A mutated glycosaminoglycan-binding domain functions as a novel probe to selectively target heparin-like epitopes on tumor cells.

The high heterogeneity and mutation rate of cancer cells often lead to the failure of targeted therapy, and therefore, new targets for multitarget therapy of tumors are urgently needed. Aberrantly expressed glycosaminoglycans (GAGs) have been shown to be involved in tumorigenesis and are promising new targets. Recently, the GAG-binding domain rVAR2 of the Plasmodium falciparum VAR2CSA protein was identified as a probe targeting cancer-associated chondroitin sulfate A-like epitopes. In this study, we found that rVAR2 could also bind to heparin (Hep) and chondroitin sulfate E. Therefore, we used rVAR2 as a model to establish a method based on random mutagenesis of the GAG-binding protein and phage display to identify and optimize probes targeting tumor GAGs. We identified a new probe, VAR2HP, which selectively recognized Hep by interacting with unique epitopes consisting of a decasaccharide structure that contains at least three HexA2S(1-4)GlcNS6S disaccharides. Moreover, we found that these Hep-like epitopes were overexpressed in various cancer cells. Most importantly, our in vivo experiments showed that VAR2HP had good biocompatibility and preferentially localizes to tumors, which indicates that VAR2HP has great application potential in tumor diagnosis and targeted therapy. In conclusion, this study provides a strategy for the discovery of novel tumor-associated GAG epitopes and their specific probes.

The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility.

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80-85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.

Rational design of a highly efficient catalytic system for the production of PAPS from ATP and its application in the synthesis of chondroitin sulfate.

The compound 3'-phosphoadenosine-5'-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of adenosine triphosphate (ATP) (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided "ADP expulsion" strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme's flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1 ) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor, and achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. Then, the PAPS catalytic system was combined with the chondroitin 4-sulfotransferase using a one-pot method. Finally, chondroitin sulfate was transformed from chondroitin at a conversion rate of 98.75%. This strategy has great potential for scale biosynthesis of PAPS and chondroitin sulfate.

Screening and surveillance of multiple solid tumours using plasma placental-like chondroitin sulfate A (pl-CSA).

Rationale: Placental-like chondroitin sulfate A (pl-CSA) is known to be exclusively synthesized in multiple cancer tissues and associated with disease severity. Here, we aimed to assess whether pl-CSA is released into bio-fluids and can serve as a cancer biomarker. Methods: A novel ELISA was developed to analyse pl-CSA content in bio-fluids using pl-CSA binding protein and an anti-pl-CSA antibody. Immunohistochemical staining of tissue chips was used as the gold standard control. Results: The developed ELISA method was specific and sensitive (1.22 µg/ml). The pl-CSA content was significantly higher in lysates and supernatants of cancer cell lines than in those of normal cell lines, in plasma from mouse cancer models than in that from control mice, and in plasma from patients with oesophageal, cervical, ovarian, or lung cancer than in that from healthy controls. Similar to the tissue chip analysis, which showed a significant difference in pl-CSA positivity between cancer tissues and normal adjacent tissues, the plasma pl-CSA analysis had 100% sensitivity and specificity for differentiating oesophageal and lung cancer patients from healthy controls. Importantly, in oesophageal and lung cancer patients, the pl-CSA content was significantly higher in late-stage disease than in early-stage disease, and it dramatically decreased after surgical resection of the tumour. Conclusion: These data indicate a direct link between plasma pl-CSA content and tumour presence, indicating that plasma pl-CSA may be a non-invasive biomarker with clinical applicability for the screening and surveillance of patients with multiple types of solid tumours.

Expression and function of chondroitin 4-sulfate and chondroitin 6-sulfate in human glioma.

