Sulfated Glycolipids Research Articles

Diazobutanone-assisted isobaric labelling of phospholipids and sulfated glycolipids enables multiplexed quantitative lipidomics using tandem mass spectrometry.

Mass spectrometry-based quantitative lipidomics is an emerging field aiming to uncover the intricate relationships between lipidomes and disease development. However, quantifying lipidomes comprehensively in a high-throughput manner remains challenging owing to the diverse lipid structures. Here we propose a diazobutanone-assisted isobaric labelling strategy as a rapid and robust platform for multiplexed quantitative lipidomics across a broad range of lipid classes, including various phospholipids and glycolipids. The diazobutanone reagent is designed to conjugate with phosphodiester or sulfate groups, while accommodating various functional groups on different lipid classes, enabling subsequent isobaric labelling for high-throughput multiplexed quantitation. Our method demonstrates excellent performance in terms of labelling efficiency, detection sensitivity, quantitative accuracy and broad applicability to various biological samples. Finally, we performed a six-plex quantification analysis of lipid extracts from lean and obese mouse livers. In total, we identified and quantified 246 phospholipids in a high-throughput manner, revealing lipidomic changes that may be associated with obesity in mice.

Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.

Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components.

Sulfatides are endogenous ligands for the TLR4–MD-2 complex

Many endogenous molecules, mostly proteins, purportedly activate the Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex, the innate immune receptor for lipopolysaccharide (LPS) derived from gram-negative bacteria. However, there is no structural evidence supporting direct TLR4-MD-2 activation by endogenous ligands. Sulfatides (3-O-sulfogalactosylceramides) are natural, abundant sulfated glycolipids that have variously been shown to initiate or suppress inflammatory responses. We show here that short fatty acid (FA) chain sulfatides directly activate mouse TLR4-MD-2 independent of CD14, trigger MyD88- and TRIF-dependent signaling, and stimulate tumor necrosis factor α (TNFα) and type I interferon (IFN) production in mouse macrophages. In contrast to the agonist activity toward the mouse receptor, the tested sulfatides antagonize TLR4-MD-2 activation by LPS in human macrophage-like cells. The agonistic and antagonistic activities of sulfatides require the presence of the sulfate group and are inversely related to the FA chain length. The crystal structure of mouse TLR4-MD-2 in complex with C16-sulfatide revealed that three C16-sulfatide molecules bound to the MD-2 hydrophobic pocket and induced an active dimer conformation of the receptor complex similar to that induced by LPS or lipid A. The three C16-sulfatide molecules partially mimicked the detailed interactions of lipid A to achieve receptor activation. Our results suggest that sulfatides may mediate sterile inflammation or suppress LPS-stimulated inflammation, and that additional endogenous negatively charged lipids with up to six lipid chains of limited length might also bind to TLR4-MD-2 and activate or inhibit this complex.

Abstract 1046: Synergistic cytotoxic effect of sulfated glycolipid PG545 in combination with ATR inhibition in ovarian cancer cells

Abstract Despite the advances in the molecular understanding of ovarian cancer (OC), improvements in therapy are still needed. PG545 is a highly sulfated small molecule and is currently in Phase I clinical investigation, which has shown promising antitumor activity in multiple tumor models, including ovarian, endometrial and pancreatic cancers. Ataxia telangiectasia and Rad3-related protein (ATR), plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. Several ATR inhibitors have been developed, but resistance is likely to occur. This study aimed to investigate the potential synergistic effect of combining PG545 with ATR inhibition in OC. Proliferation and clonogenic assay showed that combination of PG545 with ATR shRNA or ATR inhibitor (ATRi; VE-822) synergistically decreased cell survival compared with single-agent treatments in two OC cell lines, OVCAR5 and OVCAR8 cells. PG545 monotherapy induced DNA damage, activated the ATR signaling pathways as well as the S and G2/M cell cycle checkpoints. The combination of PG545 and ATRi inhibited the phosphorylation of ATR and Chk1, induced γH2AX and lead to a marked increase in pan-nuclear γH2AX-positive cells. Moreover, ATRi diminished the cell cycle effects of PG545 and markedly enhanced PG545-induced apoptosis in OC cell lines. Taken together, our results indicate that the combination of PG545 with ATR inhibition may be promising options to be considered in future clinical trials of OC. Citation Format: Yinan Xiao, Chaolin Deng, Ye Guan, Ling Jin, Upasana Ray, Prabhu Thirusangu, Julie Staub, Haotian Xu, Viji Shridhar. Synergistic cytotoxic effect of sulfated glycolipid PG545 in combination with ATR inhibition in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1046.

