Mannose Residues Research Articles

Castanea crenata Ginkbilobin-2-like Recombinant Protein Reveals Potential as an Antimicrobial against Phytophthora cinnamomi, the Causal Agent of Ink Disease in European Chestnut

The European chestnut tree (Castanea sativa Mill.) is widely cultivated throughout the world’s temperate regions. In the Mediterranean region, it has a significant economic role mainly because of the high quality of its edible nuts. The Oomycete Phytophthora cinnamomi is one of the most severe pathogens affecting European chestnuts, causing ink disease and significant losses in production. Ginkgobilobin-2 (Gnk2) in Ginkgo biloba is a secreted protein with a plant-specific cysteine-rich motif that functions as a lectin, and its carbohydrate-binding properties are closely related to its antifungal activity. The binding of lectins to mannose residues of the cell wall of Phytophthora species may disturb and disrup the cell wall structure. This work determined that the amino acid sequence has a signal peptide that directs the final protein peptide to the apoplast. The Cast_Gnk2-like expression was performed and optimized, and different in vitro antagonism tests were done against P. cinnamomi using different purified protein concentrations. As a result of one of these assays, Cast_Gnk2-like significantly reduced the mycelia growth of P. cinnamomi in liquid medium as shown by the mycelia weight (g) in control treatments was 377% higher than in the treatments. These insights reveal the potential of Cast_Gnk2-like for agricultural uses and biotechnological developments for the pathosystem chestnut/P. cinnamomi.

Narsoplimab for severe transplant-associated thrombotic microangiopathy

BackgroundTransplantation-associated thrombotic microangiopathy (TA-TMA) is an endothelial injury syndrome linked to the overactivation of complement pathways. It manifests with microangiopathic hemolytic anemia, consumptive thrombocytopenia, and microvascular thrombosis leading to ischemic tissue injury. Mannose residues on fungi and viruses activate the mannose-binding lectin complement pathway, and hence activation of the lectin pathway could be one of the reasons for triggering TA-TMA. Narsoplimab, a human monoclonal antibody targeting MASP-2 is a potent inhibitor of the lectin pathway. We describe the transplant course of a pediatric patient who developed TA-TMA following Candida-triggered macrophage activation syndrome and was treated with Narsoplimab. The data collection was performed prospectively.Case presentationThe six-year-old girl underwent a human leucocyte antigen (HLA) haploidentical hematopoietic stem cell transplant using post-transplant Cyclophosphamide for severe aplastic anemia. In the second week of the transplant, the patient developed macrophage activation syndrome necessitating treatment with steroids and intravenous immunoglobulin. Subsequently, USG abdomen and blood fungal PCR revealed the diagnosis of hepatosplenic candidiasis. Candida-triggered macrophage activation syndrome responded to antifungals, steroids, intravenous immunoglobulin, and alemtuzumab. However, the subsequent clinical course was complicated by thrombotic microangiopathy. The patient developed hypertension in the 2nd week, followed by high lactate dehydrogenase (1010 U/L), schistocytes (5 per hpf), low haptoglobin (< 5 mg/dl), thrombocytopenia, and anemia in the 3rd week. Ciclosporin was stopped, and the patient was treated with 10 days of defibrotide without response. The course was further complicated by the involvement of the gastrointestinal tract and kidneys. She had per rectal bleeding with frequent but low-volume stools, severe abdominal pain, and hypoalbuminemia with a rising urine protein:creatinine ratio. Narsoplimab was started in the 5th week of the transplant. A fall in lactate dehydrogenase was observed after starting Narsoplimab. This was followed by the resolution of gastrointestinal symptoms, proteinuria, and recovery of cytopenia. The second episode of TA-TMA occurred with parvoviraemia and was also successfully treated with Narsoplimab.ConclusionLectin pathway inhibition could be useful in treating the fatal complication of transplant-associated thrombotic microangiopathy.

Prediction of Mortality in Patients on Peritoneal Dialysis Based on the Fibrinogen Mannosylation.

