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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. [ABSTRACT FROM AUTHOR]

The typical F‐type lectin domain (FLD) has an L‐fucose‐binding motif [HX(26)RXDX(4)R/K] with conserved basic residues that mediate hydrogen bonding with alpha‐L‐fucose. About one‐third of the nonredundant FLD sequences in the publicly available databases are "atypical"; they have motifs with substitutions of these critical residues and/or variations in motif length. We addressed the question if atypical FLDs with substitutions of the critical residues retain lectin activity by performing site‐directed mutagenesis and assessing the glycan‐binding functions of typical and atypical FLDs. Site directed mutagenesis of an L‐fucose‐binding FLD from Streptosporangium roseum indicated that the critical His residue could be replaced by Ser and the second Arg by Lys without complete loss of lectin activity. Mutagenesis of His to other naturally substituting residues and mutagenesis of the first Arg to the naturally substituting residues, Lys, Ile, Ser, or Cys, resulted in loss of lectin activity. Glycan binding analysis and site‐directed mutagenesis of atypical FLDs from Actinomyces turicensis, and Saccharomonospora cyanea confirmed that Ser and Thr can assume the L‐fucose‐binding role of the critical His, and further suggested that the residue in this position is dispensable in certain FLDs. We identified, by sequence and structural analysis of atypical FLDs, a Glu residue in the complementarity determining region, CDR5 that compensates for a lack of the critical His or other appropriate polar residue in this position. We propose that FLDs lacking a typical FLD sequence motif might nevertheless retain lectin activity through the recruitment of other strategically positioned polar residues in the CDR loops. © 2018 IUBMB Life, 71(3):385–397, 2019 [ABSTRACT FROM AUTHOR]

F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor . We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewis b motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans. [ABSTRACT FROM AUTHOR]

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 β linkage with preference for α1-3, α1-4, β1-3, and β1-4 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. • The F-type lectin, Ranaspumin-4 binds to terminal D-galactose in α or β linkage. • Ranaspumin-4 displays preference for α1-3, α1-4, β1-3, and β1-4 linked D-galactose. • A methionine residue, M31 in Ranaspumin-4 supports D-galactose recognition. • M31 occurs in place of a conserved Gln residue in other F-type lectins. [ABSTRACT FROM AUTHOR]