NDP-rhamnose biosynthesis and rhamnosyltransferases: building diverse glycoconjugates in nature.

Rhamnose is an important 6-deoxy sugar present in many natural products, glycoproteins, and structural polysaccharides. Whilst predominantly found as the l-enantiomer, instances of d-rhamnose are also found in nature, particularly in the Pseudomonads bacteria. Interestingly, rhamnose is notably absent from humans and other animals, which poses unique opportunities for drug discovery targeted towards rhamnose utilizing enzymes from pathogenic bacteria. Whilst the biosynthesis of nucleotide-activated rhamnose (NDP-rhamnose) is well studied, the study of rhamnosyltransferases that synthesize rhamnose-containing glycoconjugates is the current focus amongst the scientific community. In this review, we describe where rhamnose has been found in nature, as well as what is known about TDP-ß-l-rhamnose, UDP-ß-l-rhamnose, and GDP-α-d-rhamnose biosynthesis. We then focus on examples of rhamnosyltransferases that have been characterized using both in vivo and in vitro approaches from plants and bacteria, highlighting enzymes where 3D structures have been obtained. The ongoing study of rhamnose and rhamnosyltransferases, in particular in pathogenic organisms, is important to inform future drug discovery projects and vaccine development.

RPN2 is targeted by miR-181c and mediates glioma progression and temozolomide sensitivity via the wnt/ß-catenin signaling pathway.

Accumulating evidence indicates that the dysregulation of the miRNAs/mRNA-mediated carcinogenic signaling pathway network is intimately involved in glioma initiation and progression. In the present study, by performing experiments and bioinformatics analysis, we found that RPN2 was markedly elevated in glioma specimens compared with normal controls, and its upregulation was significantly linked to WHO grade and poor prognosis. Knockdown of RPN2 inhibited tumor proliferation and invasion, promoted apoptosis, and enhanced temozolomide (TMZ) sensitivity in vitro and in vivo. Mechanistic investigation revealed that RPN2 deletion repressed ß-catenin/Tcf-4 transcription activity partly through functional activation of glycogen synthase kinase-3ß (GSK-3ß). Furthermore, we showed that RPN2 is a direct functional target of miR-181c. Ectopic miR-181c expression suppressed ß-catenin/Tcf-4 activity, while restoration of RPN2 partly reversed this inhibitory effect mediated by miR-181c, implying a molecular mechanism in which TMZ sensitivity is mediated by miR-181c. Taken together, our data revealed a new miR-181c/RPN2/wnt/ß-catenin signaling axis that plays significant roles in glioma tumorigenesis and TMZ resistance, and it represents a potential therapeutic target, especially in GBM.

Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP-ß-l-rhamnose and rhamnosylation mechanism.

Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP-ß-l-rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP-ß-l-rhamnose. The mutant H357Q exhibited activity with both UDP-ß-l-rhamnose and UDP-glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor-His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism.

Protective role of the HOG pathway against the growth defect caused by impaired biosynthesis of complex sphingolipids in yeast Saccharomyces cerevisiae.

Complex sphingolipids play critical roles in various cellular events in the yeast Saccharomyces cerevisiae. To identify genes that are related to the growth defect caused by disruption of complex sphingolipid biosynthesis, we screened for suppressor mutations and multicopy suppressor genes that confer resistance against repression of AUR1 encoding inositol phosphorylceramide synthase. From the results of this screening, we found that the activation of high-osmolarity glycerol (HOG) pathway is involved in suppression of growth defect caused by impaired biosynthesis of complex sphingolipids. Furthermore, it was found that transcriptional regulation via Msn2, Msn4 and Sko1 is involved in the suppressive effect of the HOG pathway. Lack of the HOG pathway did not enhance the reductions in complex sphingolipid levels or the increase in ceramide level caused by the AUR1 repression, implying that the suppressive effect of the HOG pathway on the growth defect is not attributed to restoration of impaired biosynthesis of complex sphingolipids. On the contrary, the HOG pathway and Msn2/4-mediated transcriptional activation was involved in suppression of aberrant reactive oxygen species accumulation caused by the AUR1 repression. These results indicated that the HOG pathway plays pivotal roles in maintaining cell growth under impaired biosynthesis of complex sphingolipids.

[Expression and functional analyses of the Arabidopsis QUA1 gene in light signal transduction].