As we already reported, fibrinogen fucosylation emerged as a prognostic marker of peritoneal membrane function in end-stage renal disease (ESRD) patients on peritoneal dialysis. After a follow-up period of 18 months, we estimated the ability of employed lectins, as well as other biochemical parameters, to serve as mortality predictors in these patients. Following a univariate Cox regression analysis, ferritin, urea clearance, residual diuresis, hyperglycemia, and an increase in the signal intensity obtained with Galanthus nivalis lectin (GNL) emerged as potential mortality predictors, but additional multivariate Cox regression analysis pointed only to glucose concentration and GNL as mortality predictors. Higher signal intensity obtained with GNL in patients that died suggested the importance of paucimannosidic/highly mannosidic N-glycan structures on fibrinogen as factors that are related to unwanted cardiovascular events and all-cause mortality and can possibly be seen as a prediction tool. Altered glycan structures composed of mannose residues are expected to affect the reactivity of mannosylated glycoproteins with mannose-binding lectin and possibly the entire cascade of events linked to this lectin. Since patients with ESRD are prone to cardiovascular complications and the formation of atherosclerotic plaques, one can hypothesize that fibrinogen with increasingly exposed mannose residues may contribute to the unwanted events.

Cell-surface protein YwfG of Lactococcus lactis binds to α-1,2-linked mannose.

Lactococcus lactis strains are used as starter cultures in the production of fermented dairy and vegetable foods, but the species also occurs in other niches such as plant material. Lactococcus lactis subsp. lactis G50 (G50) is a plant-derived strain and potential candidate probiotics. Western blotting of cell-wall proteins using antibodies generated against whole G50 cells detected a 120-kDa protein. MALDI-TOF MS analysis identified it as YwfG, a Leu-Pro-any-Thr-Gly cell-wall-anchor-domain-containing protein. Based on a predicted domain structure, a recombinant YwfG variant covering the N-terminal half (aa 28-511) of YwfG (YwfG28-511) was crystallized and the crystal structure was determined. The structure consisted of an L-type lectin domain, a mucin-binding protein domain, and a mucus-binding protein repeat. Recombinant YwfG variants containing combinations of these domains (YwfG28-270, YwfG28-336, YwfG28-511, MubR4) were prepared and their interactions with monosaccharides were examined by isothermal titration calorimetry; the only interaction observed was between YwfG28-270, which contained the L-type lectin domain, and d-mannose. Among four mannobioses, α-1,2-mannobiose had the highest affinity for YwfG28-270 (dissociation constant = 34 μM). YwfG28-270 also interacted with yeast mannoproteins and yeast mannan. Soaking of the crystals of YwfG28-511 with mannose or α-1,2-mannobiose revealed that both sugars bound to the L-type lectin domain in a similar manner, although the presence of the mucin-binding protein domain and the mucus-binding protein repeat within the recombinant protein inhibited the interaction between the L-type lectin domain and mannose residues. Three of the YwfG variants (except MubR4) induced aggregation of yeast cells. Strain G50 also induced aggregation of yeast cells, which was abolished by deletion of ywfG from G50, suggesting that surface YwfG contributes to the interaction with yeast cells. These findings provide new structural and functional insights into the interaction between L. lactis and its ecological niche via binding of the cell-surface protein YwfG with mannose.

Mixed mechanism of conformational selection and induced fit as a molecular recognition process in the calreticulin family of proteins.

The fundamental question on the mechanism of molecular recognition during ligand binding has attracted a lot of scientific scrutiny. The two competing theories of ligand binding-"induced fit" and "conformational selection" have been proposed to explain biomolecular recognition. Since exploring a family of proteins with similar structural architectures and conserved functional roles can provide valuable insight into the significance of molecular structure and function, we performed molecular dynamics simulations on the calreticulin family of proteins, which specifically recognize monoglucosylated N-glycan during the protein folding process. Atomistic simulations of lectins in free and bound forms demonstrated that they exist in several conformations spanning from favorable to unfavorable for glycan binding. Our analysis was confined to the carbohydrate recognition domain (CRD) of these lectins to demonstrate the degree of conservation in protein sequence and structure and relate them with their function. Furthermore, we computed the lectin-glycan binding affinity using the mmPBSA approach to identify the most favorable lectin conformation for glycan binding and compared the molecular interaction fields in terms of noncovalent bond interactions. We also demonstrated the involvement of Tyr and Trp residues in the CRD with the non-reducing end glucose and central mannose residues, which contribute to some of the specific interactions. Furthermore, we analyzed the conformational changes in the CRD through SASA, RMSFs and protein surface topography mapping of electrostatic and hydrophobic potentials. Our findings demonstrate a hybrid mechanism of molecular recognition, initially driven by conformational selection followed by glycan-induced fluctuations in the key residues to strengthen the glycan binding interactions.

