Three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide

The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (αHexN-(1,4)-αGalA) attached by an α1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-α-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase.     

Two-step enzymatic synthesis of UDP-N-acetylgalactosamine

UDP-GalNAc has been synthesised with high yield from GalNAc, UTP and ATP using recombinant human GalNAc kinase GK2 and UDP-GalNAc pyrophosphorylase AGX1. Both enzymes have been prepared in one step from 1 L cultures of transformed Escherichia coli and the UDP-GalNAc produced has been purified by a simple procedure. The method described is a rapid and efficient means to produce UDP-GalNAc as well as analogues like UDP-N-azidoacetylgalactosamine (UDP-GalNAz).     

A coupled assay for UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc).

UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) (i.e., core 2 GlcNAc-T) is a developmentally regulated enzyme of the O-linked oligosaccharide biosynthesis pathway. We have developed a coupled-enzyme assay for core 2 GlcNAc-T that is approximately 100 times more sensitive than the standard assay using UDP-[3H]GlcNAc as a sugar donor. Core 2 GlcNAc-T reactions were performed using unlabeled UDP-GlcNAc donor and Gal beta 1-3GalNAc alpha-paranitrophenyl (pNp) as acceptor. The product, Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha-pNp was then further reacted with purified bovine beta 1-4Gal-T and UDP-[3H]Gal to produce Gal beta 1-3([3H]Gal beta 1-4GlcNAc beta 1-6) GalNAc alpha-pNp, which was separated on an Ultrahydrogel HPLC column. Approximately 10% of the available GlcNAc-terminating acceptor was substituted in the Gal-T reaction, allowing 1 pmol of product to be readily detected. The increased sensitivity of the coupled assay should facilitate studies of core 2 GlcNAc-T activity where material is limiting or specific activity is low.     

Chick neural retina N-acetylgalactosaminyltransferase/acceptor complex: catalysis involves transfer of N-acetylgalactosamine phosphate to endogenous acceptors

Homogenates of embryonic chick neural retina prepared in 1% Triton X-100 have the ability to transfer N-acetyl[32P]galactosamine [( 32P]GalNAc) from beta-32P-labeled uridine diphosphate N-acetylgalactosamine [( beta-32P]UDP-GalNAc) to endogenous macromolecular acceptors. The phosphotransferase activity sediments as three distinct peaks upon centrifugation on sucrose gradients. These peaks are coincident with the transferase/acceptor complexes previously described [Balsamo, J., & Lilien, J. (1982) J. Biol. Chem. 257, 345-354]. The parameters of the 32P transfer reaction closely parallel those observed with UDP-[3H]GalNAc as substrate when the densest particles, H, are used as a source of transferase/acceptors. Treatment of 3H- and 32P-labeled products with alpha-N-acetylgalactosaminidase removes [3H]GalNAc residues and exposes 32P-labeled groups. These data suggest that the sugar-phosphate is transferred intact, resulting in a terminal phosphodiester linkage. The resistance of the macromolecular products to digestion by endoglycosidase F and its sensitivity to hydrolysis under mild alkaline conditions suggest that the alpha-linked sugar is transferred to an oligosaccharide chain attached to the protein core via an O-serine or threonine residue. Characterization of the 32P- and 3H-labeled H particle products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a series of coincident high molecular weight polypeptides.     

Enzymatic synthesis of blood group A and B trisaccharide analogues

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.     

Regulation of carbohydrate metabolism during Giardia encystment

Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi1 is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by "cyst wall synthase" (beta 1 --> 3 GalNAc transferase) into a highly insoluble beta 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.     

Mucin synthesis. UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase from pig and rat colon mucosa

Pig and rat colon mucosal membrane preparations catalyze the in vitro transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to GalNAc-ovine submaxillary mucin to form GlcNAc beta 1-3GalNAc-mucin. Rat colon also catalyzes the in vitro transfer of GlcNAc from UDP-GlcNAc to GlcNAc beta 1-3GalNAc-mucin to form GlcNAc beta 1-3(GlcNAc beta 1-6) GalNAc-mucin. This is the first demonstration of in vitro synthesis of the GlcNAc beta 1-3GalNAc disaccharide and of the GlcNAc beta 1-3-(GlcNAc beta 1-6)GalNAc trisaccharide, two of the four major core types found in mammalian glycoproteins of the mucin type, i.e., those containing oligosaccharides with GalNAc-alpha-serine (threonine) linkages. The activity catalyzing synthesis of the disaccharide has been named UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase (mucin core 3 beta 3-GlcNAc-transferase), while the activity responsible for synthesizing the trisaccharide has been named UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (mucin core 4 beta 6-GlcNAc-transferase). The beta 3-GlcNAc-transferase from pig colon is activated by Triton X-100, has an absolute requirement for Mn2+, and transfers GlcNAc to GalNAc-alpha-phenyl, GalNAc-alpha-benzyl, and GalNAc-ovine submaxillary mucin with apparent Km values of 5, 2, and 3 mM and Vmax values of 59, 62, and 37 nmol h-1 (mg of protein)-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of analogues of GalNAc as substrates for enzymes of the mammalian GalNAc salvage pathway

Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-Acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans, and glycoproteins; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc, which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogues of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, galactokinase 2 (GK2) and uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogues that can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway: a modification on C6 is not tolerated by GK2; AGX1 can use all products of GK2 although with various efficiencies; and all analogues transformed into UDP-GalNAc analogues except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogues that could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detection on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogues can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism, and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.     

High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.

We have expressed the Neisseria meningitidis lgtA gene at a high level in Escherichia coli. The encoded beta-N-acetylglucosaminyltransferase, referred to as LgtA, which in the bacterium is involved in the synthesis of the lacto-N-neo-tetraose structural element of the bacterial lipooligosaccharide, was obtained in an enzymatically highly active form. This glycosyltransferase appeared to be unusual in that it displays a broad acceptor specificity toward both alpha- and beta-galactosides, whether structurally related to N- or O-protein-, or lipid-linked oligosaccharides. Product analysis by one- and two-dimensional 400 MHz 1H- and 13C-NMR spectroscopy reveals that LgtA catalyzes the introduction of GlcNAc from UDP-GlcNAc in a beta 1-->3-linkage to accepting Gal residues. The enzyme can thus be characterized as a UDP-GlcNAc:Gal alpha/beta-R beta 3-N-acetylglucosaminyltransferase. Although lactose is a highly preferred acceptor substrate the recombinant enzyme also acts efficiently on monomeric and dimeric N-acetyllactosamine revealing its potential value in the synthesis of polylactosaminoglycan structures in enzyme assisted procedures. Furthermore, LgtA shows a high donor promiscuity toward UDP-GalNAc, but not toward other UDP-sugars, and can catalyze the introduction of GalNAc in beta 1-->3-linkage to alpha- or beta-Gal in the acceptor structures at moderate rates. LgtA therefore shows promise to be a useful catalyst in the preparative synthesis of both GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.     

Oxidation and transamination of the 3"-position of UDP-N-acetylglucosamine by enzymes from Acidithiobacillus ferrooxidans. Role in the formation of lipid a molecules with four amide-linked acyl chains

Lipid A, a major component of the outer membranes of Escherichia coli and other Gram-negative bacteria, is usually constructed around a beta-1',6-linked glucosamine disaccharide backbone. However, in organisms like Acidithiobacillus ferrooxidans, Leptospira interrogans, Mesorhizobium loti, and Legionella pneumophila, one or both glucosamine residues are replaced with the sugar 2,3-diamino-2,3-dideoxy-d-glucopyranose. We now report the identification of two proteins, designated GnnA and GnnB, involved in the formation of the 2,3-diamino-2,3-dideoxy-d-glucopyranose moiety. The genes encoding these proteins were recognized because of their location between lpxA and lpxB in A. ferrooxidans. Based upon their sequences, the 313-residue GnnA protein was proposed to catalyze the NAD(+)-dependent oxidation of the glucosamine 3-OH of UDP-GlcNAc, and the 369-residue GnnB protein was proposed to catalyze the subsequent transamination to form UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-d-glucopyranose (UDP-GlcNAc3N). Both gnnA and gnnB were cloned and expressed in E. coli using pET23c+. In the presence of l-glutamate and NAD(+), both proteins were required for the conversion of [alpha-(32)P]UDP-GlcNAc to a novel, less negatively charged sugar nucleotide shown to be [alpha-(32)P]UDP-GlcNAc3N. The latter contained a free amine, as judged by modification with acetic anhydride. Using recombinant GnnA and GnnB, approximately 0.4 mg of the presumptive UDP-GlcNAc3N was synthesized. The product was purified and subjected to NMR analysis to confirm the replacement of the GlcNAc 3-OH group with an equatorial NH(2). As shown in the accompanying papers, UDP-GlcNAc3N is selectively acylated by LpxAs of A. ferrooxidans, L. interrogans, and M. loti. UDP-GlcNAc3N may be useful as a substrate analog for diverse enzymes that utilize UDP-GlcNAc.     

