Luiz-Claudio F. Silva

Is there a glycosaminoglycan‐related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico‐medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non‐sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N‐ and O‐sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT‐76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan‐mediated interactions behave in the thymus. J. Cell. Physiol. 185:68–79, 2000.

Sulfated glycosaminoglycans from ovary of Rhodnius prolixus

We have characterized sulfated glycosaminoglycans from ovaries of the blood-sucking insect Rhodnius prolixus, and determined parameters of their synthesis and distribution within this organ by biochemical and histochemical procedures. The major sulfated glycosaminoglycan is heparan sulfate while chondroitin 4-sulfate is a minor component. These glycosaminoglycans are concentrated in the ovarian tissue and are not found inside the oocytes. Besides this, we detected the presence of a sulfated compound distinguished from sulfated glycosaminoglycans and possibly derived from sulfated proteins. Conversely to the compartmental location of sulfated glycosaminoglycans, the unidentified sulfated compound is located in the ovarian tissue as well as inside the oocytes. Based on these and other findings, the possible roles of ovarian sulfated glycosaminoglycans on the process of oogenesis in these insects are discussed.

THYMIC EPITHELIAL CELLS SYNTHESIZE A HEPARAN SULFATE WITH A HIGHLY SULFATED REGION

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive 35S‐Na2SO4 and 3H‐glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio 35S‐sulfate/3H‐glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin‐like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions. J Cell Physiol 178:51–62, 1999.

Membrane-associated and secreted proteoglycans from a continuous cell line derived from fibrotic schistosomal granulomas.

Proteoglycans were isolated from a continuous murine cell line (GRX) established from fibrotic granulomas induced in mouse liver by schistosomal infection, representative of liver connective tissue cells. The proteoglycans were labelled with 35SO4, extracted by guanidine-HCl + Triton X-100 in the presence of proteinase inhibitors, and purified by anion-exchange, gel-filtration and affinity-column chromatography. The major fractions of cell-associated and secreted proteoglycans are heparan sulfate proteoglycans. Gel-filtration chromatography on Sephacryl S-400 revealed Kav values of 0.20 and 0.30 for the cell-associated and secreted heparan sulfate proteoglycans, respectively. About 50% of the cell-associated heparan sulfate proteoglycans contained hydrophobic regions, as evidenced by their ability to bind to octyl-Sepharose, while only about 20% of secreted proteoglycans bound to this resin. In addition, no proteoglycan was competitively displaced from the cell surface by heparin. Taken together with other reports on proteoglycan synthesis by a variety of cell types in culture, these observations suggest that cell-surface heparan sulfate proteoglycans possibly contain a hydrophobic domain that functions as a membrane anchor in their attachment to cells. Addition of beta-D-xyloside to the cultures greatly enhanced the release of 35S-dermatan sulfate to the medium. Interestingly, dermatan sulfate is the major glycosaminoglycan found in the schistosoma-induced granuloma, from which the GRX cell line is derived. These studies provide the first biochemical description of the proteoglycans produced by a liver connective tissue cell line derived from schistosomal granulomas.

Isolation and purification of chondroitin sulfate.

Publisher Summary This chapter discusses methods for the isolation and purification of chondroitin sulfate (CS). This molecule is a sulfated glycosaminoglycan (GAG) composed of a linear polysaccharide chain with repeating disaccharides of defined structures that usually contain one hexosamine, one hexuronic acid, and a sulfate ester. The tissue CS can be extracted along with other GAG by proteolytic digestion. Once extracted, CS can be purified from other contaminant GAG by precipitation with organic solvents or enzymatic degradation of contaminant GAG species, or also by column chromatography techniques. The most frequently assays for tracking CS isolation typically utilize uronic acid content as a marker. Although a great number of methods are available for the isolation and purification of GAGs, no single set of procedures permit single‐step purification process, and hence different preparative approaches are required to purify a single species of GAG from tissue extracts. Thus, the techniques outlined in the chapter can be used as guidelines for the researchers interested in the specific isolation of CS. The identification and primary structure determination of CS have significantly contributed to elucidate its possible function on mechanisms modulating cell–cell and cell–matrix interactions.