Synthesis of antimicrobial polymers with mannose residues as binders for the FimH adhesin of Escherichia coli

AbstractRecently, antimicrobial polymers have been developed as novel antibiotics, and they are beneficial for human health. An advantage of these polymers is that they can be easily functionalized via the addition or modification of monomers. In this study, to enhance the bacterial selectivity and antimicrobial activity of a polymer, mannose monomers were copolymerized with aminopropyl monomers and isoalkyl monomers. These mannose monomers were found to mimic the mannan and N‐glycan moieties on mammalian cells interacting with the FimH adhesin of Escherichia coli. A mannose monomer with a short ethyl linker did not enhance the antimicrobial activity (minimum inhibitory concentration (MIC): 32 μg/ml) against E. coli; however, that with a longer hexyl linker enhanced the antimicrobial activity (MIC: 16 μg/ml) against E. coli selectivity (no effect on Staphylococcus aureus was observed). Further, the polymer containing the hexyl mannose residue did not develop resistance in E. coli after 15 passages. Consequently, polymers with mannose units, which have a high affinity for the FimH adhesin, can serve as effective and selective antimicrobial agents against E. coli.

Trypanosoma cruzi-Derived Molecules Induce Anti-Tumour Protection by Favouring Both Innate and Adaptive Immune Responses

Lung cancer remains the leading cause of cancer mortality worldwide. Thus, the development of strategies against this type of cancer is of high value. Parasite infections can correlate with lower cancer incidence in humans and their use as vaccines has been recently explored in preclinical models. In this study, we investigated whether immunisations with a Trypanosoma cruzi lysate from epimastigotes protect from lung tumour growth in mice. We also explore the role of parasite glycans in the induction of the protective immune response. A pre-clinical murine cancer model using the lung tumour cell line LL/2 was used to evaluate the anti-tumour potential, both in preventive and therapeutic settings, of a T. cruzi epimastigote-derived protein lysate. Immunisation with the parasite lysate prevents tumour growth and induces both humoral and cellular anti-tumour immune responses to LL-2 cancer cells. The induced immunity and tumour protection were associated with the activation of natural killer (NK) cells, the production of interferon-γ (IFN-γ) and tumour cell cytotoxicity. We also show that mannose residues in the T. cruzi lysate induce Toll-like receptor (TLR) signalling. The evaluated T. cruzi lysate possesses anti-tumour properties likely by activating innate and adaptive immunity in a process where carbohydrates seem to be essential.

An insight into new glycotherapeutics in glial inflammation: Understanding the role of glycosylation in mitochondrial function and acute to the chronic phases of inflammation.

Glycosylation plays a critical role during inflammation and glial scar formation upon spinal cord injury (SCI) disease progression. Astrocytes and microglia are involved in this cascade to modulate the inflammation and tissue remodeling from acute to chronic phases. Therefore, understating the glycan changes in these glial cells is paramount. A lectin microarray was undertaken using a cytokine-driven inflammatory mixed glial culture model, revealing considerable differential glycosylation from the acute to the chronic phase in a cytokine-combination generated inflamed MGC model. It was found that several N- and O-linked glycans associated with glia during SCI were differentially regulated. Pearson's correlation hierarchical clustering showed that groups were separated into several clusters, illustrating the heterogenicity among the control, cytokine combination, and LPS treated groups and the day on which treatment was given. Control and LPS treatments were observed to be in dense clusters. This was further confirmed with lectin immunostaining in which GalNAc, GlcNAc, mannose, fucose and sialic acid-binding residues were detected in astrocytes and microglia. However, the sialyltransferase inhibitor inhibited this modification (upregulation of the sialic acid expression), which indeed modulates the mitochondrial functions. The present study is the first functional investigation of glycosylation modulation in a mixed glial culture model, which elucidates the role of the glycome in neuroinflammation in progression and identified potential therapeutic targets for future glyco therapeutics in neuroinflammation.

