The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum-Golgi complex-trans-Golgi network by the concerted action of several glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucuronosyltransferase I, required for the formation of the linkage region, and GlcNAc N-deacetylase͞N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker ␣-mannosidase II. To study whether any of these enzymes interacted with each other, they were relocated to the endoplasmic reticulum (ER) by replacing their cytoplasmic N-terminal tails with an ER retention signal derived from the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER caused a parallel redistribution of the 2-Osulfotransferase. Transfected epimerase was also located in the ER in a cell mutant lacking the 2-O-sulfotransferase, but moved to the Golgi when the cells were transfected with 2-O-sulfotransferase cDNA. Epimerase activity was depressed in the mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their physical association was required for both epimerase stability and translocation to the Golgi. These findings provide in vivo evidence for the formation of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of heparan sulfate formation.glycosaminoglycans ͉ sulfation ͉ epimerization ͉ enzyme localization ͉ Golgi complex T he biosynthesis of heparan sulfate initiates by the translation of a proteoglycan core protein and the assembly of the so-called linkage region tetrasaccharide on specific serine residues (-GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤1-O-Ser). The chain then polymerizes by the addition of alternating N-acetylglucosamine (GlcNAc␣1,4) and glucuronic acid (GlcA␤1,4) residues. A series of modification reactions takes place simultaneously that involves at least six enzymatic activities: (i) N-deacetylation of a portion of GlcNAc residues, (ii) N-sulfation of the resulting unsubstituted amino groups to form GlcNS units, (iii) 5-epimerization of adjacent D-GlcA residues to form L-iduronic acid (IdoA), (iv) 2-O-sulfation of L-IdoA and more rarely D-GlcA residues, (v) 6-O-sulfation of glucosamine units, and (vi) occasional 3-O-sulfation of glucosamine residues (Fig. 1A). A major question concerns how these enzymes orchestrate the formation of specific oligosaccharide sequences with unique binding properties for ligands (1, 2). Multiple isozymes exist for several of the transferases that differ in substrate specificity and temporal͞spatial expression during developmen...

The lumen of the Golgi apparatus is the subcellular site where galactose is transferred, from UDP-galactose, to the oligosaccharide chains of glycoproteins, glycolipids, and proteoglycans. The nucleotide sugar, which is synthesized in the cytosol, must first be transported into the Golgi lumen by a specific UDP-galactose transporter. Previously, a mutant polarized epithelial cell (MDCKII-RCA r ) with a 2% residual rate of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., RodriguezBoulan, E., and Simons, K. (1988) J. Biol. Chem. 263, 16283-16290). The mutant has an enrichment in glucosyl ceramide and cell surface glycoconjugates bearing terminal N-acetylglucosamine, as well as a 75% reduction in sialylation of cell surface glycoproteins and glycosphingolipids.We have now studied the biosynthesis of galactose containing proteoglycans in this mutant and the corresponding parental cell line. Wild-type Madin-Darby canine kidney cells synthesize significant amounts of chondroitin sulfate, heparan sulfate, and keratan sulfate, while the above mutant synthesizes chondroitin sulfate and heparan sulfate but not keratan sulfate, the only proteoglycan containing galactose in its glycosaminoglycan polymer. The mutant also synthesizes chondroitin 6-sulfate rather than only chondroitin 4-sulfate as wild-type cells. Together, the above results demonstrate that the Golgi membrane UDP-galactose transporter is rate-limiting in the supply of UDP-galactose into the Golgi lumen; this in turn results in selective galactosylation of macromolecules. Apparently, the K m for galactosyltransferases involved in the synthesis of linkage regions of heparan sulfate and chondroitin sulfate are significantly lower than those participating in the synthesis of keratan sulfate polymer, glycoproteins, and glycolipids. The results also suggest that the 6-Osulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate.Proteoglycans are complex macromolecules consisting of a protein core to which glycosaminoglycans are covalently linked. Their strategic localization in the plasma membrane and extracellular matrix makes them important intermediates between cells and their environment (1, 2). They have been implicated to play a role in cell-cell (3) and cell-matrix interactions (4), organization of basement membranes (5), control of macromolecules' diffusion (6), and also interactions with a variety of ligands such as growth factors, hormones, and neurotransmitters (7).In most GAGs, 1 the repeating disaccharide units are composed of one amino sugar and one uronic acid, the only exception being keratan sulfate in which galactose replaces the sugar acid. Most GAGs are attached to serine of the core protein by a tetrasaccharide of xylose-galactose-galactose-glucuronic acid (8, 9). Keratan sulfate is an exception; keratan sulfate I, from cornea, is N-linked to proteins and keratan sulfate II, from skeletal tis...

