Glycosaminoglycan Assembly Research Articles

Genome‐wide Screening Uncovers Transforming Growth Factor‐β3 as Regulator of Glycosaminoglycan Assembly

Heparan sulfate (HS) and chondroitin sulfate (CS) are glycosaminoglycans covalently linked to proteoglycans (PG) present on all cell surfaces and in the extracellular matrix. They play a complex role in carcinogenesis as they are important for extracellular matrix remodeling as well as cell‐cell and cell‐matrix interactions. These processes are altered during malignancy, when tumor cells invade and systemically spread in different tissues. We have developed a genome wide screening method using CRISPR‐Cas9 gene targeting to identify novel genes involved in glycosaminoglycan metabolism. The approach utilizes agents that bind specifically to cell surface HS. One of the top hits was transforming growth factor‐β3 (TGF‐β3), a growth factor that controls multiple genes with tumor‐suppressing and tumor‐promoting effects. Cell surface levels of PG were evaluated in A375 melanoma cells after treatment with soluble TGF‐β1 or TGF‐β3 in the presence or absence of a TGF‐β type I receptor inhibitor (SB‐431542) using specific agents that bind to glycosaminoglycans, including mAbs 10E4 or CS56, guanidinylated neomycin and fibroblast growth factor‐2 (FGF2). Both TGF‐β isoforms stimulated cell surface HSPG and CSPG expression, that was blocked by SB‐431542. Inactivation of TGF‐β3 expression by CRISPR/Cas9 had the opposite effect. These findings show that TGF‐β3 is a newly identified regulatory factor of PG assembly in melanoma cells and that tonic expression of TGF‐β3 regulates PG production in a cell autonomous manner. Further exploration how TGF‐β affects disorders associated with altered PG formation in cancer might lead to innovative solutions to alter tumor cell growth and metastasis.Support or Funding InformationWe acknowledge financial support by the DFG (research fellowship, project number 420160411) and the NIH (fellowship award, K12HL141956).

Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos

Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the ext2 gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.

Aglycone Ebselen and β-d-Xyloside Primed Glycosaminoglycans Co-contribute to Ebselen β-d-Xyloside-Induced Cytotoxicity.

Most β-d-xylosides with hydrophobic aglycones are nontoxic primers for glycosaminoglycan assembly in animal cells. However, when Ebselen was conjugated to d-xylose, d-glucose, d-galactose, and d-lactose (8A-D), only Ebselen β-d-xyloside (8A) showed significant cytotoxicity in human cancer cells. The following facts indicated that the aglycone Ebselen and β-d-xyloside primed glycosaminoglycans co-contributed to the observed cytotoxicity: 1. Ebselen induced S phase cell cycle arrest, whereas 8A induced G2/M cell cycle arrest; 2. 8A augmented early and late phase cancer cell apoptosis significantly compared to that of Ebselen and 8B-D; 3. Both 8A and phenyl-β-d-xyloside primed glycosaminoglycans with similar disaccharide compositions in CHO-pgsA745 cells; 4. Glycosaminoglycans could be detected inside of cells only when treated with 8A, indicating Ebselen contributed to the unique property of intracellular localization of the primed glycosaminoglycans. Thus, 8A represents a lead compound for the development of novel antitumor strategy by targeting glycosaminoglycans.

Inactivation of Fam20B in the dental epithelium of mice leads to supernumerary incisors.

Tooth formation is tightly regulated by epithelial-mesenchymal interactions via hierarchic cascades of signaling molecules. The glycosaminoglycan (GAG) chains covalently attached to the core protein of proteoglycans (PGs) provide docking sites for signaling molecules and their receptors during the morphogenesis of tissues and organs. Although PGs are believed to play important roles in tooth formation, little is known about their exact functions in this developmental process and the relevant molecular basis. Family with sequence similarity member 20-B (FAM20B) is a newly identified kinase that phosphorylates the xylose in the common linkage region connecting the GAG with the protein core of PGs. The phosphorylation of xylose is essential for elongation of the common linkage region and the subsequent GAG assembly. In this study, we generated a Fam20B-floxed allele in mice and found that inactivating Fam20B in the dental epithelium leads to supernumerary maxillary and mandibular incisors. This finding highlights the pivotal role of PGs in tooth morphogenesis and opens a new window for understanding the regulatory mechanism of PG-mediated signaling cascades during tooth formation.

Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo

Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to- and pedicel organization on- the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1-/-) was developed, having podocyte-specific deletion of NDST1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1-/- mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate initiation: Impact of sulfation on activity and specificity

Glycosaminoglycan (GAG) assembly initiates through the formation of a linkage tetrasaccharide region serving as a primer for both chondroitin sulfate (CS) and heparan sulfate (HS) chain polymerization. A possible role for sulfation of the linkage structure and of the constitutive disaccharide unit of CS chains in the regulation of CS-GAG chain synthesis has been suggested. To investigate this, we determined whether sulfate substitution of galactose (Gal) residues of the linkage region or of N-acetylgalactosamine (GalNAc) of the disaccharide unit influences activity and specificity of chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1), a key glycosyltransferase of CS biosynthesis. We synthesized a series of sulfated and unsulfated analogs of the linkage oligosaccharide and of the constitutive unit of CS and tested these molecules as potential acceptor substrates for the recombinant human CSGalNAcT-1. We show here that sulfation at C4 or C6 of the Gal residues markedly influences CSGalNAcT-1 initiation activity and catalytic efficiency. Kinetic analysis indicates that CSGalNAcT-1 exhibited 3.6-, 1.6-, and 2.2-fold higher enzymatic efficiency due to lower K(m) values toward monosulfated trisaccharides substituted at C4 or C6 position of Gal1, and at C6 of Gal2, respectively, compared with the unsulfated oligosaccharide. This highlights the critical influence of Gal substitution on both CSGalNAcT-1 activity and specifity. No GalNAcT activity was detected toward sulfated and unsulfated analogs of the CS constitutive disaccharide (GlcA-β1,3-GalNAc), indicating that CSGalNAcT-1 was involved in initiation but not in elongation of CS chains. Our results strongly suggest that sulfation of the linkage region acts as a regulatory signal in CS chain initiation.

Podocytes require the engagement of cell surface heparan sulfate proteoglycans for adhesion to extracellular matrices

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.

Loss of heparan sulfate glycosaminoglycan assembly in podocytes does not lead to proteinuria

Podocytes synthesize the majority of the glomerular basement membrane components with some contribution from the glomerular capillary endothelial cells. The anionic charge of heparan sulfate proteoglycans is conferred by covalently attached heparan sulfate glycosaminoglycans and these are thought to provide critical charge selectivity to the glomerular basement membrane for ultrafiltration. One key component in herparan sulfate glycosaminoglycan assembly is the Ext1 gene product encoding a subunit of heparan sulfate co-polymerase. Here we knocked out Ext1 gene expression in podocytes halting polymerization of heparin sulfate glycosaminoglycans on the proteoglycan core proteins secreted by podocytes. Glomerular development occurred normally in these knockout animals but changes in podocyte morphology, such as foot process effacement, were seen as early as 1 month after birth. Immunohistochemical analysis showed a significant decrease in heparan sulfate glycosaminoglycans confirmed by ultrastructural studies using polyethyleneimine staining. Despite podocyte abnormalities and loss of heparan sulfate glycosaminoglycans, severe albuminuria did not develop in the knockout mice. We show that the presence of podocyte-secreted heparan sulfate glycosaminoglycans is not absolutely necessary to limit albuminuria suggesting the existence of other mechanisms that limit albuminuria. Heparan sulfate glycosaminoglycans appear to have functions that control podocyte behavior rather than be primarily an ultrafiltration barrier.

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.

Temperature-sensitive Glycosaminoglycan Biosynthesis in a Chinese Hamster Ovary Cell Mutant Containing a Point Mutation in Glucuronyltransferase I

