RESUMO
During pregnancy, an interpubic ligament is formed in the mouse pubic symphysis. In late stages, this ligament undergoes "relaxation" to allow proper delivery, which is expected on the 19th day. Proteoglycans and hyaluronic acid play an important role in the remodeling of the extracellular matrix in these tissues. Glycosaminoglycans and proteoglycans were studied by electron microscopic, immunohistochemical and biochemical methods in samples of mouse pubic symphysis from the 12th to 18th day of pregnancy. At the ultrastructural level, using cuprolinic blue and enzymatic digestion by chondroitin lyases, two types of proteoglycan filaments were observed in the fibrocartilage on the 12th day, as well as in D 15, D 17 and D 18 pubic ligaments. The only sulfated glycosaminoglycan in these filaments was chondroitin sulfate, as shown by chondroitin lyase treatment. Their electrophoretic mobility, before and after enzymatic degradation, corroborated this inference. The ratio of chondroitin sulfate/dry weight of symphysis showed two phases of increase: between D12 and D 15, and between D 17 and D 18. We suggest that the first corresponds mainly to an increase in decorin when the ligament is formed, and the second to versican, during "relaxation". Versican and hyaluronic acid, working as water holding molecules would be responsible for the hydration of the ligament at the end of pregnancy, allowing an increase in resiliency. The presence of hyaluronic acid was confirmed by labeling with HA-probe in the perichondrium, fibrocartilage and ligament. The role of collagen fibers as physical restrictors of the complete expansion of glycosaminoglycans and hyaluronic acid in tissue is discussed.
Assuntos
Glicosaminoglicanos/análise , Glicosaminoglicanos/ultraestrutura , Prenhez/fisiologia , Proteoglicanas/análise , Proteoglicanas/ultraestrutura , Sínfise Pubiana/metabolismo , Sínfise Pubiana/ultraestrutura , Animais , Condroitina Liases/metabolismo , Eletroforese em Gel de Ágar , Feminino , Glicosaminoglicanos/química , Ácido Hialurônico/análise , Imuno-Histoquímica , Indóis/farmacologia , Camundongos , Compostos Organometálicos/farmacologia , Gravidez , Proteoglicanas/química , Enxofre/químicaRESUMO
Heparan sulfate (HS) present on the surface of hemopoietic stromal cells has important roles in the control of adhesion and growth of hemopoietic stem and progenitor cells. Recent studies have characterized several different heparan sulfate proteoglycans (HSPGs) from both human and murine bone marrow stromal cells. In the present study, we have compared the molecular structure of HS, metabolically labeled with [(35)S]-sulfate produced by two distinct preparations of murine hemopoietic stromal cell lines. These comprised a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024. [(35)S]-HS was examined in the cell layers and in the culture medium. We identified and measured the relative proportions of the various glycosaminoglycans (GAGs) in the two stromal cell lines. Chondroitin sulfate (CS) was preponderantly secreted by the stromal cell lines, while HS was relatively more abundant in the cell-associated fractions. The two types of stromal cells differ in their HS composition, mainly due to different patterns of N- and O-sulfation. The two stromal cell lines expressed mRNA for different HSPGs. Data from reverse transcription PCR revealed that the two stromal cell lines expressed mRNA for glypican and syndecan4. Only AFT024 cell line expressed mRNA for betaglycan. There was no evidence for expression of mRNA for both syndecan1 and syndecan2. [(35)S]-sulfated macromolecules could be released from the cell surface of both stromal cell lines by phosphatidylinositol phospholipase C (PI-PLC), which is consistent with the expression of glypican detected by PCR experiments.
Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Heparitina Sulfato/metabolismo , Fígado/metabolismo , Animais , Linhagem Celular Transformada , Membrana Celular/metabolismo , Condroitina Liases/química , Condroitina Liases/metabolismo , Cromatografia por Troca Iônica/métodos , Citocinas/biossíntese , Primers do DNA , Dissacarídeos/biossíntese , Dissacarídeos/química , Eletroforese em Gel de Ágar , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/citologia , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/análise , Humanos , Fígado/citologia , Camundongos , Ácido Nitroso/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo , Radioisótopos de Enxofre , Fatores de TempoRESUMO
A dermatan sulfate-like glycosaminoglycan was isolated from the body of the ascidian Ascidia nigra (J. Biol. Chem. 270: 31027-31036, 1995). 1H NMR and fast atom bombardment mass spectrometry (FAB-MS) spectra of the tetra and disaccharides formed by chondroitin ABC lyase digestion support the proposed repeating disaccharide structure for this glycosaminoglycan, [-->4)-alpha-L-IdoA(2SO4)-(1-->3)-beta-D-GalNAc(6SO4)-(1-->] , which differ from mammalian dermatan sulfate in its sulfation at both 2-position of the alpha-L-iduronic acid and the 6-position of the N-acetyl-beta-D-galactosamine residue.
