Glycosaminoglycan Peptides Research Articles

ЭФФЕКТИВНОСТЬ И БЕЗОПАСНОСТЬ СОЧЕТАННОГО ПРИМЕНЕНИЯ ПЕПТИДНОГО КОМПЛЕКСА И ДИАЦЕРЕИНА ПРИ ОСТЕОАРТРИТЕ КОЛЕННОГО СУСТАВА

As part of a prospective study, it was evaluated the efficacy and safety of the combined use of drugs from the group of symptomatic slow acting agents (SYSADOA): Diacerein and glycosaminoglycan peptide complex (GPC) in patients with knee osteoarthritis. The dynamics of pain according to Visual Analoguе Scale (VAS), need for non-steroidal anti-inflammatory drugs, the WOMAC index, EQ-5D score, and OMERACT-OARSI criterion, the presence of adverse events were analyzed, and the effect of Diacerein on the components of the metabolic syndrome was assessed. It was shown that the concomitant use of GPC and Diacerein demonstrated high efficiency and equivalent safety in comparison with the isolated use of GPC: a decrease in the pain intensity in the early stages of therapy, a decrease in the need for NSAIDs, a tendency to improve lipid and purine metabolism.

Cleavage of the Xylosyl Serine Linkage between a Core Peptide and a Glycosaminoglycan Chain by Cellulases

We previously found that endo-beta-xylosidase from Patinopecten is an endo-type glycosidase that cleaves the xylosyl serine linkage between a glycosaminoglycan chain and its core protein (Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) J. Biol. Chem. 265, 854-860). Screening for endo-beta-xylosidase activity in several cellulases detected this activity in the enzymes from Aspergillus niger, Penicillium funiculosum, Trichoderma reesei, Trichoderma viride, and Irpex lacteus. The cellulase derived from A. niger was purified, and its molecular weight was determined to be 26,000 by SDS-PAGE. Examination of the specificity of the cellulase revealed that 1) the enzyme acts on the linkage region (xylosyl serine) between a core peptide and a glycosaminoglycan chain; 2) enzymatic activity is greater with shorter glycosaminoglycan chains; 3) the enzyme readily hydrolyzes the linkage in glycosaminoglycan peptides, but intact proteoglycan is cleaved only slowly; and 4) the activity is unaffected by the glycosaminoglycan component (chondroitin sulfate, dermatan sulfate, and heparan sulfate). Judging from these enzymatic characteristics, this cellulase is different from the endo-beta-xylosidase of Patinopecten. We believe that this cellulase will become a useful tool in the further development of glycotechnology, because, like the endo-beta-xylosidase of Patinopecten, it enables the release of intact glycosaminoglycans from glycosaminoglycan peptides.

Extraction of Glycosaminoglycan Peptide from Bovine Nasal Cartilage with 0.1 M Sodium Acetate.

Cartilage is a nonedible byproduct with little saleable value, but it is rich in glycosaminoglycan (GAG), which can be converted to a highly profitable product. In this study, GAG was extracted from bovine cartilaginous tissues including nasal cartilage, occipital articular cartilage, and temporomandibular joint disk without using the expensive exogenous proteinase commonly used to extract GAG from tissues. The GAG was extracted with highest yield ( approximately 80% of total tissue uronic acid) by incubating nasal cartilage in 0.1 M sodium acetate, pH 4.5, at 37 degrees C. Tissue autolysis was suggested to occur under the incubation condition to release GAG-peptide. The GAG-peptide purified by DEAE ion-exchange chromatography contained approximately 7% protein, 89% chondroitin sulfate, and 4% keratan sulfate and had no capability to interact with hyaluronic acid. The results suggest that the extraction method with 0.1 M sodium acetate described in this paper is useful for the preparation of GAG-peptide at a low cost.

Reversal of cytokine-induced inhibition of type II collagen gene expression by a glycosaminoglycan—peptide complex (GP-C) in cultured human chondrocytes

Reversal of cytokine-induced inhibition of type II collagen gene expression by a glycosaminoglycan—peptide complex (GP-C) in cultured human chondrocytes

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.