Abstract 11515: Mannose-Binding Lectin is Dysregulated in Cardiac Endothelial Cells of Women With Peripartum Cardiomyopathy

Introduction: Peripartum cardiomyopathy (PPCM) is a type of heart failure that presents with systolic dysfunction in the third trimester of pregnancy or early postpartum. Viral myocarditis, genetic predisposition, and hormonal changes are suggested as contributing factors to PPCM. However, the molecular basis of PPCM remained elusive. Hypothesis: Cardiac expression of genes involved in immune system response is dysregulated in PPCM. Methods: We identified women with PPCM and age/race-matched heart donors from a dataset obtained from Myocardial Applied Genomics Network (MAGNet) consortium. Left ventricular (LV) biopsies were taken at the time of heart transplantation. Bulk RNA-seq was performed on LV tissue of patients with PPCM and non-failing heart donors (NF). We used the Human Cell Atlas (HCA) reference database with accession code ERP128138 to investigate cell-specific enrichment of genes. Results: Mean age of study participants was 36.1 years and 33.3% of participants had African American ancestry. We found that LCN6 (Log2(fold_change) = -4.1, p-val = 0.0027) and FCN3 (Log2(fold_change) = -3.4, p-val = 0.0042) are downregulated in LV tissue obtained from PPCM compared to NF. LCN6 encodes lipocalin-6 and FCN3 encodes ficolin-3 which are upstream of the mannose-binding lectin complement pathway. Based on HCA dataset on adult human hearts, we observed that LCN6 and FCN3 genes are mainly expressed by endothelial cells. Conclusions: We demonstrated that expression of LCN6 and FCN3 genes, which are upstream of the mannose-binding lectin pathway, is decreased in cardiac endothelial cells of individuals with PPCM. Mannose-binding lectin is a soluble pattern recognition receptor that identifies mannose and N-acetylglucosamine residues in a wide variety of pathogens and antigen-antibody complexes and works as an opsonin that facilitates phagocytosis. Our findings suggest the possibility of impaired immune complex clearance in PPCM pathogenesis.

Synthesis & Evaluation of Novel Mannosylated Neoglycolipids for Liposomal Delivery System Applications.

Glycosylated NPs, including liposomes, are known to target various receptors involved in cellular carbohydrate transport, of which the mannoside binding receptors are attracting particular attention for their expression on various immune cells, cancers, and cells involved in maintaining central nervous system (CNS) integrity. As part of our interest in NP drug delivery, mannosylated glycoliposomal delivery systems formed from the self-assembly of amphiphilic neoglycolipids were developed, with a C12-alkyl mannopyranoside (ML-C12) being identified as a lead compoundcapable of entrapping, protecting, and improving the delivery of structurally diverse payloads. However, ML-C12 was not without limitations in both the synthesis of the glycolipids, and the physicochemical properties of the resulting glycoliposomes. Herein, the chemical syntheses of a novel series of mannosylated neoglycolipids are reported with the goal of further improving on the previous ML-C12 glyconanoparticles. The current work aimed to use a self-contingent strategy which overcomes previous synthetic limitations to produce neoglycolipids that have one exposed mannose residue, an aromatic scaffold, and two lipid tails with varied alkyl chains. The azido-ending carbohydrates and the carboxylic acid-ending lipid tails were ligated using a new one-pot modified Staudinger chemistry that differed advantageously to previous syntheses. The formation of stable neoglycoliposomes of controllable and ideal sizes (≈100-400 nm) was confirmed via dynamic light scattering (DLS) experiments and transmission electron microscopy (TEM). Beyond chemical advantages, the present study further aimed to establish potential improvements in the biological activity of the neoglycoliposomes. Concanavalin A (Con A) agglutination studies demonstrated efficient and stable cross-linking abilities dependent on the length of the linkers and lipid tails. The efficacy of the glycoliposomes in improving cytosolic uptake was investigated using Nile Red as probe in immune and cancer cell lines. Preliminary ex vivo safety assessments showed that the mannosylated glycoliposomes are hemocompatible, and non-immunogenic. Finally, using a model peptide therapeutic, the relative entrapment capacity and plasma stability of the optimal glycoliposome delivery system was evaluated and compared to the previous neoglycoliposomes. Overall, the new lead glycoliposome showed improved biological activity over ML-C12, in addition to having several chemical benefits including the lack of stereocenters, a longer linker allowing better sugar availability, and ease of synthesis using novel one-pot modified Staudinger chemistry.

