Biosynthesis Of Heparan Sulfate Proteoglycans Research Articles

Reduced production of sulfated glycosaminoglycans occurs in Zambian children with kwashiorkor but not marasmus

Background:Kwashiorkor, a form of severe malnutrition with high mortality, is characterized by edema and systemic abnormalities. Although extremely common, its pathophysiology remains poorly understood, and its characteristic physical signs are unexplained.Objective: Because kwashiorkor can develop in protein-losing enteropathy, which is caused by a loss of enterocyte heparan sulfate proteoglycan (HSPG), and previous observations suggest abnormal sulfated glycosaminoglycan (GAG) metabolism, we examined whether intestinal GAG and HSPG are abnormal in children with kwashiorkor.Design: Duodenal biopsy samples collected from Zambian children with marasmus (n = 18), marasmic kwashiorkor (n = 8), and kwashiorkor (n = 15) were examined for expression of HSPG, GAGs, and immunologic markers and compared against reference samples from healthy UK control children. GAG and HSPG expression density and inflammatory cell populations were quantitated by computerized analysis.Results: The kwashiorkor group was less wasted and had a lower HIV incidence than did the other groups. All duodenal biopsy samples showed inflammation compared with the histologically uninflamed control samples. Biopsy samples from marasmic children had greater inflammation and greater CD3+ and HLA-DR (human leukocyte antigen DR)–positive cell densities than did samples from children with kwashiorkor. Expression of both HSPG and GAGs was similar between marasmic and well-nourished UK children but was markedly lower in children with kwashiorkor in both the epithelium and lamina propria. Although underglycosylated and undersulfated, epithelial syndecan-1 protein was normally expressed in kwashiorkor, which confirmed that abnormalities arise after core protein synthesis.Conclusions: Intestinal HSPG loss occurs in kwashiorkor, which may precipitate protein-losing enteropathy to cause edema. If occurring systemically, impaired HSPG expression could cause several previously unexplained features of kwashiorkor. We speculate that a genetic predisposition to reduced HSPG biosynthesis may offer a contrasting selective advantage, by both diminishing protein catabolism during transient undernutrition and protecting against specific infectious diseases.

Toutvelu, a Regulator of Heparan Sulfate Proteoglycan Biosynthesis, Controls Guidance Cues for Germ-Cell Migration

The primitive embryonic gonad in Drosophila melanogaster is composed of germ cells and somatic gonadal precursor cells (SGPs). The assembly of a functional gonad involves a complex series of germ-cell migration events, which are thought to be guided by attractive and repulsive cues. Here, we demonstrate a novel role for toutvelu (ttv), a regulator of heparan sulfate proteoglycan biosynthesis during this process. Germline clonal analysis suggests that maternal deposition of ttv is required for proper germ-cell migration. Conversely, ectopic expression of ttv in early embryos results in severe germ-cell migration defects and inappropriate spreading of Hh protein. Moreover, overexpression of ttv in only the receiving cells, rather than in the sending cells, leads to phenotypic consequences. Finally, supporting the claim that the signaling molecule Hedgehog (Hh) may function as a chemoattractant to guide germ cells, errant germ cells are found localized near pockets containing high concentrations of Hh protein.

Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2

FGF-2 is an unconventionally secreted lectin that transmits proangiogenic signals through a ternary complex with high-affinity FGF receptors and heparan sulfate proteoglycans (HSPGs). Although FGF-2 signal transduction is understood in great detail, its mechanism of release from cells, which is independent of the classical secretory pathway, remains elusive. To test the hypothesis that FGF-2 secretion is linked to its cell-surface ligands, we studied FGF-2 release using mutants defective for HSPG binding and cells with impaired HSPG biosynthesis. Here, we report that a functional interaction between FGF-2 and HSPGs is required for net export of FGF-2 from mammalian cells. FGF-2 release requires extracellular, membrane-proximal HSPGs. We propose that extracellular HSPGs form a molecular trap that drives FGF-2 translocation across the plasma membrane.