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

Background

Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.

Results

The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit: H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.

Conclusion

Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.     

A single bifunctional UDP-GlcNAc/Glc 4-epimerase supports the synthesis of three cell surface glycoconjugates in Campylobacter jejuni

The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE might be a UDP-GlcNAc 4-epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne. The Km(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 microm, whereas those for UDP-Glc and UDP-Gal are 780 and 784 microm. The kcat and kcat/Km(app) values were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDP-GlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP sugar-binding site of Gne was modeled by using the structure of the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted, and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C. jejuni, the lipooligosaccharide was shown by capillary electrophoresis-mass spectrometry to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues with the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168.     

N-acetyl-beta-D-glucosaminyltransferases related to the synthesis of mucin-type glycoproteins in human ovarian tissue

The presence of N-acetyl-beta-D-glucosaminyltransferases in microsome preparations from human ovarian tissues was investigated with UDP-GlcNAc and several synthetic oligosaccharides as acceptors. The products were identified by paper chromatography and the linkage of the 2-acetamido-2-deoxy-beta-D-glucopyranosyl group incorporated into oligosaccharides was determined by exoglycosidase digestions, 1H-n.m.r. spectroscopy, and methylation analysis. These results showed that ovarian microsome preparations contain both beta-(1----3)- and beta-(1----6)-N-acetyl-D-glucosaminyltransferase activities which might be involved in the synthesis of mucin-type glycoproteins. Substrate competition tests suggested that both UDP-GlcNAc:-Bn glycoside of beta-D-GlcpNAc-(1----6)-alpha-D-GalpNAc [GlcNAc to GalNAc] and -Bn glycoside of beta-D-Galp-(1----3)-[beta-D-GlcNAc-(1----6)]-alpha-D-GalpNAc [GlcNAc to Gal] beta-(1----3)-N-acetyl-D-glucosaminyltransferase activities reside in a single enzyme species.     

Mucin biosynthesis: purification and characterization of a mucin beta 6N-acetylglucosaminyltransferase.

We have purified, to apparent homogeneity, a mucin beta 6N-acetylglucosaminyltransferase (beta 6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Gal beta 1-3GalNAc alpha benzyl (Bzl) to stabilize the beta 6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Superflow affinity column equilibrated with 1 mM Gal beta 1-3GalNAc alpha Bzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 mumol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the beta 6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The Km values for UDP-GlcNAc and Gal beta 1-3GalNAc alpha Bzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified beta 6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAc beta 1-3Gal beta R as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a beta 1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all beta 6GlcNAc structures found in mucin-type oligosaccharides.     

Kinetic basis for the donor nucleotide-sugar specificity of beta1, 4-N-acetylglucosaminyltransferase III

The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor.     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7

The O-antigen of lipopolysaccharide in Gram-negative bacteria plays an important role in bacterium-host interactions. Escherichia coli O86:B7 O-unit contains five sugar residues: one fucose (Fuc) and two each of N-acetylgalactosamine (GalNAc) and galactose (Gal). The entire O-antigen gene cluster was previously sequenced: orf1 was assigned the gne gene for the biosynthesis of UDP-GalNAc. To confirm this annotation, overexpression, purification, and biochemical characterization of Gne were performed. By using capillary electrophoresis, we showed that Gne can catalyze the interconversion of both UDP-GlcNAc/GalNAc and UDP-Glc/Gal almost equally well. The Km values of Gne for UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc are 370, 295, 323, and 373 microM, respectively. The comparison of kinetic parameters of Gne from Escherichia coli O86:B7 to those of other characterized UDP-GlcNAc/Glc 4-epimerases indicated that it has relaxed specificity toward the four substrates, the first characterized enzyme to have this activity in the O-antigen biosynthesis. Moreover, the calculated kcat/Km values for UDP-GalNAc and UDP-Gal are approximately 2-4 times higher than those for UDP-GlcNAc and UDP-Glc, suggesting that Gne is slightly more efficient for the epimerization of UDP-GalNAc and UDP-Gal. One mutation (S306Y) resulted in a loss of epimerase activity for non-acetylated substrates by about 5-fold but totally abolished the activity for N-acetylated substrates, indicating that residue S306 plays an important role in the determination of substrate specificity.     

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.     

Biosynthesis of the blood group P antigen-like GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure: a novel N-acetylgalactosaminyltransferase in human blood plasma.

Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase.