Characterization of the need for galactofuranose during the Neurospora crassa life cycle.

Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases.

Structure and Anticoagulant Activity of a Galactofuranose-Containing Sulfated Polysaccharide from the Green Seaweed, Codium isthmocladum.

A water-soluble sulfated polysaccharide, F2-1, was obtained from the marine green alga, Codium isthmocladum, using ion-exchange and size-exclusion chromatography. Structure analysis showed that the F2-1 was a sulfated arabinan comprising Ara, Rha, Man, Gal, and Xyl with an 18% sulfate content and a molecular weight of 100 kDa. Methylation analysis combined with desulfation, GC-MS, IR, and NMR spectroscopy showed that the backbone of F2-1 was →4)-ß-L-Arap(1→ residue. Its 2-O and/or 3-O positions showed sulfate modification; additionally, the 2-O or 3-O position showed branch points. The side chains were composed of →5)-ß-D-Galf, (1→2,6)-ß-D-Galf(1→, (1→2)-ß-L-Rhap4S, →4)-α-D-Glcp(1→, and terminal α-D-Galp(1→ and ß-D-Xylp(1→. Polysaccharides containing ß-D-galactofuranose are rarely found in seaweed. F2-1 exhibited significant anticoagulant activity in vitro. Our findings suggested that the green-tide alga, Codium isthmocladum, can be considered as a useful resource for bioactive polysaccharides.

Specific Recognition of ß-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients.

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients' blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method's limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal ß-galactofuranosyl (ß-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients' sera react specifically with synthetic ß-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfß1,3Manpα-(CH2)3SH (glycan G29SH) and Galfß1,3Manpα1,2-[Galfß1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfß1,3Manpα1,2-[Galfß1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan.

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-ß-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal ß-(1,5) and ß-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

Functional analysis of three putative galactofuranosyltransferases with redundant functions in galactofuranosylation in Aspergillus niger.

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.

The Glycan Structure of T. cruzi mucins Depends on the Host. Insights on the Chameleonic Galactose.

Trypanosoma cruzi, the protozoa that causes Chagas disease in humans, is transmitted by insects from the Reduviidae family. The parasite has developed the ability to change the structure of the surface molecules, depending on the host. Among them, the mucins are the most abundant glycoproteins. Structural studies have focused on the epimastigotes and metacyclic trypomastigotes that colonize the insect, and on the mammal trypomastigotes. The carbohydrate in the mucins fulfills crucial functions, the most important of which being the accepting of sialic acid from the host, a process catalyzed by the unique parasite trans-sialidase. The sialylation of the parasite influences the immune response on infection. The O-linked sugars have characteristics that differentiate them from human mucins. One of them is the linkage to the polypeptide chain by the hexosamine, GlcNAc, instead of GalNAc. The main monosaccharide in the mucins oligosaccharides is galactose, and this may be present in three configurations. Whereas ß-d-galactopyranose (ß-Galp) was found in the insect and the human stages of Trypanosoma cruzi, ß-d-galactofuranose (ß-Galf) is present only in the mucins of some strains of epimastigotes and α-d-galactopyranose (α-Galp) characterizes the mucins of the bloodstream trypomastigotes. The two last configurations confer high antigenic properties. In this review we discuss the different structures found and we pose the questions that still need investigation on the exchange of the configurations of galactose.

In vivo role of capsular polysaccharide in Mycoplasma mycoides.

Capsular polysaccharides have been confirmed to be an important virulence trait in many gram-positive and gram-negative bacteria. Similarly, they are proposed to be virulence traits in minimal Mycoplasma that cause disease in humans and animals. In the current study, goats were infected with the caprine pathogen Mycoplasma mycoides subsp. capri or an engineered mutant lacking the capsular polysaccharide, galactofuranose. Goats infected with the mutant strain showed only transient fever. In contrast, 5 of 8 goats infected with the parental strain reached end-point criteria after infection. These findings confirm that galactofuranose is a virulence factor in M. mycoides.