Annexin A4 inhibits sulfatide‐induced activation of coagulation factor XII

BackgroundFactor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood. ObjectiveTo test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities. MethodsRecombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain. Results and ConclusionsWe found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide‐induced plasma coagulation. Wild‐type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide‐induced auto‐activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+‐binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.

Sulfated glycolipid PG545 induces endoplasmic reticulum stress and augments autophagic flux by enhancing anticancer chemotherapy efficacy in endometrial cancer

The sulfated glycolipid PG545 shows promising antitumor activity in various cancers. This study was conducted to explore the effects and the mechanism of PG545 action in endometrial cancer (EC). PG545 exhibited strong synergy as assessed by the Chou-Talalay-Method in vitro when combined with cisplatin, or paclitaxel in both type I (Hec1B) and type II (ARK2) EC cell lines. While PG545 showed antitumor activity as monotherapy, a combination of PG545 with paclitaxel and cisplatin was highly effective in reducing the tumor burden and significantly prolonged survival of both Hec1B and ARK2 xenograft bearing mice. Mechanistically, PG545 elicits ER stress as an early response with resultant induction of autophagy. Our data demonstrated an increase in pERK, Bip/Grp78, IRE1α, Calnexin and CHOP/GADD153 within 6–24 hrs of PG545 treatment in EC cells. In parallel, PG545 also blocked FGF2 and HB-EGF mediated signaling in EC cells. Moreover, melatonin-mediated ER stress inhibition reduced PG545-mediated autophagy and PG545 in combination with cisplatin further heightened this stress response. Collectively these data indicate that PG545 exhibits strong synergistic effects with chemotherapeutics in vitro and showed promising antitumor activity in vivo. Our preclinical data indicates that in future studies PG545 can be a useful adjunct to chemotherapy in endometrial cancer.

Dextran Microparticulate Inhalable Dry Powder for the Treatment of Cystic Fibrosis and Mucopolysaccharidosis.

Cystic Fibrosis (CF) is a genetic disease which affects the patient's lungs, pancreas, liver, kidney and intestine and lacks sulfatase enzyme, leading to mucopolysaccharidosis. Colistin sulfate acts by interacting with phospholipids of bacterial cell membranes. Sulfatase enzyme reduces the high levels of sulfated glycosaminoglycans and glycolipids by the hydrolysis of sulfate esters in lysosome. The aim of the present investigation was to prepare and evaluate dextran microparticulate inhalable dry powder for the efficient targeting of colistin sulfate at affected area of lung without causing the side effects in the treatment of CF and mucopolysaccharidosis. Microparticulate dry powder was prepared by the lyophilization method and evaluated for particle size, % yield, % drug content, solid state characterization, in-vitro lung deposition study, and in-vitro drug release study. Particle size, % yield and % drug content were found to be 4.03 ± 0.196 µm, 94.02 % and 99.45 ± 0.015% respectively. Bulk density, tapped density, hausner's ratio, carr's index and angle of repose of optimized batch were found to be 0.216 ± 0.025 g/cm3, 0.236 ± 0.035 g/cm3, 1.09 ± 0.026, 8.47 ± 0.025 % and 26.10 ± 0.029˚ respectively. A fine particle fraction, fine particle dose, mass median aerodynamic diameter, geometric standard deviation and emitted dose were found to be 66.78%, 16.45 mg, 4.89 µm, 1.32 and 246.33 mg respectively. The % CDR of optimized batch was found to be 96.12 ± 0.049 % at 24 h. Based on the obtained results, we conclude that dextran microparticulate inhalable dry powder might be suitable carrier for the delivery of colistin sulfate and sulfatase in combination via pulmonary route for the treatment of cystic fibrosis and mucopolysaccharidosis.

Archaeal glycolipid adjuvanted vaccines induce strong influenza-specific immune responses through direct immunization in young and aged mice or through passive maternal immunization

Vaccine induced responses are often weaker in those individuals most susceptible to infection, namely the very young and the elderly, highlighting the need for safe and effective vaccine adjuvants. Herein we evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strainA/PuertoRico/8/1934H1N1)homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model. The impact of age (young adult vs aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. Overall, we show that archaeal lipid based adjuvants can induce potent anti-HA responses in young and aged mice that can also be passed from vaccinated mothers to pups. Furthermore, young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenge with influenza. In addition, we show that a simple admixed archaeosome formulation composed of a single sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations.