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion of D-glucuronic acid and L-iduronic acid residues encodes a truncated protein. Genome analysis and 5-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogaster and Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the fulllength enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.Heparan sulfate proteoglycans are located on the cell surface and in the extracellular matrix, where they play important roles in cell adhesion, differentiation, and growth in vitro and in vivo (1-3). To a large extent, these biological activities depend on the heparan sulfate chains attached to the core protein. Heparan sulfate, a type of glycosaminoglycan, initially assembles by the copolymerization of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA). The backbone then undergoes extensive modification initiated by the N-deacetylation and N-sulfation of subsets of GlcNAc residues. Subsequently, D-GlcA residues adjacent to the N-sulfated sugars are converted to L-IdoUA 1 by a C5-epimerase and are sulfated at C-2 by a specific sulfotransferase. The glucosamine units also can be sulfated at C-6 and to a lesser extent at C-3. The blocklike arrangement of the modified residues confers specific binding properties to the chains for protein ligands, which in turn facilitate various biological activities. Many of the enzymes involved in heparan sulfate and heparin formation seem to be members of multienzyme gene families. Two exceptions are the C5-epimerase that interconverts D-GlcA and L-IdoUA and the 2-O-sulfotransferase that adds sulfate to C-2 of IdoUA residues and to a lesser extent GlcA residues. The C5-epimerase has been partially purified from mouse mastocytoma (4) and purified to homogeneity from bovine liver (5). A bovine cDNA for the epimerase has been cloned as well (6). Kinetic studies have clarified the substrate specificity of the epimerase, showing that the enzyme will react with both D...

A methodology with high sensitivity and specificity is proposed with a cutoff value for HPA2 and Syn-1 in the immunohistochemistry assay to define the presence of tumor. It was demonstrated for the first time in the literature that HPA2 is over-expressed in colorectal carcinoma tissues compared with nonneoplastic tissues. HPA2 over-expression could be possibly related to Syn-1 shedding despite the fact that HPA2 does not present enzymatic activity as HPA1.

Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonization of distant organs. Cell adhesion plays an important role in neoplastic transformation. Tumors produce several molecules that facilitate their proliferation, invasion and maintenance, especially proteoglycans. The syndecan-4, a heparan sulfate proteoglycan, can act as a co-receptor of growth factors and proteins of the extracellular matrix by increasing the affinity of adhesion molecules to their specific receptors. It participates together with integrins in cell adhesion at focal contacts connecting the extracellular matrix to the cytoskeleton. Changes in the expression of syndecan-4 have been observed in tumor cells, indicating its involvement in cancer. This study investigates the role of syndecan-4 in the process of anoikis and cell transformation. Endothelial cells were submitted to sequential cycles of forced anchorage impediment and distinct lineages were obtained. Anoikis-resistant endothelial cells display morphological alterations, high rate of proliferation, poor adhesion to fibronectin, laminin and collagen IV and deregulation of the cell cycle, becoming less serum-dependent. Furthermore, anoikis-resistant cell lines display a high invasive potential and a low rate of apoptosis. This is accompanied by an increase in the levels of heparan sulfate and chondroitin sulfate as well as by changes in the expression of syndecan-4 and heparanase. These results indicate that syndecan-4 plays a important role in acquisition of anoikis resistance and that the conferral of anoikis resistance may suffice to transform endothelial cells.

MB-PDT is a cheap and efficient method of decreasing MM volume and thus disease progression. This reduction is mediated by downregulation of PCNA and heparanases.

BackgroundTrastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components—heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)—in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab.MethodsThe cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). The profile of sulfated glycosaminoglycans was also investigated by [35S]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate.ResultsBreast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. The addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. In addition, HS shedding was increased significantly in these resistant cells.ConclusionTrastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. In addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.