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Biosynthesis of proteoglycans and glycosaminoglycans in the presence of p-nitrophenyl-xyloside was studied using a primary rat ovarian granulosa cell culture system. Addition of p-nitrophenyl-xyloside into cell culture medium caused about a 700% increase of [³⁵S]sulfate incorporation (ED50 at 0.03 mM) into macromolecules, which included free chondroitin sulfate chains initiated on xyloside and native proteoglycans. Free chondroitin sulfate chains initiated on xyloside were almost exclusively secreted into the medium. The molecular size of chondroitin sulfate chains decreased from 40,000 to 21,000 as the total [³⁵S]sulfate incorporation was enhanced, suggesting that enhanced synthesis of chondroitin sulfate perturbed the normal mechanism of glycosaminoglycan chain termination. Biosynthesis of heparan sulfate proteoglycans was reduced by approximately 50%, likely due to competition at the level of UDP-sugar precursors. [³⁵S]Sulfate incorporation was shut down by the addition of cycloheximide with an initial half time of approximately 2 hr in the presence of xyloside, while that in the absence of xyloside was about 20 min. The difference likely reflects the turnover rate of glycosaminoglycan synthesizing capacity as a whole. The turnover rate of glycosaminoglycan synthesizing capacity observed in ovarian granulosa cells was much shorter than that observed in chondrocytes, reflecting the relative dominance of proteoglycan biosynthetic activity in the total metabolic activity of the cells.

Impact of polymorphisms in the genes encoding xylosyltransferase I and a homologue in type 1 diabetic patients with and without nephropathy

Xylosyltransferase I (XT-I) is the chain-initiating enzyme in the biosynthesis of heparan sulfate proteoglycans (HSPGs). It catalyses the transfer of xylose to specific serine residues in the core protein. The XYLT-II gene codes for a protein highly homologous to the XT-I but its biologic function is not yet known. HSPGs are thought to play an important role in the permeability properties of the glomerular basement membrane (GBM) and thus the xylotransferase genes might be potential candidate genes predisposing to diabetic nephropathy in type 1 diabetic patients. We screened all XYLT-I and XYLT-II exons and flanking intron regions in 96 Caucasians with type 1 diabetes (48 with and 48 without diabetic nephropathy) using denaturing high-performance liquid chromatography (DHPLC). We also studied a nondiabetic control group. Applying this technique we identified 13 variations in XYLT-I and 20 in XYLT-II. The variations IVS6-9T>C and IVS6-14_IVS6-13insG in XYLT-II were found with a frequency of 5.2% (5/96) in nondiabetic nephropathy patients, while all nephropathy patients were negative (P= 0.06). Nondiabetic controls also showed the single nucleotide polymorphisms (SNP) at a frequency of 1.1% (5/440). The investigation of the SNPs' influence on clinical characteristics revealed significant associations for c.1989T>C (XYLT-I) and c.2402C>G (XYLT-II) with patients' blood pressure. We detected in our cohort associations between DNA sequence variations of genes encoding xylosyltransferases and the occurrence of altered clinical characteristics.

Converting Attraction to Repulsion

Growing axons need to follow precise paths to make the proper connections. Kantor et al. investigated the mechanism by which class 5 semaphorins, specifically Sema5A, control migration of neurons of the fasciculus retroflexus (FR), a diencephalic fiber tract of axons from the habenula nucleus (Hb). Sema5A is a transmembrane semaphorin with thrombospondin-1 repeats (TSR) in the extracellular domain. Using diencephalic explants from rats, the authors found that FR axon migration was disrupted by function-blocking antibodies against Sema5A; the axons did not reach their targets and frequently crossed into the adjacent area called prosomere 2. In a membrane stripe assay in which Hb neurons were allowed to grow across cell membranes from HEK 293 cells transfected with either GFP (green fluorescent protein) or Sema5A, the neurons showed a preference for growing across the Sema5A substrate, which suggests that Sema5A acted as an attractive guidance cue. Hb neurons avoided membranes from cells expressing a version of Sema5A that was forced to form pentamers, revealing a repulsive effect of this clustered form of Sema5A. The attractive effect of Sema5A was lost if heparan sulfate proteoglycans (HSPGs) were diminished on the Hb neurons by either heparinase treatment or inhibition of HSPG biosynthesis. Sema5A coprecipitated with the HSPG syndecan-3, suggesting a direct interaction. In a more natural version of the membrane stripe assay using membranes from the prosomere 2 region, which is enriched in Sema5A, the Hb neurons avoided those membranes and function-blocking antibodies against Sema5A blocked this effect, suggesting a repulsive signal. Binding of Sema5A to the prosomere 2 region was not inhibited by heparinase or proteinase K, suggesting that another class of proteoglycan may be important for the repulsive signal. Indeed, binding to the prosomere 2 region was decreased if the tissue was treated with chondroitinase ABC. When plated onto a chondroitin sulfate proteoglycan (CSPG) surface, Hb axons fasciculated into bundles, whereas this was not observed when the neurons were plated on bovine serum albumin. In the membrane stripe assay, coating the filters with CSPG and then membranes from the Sema5A-expressing HEK 293 cells resulted in the Hb neurons avoiding the Sema5A-containing membranes. Thus, in the prosomere region, the presence of CSPG with Sema5A results in a repulsive signal, and in the FR neurons, HSPG and Sema5A are attractive. This may be how Sema5A can contribute to both fasciculation and guidance: the FR neurons fasciculate (attraction to each other mediated by Sema5A and HSPG) and avoid aberrantly crossing the prosomere region (repulsion mediated by Sema5A and CSPG). D. B. Kantor, O. Chivatakarn, K. L. Peer, S. F. Oster, M. Inatani, M. J. Hansen, J. G. Flanagan, Y. Yamaguchi, D. W. Sretavan, R. J. Giger, A. K. Kolodkin, Semaphorin 5A is a bifunctional axon guidance cue regulated by heparan and chondroitin sulfate proteoglycans. Neuron 44 , 961-975 (2004). [Online Journal]