In previous studies, we reported the isolation and characterization of a Chinese hamster ovary cell mutant (pgsG) defective in glucuronyltransferase I (GlcATI). This enzyme adds the terminal GlcA residue in the core protein-linkage tetrasaccharide (GlcAbeta1,3Galbeta1,3Galbeta1, 4Xylbeta-O-) on which glycosaminoglycan assembly occurs (Bai, X. M., Wei, G., Sinha, A., and Esko, J. D. (1999) J. Biol. Chem. 274, 13017-13024; Wei, G., Bai, X. M., Sarkar, A. K., and Esko, J. D. (1999) J. Biol. Chem. 274, 7857-7864). Here we show that incorporation of 35SO4 into glycosaminoglycans in the mutant is temperature-sensitive, with greater synthesis occurring at 33 degrees C compared with 37 degrees C. Wild-type cells show the opposite thermal dependence. Rabbit antiserum to hamster GlcATI failed to detect cross-reactive material in pgsG cells by immunofluorescence and Western blotting. Furthermore, expression of chimeric proteins composed of mutant GlcATI fused to IgG binding domain of protein A or to green fluorescent protein did not yield the proteins at the expected mass. The green fluorescent protein-tagged version appeared as a truncated protein, and immunofluorescence showed large perinuclear bodies at 30 degrees C. At 37 degrees C, the fusion protein was not readily detectable. Sequencing cDNAs from mutant and wild-type cells revealed a single base transition (G331A) in the open reading frame in pgsG cells, which resulted in a Val-111-->Met substitution. These data suggest that pgsG cells contain a labile form of GlcATI that causes conditional expression of glycosaminoglycans dependent on temperature.

Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I

The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAbeta1, 3Galbeta1,3Galbeta1,4Xylbeta-O-, attached to specific serine residues in the core protein. Studies of Chinese hamster ovary cell mutants defective in the first or second enzymes of the pathway (xylosyltransferase and galactosyltransferase I) show that the assembly of the primer occurs by sequential transfer of single monosaccharide residues from the corresponding high energy nucleotide sugar donor to the non-reducing end of the growing chain. In order to study the other reactions involved in linkage tetrasaccharide assembly, we have devised a powerful selection method based on induced resistance to a mitotoxin composed of basic fibroblast growth factor-saporin. One class of mutants does not incorporate 35SO4 and [6-3H]GlcN into glycosaminoglycan chains. Incubation of these cells with naphthol-beta-D-xyloside (Xylbeta-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galbeta1, 4Xylbeta-O-Np and Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) and sialic acid (Siaalpha2,3Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galbeta1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galbeta1,3Galbeta-O-naphthalenemethanol (<0.2 versus 3.6 pmol/min/mg). The mutation most likely lies in the structural gene encoding GlcAT-I since transfection of the mutant with a cDNA for GlcAT-I completely restored enzyme activity and glycosaminoglycan synthesis. These findings suggest that a single GlcAT effects the biosynthesis of common linkage region of both heparan sulfate and chondroitin sulfate in Chinese hamster ovary cells.

Heparan Sulfate Composition of Alternatively Spliced CD44 Fusion Proteins

Prior analyses of recombinant CD44 fusion proteins have indicated that combinatorial splicing of variant exons exerts distal effects on chondroitin sulfate content and structure, which may regulate the biological properties of the respective CD44 isoforms. The consequences of splicing of variant exons V4-7 on the heparan sulfate moieties were therefore examined, utilizing recombinant chimeras containing exons V3 and V8-10, engineered with or without exons V4-7 and expressed as Ig fusion proteins in COS cells. Splicing of exons V4-7, though they contain no consensus motifs for glycosaminoglycan assembly, resulted in markedly increased polymer sulfation levels of the heparan sulfates. The sulfate groups of both the CD44 V3-10 and V3,8-10 isoforms occurred as di- and tri-sulfated dissacharide units and were restricted to one N-sulfated block domain within the polymers. Compared to native human keratinocyte CD44, the recombinant heparan sulfates were relatively low in sulfate content. Our data indicate that variant exon V4-7 splicing exerts distal effects on the composition of this glycosaminoglycan. These effects may regulate those functions that are mediated through the heparan sulfate moieties, such as the binding of growth factors.

Formation of HNK-1 Determinants and the Glycosaminoglycan Tetrasaccharide Linkage Region by UDP-GlcUA:Galactose β1,3-Glucuronosyltransferases

