Regulation of N-glycosylation and secretion of Isthmin-1 by its C-mannosylation

BackgroundC-mannosylation is a type of protein glycosylation. Human Isthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C-mannosylation sequences at Trp²²³ and Trp²²⁶. In this study, we sought to examine the role of C-mannosylation in the secretion of ISM1.MethodsWe established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy.ResultsWe found that ISM1 is C-mannosylated at Trp²²³ and Trp²²⁶ in the TSR domain. To determine the functions of the C-mannosylation of ISM1, we established a C-mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N-glycosylated only in these C-mannosylation-defective cells.ConclusionsISM1 is C-mannosylated in its TSR domain, and the status of the C-mannosylation of ISM1 affects its N-glycosylation.General significanceThe C-mannosylation of ISM1 regulates its N-glycosylation status.     

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4)

BackgroundC-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear.MethodsWe established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan.ResultsWe identified that ADAMTS4 is C-mannosylated at Trp⁴⁰⁴ in the metalloprotease domain and at Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ in the thrombospondin type 1 repeat (TSR). The replacement of Trp⁴⁰⁴ with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity.ConclusionsOur results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity.General significanceBecause C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.     

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W¹⁵³ and W¹⁵⁶. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W¹⁵³ and W¹⁵⁶ by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W¹⁵⁶ but not W¹⁵³ based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W¹⁵⁶ and found that DPY19L3-mediated C-mannosylation of Rspo1 at W¹⁵⁶ is required for its secretion.     

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C²-Man-)Trp.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

Identification and characterization of UDP-mannose in human cell lines and mouse organs: Differential distribution across brain regions and organs

Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.     

Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

Background

Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).

Experimental

Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR.

Conclusion

This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.     

Insights into Ectodomain Shedding and Processing of Protein-tyrosine Pseudokinase 7 (PTK7)

The membrane PTK7 pseudokinase, a component of both the canonical and noncanonical/planar cell polarity Wnt pathways, modulates cell polarity and motility in biological processes as diverse as embryo development and cancer cell invasion. To determine the individual proteolytic events and biological significance of the ectodomain shedding in the PTK7 function, we used highly invasive fibrosarcoma HT1080 cells as a model system. Current evidence suggested a likely link between PTK7 shedding and cell invasion in our HT1080 cell model system. We also demonstrated that in HT1080 cells the cleavage of the PTK7 ectodomain by an ADAM proteinase was coupled with the membrane type-1 matrix metalloproteinase (MT1-MMP) cleavage of the PKP⁶²¹↓LI site in the seventh Ig-like domain of PTK7. Proteolytic cleavages led to the generation of two soluble, N-terminal and two matching C-terminal, cell-associated fragments of PTK7. This proteolysis was a prerequisite for the intramembrane cleavage of the C-terminal fragments of PTK7 by γ-secretase. γ-Secretase cleavage was predominantly followed by the efficient decay of the resulting C-terminal PTK7 fragment via the proteasome. In contrast, in HT1080 cells, which overexpressed the C-terminal PTK7 fragment, the latter readily entered the nucleus. Our data imply that therapeutic inhibition of PTK7 shedding may be used to slow cancer progression.     

Receptor for hyaluronic acid- mediated motility (RHAMM) regulates HT1080 fibrosarcoma cell proliferation via a β-catenin/c-myc signaling axis

BackgroundHigh levels of hyaluronan (HA) synthesis in various cancer tissues, including sarcomas, are correlated with tumorigenesis and malignant transformation. RHAMM (receptor for hyaluronic acid-mediated motility) is overexpressed during tumor development in different malignancies. β-Catenin is a crucial downstream mediator of the Wnt signaling cascade which facilitates carcinogenic events characterized by deregulated cell proliferation.MethodsReal-time PCR, in vitro cell proliferation assay, siRNA transfection, flow cytometry, immunoprecipitation, western blotting and immunofluorescence were utilized.ResultsThe reduction of RHAMM expression was strongly correlated with an inhibition of HT1080 fibrosarcoma cell growth (p ≤ 0.01). LMWHA, in a RHAMM-dependent manner increases cell growth of HT1080 cells ((p ≤ 0.01). Both basal and LMWHA dependent growth of HT1080 cells was attenuated by β-catenin deficiency (p ≤ 0.01). β-Catenin cytoplasmatic deposition is positively regulated by RHAMM (p ≤ 0.01). Immunoflourescence and immunoprecipitation suggest that RHAMM/β-catenin form an intracellular complex. Transfection experiments identified c-myc as candidate downstream mediator of RHAMM/β-catenin effects on HT1080 fibrosarcoma cell proliferation.ConclusionsLMWHA/RHAMM downstream signaling regulates fibrosarcoma cell growth in a β-catenin/c-myc dependent manner.General significanceThe present study suggests that RHAMM is a novel β-catenin intracellular binding partner, protecting β-catenin from degradation and supporting the nuclear translocation of this key cellular mediator, which results in c-myc activation and enhanced fibrosarcoma cell growth.     

