Biosynthesis of the linkage region of glycosaminoglycans: cloning and activity of galactosyltransferase II, the sixth member of the beta 1,3-galactosyltransferase family (beta 3GalT6)

A family of five beta1,3-galactosyltransferases has been characterized that catalyze the formation of Galbeta1,3GlcNAcbeta and Galbeta1,3GalNAcbeta linkages present in glycoproteins and glycolipids (beta3GalT1, -2, -3, -4, and -5). We now report a new member of the family (beta3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, beta3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the beta3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal beta-linked galactose residue. This product, Galbeta1,3Galbeta is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser), indicating that beta3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.     

Demonstration by heterologous expression that the Leishmania SCA1 gene encodes an arabinopyranosyltransferase

In part of the life cycle within their sand fly vector, Leishmania major parasites first attach to the fly's midgut through their main surface adhesin lipophosphoglycan (LPG) and later resynthesize a structurally distinct LPG that results in detachment and eventual transmission. One of these structural modifications requires the addition of alpha1,2-D-arabinopyranose caps to beta1,3-galactose side chains in the phosphoglycan repeat unit domain of LPG. We had previously identified two side chain arabinose genes (SCA1/2) that were involved in the alpha1,2-D-Arap capping. SCA1/2 exhibit canonical glycosyltransferase motifs, and overexpression of either gene leads to elevated microsomal alpha1,2-D-ArapT activity, resulting in arabinopyranosylation of beta1,3-Gal side chains in LPG (hereafter called side chain D-arabinopyranosyltransferase [sc-D-ArapT]). Heterologous expression in a null arabinose background was used to determine whether the SCA1 gene encodes the actual sc-D-ArapT. SCA1 expression constructs introduced into both mammalian COS-7 cells and the baculovirus-sf9 cell system exhibited considerable expression of the protein. However, functional sc-D-ArapT activity was observed only in the latter. In in vitro assays incubated with guanidine 59-diphosphate (GDP)-D-[3H]Arap as the sugar donor and utilizing exogenous LPG as an acceptor, significant sc-D-ArapT activity was observed when microsomes from the baculovirus-sf9 cells were incubated in presence of the LPG acceptor. No activity was observed in the absence of LPG. These results demonstrate that SCA1 encodes a sc-D-ArapT and provide the first example of heterologous expression of a D-ArapT gene.     

Identification of genes encoding arabinosyltransferases (SCA) mediating developmental modifications of lipophosphoglycan required for sand fly transmission of leishmania major

At key steps in the infectious cycle pathogens must adhere to target cells, but at other times detachment is required for transmission. During sand fly infections by the protozoan parasite Leishmania major, binding of replicating promastigotes is mediated by galactosyl side chain (scGal) modifications of phosphoglycan repeats of the major surface adhesin, lipophosphoglycan (LPG). Release is mediated by arabinosyl (Ara) capping of LPG scbetaGal residues upon differentiation to the infective metacyclic stage. We used intraspecific polymorphisms of LPG structure to develop a genetic strategy leading to the identification of two genes (SCA1/2) mediating scAra capping. These LPG side chain beta1,2-arabinosyltransferases (scbetaAraTs) exhibit canonical glycosyltransferase motifs, and their overexpression leads to elevated microsomal scbetaAraT activity. Although the level of scAra caps is maximal in metacyclic parasites, scbetaAraT activity is maximal in log phase cells. Because quantitative immunolocalization studies suggest this is not mediated by sequestration of SCA scbetaAraTs away from the Golgi apparatus during log phase, regulation of activated Ara precursors may control LPG arabinosylation in vivo. The SCA genes define a new family of eukaryotic betaAraTs and represent novel developmentally regulated LPG-modifying activities identified in Leishmania.     

Targeted mutations in beta1,4-galactosyltransferase I reveal its multiple cellular functions

Beta1,4-galactosyltransferase I (GalT I) is one of the most extensively studied glycosyltransferases. It is localized in the trans-Golgi compartment of most eukaryotic cells, where it participates in the elongation of oligosaccharide chains on glycoproteins and glycolipids. GalT I has also been reported in non-Golgi locations, most notably the cell surface, where it has been suggested to function non-biosynthetically as a receptor for extracellular glycoside substrates. Cloning of the GalT I cDNAs revealed that the gene encodes two similar proteins that differ only in the length of their cytoplasmic domains. Whether these different GalT I proteins, or isoforms, have similar or different biological roles is a matter of active investigation. The functions of the GalT I proteins have been addressed by targeted mutations that eliminate either both GalT I isoforms or just the long GalT I isoform. Eliminating both GalT I proteins abolishes most, but not all, GalT activity, an observation that led to the realization that other GalT family members must exist. The loss of both GalT I isoforms leads to neonatal lethality due to a wide range of phenotypic abnormalities that are most likely the result of decreased galactosylation. When the long isoform of GalT I is eliminated, galactosylation proceeds grossly normal via the short GalT I isoform, but specific defects in cell interactions occur that are thought to depend upon a non-biosynthetic function of the long GalT I isoform.     

Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP- galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled alpha-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12(+) and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the Delta mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than alpha1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S. pombe alpha1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galalpha1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S. cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S. cerevisiae.      

Identification of a Drosophila gene encoding xylosylprotein beta4-galactosyltransferase that is essential for the synthesis of glycosaminoglycans and for morphogenesis

In mammals, the xylosylprotein beta4-galactosyltransferase termed beta4GalT7 (XgalT-1, EC ) participates in proteoglycan biosynthesis through the transfer of galactose to the xylose that initiates each glycosaminoglycan chain. A Drosophila cDNA homologous to mammalian beta4-galactosyltransferases was identified using a human beta4GalT7 cDNA as a probe in a BLAST analysis of expressed sequence tags. The Drosophila cDNA encodes a type II membrane protein with 322 amino acids and shows 49% identity to human beta4GalT7. Extracts from L cells transfected with the cDNA exhibited marked galactosyltransferase activity specific for a xylopyranoside acceptor. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis in beta4GalT7-deficient Chinese hamster ovary cells. In transfectant lysates the properties of Drosophila and human beta4GalT7 resembled each other, except that Drosophila beta4GalT7 showed a less restricted specificity and was active at a wider range of temperatures. Drosophila beta4GalT7 is expressed throughout development, with higher expression levels in adults. Reduction of Drosophila beta4GalT7 levels using expressed RNA interference (RNAi) in imaginal discs resulted in an abnormal wing and leg morphology similar to that of flies with defective Hedgehog and Decapentaplegic signaling, which are known to depend on intact proteoglycan biosynthesis. Immunohistochemical analysis of tissues confirmed that both heparan sulfate and chondroitin sulfate biosynthesis were impaired. Our results demonstrate that Drosophila beta4GalT7 has the in vitro and in vivo properties predicted for an ortholog of human beta4GalT7 and is essential for normal animal development through its role in proteoglycan biosynthesis.     

Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is "selective" or "permissive", with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.     

Molecular cloning and expression of a human chondroitin synthase

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.     

Beta-(1 --> 4)-galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence

To evaluate the ability of insect cells to produce complex-type N-glycans, beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity in several insect cell lines was analyzed. For this purpose, we developed a simple and highly sensitive assay for beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity, which is based on time-resolved fluorometry of europium. Bovine serum albumin (BSA) modified with GlcNAc (GlcNAc(44)-BSA) was used as the acceptor. GlcNAc(44)-BSA was coated on a 96-well microplate, and after incubation with the enzyme sample in the presence of UDP-Gal, Eu-labeled RCA(120) (Ricinus communis aggutin I), was added. RCA(120) binds to the Galbeta(1 --> 4)GlcNAc structure in the product, and the bound Eu-RCA(120) was measured by the fluorescence of europium. When bovine beta4Gal-T-I was used as a standard reference enzyme, a linear relationship between enzyme activity and fluorescent signal was obtained over the range of 0-1000 microUnits (IU). Using this system, we were able to measure a low but significant level of beta4GalT activity in Trichoplusia ni cells ('High Five'). In contrast, no endogenous beta4GalT activity was detected in a Spodoptera frugiperda (Sf-9) cell line. However, Sf-9 cells stably transfected with the bovine beta4GalT-I gene and 'High Five' cells infected with a baculovirus containing the same gene produced activity levels that were comparable to or greater than those found in Chinese hamster ovary cells. We also showed that the beta4GalT activity level observed in the baculovirus-infected T. ni cells under the control of immediate early promoter was highly dependent on the post-infection time, suggesting that galactosylation level may also be variable during the infection period.     

Characterization of the elongating alpha-D-mannosyl phosphate transferase from three species of Leishmania using synthetic acceptor substrate analogues

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Galbeta1-4Manalpha1-P phosphosaccharide repeat structures assembled by the sequential addition of Manalpha1-P and betaGal. The synthetic acceptor substrate Galbeta1-4Manalpha1-P-decenyl and a series of analogues were used to probe Leishmania alpha-D-mannosyl phosphate transferase activity. We show that the activity detected with Galbeta1-4Manalpha1-P-decenyl is the elongating alpha-D-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPT(LPG)). Differences in the apparent K(m) values for the donor and acceptor substrates were found using L. major, L. mexicana, and L. donovani promastigote membranes, but total activity correlated with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L. mexicana amastigotes, that do not express lipophosphoglycan, lack eMPT(LPG) and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPT(LPG) activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPT(LPG) activity was determined using 24 synthetic analogues of Galbeta1-4Manalpha1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the alphaMan residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6' hydroxyl of the betaGal residue is required for substrate recognition as well as for catalysis. The rate of Manalpha1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal betaGal residue abrogates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not abrogate transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.     