Distinct and collaborative roles ofDrosophilaEXT family proteins in morphogen signalling and gradient formation

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. We have identified and isolated the other two members of the Drosophila EXT family genes, which are named sister of tout-velu (sotv) and brother of tout-velu (botv), and encode Drosophila homologues of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. We show that both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. We demonstrate further that Ttv and Sotv form a complex and are co-localized in vivo. Our results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. Our results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling.

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans.

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, we identified novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of tout-velu (ttv). The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, we found that ttv mutations also affected Dpp and Wg. These data led us to conclude that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. We propose that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients.

Role of heparan sulfate proteoglycans in cell signaling and cancer

Publisher Summary Heparan sulfate proteoglycans (HSPGs) are composed of a protein core modified on specific serine residues by the addition of heparan sulfate (HS) glucosaminoglycans (GAGs) synthesized in the Golgi. Three distinct types of proteins can serve as the HSPG cores: the transmembrane proteins encoded by the syndecan genes, the glycosylphosphatidylinositol (GPI) membrane-bound glypicans, and the extracellular matrix secreted perlecan proteins. The chapter presents the two major advances made in understanding the role of HSPGs in development and cancer. First, studies in Drosophila have identified many mutations in either the biosynthetic enzymes for example, Sugarless (Sgl), Sulfateless (Sfl), Tout velu (Ttv); or the protein cores for example, syndecans and glypicans. Analysis of the mutant phenotypes has revealed the critical roles of HSPGs in modulating various growth factor signaling pathways. Second, many mutations linked to human cancers have been isolated and shown to correspond to defects in the biosynthesis of HSPGs. The chapter explains the importance of HSPGs in cell signaling and provides insights into their functions. Modulating the activity of HSPGs could influence the development of cancers caused by aberrant cell signaling. The chapter concludes that HSPGs constitute important targets for therapeutics to treat some human tumors.

High Molecular Weight Kininogen Utilizes Heparan Sulfate Proteoglycans for Accumulation on Endothelial Cells

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of (125)I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5(H) and that the presence of Zn(2+) promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3. 3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.

Hedgehog Movement Is Regulated through tout velu–Dependent Synthesis of a Heparan Sulfate Proteoglycan

Hedgehog (Hh) molecules play critical roles during development as a morphogen, and therefore their distribution must be regulated. Hh proteins undergo several modifications that tether them to the membrane. We have previously identified tout velu ( ttv), a homolog of the mammalian EXT tumor supressor gene family, as a gene required for movement of Hh. In this paper, we present in vivo evidence that ttv is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, suggesting that HSPGs control Hh distribution. In contrast to mutants in other HSPG biosynthesis genes, the activity of the HSPG-dependent FGF and Wingless signaling pathways are not affected in ttv mutants. This demonstrates an unexpected level of specificity in the regulation of the distribution of extracellular signals by HSPGs.

A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans.