Amelioration of lapine osteoarthritis by treatment with glycosaminoglycan–peptide association complex (Rumalon)

The chondroprotective potential of glycosaminoglycan-peptide association complex (GP-C) was examined in the medial meniscectomy model of lapine osteoarthritis (OA). Prophylactic treatment with increasing doses of intramuscular GP-C (0.05-0.5 ml/kg) caused a significant reduction in OA lesion area and histologic scores, and the effect on disease activity appeared to be dose related. The DNA and uronic acid contents of OA tissue were unaffected by prophylactic treatment with GP-C. However, levels of hydroxyproline in OA cartilage increased to near control levels with prophylactic treatment. Cartilage levels of active and total metalloproteinases that digest proteoglycans were elevated in rabbits with OA; prophylactic treatment with low-dose GP-C (0.05 ml/kg) produced a significant reduction in active, but not total, enzyme. Cartilage levels of tissue inhibitor of metalloproteinases in animals with OA were comparable with control levels, but rose with increasing doses of GP-C. We also investigated GP-C as a therapeutic treatment in animals that had already developed OA lesions. Carbon black retention and histologic score returned to near-normal after therapeutic treatment with GP-C. Uronic acid and hydroxyproline levels were decreased in OA cartilage. Therapeutic treatment with GP-C had no statistically significant effect on uronic acid levels, but was associated with increased hydroxyproline content in the cartilage. The changes in metalloproteinase and metalloproteinase inhibitor were similar to those found in the studies of prophylactic treatment. The findings in this animal model may help explain some of the beneficial effects of GP-C in human OA.

Cell-associated proteoheparan sulfate from bovine arterial smooth muscle cells

Cell-associated proteoheparan sulfate has been isolated from bovine arterial smooth muscle cells preincubated with [ 35]sulfate or a combination of [ 3H]glucosamine and [ 35S]methionine. The purified proteoheparan sulfate had an apparent M r of 200,000 on calibrated Sepharose CL-2B columns. The glycosaminoglycan component ( M r ∼ 30,000) was identified as heparan sulfate by its susceptibility to specific enzymatic and chemical degradation. After degradation of the proteoheparan sulfate by microbial heparitinase the resulting protein core had an apparent M r of 92,000 on SDS-polyacrylamide gels. Its mobility was similar in the absence and presence of reducing agents indicating that the protein core consists of a single polypeptide chain. Pulse-chase experiments revealed that about 40% of the cell layer-associated proteoheparan sulfate was released into the medium, while the remainder was internalized and converted to smaller species through a series of degradation steps. Initially there was a proteolytical cleavage of the protein core generating glycosaminoglycan peptide intermediates with polysaccharides chains similar in size to the original. The half-life of the native proteoheparan sulfate was found to be about 4 h.

Future therapeutic trends in osteoarthritis.

Since cartilage contains no nerve endings, symptoms of osteoarthritis (OA) are indirect. Therapy of OA, to date, has been directed at the symptoms of pain, signs of inflammation and loss of function. A major advance has been surgical joint replacement. This benefit however, is often limited by the joint area involved and to the survival of the endoprosthesis. Orthopedic new approaches to therapy of OA include removal of abnormal tissue to stimulate repair (e.g., burring, abrasion) and grafting (e.g., osteochondral grafts, perichondrium, periosteum) to the subchondral bone. Controlled activity (e.g., passive motion) has been studied alone and with the above. Can a medication retard or reverse the degradative process of OA? Several medications are being examined for potential "chondroprotective" characteristics. Some of these agents are not new: oversulfated glycosaminoglycans (Arteparon) derived from cartilage and glycosaminoglycan peptides (Rumalon) derived from cartilage and bone marrow extracts may be prototypes of this approach to therapy. Other agents demonstrating potential benefit in retarding cartilage degradation may include non-steroidal anti-inflammatory agents, tiaprofenic acid, sodium pentosan sulfate and low dose corticosteroids. This concept of "chondroprotection" provides us a new approach to a disease in need of a new approach.

Proteoglycans synthesized by cultured mouse osteoblasts.