Plants not only use light as an energy source for photosynthesis, but also have to monitor the light quality and quantity input to execute appropriate physiological and developmental responses, such as cell differentiation, structural and functional changes, as well as the formation of tissues and organs. The process is referred to as photomorphogenesis. Arabidopsis QUA1 (QUASIMODO1), which functions in pectin synthesis, is identified as a member of glycosyltransferases. Previously, the hypocotyl elongation of the qua1-1 mutant was shown to be inhibited under dark conditions. In this study, we used the qua1-1/cry1 and qua1-1/phyB double mutants as the materials to study the function of the QUA1 gene in light signal transduction. The results showed that QUA1 not only participated in hypocotyl elongation under dark conditions, but also in blue light, red light and far red light conditions. In qua1-1 mutant seedlings, both the cell length of hypocotyl and the light-regulated gene expression were affected. Compared with cry1 and phyB mutants, qua1-1/cry1 and qua1-1/phyB double mutants had the shorter hypocotyl. Light-regulated gene expression was also affected in the double mutants. These data indicated that QUA1 might participate in the light signal transduction regulated by CRY1 and PHYB. Hence, the QUA1 gene may play multiple roles in light signal transduction by regulating the cell elongation and light-regulated gene expression.

Manic fringe promotes a claudin-low breast cancer phenotype through notch-mediated PIK3CG induction.

Claudin-low breast cancer (CLBC) is a poor prognosis disease biologically characterized by stemness and mesenchymal features. These tumors disproportionately affect younger patients and women with African ancestry, causing significant morbidity and mortality, and no effective targeted therapy exists at present. CLBC is thought to originate from mammary stem cells, but little is known on how or why these tumors express a stable epithelial-to-mesenchymal transition phenotype, or what are the driving forces of this disease. Here, we report that Manic Fringe (Mfng), which encodes an O-fucosylpeptide 3-ß-N-acetylglucosaminyltransferase known to modify EGF repeats in the Notch extracellular domain, is highly expressed in CLBC and functions as an oncogene in this context. We show that Mfng modulates Notch activation in human and mouse CLBC cell lines, as well as in mouse mammary gland. Mfng silencing in CLBC cell lines reduced cell migration, tumorsphere formation, and in vivo tumorigenicity associated with a decrease in the stem-like cell population. Mfng deletion in the Lfng(flox/flox);MMTV-Cre mouse model, in which one-third of mammary tumors resemble human CLBC, caused a tumor subtype shift away from CLBC. We identified the phosphoinositide kinase Pik3cg as a direct transcriptional target of Mfng-facilitated RBPJκ-dependent Notch signaling. Indeed, pharmacologic inhibition of PI3Kγ in CLBC cell lines blocked migration and tumorsphere formation. Taken together, our results define Mfng as an oncogene acting through Notch-mediated induction of Pik3cg. Furthermore, they suggest that targeting PI3Kγ may prove beneficial for the treatment of CLBC subtype.

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon.

In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the protein-conducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.

Oligosaccharyltransferase: the central enzyme of N-linked protein glycosylation.

N-linked glycosylation is one of the most abundant modifications of proteins in eukaryotic organisms. In the central reaction of the pathway, oligosaccharyltransferase (OST), a multimeric complex located at the membrane of the endoplasmic reticulum, transfers a preassembled oligosaccharide to selected asparagine residues within the consensus sequence asparagine-X-serine/threonine. Due to the high substrate specificity of OST, alterations in the biosynthesis of the oligosaccharide substrate result in the hypoglycosylation of many different proteins and a multitude of symptoms observed in the family of congenital disorders of glycosylation (CDG) type I. This review covers our knowledge of human OST and describes enzyme composition. The Stt3 subunit of OST harbors the catalytic center of the enzyme, but the function of the other, highly conserved, subunits are less well defined. Some components seem to be involved in the recognition and utilization of glycosylation sites in specific glycoproteins. Indeed, mutations in the subunit paralogs N33/Tusc3 and IAP do not yield the pleiotropic phenotypes typical for CDG type I but specifically result in nonsyndromic mental retardation, suggesting that the oxidoreductase activity of these subunits is required for glycosylation of a subset of proteins essential for brain development.

Chondroitin sulfate synthase-2/chondroitin polymerizing factor has two variants with distinct function.

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.

Kei1: a novel subunit of inositolphosphorylceramide synthase, essential for its enzyme activity and Golgi localization.