The intracellular amastigote form of leishmania is responsible for the cell-to-cell spread of leishmania infection in the mammalian host. In this report, we identify a high-affinity, heparin-binding activity on the surface of the amastigote form of leishmania. Amastigotes of Leishmania amazonensis bound approximately 120,000 molecules of heparin per cell, with a Kd of 8.8 x 10(-8) M. This heparin-binding activity mediates the adhesion of amastigotes to mammalian cells via heparan sulfate proteoglycans, which are expressed on the surface of mammalian cells. Amastigotes bound efficiently to a variety of adherent cells which express cell-surface proteoglycans. Unlike wild-type CHO cells, which bound amastigotes avidly, CHO cells with genetic deficiencies in heparan sulfate proteoglycan biosynthesis or cells treated with heparitinase failed to bind amastigotes even at high parasite-input dosages. Cells which express normal levels of undersulfated heparan bound amastigotes nearly as efficiently as did wild-type cells. The adhesion of amastigotes to wild-type nonmyeloid cells was almost completely inhibited by the addition of micromolar amounts of soluble heparin or heparan sulfate but not by the addition of other sulfated polysaccharides.l Binding of amastigotes to macrophages, however, was inhibited by only 60% after pretreatment of amastigotes with heparin, suggesting that macrophages have an additional mechanism for recognizing amastigotes. These results suggest that leishmania amastigotes express a high-affinity, heparin-binding activity on their surface which can interact with heparan sulfate proteoglycans on mammalian cells. This interaction may represent an important first step in the invasion of host cells by amastigotes.

Influence of Membrane-Associated Heparan Sulfate on the Internalization of the Small Proteoglycan Decorin

The small dermatan sulfate proteoglycan decorin is efficiently internalized by a variety of cells of mesenchymal origin. Previous studies had implicated the involvement of 51- and 26-kDa receptor proteins in this uptake process. The surface localization of these proteins has now been demonstrated by labeling with a membrane-impermeant, biotinylating reagent. The human keratinocyte cell line HaCaT exhibited only about 5% of the clearance rate of fibroblasts for exogeneously added decorin, although it was not deficient in the 51-and 26-kDa proteins. Evidence is presented that plasma membrane-associated heparan sulfate influences receptor trafficking and contributes to the low internalization rate of the receptors in keratinocytes: (i) Heparitinase digestion of intact keratinocytes led to an ∼10-fold increase in the efficiency of decorin endocytosis. (ii) Endocytosis of decorin was increased more than 10-fold in keratinocytes in the presence of protamine, a cationic, heparan sulfate-binding protein. This effect is considered to be caused by competition between protamine and the endocytosis receptor for cell surface-associated heparan sulfate. (iii) Preincubation of keratinocytes with heparan sulfate-degrading enzymes at 37°C led to a decrease of receptor proteins localized at the cell surface as judged by subsequent surface labeling at 0°C. (iv) An alteration of the biosynthesis of heparan sulfate proteoglycans by p -nitrophenyl-β-xyloside was accompanied by an increased yield of intracellularly located receptor proteins. Plasma membrane-associated heparan sulfate from keratinocytes differed from the corresponding species of fibroblasts in quantity and quality. It is, therefore, suggested that the intracellular trafficking of the decorin receptor proteins is influenced by the amount and/or the composition of membrane-associated heparan sulfate.

Selection of COS cell mutants defective in the biosynthesis of heparan sulfate proteoglycan

A simple procedure using human basic fibroblast growth factor (FGF) was utilized for the selection of COS cell mutants with defects in the biosynthesis or expression of heparan sulfate proteoglycan (HSPG). Our approach was based on the strong binding affinity exhibited by COS cells to human basic FGF that had been adsorbed to plastic dishes. Cell binding to basic FGF could be inhibited by heparin and heparan sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid, suggesting that the cell binding involved an interaction between basic FGF and cell surface heparin-like molecules. COS cells were treated with ethyl methanesulfonate and four stable mutants were subsequently isolated that did not bind strongly to basic FGF adsorbed to plastic. These mutants cell lines (CM-2, CM-8, CM-9, and CM-15) exhibited significantly reduced 35SO 4 incorporation into HS (40–70% depending on the cellular pool analyzed). In one of these cell lines, CM-15, the incorporation of [6- 3H]glucosamine into HS was unaltered, suggesting that the extent of oligosaccharide polymerization was equivalent to that observed for the wild-type cells. Structural analysis revealed that N-sulfated glucosamine residues were present much less frequently in HS derived from these cells as compared with that derived from wild-type cells. Furthermore, CM-15 was found to be three-fold deficient in HS N-sulfotransferase activity, but contained wild-type levels of HS O-sulfotransferase activities. These results support the observation that CM-15 is partially deficient in N-sulfotransferase activity for HS and demonstrates the utility of our selection method to identify mutant cell lines with defects in HSPG biosynthesis.

Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells: Implications for heparan sulfate proteoglycan biosynthesis

Abstract We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.

Modulation of heparan sulfate biosynthesis. Effects of 6-diazo-5-oxo-L-norleucine and low glutamine on the synthesis of heparan sulfate proteoglycan by human colon carcinoma cells.