Characterization and distribution of extracellular matrix components and receptors in GH3B6 prolactin cells.

GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50–70 % of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: α5β1 (FN receptor), α6β1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75 %) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

Patterns of sulfated glycosaminoglycan synthesis and accumulation in hepatic granulomas induced by schistosomal infection

We have characterized sulfated glycosaminoglycans of periovular granulomas induced in mouse liver by experimental infection with Schistosoma mansoni and determined parameters of their synthesis and accumulation by metabolic incorporation of 35S. The major component of glycosaminoglycans isolated from granulomas was dermatan sulfate and the minor component was heparan sulfate. A similar proportion was observed among newly synthesized 35S-labeled glycosaminoglycans, with a slight increase in the relative amount of heparan sulfate. Neither qualitative nor quantitative differences were observed between glycosaminoglycans isolated from granulomas of the acute and the chronic phase of the disease. In contrast, collagen content of granulomas increased eightfold during evolution of the disease from the acute to the chronic phase. It may be concluded that different mechanisms control glycosaminoglycan and collagen synthesis in schistosomal granulomas, as well as the ratio between these components in the extracellular matrix. This is consistent with the loose organization of the extracellular matrix in acute inflammatory reactions and its dense organization in the chronic reactions.

Biochemical characterization of heparan sulfate derived from murine hemopoietic stromal cell lines: A bone marrow‐derived cell line S17 and a fetal liver‐derived cell line AFT024

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 [35S]‐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. [35S]‐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. [35S]‐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. J. Cell. Biochem. 87: 160–172, 2002.

Proteoglycans synthesized by the hepatic granulomas isolated from schistosome-infected mice and by the granuloma-derived connective tissue cell lines

Proteoglycans synthesized in vitro by periovular granulomas isolated from livers of schistosome-infected mice were compared with those produced by granuloma-derived cell lines: the primary cell line GR and the permanent cell line GRX. Proteoglycans were metabolically labelled with 35S-sulfate and extracted with 4 M guanidine-HCl containing 2.0% Triton X-100, in the presence of proteinase inhibitors. The radiolabelled proteoglycans were purified and characterized by anion-exchange, gel-filtration and affinity-column chromatography. Heparan sulfate proteoglycans (HS-PGs) and chondroitin sulfate/dermatan sulfate-containing proteoglycans (CS/DS-PGs) were detected in both the culture medium and the cell-associated fractions obtained from GR cells. More than 90% of the cell-associated HS-PG from these cells contained a hydrophobic portion, as evidenced by their ability to bind to octyl-Sepharose. In contrast, among the secreted proteoglycans, it was the CS/DS-PG and not the HS-PG that bound to this resin. The major fractions of cell-associated and secreted proteoglycans from GRX cells were HS-PGs. Similar to HS-PGs from GR cells, 50% of the cell-associated HS-PG bound to octyl-Sepharose, while only 20% of secreted proteoglycans (HS-PGs) bound to this resin. The proteoglycans purified from the whole granuloma were composed mainly of DS-PG, of a size and hydrophobicity similar to the CS/DS-PG from GR cells. Possible correlations among the structure, secretion, distribution and function of proteoglycans in granulomatous reactions are discussed.

Heparan sulfate glycosaminoglycan expression in the intestinal tract and ovary of fully engorged adult females of the cattle tick Boophilus microplus and in their laid eggs.

Departamento de Bioqu´imica Medica, Instituto de Ciˆencias Biomedicas, Centro de Ciˆencias da Saude,Universidade Federal do Rio de Janeiro, 21941-590, Caixa Postal 68041, Rio de Janeiro, BrazilReceived 22 April 2003; received in revised form 30 May 2003; accepted 18 June 2003Keywords: Sulfated glycosaminoglycans; Heparan sulfate; Chondroitin sulfate; Tick; Boophilus microplus