Purification of an alpha-N-acetylglucosaminyltransferase from the yeast Kluyveromyces lactis and a study of mutants defective in this enzyme activity

An enzyme activity in Kluyveromyces lactis that catalyzes the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine to alpha Man(1 leads to 3) alpha Man ( 1 leads to 2) alpha Man (1 leads to 2)Man to yield alpha Man(1 leads to 3) [alpha GlcNAc(1 leads to 2)] alpha Man(1 leads to 2) alpha Man (1 leads to 2)Man, a mannoprotein side-chain unit, has been solubilized by Triton X-100 and purified 18000-fold by a combination of ion-exchange chromatography, gel filtration, hydrophobic chromatography, and adsorption to a lectin column. The enzyme activity from a K. lactis mutant (mnn2-2) that made mannoprotein lacking N-acetylglucosamine in its side chains, but that possessed a normal level of transferase activity in cell extracts, was purified and compared with the enzyme from the wild-type strain. Both transferase activities are integral membrane proteins found in particles associated with endoplasmic reticulum. The two purified enzymes had the same apparent size, heat stability, Mn2+ requirement, and Km for donor and acceptor and a similar Vmax. Wild-type and mutant cells had similar pool sizes of sugar nucleotide donor, and they incorporated labeled N-acetylglucosamine into chitin at similar rates. No evidence was obtained for an inactive enzyme precursor in mutant cells that was activated upon breaking the cells, nor did the mutant cells contain a transferase inhibitor or a hexosaminidase that could remove the sugar from the mannoprotein during processing and secretion. The mnn2-2 locus appears to be allelic with a second mutant, mnn2-1, that has the same phenotype but that lacks transferase activity in cell extracts. This suggests that the two mutations affect the structural gene for the transferase, and we conclude that the mnn2-2 mutant could contain an altered enzyme that fails to function because it is improperly localized or oriented in the membrane.     

Biosynthesis of yeast mannan. Isolation of Kluyveromyces lactis mannan mutants and a study of the incorporation of N-acetyl-D-glucosamine into the polysaccharide side chains.

One side chain in the cell wall mannan of the yeast Kluyveromyces lactis has the structure (see article). (Raschke, W. C., and Ballou, C. E. (1972) Biochemistry 11, 3807). This (Man)4GNAc unit (the N-acetyl-D-glucosamine derivative of mannotetroase) and the (Man)4 side chain, aMan(1 yields 3)aMan(1 yields 2)aMan(1 yields 2)Man, are the principle immunochemical determinants on the cell surface. Two classes of mutants were obtained which lack the N-acetyl-D-glucosamine-containing determinant. The mannan of one class, designated mmnl, lacks both the (Man)4GNAc and (Man)4 side chains. Apparently, it has a defective alpha-1 yields 3-mannosyltransferase and the (Man)4 unit must be formed to serve as the acceptor before the alpha-1 yields 2-N-acetyl-glucosamine transferase can act. The other mutant class, mnn2, lacks only the (Man)4GNAc determinant and must be defective in adding N-acetylglucosamine to the mannotetrasose side chains. Two members of this class were obtained, one which still showed a wild type N-acetylglucosamine transferase activity in cell-free extracts and the other lacking it. They are allelic or tightly linked, and were designated mnn2-1 mnn2-2. Protoplast particles from the wild type cells catalyzed a Mn2+-dependent transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the mannotetraose side chain of endogenous acceptors. Exogenous mannotetraose also served as an acceptor in a Mn2+-dependent reaction and yielded (Man)4GNAc. Related oligosaccharides with terminal alpha (1 yields 3)mannosyl units were also good acceptors. The product from the reaction with alphaMan(1 yields 3)Man had the N-acetylglucosamine attached to the mannose unit at the reducing end, which supports the conclusion that the cell-free glycosyltransferase activity is identical with that involved in mannan synthesis. The reaction was inhibited by uridine diphosphate. Protoplast particles from the mmnl mutants showed wild type N-acetylglucosamine transferase activity with exogenous acceptor, but they had no endogenous activity because the endogenous mannan lacked acceptor side chains. Particles from the mnn2-1 mutant failed to catalyze N-acetylglucosamine transfer. In contrast, particles from the mnn2-2 mutant were indistinguishable from wild type cells in their transferase activity. Some event accompanying cell breakage and assay of the mnn2-2 mutant allowed expression of a latent alpha-1 yields 2-N-acetylglucosamine transferase with kinetic properties similar to those of the wild type enzyme.     