Tgat oncoprotein functions as a inhibitor of RECK by association of the unique C-terminal region

We identified RECK, a membrane-anchored glycoprotein negatively regulating the activities of MMPs, as a molecule interacting with Tgat oncoprotein consisting of RhoGEF domain and the unique C-terminal 15 amino acids. The Tgat increased the invasive potential of NIH3T3 cells expressing endogenous mouse RECK and this effect was partially inhibited by the co-expression of human RECK. On the contrary, the expression of exogenous human RECK in HT1080 cell line lacking the endogenous RECK expression reduced its invasive activity, which was recovered by the Tgat co-expression. Moreover, a Tgat mutant lacking the C-terminal region lost the potential to compete the function of RECK in HT1080 cells. These findings indicate that Tgat is the functional inhibitor of RECK, and the activation of MMPs induced by Tgat is likely to enhance invasive activities of cancer cells expressing Tgat.     

Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan

Protein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the ³⁶WDAWGPWSECSRTC⁴⁹ sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp³⁹ and Trp⁴². C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-³H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion.The ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type-1 repeats) superfamily (1) consists of 19 secreted metalloproteases (ADAMTS proteases) and six ADAMTS-like proteins in humans. ADAMTS-like proteins closely resemble the ancillary domains of ADAMTS proteases and like them have a conserved modular organization containing one or more thrombospondin type-1 repeats (TSRs)² (2–5). TSRs are modules of ∼50 amino acids having a characteristic six-cysteine signature. The prototypic ADAMTSL, ADAMTSL1, also referred to as punctin-1 because of its punctate distribution in the substratum of transfected cells, is a 525-residue glycoprotein containing four TSRs (4). A longer punctin-1 variant arising from alternative splicing, containing 13 TSRs and homologous to ADAMTSL3, is predicted by the human genome sequencing project ({"type":"entrez-nucleotide","attrs":{"text":"NM_001040272","term_id":"334688819","term_text":"NM_001040272"}}NM_001040272) but has not yet been physically cloned and expressed. The function of ADAMTSL1/punctin-1 is unknown. Recently, ADAMTSL2 and ADAMTSL4 mutations were identified in the genetic disorders geleophysic dysplasia (6) and recessive isolated ectopia lentis, respectively (2). In genome-wide analysis, the ADAMTSL3 locus has been associated with variation in human height (7). Thus, in addition to known genetic disorders caused by ADAMTS mutations (8, 9), ADAMTSL family members are now also implicated in human disease. Among the ADAMTS proteases, ADAMTS5 and ADAMTS4 are strongly associated with cartilage destruction in arthritis (10–12).Like most secreted proteins, the ADAMTS superfamily members undergo post-translational modification and are predicted to contain N-linked oligosaccharides. In addition, TSRs of ADAMTS superfamily members, by virtue of high sequence similarity to the corresponding motifs in thrombospondin 1 and properdin, are predicted to contain two uncommon types of glycosylation. Specifically, TSRs often contain the sequence motifs W⁰XXW⁺³ and C¹X_2–3(S/T)C²XXG, which are consensus sites for protein C-mannosylation of the W⁰ residue and O-fucosylation (of Ser/Thr) respectively, in close proximity to each other (13, 14). In recently published work, it was shown that ADAMTSL1 and ADAMTS13 are modified by O-fucosylation, the covalent attachment to Ser or Thr residues of fucose or a fucose-glucose disaccharide (15, 16). Punctin-1 contains consensus sequences for O-fucosylation in all four of its TSRs, but the presence of the glycans was previously only confirmed on TSR2, -3, and -4 (16). Addition of O-fucose is mediated by protein O-fucosyltransferase 2 (POFUT2), which is a distinct transferase from that which catalyzes addition of O-linked fucose to epidermal growth factor-like repeats (POFUT1) (17, 18). A β3-glucosyltransferase subsequently adds glucose to the 3′-OH of the fucose (19, 20). It was further demonstrated that O-fucosylation, which occurs after completion of TSR folding, was rate-limiting for secretion of punctin-1 and ADAMTS13 (15, 16). This role was inferred from the following two experimental observations. 