A dot blot assay for sulfated glycosaminoglycans

A dot blot assay for detection of low amounts of heparin and sulfated glycosaminoglycans (GAGs) is described. The detection range is between 25 ng/ml and 1000 ng/ml of heparin. The assay is based on the interference of sulfated GAGs with the binding of a synthetic ligand (described in this paper) to defined receptors like collagen type V and histones. Ligand binding to type V collagen was suppressed specifically by heparin, but not by other sulfated GAGs like heparin sulfate and chondroitin sulfate. Ligand binding to histones was suppressed most strongly by heparin, but also by chondroitin sulfate. Hyaluronic acid did not interfere.     

Sulfated mucopolysaccharides from normal Swiss 3T3 cell line and its tumorigenic mutant ST1: possible role of chondroitin sulfates in neoplastic transformation.

The sulfated mucopolysaccharide composition of normal Swiss 3T3 cell line and its tumorigenic mutant ST1 is reported. It is shown that chondroitin sulfate B and heparitin sulfate are the sulfated mucopolysaccharides of the normal 3T3 line whereas chondroitin sulfate A and heparitin sulfate are the major ones of the ST1 variant. Degradation of the chondroitin sulfates derived from both cell lines with chondroitinases B and ABC have shown that they contain only 4-sulfated disaccharides differing from each other by the type of uronic acid residue. It is also shown that the chondroitin sulfate A from the tumorigenic variant is mostly located at the cell surface whereas the chondroitin sulfate B from the normal line is less accessible to trypsinization. A relative increase of chondroitin sulfate A was also observed in 3T3 that had lost contact inhibition after successive subcultures, and in the 3T6 cell line. These combined results are in agreement with the earlier proposal that glucuronic acid-containing chondroitin sulfate plays a role in the stimulation of cell division in neoplastic and embryonic tissues.     

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.     

Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts.

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.     

Surface sulfated mucopolysaccharides of primary and permanent mammalian cell lines.

The sulfated mucopolysaccharide composition of the mammalian cell lines: HeLa, H.Ep.2, AV₃, WI-38, BHK and a cell culture of rabbit lung tissue is reported. It is shown that chondroitin sulfate AC and heparitin sulfate are the main mucopolysaccharides of the permanent cell lines whereas chondroitin sulfate B and heparitin sulfate are the major ones in the primary cultures, with no significant change in their relative concentrations up to seven generations. It is also shown that besides heparitin sulfate, chondroitin sulfate AC and chondroitin sulfate B are located at the surface of the cells. These results are in agreement with the earlier proposals that heparitin sulfate and chondroitin sulfate B might play a role in cell recognition and adhesiveness and that chondroitin sulfate AC might act as a stimulant of cell division.     

Tissue specific distribution of sulfated mucopolysaccharides in mammals.

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied. The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.     

Tissue specific distribution of sulfated mucopolysaccharides in mammals

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied.The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.     

Ultrastructural and immunocytochemical study of bone-derived cells cultured in three-dimensional matrices: Influence of chondroitin-4 sulfate on mineralization

Abstract. Bone-derived cells were cultured in three-dimensional reconstituted matrices made of type I collagen or type I collagen chondroitin-4-sulfate. As observed by microscope, their characteristics were as follows: The cells deposited a faint extracellular matrix mainly composed of type I collagen. In the collagen-chondroitin-sulfate sponge fibers, a calcification process, which involved the deposition of hydroxyapatite crystals, was demonstrated. Mineralization occurred only in collagen chondroitin sulfate sponge fibers when seeded with bone-derived cells and was not seen with nonosteogenic cells, such as gingival fibroblasts. Gla protein was intracellularly visualized in both types of sponges seeded with bone-derived cells while an extracellular secretion was seen only in the collagen chondroitin sulfate sponge fibers where calcification occurred. These results suggest that collagen chondroitin sulfate promotes in vitro mineralization of three-dimensional collagen matrices when seeded with bone-derived cells.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of monoclonal antibody 155.8 and partial characterization of its proteoglycan antigen on human melanoma cells

Immunization of mice with a plasma membrane-enriched fraction from human malignant melanoma cells and subsequent generation of hybridomas resulted in the isolation of an IgG1 monoclonal antibody, 155.8, that recognizes chondroitin sulfate proteoglycans. By cell binding analysis, 155.8 was shown to react with seven of eight cultured melanoma cell lines, but not with a variety of lymphoblastoid cell lines or cultured tumor cells derived from other solid tumor types. Indirect immunoprecipitation of the 155.8 antigen from intrinsically labeled melanoma cells revealed a glycoprotein of Mr = 250,000 and a sulfated molecule of Mr greater than 400,000. The antigen was identified as a chondroitin sulfate type A/C proteoglycan synthesized by melanoma cells on the basis of its sensitivity to chondroitinase ABC digestion and the identification of sulfated glycosaminoglycans released from the antigen immunoprecipitated by 155.8. The determinants recognized by antibodies 155.8 and 9.2.27, another anti-chondroitin sulfate proteoglycan, immunoprecipitate only a proteoglycan from high density cesium chloride gradient fractions, (1.487 g/liter); however, they immunoprecipitate a free glycoprotein of Mr = 250,000 from low density fractions (1.317 g/liter). This demonstrated that the 155.8 and 9.2.27 determinants, both of which reside on the glycoprotein of Mr = 250,000, are also present in the proteoglycan, suggesting that this glycoprotein is the proteoglycan core protein. Monoclonal antibody 155.8 reacts with a determinant on the core protein distinct from that recognized by 9.2.27. Proteoglycans bearing 155.8 determinants are distributed on the surface of cultured melanoma cells in a punctated fashion that apparently resolves to short, filamentous structures at high magnification. Immunohistochemical analysis demonstrated that 155.8-defined proteoglycans are found in freshly biopsied melanoma tissue, suggesting that these antigens are also synthesized in vivo by melanoma cells.     