Key molecules promoting migration and invasion exist in the extracellular matrix, and include chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), functionally important carbohydrate chains of chondroitin sulfate proteoglycans that participate in regulating cancer development. Here, we show that C4S and C6S expression is upregulated in human glioma tissues, when compared to normal brain tissue, and that the extent of upregulation positively correlated with glioma malignancy. Treatment of cultured glioma cells with C4S and C6S enhanced cell viability, migration, and invasion, increased MMP-2 and MMP-9 levels, enhanced N-cadherin, but reduced E-cadherin expression. Inhibition of expression of the two CS synthetic enzymes chondroitin 4-O-sulfotransferase-1 (C4ST-1/CHST11) and chondroitin 6-O-sulfotransferase-1 (C6ST-1/CHST3) suppressed cell viability, migration and invasion, reduced MMP-2 and MMP-9 expression, and reduced N-cadherin expression, but increased E-cadherin levels. The C4S- and C6S-enhanced epithelial-to-mesenchymal transition and expression of MMP-2 occurred via activation of the PI3K/AKT signaling pathway, known to be involved in promoting cell migration and invasion. In immune-deficient larval zebrafish, C4S and C6S increased the numbers of viable tumor cells, thereby promoting glioma cell proliferation. The present observations point to a novel role of C4S and C6S in human glioma cell functions, thus possibly representing targets in glioma therapy.

NMR and molecular modeling reveal specificity of the interactions between CXCL14 and glycosaminoglycans.

CXCL14, chemokine (C-X-C motif) ligand 14, is a novel highly conserved chemokine with unique features. Despite exhibiting the typical chemokine fold, it has a very short N-terminus of just two amino acid residues responsible for chemokine receptor activation. CXCL14 actively participates in homeostatic immune surveillance of skin and mucosae, is linked to metabolic disorders and fibrotic lung diseases and possesses strong anti-angiogenic properties in early tumor development. In this work, we investigated the interaction of CXCL14 with various glycosaminoglycans (GAGs) by nuclear magnetic resonance spectroscopy, microscale thermophoresis, analytical heparin (HE) affinity chromatography and in silico approaches to understand the molecular basis of GAG-binding. We observed different GAG-binding modes specific for the GAG type used in the study. In particular, the CXCL14 epitope for HE suggests a binding pose distinguishable from the ones of the other GAGs investigated (hyaluronic acid, chondroitin sulfate-A/C, -D, dermatan sulfate). This observation is also supported by computational methods that included molecular docking, molecular dynamics and free energy calculations. Based on our results, we suggest that distinct GAG sulfation patterns confer specificity beyond simple electrostatic interactions usually considered to represent the driving forces in protein-GAG interactions. The CXCL14-GAG system represents a promising approach to investigate the specificity of GAG-protein interactions, which represents an important topic for developing the rational approaches to novel strategies in regenerative medicine.

Adherence of Lactobacillus salivarius to HeLa Cells Promotes Changes in the Expression of the Genes Involved in Biosynthesis of Their Ligands.

The attachment of a variety of Lactobacilli to the mucosal surfaces is accomplished through the interaction of OppA, a superficial bacterial protein also involved in oligopeptide internalization, and the glycosaminoglycan moiety of the proteoglycans that form the epithelial cell glycocalyx. Upon the interaction of the vaginal isolate Lactobacillus salivarius Lv72 and HeLa cell cultures, the expression of oppA increased more than 50-fold over the following 30 min, with the overexpression enduring, albeit at a lower rate, for up to 24 h. Conversely, transcriptional analysis of 62 genes involved in proteoglycan biosynthesis revealed generalized repression of genes whose products catalyze different steps of the whole pathway. This led to decreases in the superficial concentration of heparan (60%) and chondroitin sulfate (40%), although the molecular masses of these glycosaminoglycans were higher than those of the control cultures. Despite this lowering in the concentration of the receptor, attachment of the Lactobacilli proceeded, and completely overlaid the underlying HeLa cell culture.

Potential involvement of chondroitin sulfate A in the pathogenesis of ameloblastoma.