1) Expression of wild-type punctin-1 and ADAMTS13 in Lec13 cells, which do not fucosylate proteins, led to their decreased secretion (15, 16). 2) Mutation of the modified Ser or Thr residues greatly reduced secretion of punctin-1 and ADAMTS13 (15, 16).Protein C-mannosylation is the attachment of an α-mannopyranosyl residue to the indole C-2 of tryptophan via a C-C linkage (14, 21). Unlike O-fucosylation, it can utilize protein primary structure rather than tertiary structure as the determinant, i.e. mannose is added to unfolded polypeptides or unstructured synthetic peptides (22). C-Mannosylation uses dolichyl-phosphate mannose (Dol-P-Man) as the precursor and appears to be enzyme-catalyzed within the endoplasmic reticulum (23), but the responsible mannosyltransferase has not yet been identified. A variety of mammalian cell lines can perform this modification (24). Proteins known to be C-mannosylated include human RNase 2, interleukin 12, the mucins MUC5AC and MUC5B, and several proteins containing TSRs, such as thrombospondin-1, F-spondin, and components of complement (C6 and C7) and properdin (13, 21, 25–27).Krieg et al. (22) proposed a model in which the C-mannosyltransferase bound directly to the WXXW⁺³ motif, analogous to the Asn-X-(Thr/Ser) motif for N-glycosylation, and later analysis showed that both the Trp residues in the W⁰XXW⁺³XXX motif and the sole Trp residue in a (F/Y¹)XXW⁺³ motif could be modified (13). Based on meta-analysis of the C-mannosylation literature, Julenius (28) used a neural network approach to develop a prediction algorithm for protein C-mannosylation, termed NetCGlyc. This analysis suggested that Cys was an acceptable substitute for Trp at the +3 position (i.e. permitting C-mannosylation of W⁰ in a W⁰SSC motif). Julenius (28) reported a clear preference for small and/or polar residues (Ser, Ala, Gly, and Thr) at the +1 position, whereas Phe and Leu were not allowed. The NetCGlyc algorithm provides a useful guide for prediction of C-mannosylation sites, especially in the ADAMTS superfamily, which has a large number of TSRs (27). Here we specifically inquired whether the short form of punctin-1, the prototypic ADAMTSL, is modified by C-mannosylation, analyzed the role of Trp residues in the punctin TSRs, and investigated its possible functional significance in punctin-1 biosynthesis. By demonstrating that TSR1 of ADAMTS5 is also C-mannosylated, we extended the analysis to identify this unusual modification in an ADAMTS protease.

TABLE 1

Predicted C-mannosylation sites^a in the ADAMTS superfamilyOpen in a separate window^aThe full-length human reference ADAMTS sequences from GenBank™ were analyzed at the NetCGly 1.0 server for prediction of C-mannosylation sites. For prediction of O-fucosylation sites in the same peptide, the consensus sequence C¹X_2–3(S/T)C² XXG was utilized.^bThe sequence context in which the predicted modified Trp residue occurs is provided, and the residue with predicted modification is numbered. Ser/Thr residues predicted to be O-fucosylated based on the consensus sequence CXX(S/T)C are underlined.^cSequences containing predicted C-mannosylation sites that are not within TSRs are shown in italics.     

Resveratrol with antioxidant activity inhibits matrix metalloproteinase via modulation of SIRT1 in human fibrosarcoma cells