Self-Referenced Ratiometric Detection of Sulfatase Activity with Dual-Emissive Urease-Encapsulated Gold Nanoclusters.

In this study, on the basis of the biomineralization capability of urease, a facile, one-step, and green synthetic method has been proposed for the fabrication of gold nanoclusters (AuNCs). The prepared urease-encapsulated AuNCs (U-AuNCs) exhibited strong red fluorescence emission (λem = 630 nm) with a quantum yield as high as 17%. Interestingly, at a low concentration, the U-AuNC solution was found to be a dual-emissive system with the blue emission of the dityrosine (diTyr) residues of urease and the red emission of the embedded AuNCs. Further experiments demonstrated that p-nitrophenol (PNP) can selectively suppress the 410 nm emission of the diTyr residues of U-AuNCs without affecting the red emission of the U-AuNCs. The fluorescence quenching mechanism between U-AuNCs and PNP was systematically studied, and the leading role of the inner filter effect (IFE) was identified. Additionally, based on the sulfatase-catalyzed hydrolysis of p-nitrophenyl sulfate (PNPS) to release PNP, a self-referenced ratiometric detection method for sulfatase, which plays a crucial role in sulfur cycling, degradation of sulfated glycosaminoglycans and glycolipids, and extracellular remodeling of sulfated glycosaminoglycans, was developed by using dual-emissive U-AuNCs as the signal readout, in which the diTyr residues served as the probe and the AuNCs functioned as the internal reference. This IFE-based ratiometric sensing strategy showed a good linear relationship over the range of 0.01-1 U/mL ( R2 = 0.997). The detection limit for sulfatase activity was 0.01 U/mL. The developed protocol was successfully used to detect sulfatase activity in human serum samples. The simplicity, rapidity, low cost, high credibility, good reproducibility, and excellent selectivity of the detection platform serve as an inspiration for further applications of fluorescent AuNCs in chemo/biosensing.

Safety and biodistribution of sulfated archaeal glycolipid archaeosomes as vaccine adjuvants

ABSTRACTArchaeosomes are liposomes comprised of ether lipids derived from various archaea. Unlike conventional ester-linked liposomes, archaeosomes exhibit high pH and thermal stability. As adjuvants, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses and enhance protection in murine models of infectious disease and cancer. Archaeosomes constituted with total polar lipids (TPL) of various archaea are relatively complex, comprising >10 different lipid compounds. Archaeosomes can be constituted with semi-synthetic glycerolipids built on ether-linked isoprenoid phytanyl cores with varied synthetic glycol- and amino-head groups. However, such semi-synthetic archaeosomes involve many synthetic steps to arrive at the final desired glycolipid composition. We have developed a novel archaeosome formulation comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA) mixed with uncharged glycolipid (lactosylarchaeol, LA). This new class of adjuvants can be easily synthesized and retains strong immunostimulatory activity for induction of cell-mediated immunity following systemic immunization. Herein, we demonstrate the safety of SLA/LA archaeosomes following intramuscular injection to mice and evaluate the immunogenicity, in vivo distribution and cellular uptake of antigen (ovalbumin) encapsulated into SLA/LA archaeosomes. Overall, we have found that semi-synthetic sulfated glycolipid archaeosomes are a safe and effective novel class of adjuvants capable of inducing strong antigen-specific immune responses in mice and protection against subsequent B16 melanoma tumor challenge. A key step in their mechanism of action appears to be the recruitment of immune cells to the injection site and the subsequent trafficking of antigen to local draining lymph nodes.