Structure, Function and Mechanism of N‐Glycan Processing Enzymes: endo‐α‐1,2‐Mannanase and endo‐α‐1,2‐Mannosidase

AbstractWhile most glycosidases that act on N‐linked glycans remove a single sugar residue at a time, endo‐α‐1,2‐mannosidases and endo‐α‐1,2‐mannanases of glycoside hydrolase family GH99 cut within a chain and remove two or more sugar residues. They are stereochemically retaining enzymes that use an enzymatic mechanism involving an epoxide intermediate. Human endo‐α‐1,2‐mannosidase (MANEA) trims glucosylated mannose residues; the endomannosidase pathway provides a glucosidase‐independent pathway for glycoprotein maturation. Cell‐active MANEA inhibitors alter N‐glycan processing and reduce infectivity of dengue virus, demonstrating that MANEA has potential as a host‐directed antiviral target. Sequence‐related enzymes from gut Bacteroides spp. exhibit endo‐α‐1,2‐mannosidase activity and are a fruitful test bed for structure‐guided inhibitor development. The genes encoding the Bacteroides spp. enzymes sit within polysaccharide utilization loci and are preferential endo‐α‐1,2‐mannanases.

Isolation and characterization of high-mannose type glycans containing five or six mannose residues from hen egg yolk

High-mannose type glycans play important roles in biosynthesis of glycoproteins including glycoprotein quality control system. In the endoplasmic reticulum (ER), α1,2-mannosidases cleave several mannose (Man) residues to give small high-mannose type glycans, such as glycans containing five or six mannose residues (M5-glycan or M6-glycan). These glycans are reported to act as a signal for degradation processes of glycoproteins in the ER. In this work, we isolated the M5-glycan and the M6-glycan from delipidated egg yolk and confirmed that their structures were identical to human type glycans based on rigorous NMR experiments, suggesting the potential use for semisynthesis of glycoconjugates and glycan analysis.

High mannose level in bladder cancer enhances type 1 fimbria-mediated attachment of uropathogenic E. coli.

Bladder cancer is the 4th leading cancer in men. Tumor resection followed by bladder instillation of Bacillus Calmette-Guérin (BCG) is the primary treatment for high-risk patients with Non-Muscle Invasive Bladder Cancer (NMIBC) to prevent recurrence and progression to muscle-invasive disease. This treatment, however, lacks efficiency and causes severe adverse effects. Mannose residues are expressed on bladder surfaces and their levels were indicated to be higher in bladder cancer. Intravesical instillations of a recombinant Pseudomonas aeruginosa (PA) overexpressing the mannose-sensitive hemagglutination fimbriae (PA-MSHA), and of a mannose-specific lectin-drug conjugate showed efficiency against NMIBC in murine models of bladder cancer. Urothelial mannosylation facilitates bladder colonization by Uropathogenic E. coli (UPEC) via the interaction with the FimH mannose lectin, positioned at the tip of type 1 fimbria. A recombinant BCG strain overexpressing FimH on its outer surface, exhibited higher attachment and internalization to bladder cancer cells and increased effectivity in treating bladder cancer in mice. Investigating the pattern of mannose expression in NMIBC is important for improving treatment. Here, using tissue microarrays containing multiple normal and cancerous bladder samples, and lectins, we confirm that human bladder cancer cells express high mannose levels. Using UPEC mutants lacking or overexpressing type 1 fimbria, we also demonstrate that tumor-induced hypermannosylation increases type 1 fimbria mediated UPEC attachment to human and mouse bladder cancer. Our results provide an explanation for the effectiveness of PA-MSHA and the FimH-overexpressing BCG and support the hypothesis that mannose-targeted therapy holds potential for improving bladder cancer treatment.