Ameloblastoma is classified as a benign odontogenic tumor characterized by locally invasive behavior and high risk of recurrence. Here, we evaluate a potential role for glycosaminoglycan, a structural component of cell membranes and extracellular matrix, in ameloblstoma pathogenesis. We subjected formalin-fixed, paraffin-embedded tissue sections of 34 cases of ameloblastoma, 10 of odontogenic keratocyst, and 17 of dentigerous cyst to immunohistochemistry using monoclonal antibodies recognizing chondroitin sulfate A (CS-A), heparan sulfate (HS), and keratan sulfate (KS). Expression levels of CS-A in epithelial component and stroma of ameloblastoma were significantly higher than those in odontogenic keratocyst and dentigerous cyst. Moreover, CS-A in ameloblastoma was more strongly expressed in stellate reticulum-like cells than in amelobast-like cells with statistical significance. On the other hand, expression levels of HS and KS in epithelial component and stroma of ameloblastoma were lower compared with CS-A. These results overall reveal that among these odontogenic lesions, CS-A is preferentially expessed in ameloblastoma, suggesting potential pathogenetic role probably in cytodifferention of tumor cells to stellate reticulum-like cells.

MDA-MB-231 breast cancer cell viability, motility and matrix adhesion are regulated by a complex interplay of heparan sulfate, chondroitin-/dermatan sulfate and hyaluronan biosynthesis.

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (ß4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of ß4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in ß4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.

Altering the Proteoglycan State of Transforming Growth Factor ß Type III Receptor (TßRIII)/Betaglycan Modulates Canonical Wnt/ß-Catenin Signaling.

Hyperactive Wnt/ß-catenin signaling is linked to cancer progression and developmental abnormalities, making identification of mechanisms controlling Wnt/ß-catenin signaling vital. Transforming growth factor ß type III receptor (TßRIII/betaglycan) is a transmembrane proteoglycan co-receptor that exists with or without heparan and/or chondroitin sulfate glycosaminoglycan (GAG) modifications in cells and has established roles in development and cancer. Our studies here demonstrate that TßRIII, independent of its TGFß co-receptor function, regulates canonical Wnt3a signaling by controlling Wnt3a availability through its sulfated GAG chains. Our findings revealed, for the first time, opposing functions for the different GAG modifications on TßRIII suggesting that Wnt interactions with the TßRIII heparan sulfate chains result in inhibition of Wnt signaling, likely via Wnt sequestration, whereas the chondroitin sulfate GAG chains on TßRIII promote Wnt3a signaling. These studies identify a novel, dual role for TßRIII/betaglycan and define a key requirement for the balance between chondroitin sulfate and heparan sulfate chains in dictating ligand responses with implications for both development and cancer.

Oncofetal Chondroitin Sulfate Glycosaminoglycans Are Key Players in Integrin Signaling and Tumor Cell Motility.

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion, and anchorage-independent growth of tumor cells in vitro Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-ß1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes ß-1,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. IMPLICATIONS: The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR.

Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVß3 and Inhibits Osteoclast Differentiation.

Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVß3, and inhibited the direct binding of OA to integrin αVß3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVß3 and HSPG.

Heavy chain transfer by tumor necrosis factor-stimulated gene 6 to the bikunin proteoglycan.

We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors.

Increased expression of colonic Wnt9A through Sp1-mediated transcriptional effects involving arylsulfatase B, chondroitin 4-sulfate, and galectin-3.

In cultured human colonic epithelial cells and mouse colonic tissue, exposure to the common food additive carrageenan leads to inflammation, activation of Wnt signaling, increased Wnt9A expression, and decline in the activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase). In this study, the novel transcriptional mechanism by which carrageenan and decline in ARSB increase Wnt9A expression in NCM460 and HT-29 human colonic epithelial cells and in mouse colon is presented. Increased expression of Wnt9A has been associated with multiple malignancies, including colon carcinoma, and with ectodermal and mesoendodermal morphogenesis. When ARSB activity was reduced by siRNA or by exposure to carrageenan (1 µg/ml for 24 h), degradation of chondroitin 4-sulfate (C4S) was inhibited, leading to accumulation of more highly sulfated C4S, which binds less galectin-3, a ß-galactoside-binding protein. Nuclear galectin-3 increased and mediated increased binding of Sp1 to the Sp1 consensus sequence in the Wnt9A promoter, shown by oligonucleotide-binding assay and by chromatin immunoprecipitation assay. When galectin-3 was silenced, the increases in Sp1 binding to the Wnt9A promoter and in Wnt9A expression, which followed carrageenan or ARSB silencing, were inhibited. Mithramycin A, a specific inhibitor of Sp1 oligonucleotide binding, and Sp1 siRNA blocked the carrageenan- and ARSB siRNA-induced increases in Wnt9A expression. These studies reveal how carrageenan exposure can lead to transcriptional events in colonic epithelial cells through decline in arylsulfatase B activity, with subsequent impact on C4S, galectin-3, Sp1, and Wnt9A and can exert significant effects on Wnt-initiated signaling and related vital cell processes.