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.     

Regulation of haemopoiesis in long-term bone marrow cultures. IV. Glycosaminoglycan synthesis and the stimulation of haemopoiesis by beta- D-xylosides

Sulfated glycosaminoglycans (GAGs) are distributed in consistent and distinctive patterns between the cell surface and the growth medium of haemopoietically active long-term bone marrow cultures. Heparan sulfate is the main cell surface component and chondroitin sulfate is the major sulfated species in the medium. When the cultures are supplemented with beta-D-xylosides a significant increase in chondroitin sulfate synthesis is observed but no stimulation of heparan sulfate synthesis occurs. The chondroitin sulfate accumulates in the culture medium in beta-D-xyloside-treated cultures but the composition of sulfated GAGs in cell-surface derived material is unaffected. beta-D-xylosides also stimulate the production of haemopoietic cells without any apparent alteration in the adherent stromal cells of the marrow cultures. Equivalent increases are obtained in cells at all stages of development so that a fivefold increase in pluripotent stem cells (CFU-S) is matched by fivefold increase in the granulocyte-macrophage progenitors (GM-CFC) and in mature granulocytes. The stimulation persists for many weeks in beta-D-xyloside-treated cultures. These results indicate that the sulfated GAGs may play an important role in the regulation of haemopoiesis.     

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.     

Characterization of a heparan sulfate and a peculiar chondroitin 4-sulfate proteoglycan from platelets. Inhibition of the aggregation process by platelet chondroitin sulfate proteoglycan

A high molecular weight chondroitin sulfate proteoglycan (Mr 240,000) is released from platelet surface during aggregation induced by several pharmacological agents. Some details on the structure of this compound are reported. beta-Elimination with alkali and borohydride produces chondroitin sulfate chains with a molecular weight of 40,000. The combined results indicate a proteoglycan molecule containing 5-6 chondroitin sulfate chains and a protein core rich in serine and glycine residues. Degradation with chondroitinase AC shows that a 4-sulfated disaccharide is the only disaccharide released from this chondroitin sulfate, characterizing it as a chondroitin 4-sulfate homopolymer. It is shown that this proteoglycan inhibits the aggregation of platelets induced by ADP. Analysis of the sulfated glycosaminoglycans not released during aggregation revealed the presence of a heparan sulfate in the platelets. Degradation by heparitinases I and II yielded the four disaccharide units of heparan sulfates: N,O-disulfated disaccharide, N-sulfated disaccharide, N-acetylated 6-sulfated disaccharide, and N-acetylated disaccharide. The possible role of the sulfated glycosaminoglycans on cell-cell interaction is discussed in view of the present findings.     

Cell recognition and adhesiveness: a possible biological role for the sulfated mucopolysaccharides.

The sulfated mucopolysaccharide composition of different neonate, adult and tumoral tissues is reported. It is shown that each tissue has a characteristic composition with respect to the relative amount, type and molecular size of chondroitin sulfate $\text{A}\text{C}$, chondroitin sulfate B and heparitin sulfate. Neonate and tumor tissues contain large amounts of chondrotin sulfate $\text{A}\text{C}$ which is nearly absent in most adult and normal tissues respectively. Based on these and other results a possible role for the sulfated mucopolysaccharides in cell recognition and adhesiveness is proposed.     

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm²) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm², respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm²). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm² at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

Turnover, change of composition with rate of cell growth and effect of phenylxyloside on synthesis and structure of cell surface sulfated glycosaminoglycans of normal and transformed cells

The synthesis of sulfated glycosaminoglycans was analysed in mouse fibroblasts during the transition from exponential growth to quiescent monolayers. 'Normal' Swiss 3T3 fibroblasts were compared with SV40 transformed 3T3, C6, ST1 and HeLa cells. p-Nitrophenyl-beta-D-xyloside, an artificial acceptor for glycosaminoglycans synthesis, was used as a probe. Exponentially growing 'normal' 3T3 cells synthesized both dermatan sulfate and chondroitin 4-sulfate, retaining the latter and releasing the former to the medium. Upon reaching quiescence these cells switched to retention of dermatan sulfate and release of chondroitin 4-sulfate. SV3T3 cells synthesized several fold less sulfated glycosaminoglycans than 'normal' 3T3. Even though SV3T3 cells are able to synthesize dermatan sulfate, they only retained chondroitin 4-sulfate, never switching to retention of dermatan sulfate. These results indicated that the transition from rapidly proliferating to resting G0 state in normal cells is accompanied by a switch from chondroitin-sulfate rich to dermatan-sulfate-rich cells. This switching was not observed with transformed cells, which are unable to enter the G0 state. Phenylxyloside caused a several fold increase in glycosaminoglycans released to the medium in both cell types, but it did not interfere with either growth rate or cell morphology. Particularly the phenylxyloside treatment led to an increase of more than 10-fold in production of dermatan and chondroitin sulfate by SV3T3, C6, ST1 and HeLa cells. This demonstrated that transformed cells have a high capacity for glycosaminoglycan synthesis. Analysis of enzymatic degradation products of glycosaminoglycans, synthesized in the presence of phenylxyloside, by normal and transformed cells, led to the finding of 4- and 6-sulfated iduronic and glucuronic acid-containing disaccharides. This result indicated that the xyloside causes the synthesis of a peculiar chondroitin sulfate/dermatan sulfate, in both normal and transformed cells.     

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.     

Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.