Proteoglycan synthesis in nonmineralizing osteoblast cultures was investigated. Cultures were labeled with [35S]sulfate or [3H]serine, and proteoglycans were extracted from medium and cell layer with 4 M guanidine HCl. Labeled material was subjected to Sepharose CL-4B and DEAE-Sephacel chromatography and polyacrylamide gel electrophoresis. The size and composition of the glycosaminoglycan chains and the protein core size were determined. Two proteoglycan populations were isolated by Sepharose CL-4B chromatography: a minor excluded species with chondroitin sulfate chains of apparent Mr 25,000 and a smaller population (Kav = 0.43) accounting for 80% of the total labeled material. This small population resolved into two species by polyacrylamide gel electrophoresis. Both species contain dermatan sulfate chains of apparent Mr 40,000 and a core protein with Mr 45,000 on sodium dodecyl sulfate gels. With the exception of their glycosaminoglycan composition these species appear similar to those extracted from bone. In addition, high molecular weight hyaluronic acid and glycosaminoglycan peptides were found in cell extracts.

Extraction and purification of proteoglycans from mature bovine bone.

Proteoglycans were extracted in good yields from the mineralized matrix of ground bovine bone, by using a two-step extraction procedure. Proteoglycans (8% of total), not associated with the bone mineral, were extracted at - 20 degrees C with 4M-guanidinium chloride containing proteinase inhibitors. Proteoglycans associated with the mineral, which accounted for 60% of the total, were then solubilized when EDTA was added to the extraction solvent. They were fractionated and purified in the presence of 4M-guanidinium chloride by CsCl-density-gradient centrifugations followed by chromatography on Sepharose CL-4B. Further purification was obtained by chromatography on DEAE-cellulose and hydroxyapatite in the presence of 7 M-urea. Three populations of proteoglycans and additional glycosaminoglycan peptides were obtained. The molecular dimensions of both intact molecules and of their side chains as well as their amino acid composition were different, indicating that they represent separate molecular entities. The main proteoglycan self-aggregated in the absence of 4M-guanidinium chloride or 7 M-urea, a property that was abolished when the proteoglycan core protein was fragmented.

Etude biochimique comparative des glycosaminoglycannes peptides du cartilage articulaire de la tete femorale humaine normale et arthrosique

Glycosaminoglycan peptides prepared by papain hydrolysis of different regions were obtained from osteoarthrotic and normal human femoral heads. Data obtained in these experiments show that in osteoarthrosis a decrease in keratan sulfate and an increase in chondroitin sulfate are observed. Since keratan sulfate appeared to play an important role in proteoglycan aggregation, we suggest that the keratan sulfate decrease is one of the factors involved in the cartilage disorder observed in patients suffering from osteoarthrosis.

Sulfated glycopeptides and glycosaminoglycan peptides isolated from intestinal mucosae of rabbit

1. 1. Two sulfated glycopeptides and a highly sulfated uronic acid-containing glycosaminoglycan peptide were isolated in an highly pure state from protease-digested mucosal scrapings from either rabbit small intestine or colon. 2. 2. Small intestinal mucosa yielded much more highly sulfated fractions than did colonic mucosa. 3. 3. All the sulfated glycopeptides were similar in having galactose, equimolar ratios of glucosamine and galactosamine, the major carbohydrate components, with fucose and sialic acid in addition. Sulfate, present in each sulfated glycopeptide, had a molar ratio to hexosamine ranging from 0.11 to 0.41; this ratio increased with increasing molarity of eluting salt. 4. 4. The most acidic fraction, highly sulfated uronic acid-containing glycosamino-glycan peptide, contained a large quantity of sulfate and an appreciable quantity of uronic acid, in addition to galactose, hexosamine (glucosamine/galactosamine = 0.5), fucose and peptide. 5. 5. Threonine, proline and serine were the predominant amino acids in all the purified fractions. The results of the alkali treatment suggested that the linkages were through the hydroxyl group of threonine and serine to carbohydrates. 6. 6. All the purified fractions showed dextrorotation. The optical activity and sedimentation coefficient of the corresponding fractions from the two different tissues were similar.