AimsResveratrol, a silent information regulator 1 (SIRT1) activator, has been reported to act as an antioxidant contained in red wine and prevent the development of cardiovascular diseases. Histone deacetylase such as SIRT1 is involved in the regulation of lifespan extension. In this study, the effect of resveratrol on matrix metalloproteinases (MMPs) that play an important role in metastasis was examined in human fibrosarcoma cell line, HT1080.Main methodsThe effect of resveratrol on MMPs' activity was evaluated using gelatin zymography. Western blot analysis and RT-PCR assay were used to determine the effect of resveratrol on the expression level of MMP-9, MAPK and SIRT1 proteins and genes, respectively.Key findingsIt was observed that resveratrol exhibited not only antioxidant effects on lipid peroxidation and DNA oxidation but also inhibitory effects on the expression of MMP-2 and 9 in HT1080 cells stimulated with either phorbol myristate acetate or phenazine methosulfate. Furthermore, it was found that treatment with resveratrol decreased the level of MMP-9 expression via down-regulation of p-ERK, c-fos and p65. In addition, the level of SIRT1 gene expression was also enhanced by treatment of resveratrol alone but the level of MMP-9 gene expression was decreased.SignificanceThese results suggest that the activation of SIRT1 in the presence of resveratrol especially inhibits the expression of MMP-9 in HT1080 cells, providing evidence that resveratrol can be a potential candidate for chemoprevention of cancer.     

Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor

The 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) acting as a receptor for prions and viruses is overexpressed in various cancer cell lines, and their metastatic potential correlates with LRP/LR levels. We analyzed the tumorigenic fibrosarcoma cell line HT1080 regarding 37-kDa/67-kDa LRP/LR levels and its invasive potential. Compared to the less invasive embryonic fibroblast cell line NIH3T3, the tumorigenic HT1080 cells display approximately 1.6-fold higher cell-surface levels of LRP/LR. We show that anti-LRP/LR tools interfere with the invasive potential of HT1080 cells. Anti-LRP/LR single-chain variable fragment antibody (scFv) iS18 generated by chain shuffling from parental scFv S18 and its full-length version immunoglobulin G1-iS18 reduced the invasive potential of HT1080 cells significantly by 37% and 38%, respectively. HT1080 cells transfected with lentiviral plasmids expressing small interfering RNAs directed against LRP mRNA showed reduced LRP levels by approximately 44%, concomitant with a significant decrease in the invasive potential by approximately 37%. The polysulfated glycans HM2602 and pentosan polysulfate (SP-54), both capable of blocking LRP/LR, reduced the invasive potential by 20% and 35%, respectively. Adhesion of HT1080 cells to laminin-1 was significantly impeded by scFv iS18 and immunoglobulin G1-iS18 by 60% and 68%, respectively, and by SP-54 and HM2602 by 80%, suggesting that the reduced invasive capacity achieved by these tools is due to the perturbation of the LRP/LR-laminin interaction on the cell surface. Our in vitro data suggest that reagents directed against LRP/LR or LRP mRNA such as antibodies, polysulfated glycans, or small interfering RNAs, previously shown to encompass an anti-prion activity by blocking or downregulating the prion receptor LRP/LR, might also be potential cancer therapeutics blocking metastasis by interfering with the LRP/LR-laminin interaction in neoplastic tissues.     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.     

Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin

Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin.     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

Photodynamic anti-cancer effects of fullerene [C60]–PEG complex on fibrosarcomas preferentially over normal fibroblasts in terms of fullerene uptake and cytotoxicity

A water-soluble complex of fullerene [C₆₀]:polyethylene glycol (PEG) (1:350 wt/wt) (C₆₀–PEG), but not PEG alone, was found in the present study by ESR/DMPO spin-trap method to generate hydroxyl radicals 6.5-fold as abundant as the non-irradiation level, when irradiated with visible light (400–600 nm, 140 J/cm²: 450-fold as intense as in average outdoor), but not to generate without irradiation. At 3 h after irradiation with C₆₀–PEG, human fibrosarcoma cells HT1080 were obviously degenerated together with diminished microvilli, cell shrinkage and cell fragmentation as observed by SEM and were shown either for increased cytotoxicity by dual stains with calcein-AM and propidium iodide or for nuclear condensation and fragmentation by Hoechst 33342 stain, any of which were, in contrast, scarcely changed in normal human fibroblastic cells DUMS16 derived from the same connective tissue type as HT1080 cells. Under the conditions, the maximum intracellular uptake amount was more abundant for HT1080 cells than for DUMS16 cells, either by immunostain/fluorography using polyclonal antibody against fullerene [C₆₀], or by HPLC method indicating the 2.4-fold preferential uptake of C₆₀–PEG into HT1080 cells, suggested to greater phagocytotic ability characteristic of cancer cells, over DUMS16 cells being non-macrophage-like normal cells. Thus, C₆₀–PEG is expected as a photosensitizer for photodynamic therapy with scarce side effects to normal cells and preferential reactive oxygen species generation in cancer cells.