Characterization and N-terminal sequence of human platelet proteoglycan.
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ABSTRACT:
SUBMITTER: Perin JP
PROVIDER: S-EPMC1135341 | biostudies-other | 1988 Nov
REPOSITORIES: biostudies-other
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Project description:Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.
| S-EPMC1152987 | biostudies-other
Project description:Proteoglycans (PGs) are among the most structurally complex biomacromolecules in nature. They are present in all animal cells and frequently exert their critical biological functions through interactions with protein ligands and receptors. PGs are comprised of a core protein to which one or multiple, heterogeneous, and polydisperse glycosaminoglycan (GAG) chains are attached. Proteins, including the protein core of PGs, are now routinely sequenced either directly using proteomics or indirectly using molecular biology through their encoding DNA. The sequencing of the GAG component of PGs poses a considerably more difficult challenge because of the relatively underdeveloped state of glycomics and because the control of their biosynthesis in the endoplasmic reticulum and the Golgi is poorly understood and not believed to be template driven. Recently, the GAG chain of the simplest PG has been suggested to have a defined sequence based on its top-down Fourier transform mass spectral sequencing. This review examines the advances made over the past decade in the sequencing of GAG chains and the challenges the field face in sequencing complex PGs having critical biological functions in developmental biology and pathogenesis.
| S-EPMC3425375 | biostudies-literature
Project description:We have isolated from the conditioned medium of an established endothelial cell line a heparan sulphate proteoglycan whose involvement in the inhibition of the extrinsic coagulation pathway was reported in previous studies [Colburn & Buonassisi (1982) Biochem. Biophys. Res. Commun. 104, 220-227]. The proteoglycan was purified by gel filtration and ion-exchange chromatography, and appears to be free of contaminating proteins as determined by polyacrylamide-gel electrophoresis of the radioiodinated protein core before and after removal of the glycosaminoglycan chains by treatment with heparitinase. By this procedure the Mr of the protein core was estimated to be 22000. The N-terminal end was sequenced up to amino acid 25. The 21st residue is likely to be glycosylated. Analysis of the purified proteoglycan by gel-filtration chromatography yielded Kd values of 0.2 for the whole molecule and 0.35 for the glycosaminoglycan chains. The structure that emerges from these data is that of a heparan sulphate proteoglycan characterized by a relatively small protein core and few glycosaminoglycan chains.
| S-EPMC1148467 | biostudies-other
Project description:BackgroundRheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.MethodsCitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.FindingsSera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.ConclusionsCitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.
| S-EPMC4778950 | biostudies-literature
Project description:Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan, with a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike nucleic acids and proteins, whose synthesis is template driven, Golgi-synthesized glycosaminoglycans are not believed to have predictable or deterministic sequences. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size-similar and charge-similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular ions corresponding to monocompositional peptidoglycosaminoglycans. Fragmentation using collision-induced dissociation unexpectedly afforded a single sequence for each monocompositional parent ion, unequivocally demonstrating the presence of a defined sequence. The biosynthetic pathway common to all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment in the understanding of information storage in complex glycans.
| S-EPMC3197799 | biostudies-literature
Project description:This study consists of (1) the extraction of proteoglycan from the human meniscus under dissociative conditions, (2) an investigation of the changes that occur in the abundance and structure of this proteoglycan with age and (3) a comparison of these findings with those for human articular-cartilage proteoglycan. Adult meniscus was found to possess proteoglycan molecules of similar size and glycosaminoglycan content to those present in cartilage, although tissue concentrations were considerably lower. In addition, age-related changes, with respect to the occurrence of keratan sulphate and the sulphation of chondroitin sulphate chains, were common to both tissues. The presence of aggregated proteoglycan was demonstrated, although specific interaction with hyaluronic acid was not conclusively shown biochemically. Differences were, however, noted in the structure of the proteoglycan between the two tissues: dermatan sulphate was found in the meniscus proteoglycan preparation and the core proteins exhibited some dissimilarities. A proteoglycan structure of this type would be compatible with its participation in meniscus elasticity, especially as the material is localized in a specific area.
| S-EPMC1161448 | biostudies-other
Project description:A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200,000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15,000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24,000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.
| S-EPMC1150154 | biostudies-other
Project description:Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.
| S-EPMC8622445 | biostudies-literature
Project description:Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C.R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.
| S-EPMC1219281 | biostudies-other
Project description:Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.
| S-EPMC1154117 | biostudies-other