Hyaluronic acid receptor for endocytosis (HARE)-mediated endocytosis of hyaluronan, heparin, dermatan sulfate, and acetylated low density lipoprotein (AcLDL), but not chondroitin sulfate types A, C, D, or E, activates NF-κB-regulated gene expression.

The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638-648). Unique intermediate size Select-HA(TM), heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10-25 nM. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by ∼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.

Clathrin pit-mediated endocytosis of neutrophil elastase and cathepsin G by cancer cells.

Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1(+) endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized (125)I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (K(d)) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.

Glycosaminoglycans and sialylated glycans sequentially facilitate Merkel cell polyomavirus infectious entry.

Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus.

Use of gene-modified keratinocytes and fibroblasts to enhance regeneration in a full skin defect.

BACKGROUND: With the development of cell-based gene transfer techniques, genetically modified human keratinocytes (Kc) and fibroblasts (Fb) have been proven to be a better choice in wound repair. METHODS: This study was designed to construct in one step a gene-modified artificial skin by a genetically engineered Kc expressing PDGF-BB and Fb expressing VEGF(165) and bFGF. The wound healing effect in a full-thickness wound model was then observed. Unmodified artificial skin served as control. On the post-operative days 7, 14, and 21, residual wound area was calculated and skin wound tissues were subjected to biopsy for further investigation. RESULTS: Compared with unmodified artificial skin, gene-modified artificial skin resulted in a reduced wound contraction and a well-organized human epidermis and better formed dermis. CONCLUSIONS: The results suggest that our two-layer, gene-modified artificial skin improved both vascularization and epidermalization for skin regeneration. This technique could bring about a new approach in the treatment of burns and chronic wounds.

Distinct effects of N-acetylgalactosamine-4-sulfatase and galactose-6-sulfatase expression on chondroitin sulfates.

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.

Functional analysis of proteoglycan galactosyltransferase II RNA interference mutant flies.

Heparan sulfate proteoglycan plays an important role in developmental processes by modulating the distribution and stability of the morphogens Wingless, Hedgehog, and Decapentaplegic. Heparan and chondroitin sulfates share a common linkage tetrasaccharide structure, GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser. In the present study, we identified Drosophila proteoglycan galactosyltransferase II (dbeta3GalTII), determined its substrate specificity, and performed its functional analysis by using RNA interference (RNAi) mutant flies. The enzyme transferred a galactose to Galbeta1,4Xyl-pMph, confirming that it is the Drosophila ortholog of human proteoglycan galactosyltransferase II. Real-time PCR analyses revealed that dbeta3GalTII is expressed in various tissues and throughout development. The dbeta3GalTII RNAi mutant flies showed decreased amounts of heparan sulfate proteoglycans. A genetic interaction of dbeta3GalTII with Drosophila beta1,4-galactoslyltransferase 7 (dbeta4GalT7) or with six genes that encode enzymes contributing to the synthesis of glycosaminoglycans indicated that dbeta3GalTII is involved in heparan sulfate synthesis for wing and eye development. Moreover, dbeta3GalTII knock-down caused a decrease in extracellular Wingless in the wing imaginal disc of the third instar larvae. These results demonstrated that dbeta3GalTII contributes to heparan sulfate proteoglycan synthesis in vitro and in vivo and also modulates Wingless distribution.