected in the denatured, trimetric γ-component of Type III collagen as judged by the failure to form derivative with the alkylating reagents iodo- [ 14C]acetic acid and 4-vinylpyridine. This absence of sulfhydryl group reactivity did not appear to result from disulfide-binding between Type III collagen and noncollagenous peptides. Collectively, the results indicate that all of the six sulfhydryl groups of the Type III collagen molecule participate in interchain disulfide-bonding.

Identification and partial characterisation of the non-collagenous amino- and carboxyl-terminal extension peptides of cartilage procollagen

Procollagen secreted by embryonic chick cartilage cells has been partially purified by (NH 4) 2SO 4 precipitation and DEAE-cellulose chromatography. Analyses of the products of digestion by human rheumatoid synovial collagenase and bacterial collagenase indicated the presence of non-collagenous peptide sequences at the N- and C-termini. Both regions were found to incorporate [ 35S]cystine but inter-chain disulphide bonds were restricted to a C-terminal location. Electrophoretic analysis gave apparent molecular weights of 13000 and 36000 daltons for the respective N- and C-terminal extensions.

Differences between CNBr peptides of soluble and insoluble bovine collagens

The compositions of the cyanogen bromide peptides of insoluble bovine skin and dentin collagens are nearly identical with those obtained from the soluble collagen. However, some qualitative and quantitative differences are present. The insoluble collagens do not go completely in solution after the treatment. The insoluble residues have compositions showing a firm association of collagen with non-collagenous peptides. The content of peptide α1-CB6 is sharply reduced in both insoluble skin and dentin collagen, indicating involvement of this peptide in the formation of intermolecular cross-links. Several heterogeneities were noted regarding many peptides. In particular, two forms of α1-CB3 and of α1-CB6 were isolated in agreement with the idea that two types of α1-chains are present. The situation is more complex, in that the second form of α1-CB3 from skin and dentin are not identical.