Characterization and biological evaluation of Crataegus azarolus fruit polysaccharides

The polysaccharide obtained from Crataegus azarolus fruit, by water extraction followed by ethanol precipitation and fractionation using copper complex, shows physiological activities: prebiotic and antioxidant. The total acid hydrolysis shows that the polysaccharide consists of galactose, glucose, mannose and rhamnose. The employed physico-chemical techniques of the polysaccharide sequences approach (FT-IR, MALDI-TOF-MS spectrometry, 1H and 13C NMR) indicate that it is a Galactoglucomannan, the main chain consists of major amounts of β-(1→4)-linked glucose and mannose residues with monosaccharide branches of α-(1→6) galactose and O-acetyl substituents. The properties of this new glucomannan suggest that it could find an application as a prebiotic in the food industry.

Inositol acylation of phosphatidylinositol mannosides: a rapid mass response to membrane fluidization in mycobacteria

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of the plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) are rapidly remodeled upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a triacylated form: two acyl chains of the PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, we determined the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. Additionally, we show that PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol pre-exposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.

Unusual β1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell–pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected β1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel β1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal β1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.

Mnt1, an α-(1→2)-mannosyltransferase responsible for the elongation of N-glycans and O-glycans in Aspergillus fumigatus.

The fungal cell wall is necessary for survival as it serves a barrier for physical protection. Therefore, glycosyltransferases responsible for the synthesis of cell wall polysaccharides may be suitable targets for drug development. Mannose is a monosaccharide that is commonly found in sugar chains in the walls of fungi. Mannose residues are present in fungal-type galactomannan, O-glycans, N-glycans, glycosylphosphatidylinositol anchors, and glycosyl inositol phosphorylceramides in Aspergillus fumigatus. Three genes that are homologous to α-(1→2)-mannosyltransferase genes and belong to the glycosyltransferase family 15 were found in the A. fumigatus strain, Af293/A1163, genome: cmsA/ktr4, cmsB/ktr7, and mnt1. It is reported that the mutant ∆mnt1 strain exhibited a wide range of properties that included high temperature and drug sensitivity, reduced conidia formation, leakage at the hyphal tips, and attenuation of virulence. However, it is unclear whether Mnt1 is a bona fide α-(1→2)-mannosyltransferase and which mannose residues are synthesized by Mnt1 in vivo. In this study, we elucidated the structure of the Mnt1 reaction product, the structure of O-glycan in the Δmnt1 strain. In addition, the length of N-glycans attached to invertase was evaluated in the Δmnt1 strain. The results indicated that Mnt1 functioned as an α-(1→2)-mannosyltransferase involved in the elongation of N-glycans and synthesis of the second mannose residue of O-glycans. The widespread abnormal phenotype caused by the disruption of the mnt1 gene is the combined result of the loss of mannose residues from O-glycans and N-glycans. We also clarified the enzymatic properties and substrate specificity of Mnt1 based on its predicted protein structure.

A novel mannose-containing sialoprotein adhesin involved in the binding of Candida albicans cells to DMBT1.