Fungal sphingolipids have inositol-phosphate head groups, which are essential for the viability of cells. These head groups are added by inositol phosphorylceramide (IPC) synthase, and AUR1 has been thought to encode this enzyme. Here, we show that an essential protein encoded by KEI1 is a novel subunit of IPC synthase of Saccharomyces cerevisiae. We find that Kei1 is localized in the medial-Golgi and that Kei1 is cleaved by Kex2, a late Golgi processing endopeptidase; therefore, it recycles between the medial- and late Golgi compartments. The growth defect of kei1-1, a temperature-sensitive mutant, is effectively suppressed by the overexpression of AUR1, and Aur1 and Kei1 proteins form a complex in vivo. The kei1-1 mutant is hypersensitive to aureobasidin A, a specific inhibitor of IPC synthesis, and the IPC synthase activity in the mutant membranes is thermolabile. A part of Aur1 is missorted to the vacuole in kei1-1 cells. We show that the amino acid substitution in kei1-1 causes release of Kei1 during immunoprecipitation of Aur1 and that Aur1 without Kei1 has hardly detectable IPC synthase activity. From these results, we conclude that Kei1 is essential for both the activity and the Golgi localization of IPC synthase.

Structural analysis of the core region of O-lipopolysaccharide of Porphyromonas gingivalis from mutants defective in O-antigen ligase and O-antigen polymerase.

Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, Delta PG1051 (WaaL, O-antigen ligase) and Delta PG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an "uncapped core," which is devoid of O polysaccharide (O-PS), and a "capped core," which contains the site of O-PS attachment. The inner core region lacks L(D)-glycero-D(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-D-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the "uncapped core" is attached to the glycerol and is composed of a linear alpha-(1-->3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, alpha-D-allosamine, is attached to the glycerol at position 3. In the "capped core," there is a three- to five-residue extension of alpha-(1-->3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. beta-D-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.

The chondroitin polymerase K4CP and the molecular mechanism of selective bindings of donor substrates to two active sites.

Bacterial chondroitin polymerase K4CP is a multifunctional enzyme with two active sites. K4CP catalyzes alternative transfers of glucoronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to elongate a chain consisting of the repeated disaccharide sequence GlcAbeta1-3GalNAcbeta1-4. Unlike the polymerization reactions of DNA and RNA and polypeptide synthesis, which depend upon templates, the monosaccharide polymerization by K4CP does not. To investigate the catalytic mechanism of this reaction, we have used isothermal titration calorimetry to determine the binding of the donor substrates UDP-GlcA and UDP-GalNAc to purified K4CP protein and its mutants. Only one donor molecule bound to one molecule of K4CP at a time. UDP-GlcA bound only to the C-terminal active site at a high affinity (K(d)=6.81 microm), thus initiating the polymerization reaction. UDP-GalNAc could bind to either the N-terminal or C-terminal active sites at a low affinity (K(d)=266-283 microm) but not to both sites at the same time. The binding affinity of UDP-GalNAc to a K4CP N-terminal fragment (residues 58-357) was profoundly decreased, yielding the average K(d) value of 23.77 microm, closer to the previously reported K(m) value for the UDP-GalNAc transfer reaction that takes place at the N-terminal active site. Thus, the first step of the reaction appears to be the binding of UDP-GlcA to the C-terminal active site, whereas the second step involves the C-terminal region of the K4CP molecule regulating the binding of UDP-GalNAc to only the N-terminal active site. Alternation of these two specific bindings advances the polymerization reaction by K4CP.

Characterization of cyclofructans from inulin by Saccharomyces cerevisiae Strain displaying cell-surface cycloinulooligosaccharide fructanotransferase.

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans (GenBank access code AF222787) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein Aga1p. The surface display of CFTase was confirmed by immunofluorescence microscopy and enzymatic assay. The optimized reaction conditions of surface-displayed CFTase were as follows; pH, 8.0; temperature, 50 degrees C; enzyme amount, 30 milliunit; substrate concentration, 5%; inulin source, Jerusalem artichoke. As a result of the reaction with inulin, cycloinulohexaose was produced as a major product along with cycloinuloheptaose and cycloinulooctaose as minor products.

Structural and functional insights into intramolecular fructosyl transfer by inulin fructotransferase.

Inulin fructotransferase (IFTase), a member of glycoside hydrolase family 91, catalyzes depolymerization of beta-2,1-fructans inulin by successively removing the terminal difructosaccharide units as cyclic anhydrides via intramolecular fructosyl transfer. The crystal structures of IFTase and its substrate-bound complex reveal that IFTase is a trimeric enzyme, and each monomer folds into a right-handed parallel beta-helix. Despite variation in the number and conformation of its beta-strands, the IFTase beta-helix has a structure that is largely reminiscent of other beta-helix structures but is unprecedented in that trimerization is a prerequisite for catalytic activity, and the active site is located at the monomer-monomer interface. Results from crystallographic studies and site-directed mutagenesis provide a structural basis for the exolytic-type activity of IFTase and a functional resemblance to inverting-type glycosyltransferases.

