Being recognized as an important constituent of the glycoprotein folding cycle, uridine diphosphate-glucose:glycoprotein glucosyltransferase (UGGT) has been a subject of intense study. Up to now, it is two isoforms, UGGT1 and 2 have been identified, which share ∼ 50% amino acid identity. UGGT1 is a well-documented enzyme which functions as a folding sensor in the endoplasmic reticulum, by the virtue of its ability to transfer a glucose residue to non-glucosylated high-mannose-type glycans of immature glycoproteins exhibiting non-native conformation. On the other hand, direct evidence to support the glucosyltransferase activity of UGGT2 has been lacking, leaving it unclear as to whether it has any function in the glycoprotein folding process. This study aimed to reveal the property of human UGGT2 by using synthetic substrates such as fluorescently labeled glycans and N-glycosylated proteins. The analysis, for the first time, revealed the glucosyltransferase activity of UGGT2, whose specificity was shown to be quite similar to UGGT1, in terms of both glycan specificity and preferential recognition of proteins having non-native conformations. Finally, Sep15 was found to form the heterodimeric complex with both isoforms of UGGT and markedly enhanced its glucosyltransferase activity.

Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. α-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of β-Kdo residues links "group 2" capsular polysaccharides to (lyso)phosphatidylglycerol. β-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo-dependent α-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the β-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype β-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena. The β-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal α/β domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities. microbial glycobiology | 3-deoxy-D-manno-oct-2-ulosonic acid | Kdo | glycosyltransferase | polysaccharide

From the stacks: A novel method for construction of a high-mannose-type glycan library by systematic enzymatic trimming of a single synthetic Man9-based precursor was developed. Efficient chemical synthesis of the tetradecasaccharide common precursor and orthogonal enzymatic trimming to obtain all M(8-9) and G(1)M(8-9) derivatives was demonstrated. G = glucose, M = mannose.

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Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-β-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a β-linked Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering.microbial glycobiology | lipopolysaccharides | glycosyltransferases | glycan biosynthesis | molecular ruler B acterial surfaces possess an array of complex glycoconjugates (sugar-containing macromolecules) that play varied and vital roles in the biology of these organisms. In pathogens, glycoconjugates participate in adhesion, biofilm formation, and interaction with innate and adaptive immune responses. For example, in gram-negative bacteria such as Escherichia coli, long glycan chains in capsular polysaccharides and the O-antigen polysaccharide (OPS) components of lipopolysaccharide (LPS) molecules typically confer resistance to opsonophagocytosis and complement-mediated killing (1, 2). Effective protection depends on the amount and surface distribution of glycan, as well as glycan chain lengths suited for a given purpose. As an example, shorter OPS chains offer no resistance to complementmediated killing, whereas chains longer than an optimal size potentially represent an unnecessary energy cost while offering no additional advantage (3, 4). Despite its fundamental importance in colonization and virulence of bacterial pathogens, the molecular mechanisms of glycan chain-length regulation are often poorly understood (5). However, OPS biosynthesis provides influential prototypes for understanding the guiding principles underpinning chainlength regulatory mechanisms in bacterial glycoconjugates in general.The LPS glycolipid is a major component of the outer membrane of gram-negative bac...

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