STD-NMR studies of two acceptor substrates of GlfT2, a galactofuranosyltransferase from Mycobacterium tuberculosis: Epitope mapping studies

The major structural component of the mycobacterial cell wall, the mycolyl–arabinogalactan–peptidoglycan complex, possesses a galactan core composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating β-(1→6) and β-(1→5) linkages. Recent studies have shown that the entire galactan is synthesized by two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation transfer difference (STD) NMR studies GlfT2 using two trisaccharide acceptor substrates, β-d-Galf-(1→6)-β-d-Galf-(1→5)-β-d-Galf-O(CH₂)₇CH₃ (2) and β-d-Galf-(1→5)-β-d-Galf-(1→6)-β-d-Galf-O(CH₂)₇CH₃ (3), as well as the donor substrate for the enzyme, UDP-Galf. Epitope mapping demonstrated a greater enhancement toward the ‘reducing’ ends of both trisaccharides, and that UDP-galactofuranose (UDP-Galf) made more intimate contacts through its nucleotide moiety. This observation is consistent with the greater flexibility required within the active site of the reaction between the growing polymer acceptor and the UDP-Galf donor. The addition of UDP-Galf to either 2 or 3 in the presence of GlfT2 generated a tetrasaccharide product, indicating that the enzyme was catalytically active.     

A processive carbohydrate polymerase that mediates bifunctional catalysis using a single active site

Even in the absence of a template, glycosyltransferases can catalyze the synthesis of carbohydrate polymers of specific sequence. The paradigm has been that one enzyme catalyzes the formation of one type of glycosidic linkage, yet certain glycosyltransferases generate polysaccharide sequences composed of two distinct linkage types. In principle, bifunctional glycosyltransferases can possess separate active sites for each catalytic activity or one active site with dual activities. We encountered the fundamental question of one or two distinct active sites in our investigation of the galactosyltransferase GlfT2. GlfT2 catalyzes the formation of mycobacterial galactan, a critical cell-wall polymer composed of galactofuranose residues connected with alternating, regioisomeric linkages. We found that GlfT2 mediates galactan polymerization using only one active site that manifests dual regioselectivity. Structural modeling of the bifunctional glycosyltransferases hyaluronan synthase and cellulose synthase suggests that these enzymes also generate multiple glycosidic linkages using a single active site. These results highlight the versatility of glycosyltransferases for generating polysaccharides of specific sequence. We postulate that a hallmark of processive elongation of a carbohydrate polymer by a bifunctional enzyme is that one active site can give rise to two separate types of glycosidic bonds.     

The mycelial cell wall of Penicillium charlesii G. Smith NRRL 1887

Mycelial cell wall preparations of Penicillium charlesii G. Smith have been shown to have a total neutral carbohydrate content of 69%. Glucosamine which comprises 12% of the wall is present principally as chitin. Lipids comprise 37.5% of the wall while polypeptides comprise 19.5%. No galactofuranose units are present in the wall of this organism which is known to produce an extracellular β-1,5-galactofuranose containing polymer.     

Galactosyl transferases in mycobacterial cell wall synthesis

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual β-(1→4) and β-(1→5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.     

Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal β-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C₄-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating β-(1→5) and β-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs.     

Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation

A variety of Southern African resurrection plants were surveyed using high-throughput cell wall profiling tools. Species evaluated were the dicotyledons, Myrothamnus flabellifolia and Craterostigma plantagineum; the monocotyledons, Xerophyta viscosa, Xerophyta schlecterii, Xerophyta humilis and the resurrection grass Eragrostis nindensis, as well as a pteridophyte, the resurrection fern, Mohria caffrorum. Comparisons were made between hydrated and desiccated leaf and frond material, with respect to cell wall composition and polymer abundance, using monosaccharide composition analysis, FT-IR spectroscopy and comprehensive microarray polymer profiling in combination with multivariate data analysis. The data obtained suggest that three main functional strategies appear to have evolved to prepare plant cell walls for desiccation. Arabinan-rich pectin and arabinogalactan proteins are found in the resurrection fern M. caffrorum and the basal angiosperm M. flabellifolia where they appear to act as ‘pectic plasticizers’. Dicotyledons with pectin-rich walls, such as C. plantagineum, seem to use inducible mechanisms which consist of up-regulating wall proteins and osmoprotectants. The hemicellulose-rich walls of the grass-like Xerophyta spp. and the resurrection grass E. nindensis were found to contain highly arabinosylated xylans and arabinogalactan proteins. These data support a general mechanism of ‘plasticising’ the cell walls of resurrection plants to desiccation and implicate arabinose-rich polymers (pectin-arabinans, arabinogalactan proteins and arabinoxylans) as the major contributors in ensuring flexibility is maintained and rehydration is facilitated in these plants.     

Contribution of galactofuranose to the virulence of the opportunistic pathogen Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.     

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.     

Elucidation of Substrate Specificity in Aspergillus nidulans UDP-Galactose-4-Epimerase

The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD⁺ and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K _m and k _cat for the enzyme were determined to be 0.11 mM and 12.8 s^-1, respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.     

Synthesis of sugar-amino acid-nucleosides as potential glycosyltransferase inhibitors

Sugar-amino acid-nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner similar to the diphosphate metal ion complex. C-Glycoside analogues of the d-galactopyranose or l-arabinofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were chosen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.     

Structure of hexasaccharide 1-phosphate polymer from Arthrobacter uratoxydans VKM Ac-1979T cell wall

A hexasaccharide 1-phosphate polymer of original structure and two teichoic acids (TA) belonging to different structural types were found in Arthrobacter uratoxydans VKM Ac-1979^T cell wall. The poly(hexasaccharide 1-phosphate) combines features of teichuronic acids and glycosyl 1-phosphate polymers, and its structure has never been reported earlier. Its composition includes residues of α- and β-D-glucuronic acid as well as α-D-galacto-, β-D-gluco-, α-D-mannopyranose, and 6-O-acetylated 2-acetamido-2-deoxy-α-D-glucopyranose. The phosphodiester bond in the polymer joins the glycoside hydroxyl of α-D-glucuronic acid and O6 of α-D-galactopyranose. TA 1 is β-D-glucosylated 1,3-poly(glycerol phosphate), and TA 2 is 3,6-linked poly[α-D-glucosyl-(1→2)-glycerol phosphate]. The phosphate-containing polymers were studied by chemical methods and on the basis of one-dimensional ¹H-, ¹³C-, and ³¹P-NMR spectra, homonuclear two-dimensional ¹H/¹H COSY, TOCSY, ROESY, and heteronuclear ¹H/¹³C HSQC, HSQC-TOCSY, HMBC, and ¹H/³¹P HMBC experiments. The set and structure of the polymers revealed as well as the cell wall sugars (galactose, glucose, mannose, glucosamine) and glycerol can be used in microbiological practice for taxonomic purposes.     

Development of a plate-based scintillation proximity assay for the mycobacterial AftB enzyme involved in cell wall arabinan biosynthesis

A number of mycobacterial arabinosyltransferases, such as the Emb proteins, AftA, AftB, AftC, and AftD have been characterized and implicated to be involved in the cell wall arabinan assembly. These arabinosyltransferases are essential for the viability of the organism and are logically valid targets for developing new anti-tuberculosis agents. For instance, Ethambutol, a first line anti-tuberculosis drug, targets the Emb proteins involved in the formation of the arabinan of cell wall arabinogalactan. Among these arabinosyltransferases, the terminal β-(1→2) arabinosyltransferase activity has been associated with AftB. The predicted topology of AftB in Mycobacterium tuberculosis has 10 N terminal transmembrane domains and a C terminal hydrophilic domain similar to the Emb proteins. It has a conserved GT-C motif and is difficult to express. In a cell free assay, synthetic disaccharide, α-d-Araf-(1→5)-α-d-Araf-octyl, has been used as a substrate to explore the function of AftB. In our work, the disaccharide was synthesized in its pentenylated and biotinylated form, and the enzymatic product formed was identified as the β-(1→2) arabinofuranose adduct. When synthetic tri- and tetra-saccharides were used as substrates, a mixture of products containing both β-(1→2) and α-(1→5) linkages were formed. Therefore, the biotinylated disaccharide was selected to develop a scintillation proximity assay.     