Structural analysis of α1,3-linked galactose-containing oligosaccharides in Schizosaccharomyces pombe mutants harboring single and multiple α-galactosyltransferase genes disruptions

In the fission yeast Schizosaccharomyces pombe, galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases in the lumen of the Golgi apparatus. In S. pombe, the major in vitro α1,2-galactosyltransferase activity has been purified, the gma12(+) gene has been cloned, and three α-galactosyltransferase genes (gmh1(+)-gmh3(+)) have also been partially characterized. In this study, we found three additional uncharacterized genes with homology to gmh1(+) (gmh4(+)-gmh6(+)) in the fission yeast genome sequence. All possible single disruption mutants and the septuple disruption strain were constructed and characterized. The electrophoretic mobility of acid phosphatase prepared from gma12Δ, gmh2Δ, gmh3Δ and gmh6Δ mutants was higher than that from wild type, indicating that Gma12p, Gmh2p, Gmh3p and Gmh6p are required for the galactosylation of N-linked oligosaccharides. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides from each single mutant showed that Gma12p, Gmh2p and Gmh6p are involved in galactosylation of O-linked oligosaccharides. The septuple mutant exhibited similar drug and temperature sensitivity as a gms1Δ mutant that is incapable of galactosylation. Oligosaccharide structural analysis based on HPLC and methylation analysis revealed that the septuple mutant still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating that an unknown α1,3-galactosyltransferase activity was still present in the septuple mutant.     

Mice Overexpressing β-1,4-Galactosyltransferase I Are Resistant to TNF-Induced Inflammation and DSS-Induced Colitis

Glycosylation is an essential post-translational modification, which determines the function of proteins and important processes such as inflammation. β-1,4-galactosyltransferase I (βGalT1) is a key enzyme involved in the addition of galactose moieties to glycoproteins. Intestinal mucins are glycoproteins that protect the gut barrier against invading pathogens and determine the composition of the intestinal microbiota. Proper glycosylation of mucus is important in this regard. By using ubiquitously expressing βGalT1 transgenic mice, we found that this enzyme led to strong galactosylation of mucus proteins, isolated from the gut of mice. This galactosylation was associated with a drastic change in composition of gut microbiota, as TG mice had a significantly higher Firmicutes to Bacteroidetes ratio. TG mice were strongly protected against TNF-induced systemic inflammation and lethality. Moreover, βGalT1 transgenic mice were protected in a model of DSS-induced colitis, at the level of clinical score, loss of body weight, colon length and gut permeability. These studies put βGalT1 forward as an essential protective player in exacerbated intestinal inflammation. Optimal galactosylation of N-glycans of mucus proteins, determining the bacterial composition of the gut, is a likely mechanism of this function.     

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.     

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower ??1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.     

Molecular characterization of β1,4-galactosyltransferase 7 genetic mutations linked to the progeroid form of Ehlers-Danlos syndrome (EDS)

β1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme initiating glycosaminoglycan (GAG) synthesis. Based on in vitro and ex vivo kinetics studies and structure-based modelling, we molecularly characterized β4GalT7 mutants linked to the progeroid form of Ehlers-Danlos syndrome (EDS), a severe connective tissue disorder. Our results revealed that loss of activity upon L206P substitution due to altered protein folding is the primary cause for the GAG synthesis defect in patients carrying the compound A186D and L206P mutations. We showed that R270C substitution strongly reduced β4GalT7 affinity towards xyloside acceptor, thus affecting GAG chains formation. This study establishes the molecular basis for β4GalT7 defects associated with altered GAG synthesis in EDS.     

Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.

alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.     

A beta-1,3-galactosyltransferase and brainiac/bre5 homolog expressed in the midgut did not contribute to a Cry1Ab toxin resistance trait in Ostrinia nubilalis

Post-translational glycosylation of midgut epithelial protein and lipid receptors may be required prior to binding of activated Bacillus thuringiensis (Bt) Cry toxins. A 931bp cDNA encoding a putative 297-residue beta-1,3-galactosyltransferase (beta3GalT5) was cloned from larval Ostrinia nubilalis midgut tissue, and showed homology to Drosophila brainiac (brn) and Caenorhabditis elegans bre5 proteins. Single nucleotide polymorphisms (SNPs) were detected in coding and promoter regions of O. nubilalis beta3GalT5 (Onb3GalT5), of which 3 of 31 CDS SNPs were non-synonymous. SNPs within HaeIII and MspI recognition sites were confirmed by PCR-RFLP, and are Mendelian inherited. Analysis of F(2) pedigrees suggested an Onb3GalT5 SNP C660 fixed within a Cry1Ab-resistant colony was not correlated with Cry1Ab resistance traits, as measured by higher larval O. nubilalis weights when fed toxin-containing diet.