Correction: Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0274415.].

Submicron Emitters Enable Reliable Quantification of Weak Protein-Glycan Interactions by ESI-MS.

Interactions between carbohydrates (glycans) and glycan-binding proteins (GBPs) regulate a wide variety of important biological processes. However, the affinities of most monovalent glycan-GBP complexes are typically weak (dissociation constant (Kd) > µM) and difficult to reliably measure with conventional assays; consequently, the glycan specificities of most GBPs are not well established. Here, we demonstrate how electrospray ionization mass spectrometry (ESI-MS), implemented with nanoflow ESI emitters with inner diameters of ∼50 nm, allows for the facile quantification of low-affinity glycan-GBP interactions. The small size of the droplets produced from these submicron emitters effectively eliminates the formation of nonspecific glycan-GBP binding (false positives) during the ESI process up to ∼mM glycan concentrations. Thus, interactions with affinities as low as ∼5 mM can be measured directly from the mass spectrum. The general suppression of nonspecific adducts (including nonvolatile buffers and salts) achieved with these tips enables ESI-MS glycan affinity measurements to be performed on C-type lectins, a class of GBPs that bind glycans in a calcium-dependent manner and are important regulators of immune response. At physiologically relevant calcium ion concentrations (2-3 mM), the extent of Ca2+ nonspecific adduct formation observed using the submicron emitters is dramatically suppressed, allowing glycan affinities, and the influence of Ca2+ thereon, to be measured. Finally, we show how the use of submicron emitters and suppression of nonspecific binding enable the quantification of labile (prone to in-source dissociation) glycan-GBP interactions.

1-C-phosphonomethyl- and 1-C-difluorophosphonomethyl-1,4-imino-l-arabinitols as Galf transferase inhibitors: A comparison.

The convenient preparation of iminopentitol derivatives, based on a 1,4-dideoxy-1,4-imino-l-arabinitol scaffold carrying ß-phosphono(difluoromethyl) or ß-phosphonomethyl appendages, as Galf-1P mimics, is reported. The compounds were tested for their ability to inhibit GlfT2, a vital galactofuranosyltransferase involved in the cell wall biosynthesis of mycobacteria. Interestingly, the Galf-1P mimics lacking a fluorine atom (7 and 8) were very poor inhibitors, showing less than 20% inhibition of GlfT2, whereas compounds 2 and 3, which contains a difluoromethylenephosphonate moiety were more potent inhibitors. Compound 3 that is fully deprotected was the most potent showing a significant IC50 value (0.9 mm), despite the absence of the diphosphate linkage present in the parent sugar nucleotide. This study paves the way to the synthesis of more complex ß-phosphonomethyl-imino-l-arabinitol derivatives as simplified mimics of UDP-α-d-Galf.

Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site.

The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase (GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step of the closely related human ABO(H) blood group A and B antigens. The catalytic mechanism of these model retaining enzymes remains under debate, where Glu303 has been suggested to act as a putative nucleophile in a double displacement mechanism, a local dipole stabilizing the intermediate in an orthogonal associative mechanism or a general base to stabilize the reactive oxocarbenium ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes having nearly identical sequences, the corresponding mutants of GTA/GTB have up to a 13-fold difference in their residual activities relative to wild type. High-resolution single crystal X-ray diffraction studies reveal, surprisingly, that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å further from the anomeric carbon of donor substrate compared to wild type. However, complicating the analysis is the observation that Glu303 itself plays a critical role in maintaining the stability of a strained "double-turn" in the active site through several hydrogen bonds, and any mutation other than E303Q leads to significantly higher thermal motion or even disorder in the substrate recognition pockets. Thus, there is a remarkable juxtaposition of the mutants E303C and E303D, which retain significant activity despite disrupted active site architecture, with GTB/E303Q, which maintains active site architecture but exhibits zero activity. These findings indicate that nucleophilicity at position 303 is more catalytically valuable than active site stability and highlight the mechanistic elasticity of these enzymes.

Synthesis of Unprecedented Sulfonylated Phosphono-exo-Glycals Designed as Inhibitors of the Three Mycobacterial Galactofuranose Processing Enzymes.

This study reports a new methodology to synthesize exo-glycals bearing both a sulfone and a phosphonate. This synthetic strategy provides a way to generate exo-glycals displaying two electron-withdrawing groups and was applied to eight different carbohydrates from the furanose and pyranose series. The Z/E configurations of these tetrasubstituted enol ethers could be ascertained using NMR spectroscopic techniques. Deprotection of an exo-glycal followed by an UMP (uridine monophosphate) coupling generated two new UDP (uridine diphosphate)-galactofuranose analogues. These two Z/E isomers were evaluated as inhibitors of UGM, GlfT1, and GlfT2, the three mycobacterial galactofuranose processing enzymes. Molecule 46-(E) is the first characterized inhibitor of GlfT1 reported to date and was also found to efficiently inhibit UGM in a reversible manner. Interestingly, GlfT2 showed a better affinity for the (Z) isomer. The three enzymes studied in the present work are not only interesting because, mechanistically, they are still the topic of intense investigations, but also because they constitute very important targets for the development of novel antimycobacterial agents.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner.

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the "tucked under" conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.

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 beta-(1-->6) and beta-(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, beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-O(CH2)7CH3 (2) and beta-D-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-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.