A mannitol teichoic acid containing rhamnose and pyruvic acid acetal from the cell wall of Brevibacterium permense VKM Ac-2280

The cell wall of Brevibacterium permense VKM Ac-2280 contains two teichoic acids. The major polymer represents a 1,6-poly(mannitol phosphate) substituted wirh either L-rhamnose (approximately 70%, unit A) or (S)-acetal of pyruvic acid (approximately 30%, unit B) with the overall chain length approximately 10 mannitol phosphate units. [carbohydrate structure: see text] The other polymer is an unsubstituted 1,3-poly(glycerol phosphate). The structures of the polymers were established using chemical degradations and NMR spectroscopy. The data obtained may be helpful in determination of the species-specific status of newly isolated Brevibacterium strains.     

Kinetics of intracellular carbon allocation in a marine diatom

To test models of intracellular carbon flow we measured the labelling kinetics (from ¹⁴CO₂) of major classes of cell polymers (carbohydrate, protein, lipid) and of dissolved organic carbon produced by the marine diatom Thalassiosira pseudonana Hustedt, grown at rates of 0.2 to 2.0·day^?1 under nitrogen or light limitation. Compartmental analysis indicated that tracer carbon quickly entered respiratory and excretory streams, accumulating in the cells at the rate of net production after only 25–50% of cell generation (doubling) time. Respiration rates were low (≤ 0.1 · day^?1) and suggested that illuminated cells in steady-state growth made only minor use of oxidative respiration to support cell synthesis. The tracer was quick to enter all polymers; compartmental analysis indicated that polymer labelling rates were close to the rates of mass synthesis after several hours of incubation with ¹⁴C. Polymer labelling also showed a reallocation of photosynthate from protein to carbohydrate within a few hours of perturbation (shift-down) of nutrient supply in a N-limited chemostat. In steady-state growth, the protein: carbohydrate ratio increased directly with N-limited growth rate but attained its maximum under extreme light-limitation. Carbon flow into the metabolic processes of respiration, excretion and polymer synthesis appeared to be mediated by a small and rapidly cycled pool of substrates under all steady-state growth conditions.     

A new polymer of 8,9-Di-O-Glucosylated 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid from a cell wall of Brevibacterium casei ACM Ac-2114T

A polysaccharide containing the residues of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) was found in the cell wall of the Brevibacterium casei strain ACM Ac-2114^T. The polymer structure was elucidated by analyzing one-dimensional spectra of ¹H and ¹³C NMR and bidimentional experiments ¹H/¹H-COSY, TOCSY, ¹H/¹³C-gHSQC, and ¹H/¹³C-gHMBC. The polymer is built up of the 2 → 4-linked Kdn residues substituted by β-D-Glcp residues at 8- and 9-hydroxyls; such a polymer with disubstituted Kdn residues was found for the first time. A glycosylated teichoic acid of the 1,3-poly(glycerolphosphate) type was also identified among other anionic polymers of cell wall.     

Aggregation during coniferyl alcohol polymerization in pectin solution: a biomimetic approach of the first steps of lignification

Coniferyl alcohol was polymerized in pectin solution in order to mimic the lignification that is the final step of biosynthesis of plant cell wall. Dehydrogenated polymers (DHP = coniferyl alcohol polymers = synthetic lignin) interact with pectin to form hydrophobic clusters as monitored by pyrene fluorescence spectroscopy. The structure of these clusters was studied during the polymerization of synthetic lignin by static and quasielastic light scattering and small angle neutron scattering experiments. We show that synthetic lignin and pectin contribute to the same clusters, but the inner structure of these clusters is very heterogeneous and displays three phases. One observes a segregation between well separated pectin and lignin rich phases at length scales below approximately 30 nm. As a corollary of this segregation, clusters embody a large amount of solvent. On average, the density of the polymer rich phase (lignin plus pectin) inside clusters increases while its specific surface area decreases throughout the polymerization process. These results are discussed with respect to in vivo lignification of the plant cell wall.     

Study of the Carbohydrate Specificity of Antibodies Against <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Using the Library of Synthetic Mycoantigens

The carbohydrate specificity of antibodies obtained by immunizing laboratory animals with immunogens prepared from the Aspergillus fumigatus cell wall was analyzed using the library of biotinylated synthetic oligosaccharides. It has been shown that the main part of antipolysaccharide antibodies recognizes galactomannan in the studied immunogens with preference to its fragments containing more than two β-(1→5)-linked galactofuranosyl units. The data obtained can form the basis for the development of enzyme immunoassay for the detection of this dangerous fungal pathogen.     

Regulation of the Contribution of Integrin to Cell Attachment on Poly(2-Methoxyethyl Acrylate) (PMEA) Analogous Polymers for Attachment-Based Cell Enrichment

Cell enrichment is currently in high demand in medical engineering. We have reported that non-blood cells can attach to a blood-compatible poly(2-methoxyethyl acrylate) (PMEA) substrate through integrin-dependent and integrin-independent mechanisms because the PMEA substrate suppresses protein adsorption. Therefore, we assumed that PMEA analogous polymers can change the contribution of integrin to cell attachment through the regulation of protein adsorption. In the present study, we investigated protein adsorption, cell attachment profiles, and attachment mechanisms on PMEA analogous polymer substrates. Additionally, we demonstrated the possibility of attachment-based cell enrichment on PMEA analogous polymer substrates. HT-1080 and MDA-MB-231 cells started to attach to poly(butyl acrylate) (PBA) and poly(tetrahydrofurfuryl acrylate) (PTHFA), on which proteins could adsorb well, within 1 h. HepG2 cells started to attach after 1 h. HT-1080, MDA-MB-231, and HepG2 cells started to attach within 30 min to PMEA, poly(2-(2-methoxyethoxy) ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe2A) and poly(2-(2-methoxyethoxy) ethoxy ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe3A), which suppress protein adsorption. Moreover, the ratio of attached cells from a cell mixture can be changed on PMEA analogous polymers. These findings suggested that PMEA analogous polymers can be used for attachment-based cell enrichment.     

Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase

As a key constituent of their protective cell wall all mycobacteria produce a large structural component, the mycolyl-arabinogalactan (mAG) complex, which has at its core a galactan moiety of alternating beta-(1-->5) and beta-(1-->6) galactofuranosyl residues. Galactan biosynthesis is essential for mycobacterial viability and thus inhibitors of the enzymes involved in its assembly are potential drugs for the treatment of mycobacterial diseases, including tuberculosis. Only two galactofuranosyltransferases, GlfT1 and GlfT2, are responsible for the biosynthesis of the entire galactan domain of the mAG and we report here the first high-throughput assay for GlfT2. Successful implementation of the assay required the synthesis of multi-milligram amounts of the donor for the enzyme, UDP-Galf, 1, which was achieved using a chemoenzymatic approach. We also describe an improved expression system for GlfT2, which provides a larger amount of active protein for the assay. Kinetic analysis of 1 and a known trisaccharide acceptor for the enzyme, 2, have been carried out and the apparent K(m) and k(cat) values obtained for the latter are in agreement with those obtained using a previously reported radiochemical assay. The assay has been implemented in 384-well microtiter plates, which will facilitate the screening of large numbers of potential GlfT2 inhibitors, with possible utility as novel anti-TB drugs.