Sulfated archaeal glycolipid archaeosomes as a safe and effective vaccine adjuvant for induction of cell-mediated immunity

ABSTRACTArchaeosomes are liposomal vesicles composed of ether glycerolipids unique to the domain of Archaea. Unlike conventional ester-linked liposomes, archaeosomes exhibit high stability and possess strong adjuvant and immunostimulatory properties making them an attractive vaccine delivery vehicle. Traditionally comprised of total polar lipids (TPL) or semi-synthetic phospho-glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups, archaeosomes can induce robust and long-lasting humoral and cell-mediated immune responses against antigenic cargo and provide protection in murine models of infectious disease and cancer. However, traditional TPL archaeosome formulations are relatively complex comprising several lipid species. Semi-synthetic archaeosomes tested previously contain a combination of several phospho-glycolipids (negative and neutral charged) to produce a stable, uniform-sized liposome formulation. Moreover, they involve many synthetic steps to arrive at the final desired glycolipid composition. Herein, we present a novel adjuvant formulation comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA). SLA individually or mixed with uncharged glyolipid (lactosylarchaeol, LA) constituted efficacious carrier vesicles for entrapped antigens (ovalbumin or melanoma associated tyrosinase-related protein 2 [TRP-2]) and induction of strong cell-mediated responses in mice and protection against subsequent B16 melanoma tumor challenge. Thus, semi-synthetic sulfated glycolipid archaeosomes represent a new class of adjuvants that will potentially ease manufacturing and scale-up, while retaining immunostimulatory activity.

Correction: Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri

[This corrects the article DOI: 10.1371/journal.pone.0083703.].

Basement membranes

Basement membranes (BMs) are thin, dense sheets of specialized, self-assembled extracellular matrix that surround most animal tissues (Figure 1, top). The emergence of BMs coincided with the origin of multicellularity in animals, suggesting that they were essential for the formation of tissues. Their sheet-like structure derives from two independent polymeric networks - one of laminin and one of type IV collagen (Figure 1, bottom). These independent collagen and laminin networks are thought to be linked by several additional extracellular matrix proteins, including nidogen and perlecan (Figure 1, bottom). BMs are usually associated with cells and are anchored to cell surfaces through interactions with adhesion receptors and sulfated glycolipids (Figure 1, bottom). Various combinations of other proteins, glycoproteins, and proteoglycans - including fibulin, hemicentin, SPARC, agrin, and type XVIII collagen - are present in BMs, creating biochemically and biophysically distinct structures that serve a wide variety of functions. BMs have traditionally been viewed as static protein assemblies that provide structural support to tissues. However, recent studies have begun to uncover dynamic, active roles for BMs in many developmental processes. Here, we discuss established and emerging roles of BMs in development, tissue construction, and tissue homeostasis. We also explore how cells traverse BM barriers, the roles of BMs in human diseases, and future directions for the field.

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies.

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.

Genotypic characterization of Brazilian patients with infantile and juvenile forms of metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder inherited as an autosomal recessive trait. MLD is caused by the deficiency of arylsulfatase A (ARSA), a lysosomal enzyme that catalyzes the first step in the degradation of sulfated glycolipids, which are essential components of the myelin sheet. Notably, between 7% and 15% of healthy individuals show in vitro deficiency of ARSA, a condition called ARSA pseudodeficiency (ARSA-PD). To date, 151 ARSA-MLD mutations have been reported in the gene encoding ARSA (ARSA), among which IVS2+1G>A and P426L occur at high frequencies in most of the studied populations. The aim of this work was to identify ARSA mutant alleles in a cohort of 27 unrelated Brazilian MLD patients. The most frequent ARSA-MLD mutation, IVS2+1G>A, and the ARSA-PD polymorphisms, N350S and 1524+95A>G, were detected using real-time PCR, while the remaining mutations were detected using direct sequencing of ARSA. In concordance with previous reports, IVS2+1G>A and P426L were the most common ARSA-MLD mutations in our cohort of MLD patients, found at frequencies of 0.05 and 0.08, respectively. Interestingly, two mutations previously reported as rare, 103_110del8 and 1190_1191insC, were found at higher frequencies in our cohort of MLD patients, 0.08 and 0.06, respectively. Additionally, 11 other rare ARSA-MLD mutations were found at lower frequencies in our cohort of MLD patients. To our knowledge, this is the first systematic genotypic characterization of MLD patients from Latin America. This work highlights the genetic heterogeneity of MLD, and supports genotype–phenotype associations, which become more important as specific treatments are being developed for this devastating disorder.

Mass Spectrometry in Pharmacokinetic Studies of a Synthetic Compound for Spinal Cord Injury Treatment.