We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue. Following a 24-h incubation with 100 micrograms/ml DON (0.58 mM) or without L-glutamine, the incorporation of [35S]sulfate was maximally inhibited to about 50%, whereas the incorporation of [3H]leucine or [3H]serine and their specific activity were not significantly affected. Several lines of evidence indicate that the inhibition of [35S]sulfate incorporation was mediated by a reduction in the intracellular pool of UDP-N-acetylhexosamine: the intracellular hexosamine levels were reduced by about 50%; taking into account the changes in specific activity, the incorporation of [3H]glucosamine was also significantly inhibited; and exogenous D-glucosamine (2.3 mM) was capable of substantially reversing the inhibitory effects of DON. This decrease in endogenous sugar supply resulted in the generation of an underglycosylated, lower buoyant density proteoglycan which contained significantly fewer heparan sulfate chains of otherwise normal size and sulfation and even fewer O-linked oligosaccharides. These biochemical changes were corroborated ultrastructurally by the appearance of smaller ruthenium red-stained proteoglycans on the surface of the cells. DON also caused a marked inhibition of cell proliferation and profound morphological changes, both of which were reversible upon culturing in DON-free medium. These results demonstrate that perturbations in glutamine metabolism have profound effects on the structure of heparan sulfate proteoglycan and on the phenotype of human colon carcinoma cells and indicate that DON treatment could be useful for studying post-translational modifications of proteoglycans in various cell systems.

Biosynthesis of heparan sulfate proteoglycans of developing chick breast skeletal muscle in vitro

Previous studies have reported an increase in heparan sulfate glycosaminoglycan (HSGAG) during skeletal muscle differentiation in culture. We have investigated this phenomenon further in relation to the heparan sulfate proteoglycans (HSPG) produced by myogenic cultures. Pulse-chase analysis indicated an approx. 3-fold increase in heparan sulfate synthesis in myotube cultures over that in proliferating or aligning myoblast cultures. Muscle fibroblast culture heparan sulfate synthesis was higher than that of myoblasts but was lower than myotubes. The turnover rates appeared to be the same for all stages of development, with a t 1 2 of approx. 5 h. Enrichment for heparan sulfate by Sepharose CL-4B and DEAE-Sephacel chromatography indicated an increase in the hydrodynamic size of the proteoglycan produced by myotubes over that from myoblasts, with a shift in K av from 0.14–0.19 to 0.07. Fibroblasts synthesized the smallest proteoglycan, with a K av of 0.22. All of the proteoglycans contained similar sized glycosaminoglycan chains with an estimated molecular weight of 30 000–40 000. Localization of the heparan sulfate proteoglycan in myotube cultures by trypsin sensitivity indicated much of the intact proteoglycan to be closely associated with the cell surface, while internalized material appeared in a degraded form.

Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface.

After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.

Stacking faults in steam-oxidizing silicon : M. L. Joshi, Acta Metall.14, No. 10, October (1966), p. 1157.

A simple procedure using human basic fibroblast growth factor (FGF) was utilized for the selection of COS cell mutants with defects in the biosynthesis or expression of heparan sulfate proteoglycan (HSPG). Our approach was based on the strong binding affinity exhibited by COS cells to human basic FGF that had been adsorbed to plastic dishes. Cell binding to basic FGF could be inhibited by heparin and heparan sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid, suggesting that the cell binding involved an interaction between basic FGF and cell surface heparin-like molecules. COS cells were treated with ethyl methanesulfonate and four stable mutants were subsequently isolated that did not bind strongly to basic FGF adsorbed to plastic. These mutants cell lines (CM-2, CM-8, CM-9, and CM-15) exhibited significantly reduced 35SO4 incorporation into HS (40–70% depending on the cellular pool analyzed). In one of these cell lines, CM-15, the incorporation of [6-3H]glucosamine into HS was unaltered, suggesting that the extent of oligosaccharide polymerization was equivalent to that observed for the wild-type cells. Structural analysis revealed that N-sulfated glucosamine residues were present much less frequently in HS derived from these cells as compared with that derived from wild-type cells. Furthermore, CM-15 was found to be three-fold deficient in HS N-sulfotransferase activity, but contained wild-type levels of HS O-sulfotransferase activities. These results support the observation that CM-15 is partially deficient in N-sulfotransferase activity for HS and demonstrates the utility of our selection method to identify mutant cell lines with defects in HSPG biosynthesis.