Candida albicans colonizes the oral cavity and causes oral candidiasis and early childhood caries synergistically with cariogenic Streptococcus mutans. Colonization of oral tissues with C. albicans is an essential step in the initiation of these infectious diseases. Deleted in malignant brain tumors 1 (DMBT1), also known as salivary agglutinin or gp-340, belongs to the scavenger receptor cysteine-rich (SRCR) superfamily and has important functions in innate immunity. In the oral cavity, DMBT1 causes microbial adherence to tooth enamel and oral mucosa surfaces, but the adherence of C. albicans to DMBT1 has not been examined. In this study, we investigated the binding of C. albicans to DMBT1 and isolated the fungal components responsible for the binding. Candida albicans specifically bound to DMBT1 and strongly bound to the peptide domain SRCRP2. Binding to SRCRP2 was inhibited by N-acetylneuraminic acid and mannose and by lectins recognizing these sugars. The isolated component had a molecular mass of 25kDa, contained sialic acid and mannose residues, and inhibited C. albicans binding to SRCRP2. The localization of the 25-kDa protein on the surface of C. albicans cell walls was confirmed by immunostaining and a cell ELISA using an antiserum to the protein, and Western blotting revealed the presence of the 25-kDa protein in the cell wall fraction of C. albicans. These results suggest that the isolated adhesin is localized on the surface of C. albicans cell walls and that sialic acid and mannose residues in the adhesin play a significant role in the binding reaction.

Application of Glucomannan

Glucomannan (GM) a water-soluble polysaccharide possesses mannose residues which are extracted from tubers, bulbs, softwoods and roots of many plants. Glucomannan is considered to be a dietary fibre which is actually present in some plant species in the form of hemicelluloses component in the cell wall. Its structure consists of linear chain of mixed residues of α-1,4 linked D-mannose and D-glucose monomers arranged in blocks. The molecular weight of native GM lies between 1×104– 2×106. Low molecular weight GM could be obtained by de-polymerization technique. The average molecular weight of Konjac Glucomannan (KGM) is 500,000 – 2,000,000 and varies with species, growing area, storage time and processing methods. The glucomannan from different sources vary in its mannose to glucose ratio. Konjac tuber glucomannan has a molar ratio of 1.6:1 or 1.4:1 (ratio differs with konjac breeds), Orchid tubers and Scotch pine have molar ratios of 3.6:1 and 2.1:1 respectively. Diversity of glucomannan also depends on degree of acetylation in GM chain. Values of degree of acetylation are 5to10% or every 19th sugar residue (attached randomly at C-6 position in KGM), is actually responsible to facilitate dispersion and solubility by inhibiting the intra-molecular hydrogen bonds. This solubility function is attractive for multiple pharmaceutical applications. However increase in acetylation degree in GM slows the gelation process. The intrinsic viscosity of KGM solution is highest among the polysaccharides that facilitate swelling behaviour and hence gel formation that finds application in food industry. Gels have good stability, films, hydrogel, beads, micro and nanoparticles of Glucomannan may have potential usage in therapeutic drug delivery systems without causing toxicity. It may also be useful in treatment of chronic constipation, decreasing serum cholesterol, and increasing insulin sensitivity. As a food supplement it could play a significant role in weight loss. Carboxymethylated glucomannan improves the properties of paper, such as burst index, dry tensile index, and wet tensile index. These diverse applications make Glucomannan a most sought after biomolecule. Keywords: Biomolecule, Glucomannan, Diverse application

Electrical sensor system for in vitro bacteria biofilm diagnostics

Bacterial biofilm is a complex community of microbes that contributes to the formation of bacterial resistance to adverse environmental factors. Detection and analysis of biologically active compounds indicative of biofilm development is a promising direction in the diagnosis of biofilm formation. This paper describes the possibility of detecting surface changes in Pseudomonas bacteria when they change from planktonic to biofilm form. These surface changes were detected through the conformational changes in the outer membrane components, including lipopolysaccharide (LPS). The electrophysical profile of the same strain differed largely between planktonic and biofilm cultures. The monosaccharide and fatty acid composition and the electrophoretic mobility of cell surface LPS and the biofilm matrix polysaccharides of P. putida TSh-18 were characterized. In the LPS of biofilm cells, rhamnose and glucose residues prevailed; in the biofilm matrix polysaccharides, rhamnose, mannose, and glucose residues prevailed. The fatty acid composition of the LPS of biofilms was similar to that of the LPS of planktonic cells. The biofilms contained both S- and R-form LPS. The results are promising for the development of an electrical sensor method for the rapid in vitro diagnostics of the initial phase of bacterial biofilm formation and timely blocking of biofilm communities.