Chemical synthesis of the pentasaccharide repeating unit of the O-specific polysaccharide from Escherichia coli O132 in the form of its 2-aminoethyl glycoside.

The total chemical synthesis of the pentasaccharide repeating unit of the O-polysaccharide from E. coli O132 is accomplished in the form of its 2-aminoethyl glycoside. The 2-aminoethyl glycoside is particularly important as it allows further glycoconjugate formation utilizing the terminal amine without affecting the stereochemistry of the reducing end. The target was achieved through a [3 + 2] strategy where the required monosaccharide building blocks are prepared from commercially available sugars through rational protecting group manipulation. The NIS-mediated activation of thioglycosides was used extensively for the glycosylation reactions throughout.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

Identification of novel inhibitors against UDP-galactopyranose mutase to combat leishmaniasis.

Leishmania, a protozoan parasite that causes leishmaniasis, affects 1-2 million people every year worldwide. Leishmaniasis is a vector born disease and characterized by a diverse group of clinical syndromes. Current treatment is limited because of drug resistance, high cost, poor safety, and low efficacy. The urgent need for potent agents against Leishmania has led to significant advances in the development of novel antileishmanial drugs. ß-galactofuranose (ß-Galf) is an important component of Leishmanial cell surface matrix and plays a critical role in the pathogenesis of parasite. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) which acts as the precursor for ß-Galf synthesis. Due to its absence in human, this enzyme is selected as the potential target in search of new antileishmanial drugs. Three dimensional protein structure model of Leishmania major UGM (LmUGM) has been homology modeled using Trypanosoma cruzi UGM (TcUGM) as a template. The stereochemistry was validated further. We selected already reported active compounds from PubChem database to target the LmUGM. Three compounds (6064500, 44570814, and 6158954) among the top hit occupied the UDP binding site of UGM suggested to work as a possible inhibitor for it. In vitro antileishmanial activity assay was performed with the top ranked inhibitor, 6064500. The 6064500 molecule has inhibited the growth of Leishmania donovani promastigotes significantly. Further, at similar concentrations it has exhibited significantly lesser toxicity than standard drug miltefosine hydrate in mammalian cells.

Biosynthesis of galactomannans found in filamentous fungi belonging to Pezizomycotina.

The galactomannans (GMs) that are produced by filamentous fungi belonging to Pezizomycotina, many of which are pathogenic for animals and plants, are polysaccharides consisting of α-(1→2)-/α-(1→6)-mannosyl and ß-(1→5)-/ß-(1→6)-galactofuranosyl residues. GMs are located at the outermost layer of the cell wall. When a pathogenic fungus infects a host, its cell surface must be in contact with the host. The GMs on the cell surface may be involved in the infection mechanism of a pathogenic fungus or the defense mechanism of a host. There are two types of GMs in filamentous fungi, fungal-type galactomannans and O-mannose type galactomannans. Recent biochemical and genetic advances have facilitated a better understanding of the biosynthesis of both types. This review summarizes our current information on their biosynthesis.

Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition.

Intelectins (X-type lectins), broadly distributed throughout chordates, have been implicated in innate immunity. Xenopus laevis embryonic epidermal lectin (XEEL), an intelectin secreted into environmental water by the X. laevis embryo, is postulated to function as a defense against microbes. XEEL is homologous (64% identical) to human intelectin-1 (hIntL-1), which is also implicated in innate immune defense. We showed previously that hIntL-1 binds microbial glycans bearing exocyclic vicinal diol groups. It is unknown whether XEEL has the same ligand specificity. Also unclear is whether XEEL and hIntL-1 have similar quaternary structures, as XEEL lacks the corresponding cysteine residues in hIntL-1 that stabilize the disulfide-linked trimer. These observations prompted us to further characterize XEEL. We found that hIntL-1 and XEEL have similar structural features. Even without the corresponding intermolecular disulfide bonds present in hIntL-1, the carbohydrate recognition domain of XEEL (XEELCRD) forms a stable trimer in solution. The structure of XEELCRD in complex with d-glycerol-1-phosphate, a residue present in microbe-specific glycans, indicated that the exocyclic vicinal diol coordinates to a protein-bound calcium ion. This ligand-binding mode is conserved between XEEL and hIntL-1. The domain architecture of full-length XEEL is reminiscent of a barbell, with two sets of three glycan-binding sites oriented in opposite directions. This orientation is consistent with our observation that XEEL can promote the agglutination of specific serotypes of Streptococcus pneumoniae. These data support a role for XEEL in innate immunity, and they highlight structural and functional conservation of X-type lectins among chordates.