While expression-cloning enzymes involved in heparan sulfate biosynthesis, we isolated a cDNA that encodes a protein 65% identical to the UDP-GlcUA:glycoprotein beta1, 3-glucuronosyltransferase (GlcUAT-P) involved in forming HNK-1 carbohydrate epitopes (3OSO3GlcUAbeta1,3Gal-) on glycoproteins. The cDNA contains an open reading frame coding for a protein of 335 amino acids with a predicted type II transmembrane protein orientation. Cotransfection of the cDNA with HNK-1 3-O-sulfotransferase produced HNK-1 carbohydrate epitopes in Chinese hamster ovary (CHO) cells and COS-7 cells. In vitro, a soluble recombinant form of the enzyme transferred GlcUA in beta-linkage to Galbeta1,3/4GlcNAcbeta-O-naphthalenemethanol, which resembles the core oligosaccharide on which the HNK-1 epitope is assembled. However, the enzyme greatly preferred Galbeta1, 3Galbeta-O-naphthalenemethanol, a disaccharide component found in the linkage region tetrasaccharide in chondroitin sulfate and heparan sulfate. During the course of this study, a human cDNA clone was described that was thought to encode UDP-GlcUA:Galbeta1,3Gal-R glucuronosyltransferase (GlcUAT-I), involved in the formation of the linkage region of glycosaminoglycans (Kitagawa, H., Tone, Y., Tamura, J., Neumann, K. W., Ogawa, T., Oka, S., Kawasaki, T., and Sugahara, K. (1998) J. Biol. Chem. 273, 6615-6618). The deduced amino acid sequences of the CHO and human cDNAs are 95% identical, suggesting that they are in fact homologues of the same gene. Transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDNA restored glycosaminoglycan assembly in vivo, confirming its identity. Interestingly, transfection of the mutant with GlcUAT-P also restored glycosaminoglycan synthesis. Thus, both GlcUAT-P and GlcUAT-I have overlapping substrate specificities. However, the expression of the two genes was entirely different, with GlcUAT-I expressed in all tissues tested and GlcUAT-P expressed only in brain. These findings suggest that, in neural tissues, GlcUAT-P may participate in both HNK-1 and glycosaminoglycan production.

Primers of Glycosaminoglycan Biosynthesis from Peruvian Rain Forest Plants

We have developed a rapid, high throughput screening assay for compounds that alter the assembly of glycosaminoglycan chains in Chinese hamster ovary cells. The assay uses autoradiography to measure the binding of newly synthesized [35S]proteoglycans and [35S]glycosaminoglycans to a positively charged membrane. Screening over 1000 extracts from a random plant collection obtained from the Amazon rain forest yielded five plants that stimulated glycosaminoglycan assembly in both wild-type cells and a mutant cell line defective in xylosyltransferase (the first committed enzyme involved in glycosaminoglycan biosynthesis). Fractionation of an extract of Maieta guianensis by silica gel and reverse-phase chromatography yielded two pure compounds with stimulatory activity. Spectroscopic analysis by NMR and mass spectrometry revealed that the active principles were xylosides of dimethylated ellagic acid. One of the compounds also contained a galloyl group at C-3 of the xylose moiety. These findings suggest that plants and other natural products may be a source of agents that can potentially alter glycosaminoglycan and proteoglycan formation in animal cells.

Isolation and characterization of an endodermally derived, proteoglycan-like extracellular matrix molecule that may be involved in larval starfish digestive tract morphogenesis.

A monoclonal antibody, anti-Pisaster matrix-1 (anti-PM1) has been developed against an extracellular matrix antigen, Pisaster matrix-1 (PM1) found in embryos and larvae of the starfish Pisaster ochraceus. Pisaster matrix-1 was first observed in endodermal cells of the early gastrula, and shortly thereafter it was secreted into the blastocoel where it accumulated steadily during gastrulation. During the late gastrula stage it also appeared in the extracellular matrix (ECM) of the gut lumen. Immunogold electron microscopy with anti-PM1 revealed that PM1 was found in condensations of ECM associated with blastocoel matrix fibers, in the trans Golgi network, in Golgi-associated vesicles in endoderm and mesenchyme cells and throughout the ECM lining the digestive tract of late gastrula and bipinnaria larvae. When blastula or early gastrula stage embryos were grown in the presence of the PM1 antibody, archenteron elongation, bending and mouth formation failed to occur. Pisaster matrix-1 stained with alcian blue and its assembly could be disrupted with the common inhibitor of O-linked glycosaminoglycan assembly, beta-xyloside but not by tunicamycin. It was not sensitive to enzymes that degrade vertebrate proteoglycans. Pisaster matrix-1 is a large (600 kDa) proteoglycan-like glycosaminoglycan, secreted exclusively by endodermal and/or endodermally derived cells that may be necessary for morphogenesis of the mouth and digestive tract of Pisaster ochraceus embryos/larvae.