Assuntos
Condroitina Liases/metabolismo , Dermatan Sulfato/metabolismo , Urocordados/química , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animais , Dermatan Sulfato/química , Dermatan Sulfato/isolamento & purificação , Dissacarídeos/química , Dissacarídeos/metabolismo , Ácido Idurônico/química , Ácido Idurônico/metabolismo , Espectroscopia de Ressonância Magnética , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Sulfatos/metabolismoRESUMO
A dermatan sulfate, similar to the mammalian glycosaminoglycans but not identical with any of them, has been isolated from the body of the ascidian Ascidia nigra. Degradation with chondroitin ABC lyase, analysis of the disaccharide products by digestion with chondro-4- and -6-sulfatases, and 1H and 13C NMR data confirm that the predominant structure is [4-alpha-L-IdoA-(2SO4)-1-->3-beta-D-GalNAc(6SO4)-1]n. Mammalian dermatan sulfate is an anticoagulant due to its ability to potentiate inhibition of thrombin by heparin cofactor II. The structure in dermatan sulfate which binds to heparin cofactor II is [4-alpha-L-IdoA-(2SO4)-1-->3-beta-D-GalNAc(4SO4)-1]n, where n > or = 3. We have compared the ascidian dermatan sulfate with mammalian dermatan sulfate and with chemically oversulfated mammalian dermatan sulfate for anticoagulant activity as measured by the activated partial thromboplastin time assay and for its ability to potentiate heparin cofactor II. In spite of its high content of 2-O-sulfated alpha-L-iduronic acid residues, the ascidian compound had no discernible anticoagulant activity and had low ability to potentiate heparin cofactor II. These results suggest that 4-O-sulfation of the N-acetyl-beta-D-galactosamine residues is essential for the anticoagulant activity of dermatan sulfate.
Assuntos
Anticoagulantes/química , Dermatan Sulfato/química , Sulfatos/química , Acetilgalactosamina/química , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Sequência de Carboidratos , Condroitina Liases/metabolismo , Cromatografia DEAE-Celulose , Cromatografia em Papel , Dermatan Sulfato/isolamento & purificação , Dermatan Sulfato/farmacologia , Cofator II da Heparina/química , Humanos , Hidrólise , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , UrocordadosRESUMO
A sulfatase acting upon chondroitin sulfate polymers, free of beta-glucuronidase and beta-N-acetylhexosaminidases, was isolated from extracts of the mollusc Anomalocardia brasiliana. The enzyme totally desulfates both chondroitin 4- and 6-sulfates without concomitant depolymerization of the compounds. It has no activity upon heparan sulfate, heparin, dermatan sulfate, and chondroitin sulfate disaccharides. It shows a pH of 5.0 and a temperature of 37 degrees C for optimum activity with a Km of 4 x 10(-5) M. The sulfatase is inhibited by sulfate and phosphate ions and HgCl2. The latter inhibition is reverted by sodium tetrathionate. Contrary to the sulfatases described so far the enzyme is activated by the lactone of D-saccharic acid when in the presence of beta-glucuronidase and beta-N-acetylgalactosaminidase. Several experiments indicate that the sulfatase is the first enzyme in the sequential degradation of chondroitin sulfate in the mollusc. This differs from the pathway of degradation of this compound in vertebrates and bacteria.
Assuntos
Condroitina Liases/metabolismo , Sulfatos de Condroitina/metabolismo , Glicosídeo Hidrolases/metabolismo , Moluscos/enzimologia , Animais , Sequência de Carboidratos , Bovinos , Condroitina Liases/isolamento & purificação , Cromatografia em Gel , Mucosa Gástrica/química , Glicosaminoglicanos/metabolismo , Glicosídeo Hidrolases/isolamento & purificação , Heparina/isolamento & purificação , Heparina/metabolismo , Hidrólise , Cinética , Dados de Sequência Molecular , Oligossacarídeos/metabolismo , Especificidade por SubstratoRESUMO
Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/análise , Condroitina/análogos & derivados , Dermatan Sulfato/análise , Mucosa Intestinal/análise , Mastócitos/análise , Infecções por Nematoides/metabolismo , Proteoglicanas/análise , Animais , Condroitina Liases/metabolismo , Sulfatos de Condroitina/análise , Dissacarídeos/análise , Glicosaminoglicanos/análise , Nippostrongylus , Peptídeo Hidrolases/farmacologia , Ratos , Ratos EndogâmicosRESUMO
1. The distribution chondroitin 4- and 6-sulfates in the epiphysial cartilages of several mammals are reported. 2. Chondroitin 6-sulfate is present in higher relative proportion in articular surfaces of young and adult epiphysial cartilages in most of the mammals studied. 3. Exception to this was found in some species of the order Rodentia in which chondroitin 4-sulfate was almost the only chondroitin present in young and adult cartilages. 4. These and other results suggest that chondroitin 4-sulfate may be an important component for the calcification process, whereas chondroitin 6-sulfate seems to be related to the integrity of the articular surfaces.