GfsA is a ß1,5-galactofuranosyltransferase involved in the biosynthesis of the galactofuran side chain of fungal-type galactomannan in Aspergillus fumigatus.

Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-d-galactofuranose as a sugar donor, and p-nitrophenyl-ß-d-galactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the ß-galactofuranose residue via a ß-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-d-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the ∆AfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of ß1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-d-galactofuranose: ß-d-galactofuranoside ß1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.

Distribution of the O-acetyl groups and ß-galactofuranose units in galactoxylomannans of the opportunistic fungus Cryptococcus neoformans.

Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that ß-galactofuranose (ß-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.

Multiple functionalities of reduced flavin in the non-redox reaction catalyzed by UDP-galactopyranose mutase.

Flavin cofactors are widely used by enzymes to catalyze a broad range of chemical reactions. Traditionally, flavins in enzymes are regarded as redox centers, which enable enzymes to catalyze the oxidation or reduction of substrates. However, a new class of flavoenzyme has emerged over the past quarter century in which the flavin functions as a catalytic center in a non-redox reaction. Here we introduce the unifying concept of flavin hot spots to understand and categorize the mechanisms and reactivities of both traditional and noncanonical flavoenzymes. The major hot spots of reactivity include the N5, C4a, and C4O atoms of the isoalloxazine, and the 2' hydroxyl of the ribityl chain. The role of hot spots in traditional flavoenzymes, such as monooxygenases, is briefly reviewed. A more detailed description of flavin hot spots in noncanonical flavoenzymes is provided, with a focus on UDP-galactopyranose mutase, where the N5 functions as a nucleophile that attacks the anomeric carbon atom of the substrate. Recent results from mechanistic enzymology, kinetic crystallography, and computational chemistry provide a complete picture of the chemical mechanism of UDP-galactopyranose mutase.

Distinct galactofuranose antigens in the cell wall and culture supernatants as a means to differentiate Fusarium from Aspergillus species.

Detection of carbohydrate antigens is an important means for diagnosis of invasive fungal infections. For diagnosis of systemic Aspergillus infections, galactomannan is commonly used, the core antigenic structure of which consists of chains of several galactofuranose moieties. In this study, we provide evidence that Fusarium produces at least two distinct galactofuranose antigens: Smaller amounts of galactomannan and larger quantities of a novel antigen recognized by the monoclonal antibody AB135-8. In A. fumigatus, only minor amounts of the AB135-8 antigen are found in supernatants and in the apical regions of hyphae. A galactofuranose-deficient A. fumigatus mutant lacks the AB135-8 antigen, which strongly suggests that galactofuranose is an essential constituent of this antigen. Using a combination of AB135-8 and a galactomannan-specific antibody, we were able to unambiguously differentiate A. fumigatus and Fusarium hyphae in immunohistology. Moreover, since Fusarium releases the AB135-8 antigen, it appears to be a promising target antigen for a serological detection of Fusarium infections.

Synthesis and biological evaluation of nonionic substrate mimics of UDP-Galp as candidate inhibitors of UDP galactopyranose mutase (UGM).