Influence of core protein sequence on glycosaminoglycan assembly

Recent studies have revealed a correlation between amino acid sequences around glycosylation sites in proteoglycans and the ability of cells to initiate and process glycosaminoglycan chains. Initiation depends on Ser—Gly/Ala dipeptides that have one or more acidic amino acids in close proximity. The formation of heparan sulfate chains depends on a nearby cluster of acidic residues, hydrophobic amino acids, and the close spacing of glycosylation sites.

Heparan sulfate: a piece of information.

The sulfated glycosaminoglycans, heparan sulfate and heparin, are increasingly implicated in cell-biological processes such as cytokine action, cell adhesion, and regulation of enzymic catalysis. These activities generally depend on interactions of the polysaccharides with proteins, mediated by distinct saccharide sequences, and expressed at various levels of specificity, selectivity, and molecular organization. The formation of heparin/ heparan sulfate in the cell requires an elaborate biosynthetic machinery, that is conceived in terms of a novel model of glycosaminoglycan assembly and processive modification. Recent advances in the identification and molecular analysis of the enzymes and other proteins involved in the biosynthesis provide novel tools to study the regulation of the process, presently poorly understood, at the subcellular and cellular levels. The potential medical importance of heparin-related compounds is likely to promote the biotechnological exploitation of components of the biosynthetic machinery.

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.

Recycling of a glycosylphosphatidylinositol-anchored heparan sulphate proteoglycan (glypican) in skin fibroblasts.

We have used suramin and brefeldin A to investigate the nature of a heparan sulphate proteoglycan that appears to recycle from the cell surface to intracellular compartments which synthesize new heparan sulphate chains. Suramin, which would block internalization and deglycanation of a putative recycling cell surface proteoglycan, markedly increases the yield of a membrane-bound proteoglycan with a core protein of 60-70 kDa and unusually long heparan sulphate side chains. When transport of newly made core proteins to their Golgi sites for glycosaminoglycan assembly is blocked, by using brefeldin A, [3H]glucosamine and [35S]sulphate incorporation into cell surface-bound heparan sulphate proteoglycan can still take place. After chemical biotinylation of cell surface proteins in brefeldin A-treated cells, followed by metabolic [35S]sulphation in the presence of the same drug, biotin-tagged [35S]proteoglycan can be demonstrated, indicating the presence of recycling proteoglycan species. By pre-labelling cells with [3H]leucine or [3H]inositol in the presence of suramin, followed by chase labelling with [35S]sulphate in the presence of brefeldin A, a 3H- and 35S-labelled, hydrophobic heparan sulphate proteoglycan with a core protein of 60-65 kDa is obtained. The proteoglycan loses its hydrophobicity when glucosamine-inositol bonds are cleaved, indicating that it is membrane bound via a glycosylphosphatidylinositol anchor. However, treatment with phosphatidylinositol-specific phospholipase C has no effect, suggesting that the inositol moiety may be acylated. We propose that a portion of the lipid-anchored proteoglycan glypican is internalized, recycled via the Golgi, where heparan sulphate chains are added, and finally re-deposited at the cell surface.

Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts

(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl β- d-xylopyranoside (and [ 3H]galactose) or 125I-labelled p-hydroxyphenyl β- d-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception: Cell-associated heparan sulphate-proteoglycan was still produced at 20% of the control level. (3) Treatment with suramin or heparin resulted in decreased deposition of proteoglycan in the pericellular matrix but increased accumulation of cell-associated proteoglycan. Primaquine blocked all proteoglycan synthesis. (4) In the presence of cycloheximide, exogenous β- d-xyloside initiated galactosaminoglycan production. In contrast, in brefeldin A-treated cells, synthesis was completely abolished. Not even formation of the linkage-region trisaccharide could be detected. (5) These results suggest that exogenous xyloside enters the endoplasmic reticulum and is subsequently transported to the trans-Golgi complex where all further steps involved in glycosaminoglycan assembly takes place. (6) Heparan sulphate proteoglycan produced by brefeldin A-treated cells could be derived from (a) an intracellular pool of preformed core protein located to the trans-Golgi complex, or (b) resident proteoglycan that was either deglycanated/reglycanated or chain-extended. As combined treatment with suramin and brefeldin A markedly reduced cell-associated proteoglycan production, the latter possibility is favoured.