Novel serine/threonine-O-glycosylation with N-acetylneuraminic acid and 3-deoxy-D-manno-octulosonic acid by bacterial flagellin glycosyltransferases.

Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.

Amino acid residues important for D-galactose recognition by the F-type lectin, Ranaspumin-4.

F-type lectins are typically L-fucose binding proteins with characteristic L-fucose-binding and calcium-binding sequence motifs, and an F-type lectin fold. An exception is Ranaspumin-4, an F-type lectin of the Tungra frog, Engystomops pustulosus. Ranaspumin-4 is D-galactose specific and does not bind to L-fucose although it has the conserved L-fucose binding sequence motif and shares overall sequence similarity with other F-type lectins. Here, we report the detailed glycan-binding profile of wild-type Ranaspumin-4 using hemagglutination inhibition assays, flow cytometry assays and enzyme-linked lectin assays, and identify residues important for D-galactose recognition using rational site-directed mutagenesis. We demonstrate that Ranaspumin-4 binds to terminal D-galactose in α or ß linkage with preference for α1-3, α1-4, ß1-3, and ß1-4 linkages. Further, we find that a methionine residue (M31) in Ranaspumin-4 that occurs in place of a conserved Gln residue (in other F-type lectins), supports D-galactose recognition. Resides Q42 and F156 also likely aid in D-galactose recognition.

Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras.

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).

New carbohydrate binding domains identified by phage display based functional metagenomic screens of human gut microbiota.

Uncultured microbes represent a huge untapped biological resource of novel genes and gene products. Although recent genomic and metagenomic sequencing efforts have led to the identification of numerous genes that are homologous to existing annotated genes, there remains, yet, an enormous pool of unannotated genes that do not find significant sequence homology to existing annotated genes. Functional metagenomics offers a way to identify and annotate novel gene products. Here, we use functional metagenomics to mine novel carbohydrate binding domains that might aid human gut commensals in adherence, gut colonization, and metabolism of complex carbohydrates. We report the construction and functional screening of a metagenomic phage display library from healthy human fecal samples against dietary, microbial and host polysaccharides/glycoconjugates. We identify several protein sequences that do not find a hit to any known protein domain but are predicted to contain carbohydrate binding module-like folds. We heterologously express, purify and biochemically characterize some of these protein domains and demonstrate their carbohydrate-binding function. Our study reveals several previously unannotated carbohydrate-binding domains, including a levan binding domain and four complex N-glycan binding domains that might be useful for the labeling, visualization, and isolation of these glycans.

Biofilm inhibitory effect of alginate lyases on mucoid P. aeruginosa from a cystic fibrosis patient.

Chronic mucoid Pseudomonas aeruginosa infections are a major scourge in cystic fibrosis patients. Mucoid P. aeruginosa displays structured alginate-rich biofilms that are resistant to antibiotics. Here, we have assessed the efficacy of a panel of alginate lyases in combating mucoid P. aeruginosa biofilms in cystic fibrosis. Albeit we could not demonstrate alginate degradation by alginate lyases in sputum, we demonstrate that the endotypic alginate lyases, CaAly (from Cellulophaga algicola) and VspAlyVI (from Vibrio sp. QY101) and the exotypic alginate lyases, FspAlyFRB (from Falsirhodobacterium sp. alg1), and SA1-IV (from Sphingomonas sp. A1), indeed inhibit biofilm formation by a mucoid P. aeruginosa strain isolated from the sputum of a cystic fibrosis patient with comparative effect to that of the glycoside hydrolase PslG, a promising candidate for biofilm treatment. We believe that these enzymes should be explored for in vivo efficacy in future studies.