The synthesis of 1-[5-O-(α-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione and 1-[(5-O-(ß-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione as non-ionic substrate mimics of UDP-Galp are described. UDP-Galp is a precursor of Galf, which is a primary component of the cell-wall glycans of several microorganisms. The interconversion of UDP-Galp and UDP-Galf is catalyzed by UDP galactopyranose mutase (UGM); its inhibition comprises a mode of compromising the microorganisms. The nonionic polyhydroxylated chain was intended to mimic the ionic pyrophosphate group and the ribose moiety in UDP-Galp and increase the bioavailabilities of the candidate inhibitors. Inhibition assays with UGM of Mycobacterium tuberculosis showed only weak inhibition of the enzyme by these compounds.

Synthesis of the O-linked hexasaccharide containing ß-D-Galp-(1→2)-D-Galf in Trypanosoma cruzi mucins. Differences on sialylation by trans-sialidase of the two constituent hexasaccharides.

The hexasaccharide ß-D-Galp-(1→2)-[ß-D-Galp-(1→3)]-ß-D-Galp-(1→6)-[ß-D-Galp(1→2)-ß-D-Galf(1→4)]-D-GlcNAc (10) and its ß-D-Galf-(1→2)-ß-D-Galf containing isomer (7) are the largest carbohydrates in mucins of some strains of Trypanosoma cruzi. The terminal ß-D-Galp units are sites of sialylation by the parasite trans-sialidase. Hexasaccharide 10 was chemically synthesized for the first time by a [3+3] nitrilium based convergent approach, using the trichloroacetimidate method of glycosylation. The (1)H NMR spectrum of its alditol was identical to the spectrum of the product released by ß-elimination from the parasite mucin. The trans-sialylation reaction studied on the benzyl glycoside of 10 showed two monosialylated products whose relative abundance changed with time. On the other hand, only one product was produced by sialylation of the benzyl glycoside of 7. A preparative synthesis of the latter and spectroscopic analysis of the product unequivocally established the sialylation site at the less hindered (1→3)-linked galactopyranose.

Structure, mechanism, and dynamics of UDP-galactopyranose mutase.

The flavoenzyme UDP-galactopyranose mutase (UGM) is a key enzyme in galactofuranose biosynthesis. The enzyme catalyzes the 6-to-5 ring contraction of UDP-galactopyranose to UDP-galactofuranose. Galactofuranose is absent in humans yet is an essential component of bacterial and fungal cell walls and a cell surface virulence factor in protozoan parasites. Thus, inhibition of galactofuranose biosynthesis is a valid strategy for developing new antimicrobials. UGM is an excellent target in this effort because the product of the UGM reaction represents the first appearance of galactofuranose in the biosynthetic pathway. The UGM reaction is redox neutral, which is atypical for flavoenzymes, motivating intense examination of the chemical mechanism and structural features that tune the flavin for its unique role in catalysis. These studies show that the flavin functions as nucleophile, forming a flavin-sugar adduct that facilitates galactose-ring opening and contraction. The 3-dimensional fold is novel and conserved among all UGMs, however the larger eukaryotic enzymes have additional secondary structure elements that lead to significant differences in quaternary structure, substrate conformation, and conformational flexibility. Here we present a comprehensive review of UGM three-dimensional structure, provide an update on recent developments in understanding the mechanism of the enzyme, and summarize computational studies of active site flexibility.

Expedient synthesis of 1,6-anhydro-α-D-galactofuranose, a useful intermediate for glycobiological tools.

A new and efficient three-step procedure for the synthesis of 1,6-anhydro-α-D-galactofuranose is described. The key step involves the formation of the galactofuranosyl iodide by treatment of per-O-TBS-D-Galf with TMSI, the selective 6-O-desilylation by an excess of TMSI, and the simultaneous nucleophilic attack of the 6-hydroxy group on the anomeric carbon, with the iodide as a good leaving group. This compound is a good precursor for building blocks for the construction of 1→6 linkages.