The studies of drugs that could constitute a palliative to spinal cord injury (SCI) are a continuous and increasing demand in biomedicine field from developed societies. Recently we described the chemical synthesis and antiglioma activity of synthetic glycosides. A synthetic sulfated glycolipid (here IG20) has shown chemical stability, solubility in polar solvents, and high inhibitory capacity over glioma growth. We have used mass spectrometry (MS) to monitor IG20 (m/z = 550.3) in cells and tissues of the central nervous system (CNS) that are involved in SCI recovery. IG20 was detected by MS in serum and homogenates from CNS tissue of rats, though in the latter a previous deproteinization step was required. The pharmacokinetic parameters of serum clearance at 24 h and half-life at 4 h were determined for synthetic glycoside in the adult rat using MS. A local administration of the drug near of spinal lesion site is proposed.

Deciphering archaeal glycolipids of an extremely halophilic archaeon of the genus Halobellus by MALDI-TOF/MS

Polar membrane lipids of an archaeal microorganism recently isolated from the natural salt lake Fuente de Piedra (Málaga, Spain) have been studied by means of TLC in combination with MALDI-TOF mass spectrometry. The major phospholipids are the ether lipids phosphatidylglycerophosphate methyl ester and phosphatidylglycerosulfate, while phosphatidylglycerol is barely detectable; in addition the bisphosphatidylglycerol (archaeal cardiolipin) has been detected for the first time in a representative of the genus Halobellus. The structures of glycolipids, including a glycosyl-cardiolipin, have been elucidated by post source decay (PSD) mass spectrometry analysis. Besides the monosulfated diglycosyl diphytanylglyceroldiether, two variants of a bis-sulfated diglycosyl diphytanylglyceroldiether have been identified; furthermore the glycosyl-cardiolipin is found to have the same structure of the analogue present in Halorubrum trapanicum and Haloferax volcanii. The role of the abundant sulfated glycolipids in facing high extracellular salinity is discussed.

Roles of galactose 3'-O- sulfation in signaling.

Galactose-3'-O- sulfation is specific and exists in many important molecules from various human tissues, and the sulfation modification results in alteration of host molecule recognition and interaction with partner molecules which lead to signaling. The modification is thus associated with the regulation of cellular adhesion and interaction, and involved in cell recognition and even in tumor metastasis process since the binding affinity to their extracellular ligands has changed. Sulfated glycoproteins or glycolipids may also trigger signaling in the cells, which is important in regulating cell-cell and cell-extracellular matrix interaction, and in adhesion molecule transcription activation.

Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri

We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6×Histidine-fused EF-Tu (His6-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His6-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His6-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His6-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces.

Enhanced Expression of Sulfatide, a Sulfated Glycolipid, in Well-Differentiated Endometrial Adenocarcinoma

It is well known that a poorly differentiated endometrial adenocarcinoma shows more rapid progression and a worse response to therapy than a well-differentiated endometrial adenocarcinoma. Qualitative and quantitative changes of cell surface glycolipids occur during neoplastic transformation. Sulfatide is one of the sulfated glycolipids in the cell membrane that may have an important role in various functions such as cell adhesion. To examine the molecular background of the morphological and biological features of well-differentiated and poorly differentiated cancer, we measured the levels of lipids, especially glycolipids, in tumor tissues from patients with endometrial carcinoma. We determined the composition of lipids and glycolipids in tumor tissues, investigated glycosyltransferase messenger RNA expression by the reverse transcription-polymerase chain reaction, and assessed the localization of galactosylceramide sulfotransferase (an enzyme involved in sulfatide biosynthesis) by immunohistochemical staining. No significant differences were observed between well-differentiated and poorly differentiated cancer with respect to the levels of cholesterol ester, cholesterol, phospholipids, cholesterol sulfate, ceramides, neutral glycolipids of the globo series, and GM3 ganglioside. However, the amount of sulfatides in well-differentiated tumors was significantly greater than that in poorly differentiated tumors, which was confirmed by thin-layer chromatography and immunostaining with a monoclonal antisulfatide antibody. Altered expression of sulfatide was found to be secondary to a change of galactosylceramide sulfotransferase messenger RNA expression. Immunohistochemical staining revealed that galactosylceramide sulfotransferase expression was characteristically observed in glandular areas but not in solid areas. These findings suggest that sulfatide contributes to the well-differentiated phenotype of endometrial adenocarcinoma and that it is being expressed in normal uterine endometrium at sites of gland formation during the luteal phase, as we have previously reported.