Role of lgtC in resistance of nontypeable Haemophilus influenzae strain R2866 to human serum.

We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

Identification of mouse sphingomyelin synthase 1 as a suppressor of Bax-mediated cell death in yeast.

We have identified mouse sphingomyelin synthase 1 as a novel suppressor of the growth inhibitory effect of heterologously expressed Bax. Yeast cells expressing sphingomyelin synthase 1 were also found to show an increased resistance to a variety of cytotoxic stimuli including hydrogen peroxide, osmotic stress and elevated temperature. Sphingomyelin synthase 1 functions by catalyzing the conversion of ceramide and phosphatidylcholine to sphingomyelin and diacylglycerol. Ceramide is an antiproliferative and proapoptotic sphingolipid whose level increases in response to a variety of stresses. Consistent with its biochemical function, yeast cells expressing sphingomyelin synthase 1 have an enhanced ability to grow in media containing the cell-permeable C2-ceramide analog as well as the ceramide precursor phytosphingosine. We also show that overexpression of AUR1, a potential yeast functional homolog of sphingomyelin synthase, also protects cells from osmotic stress. Taken together, these results suggest that sphingomyelin synthase 1 likely prevents cell death by counteracting stress-mediated accumulation of endogenous sphingolipids.

Single amino acid residue changes in subsite -1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized.

Bacterial fructansucrase enzymes belong to glycoside hydrolase family 68 and catalyze transglycosylation reactions with sucrose, resulting in the synthesis of fructooligosaccharides and/or a fructan polymer. Significant differences in fructansucrase enzyme product specificities can be observed, i.e. in the type of polymer (levan or inulin) synthesized, and in the ratio of polymer versus fructooligosaccharide synthesis. The Lactobacillus reuteri 121 inulosucrase enzyme produces a diverse range of fructooligosaccharide molecules and a minor amount of inulin polymer [with beta(2-1) linkages]. The three-dimensional structure of levansucrase (SacB) of Bacillus subtilis revealed eight amino acid residues interacting with sucrose. Sequence alignments showed that six of these eight amino acid residues, including the catalytic triad (D272, E523 and D424, inulosucrase numbering), are completely conserved in glycoside hydrolase family 68. The other three completely conserved residues are located at the -1 subsite (W271, W340 and R423). Our aim was to investigate the roles of these conserved amino acid residues in inulosucrase mutant proteins with regard to activity and product profile. Inulosucrase mutants W340N and R423H were virtually inactive, confirming the essential role of these residues in the inulosucrase active site. Inulosucrase mutants R423K and W271N were less strongly affected in activity, and displayed an altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer. Our data show that the -1 subsite is not only important for substrate recognition and catalysis, but also plays an important role in determining the size of the products synthesized.

Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase).

In the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination of d-glycopyranoside residues in (4)C(1) conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the beta-(1-->2)-fructosyl transfer to 2-OH of L-sugars (L-glucose, L-rhamnose, L-galactose, L-fucose, L-xylose) presumably in a (1)C(4) conformation. In those conformations, the L-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis.

Specific amino acids of the glycosyltransferase LpsA direct the addition of glucose or galactose to the terminal inner core heptose of Haemophilus influenzae lipopolysaccharide via alternative linkages.

Lipopolysaccharide is the major glycolipid of the cell wall of the bacterium Haemophilus influenzae, a Gram-negative commensal and pathogen of humans. Lipopolysaccharide is both a virulence determinant and a target for host immune responses. Glycosyltransferases have high donor and acceptor substrate specificities that are generally limited to catalysis of one unique glycosidic linkage. The H. influenzae glycosyltransferase LpsA is responsible for the addition of a hexose to the distal heptose of the inner core of the lipopolysaccharide molecule and belongs to the glycosyltransferase family 25. The hexose added can be either glucose or galactose and linkage to the heptose can be either beta1-2 or beta1-3. Each H. influenzae strain uniquely produces only one of the four possible combinations of linked sugar in its lipopolysaccharide. We show that, in any given strain, a specific allelic variant of LpsA directs the anomeric linkage and the added hexose, glucose, or galactose. Site-directed mutagenesis of a single key amino acid at position 151 changed the hexose added in vivo from glucose to galactose or vice versa. By constructing chimeric lpsA gene sequences, it was shown that the 3' end of the gene directs the anomeric linkage (beta1-2 or beta1-3) of the added hexose. The lpsA gene is the first known example where interstrain variation in lipopolysaccharide core structure is directed by the specific sequence of a genetic locus encoding enzymes directing one of four alternative possible sugar additions from the inner core.