Effects of mannoprotein mutations on Saccharomyces cerevisiae core oligosaccharide structure

By the combined actions of an endo-alpha-1 leads to 6-mannanase and an endo-beta-N-acetylglucosaminidase, the core oligosaccharides can be released from Saccharomyces cerevisiae X2180 mnn2 mannoproteins. The effects of various mannoprotein mutations were evaluated by structural comparison of these core oligosaccharides with those prepared from double mutant strains with the genotypes mnn1 mnn2, mnn2 mnn3, mnn2 mnn4, and mnn2 mnn5. The results indicate that only the mnn1 lesion has a major effect on the mannoprotein core structure. Whereas the mnn2 mannoprotein yields a core composed of 6 fragments that differ in size from each other by single mannose units, only the two smallest species predominate in the mnn1 mnn2 preparation. This change is correlated with a loss of terminal alpha 1 leads to 3-mannosyl residues, an effect on the mnn1 lesion that is found also in the polysaccharide outer chain and hydroxyamino acid-linked mannooligosaccharides. The mnn3 and mnn5 mutations also had slight effects on the core size, but clear differences in linkage composition were not apparent. The results suggest that core oligosaccharides have an average composition of Man11GlcNAc, whereas Man9GlcNAc is the major oligosaccharide in strains containing the mnn1 defect. These values are 2 to 3 sugars less than those estimated previously (Nakajima, T., and Ballou, C. E. (1975) Biochem. Biophys. Res. Commun. 66, 870-879). Detailed analysis of the major core oligosaccharide from the mnn1 mnn2 mutant revealed that the two mannoses in alpha 1 leads to 3 linkage to the backbone were adjacent to each other and that the oligosacccharide is nearly identical with one isolated from chinese hamster ovary cell membranes (Li, E., and Kornfeld, S. (1979) J. Biol. Chem. 254, 1600-1605). This finding provides strong evidence for the evolutionary conservation of this structural feature of the high mannose core oligosaccharides.     

Functional analysis of a <Emphasis Type="Italic">Hansenula polymorpha MNN2-2</Emphasis> homologue encoding a putative UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine transporter localized in the endoplasmic reticulum

The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2Δ) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2Δ strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.     

The presence of N-acetyllactosamine and lactose: beta (1-3)N-acetylglucosaminyltransferase activity in human urine

Normal human urine was found to contain beta (1-3)N-acetylglucosaminyltransferase catalyzing the transfer of N-acetylglucosamine from UDP-GlcNAc to N-acetyllactosamine and lactose. Lacto-N-tetraose which carries the terminal Gal beta (1-3)GlcNAc structure was a poor acceptor. The product of the transferase reaction with N-acetyllactosamine as acceptor was identified by methylation analysis as GlcNAc beta (1-3)Gal beta (1-4)GlcNAc. The beta-linkage of the GlcNAc in the synthesized trisaccharide was confirmed by the action of the specific beta-N-acetylhexosaminidase. The enzyme requires Mn2+ ions for its activity, shows a broad pH optimum from 7 to 9, and appears to have a molecular weight of about 200,000 as estimated by Sephadex gel filtration.     

The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles.

In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes.     

A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure

We find that the N-linked Man8GlcNAc2- core oligosaccharide of Saccharomyces cerevisiae mnn mutant mannoproteins is enlarged by the addition of the outer chain to the alpha 1----3-linked mannose in the side chain that is attached to the beta 1----4-linked mannose rather than by addition to the terminal alpha 1----6-linked mannose. This conclusion is derived from structural studies on a phosphorylated oligosaccharide fraction and from mass spectral fragment analysis of neutral core oligosaccharides.     

Structure of the phosphorylated N-linked oligosaccharides from the mnn9 and mnn10 mutants of Saccharomyces cerevisiae

The N-linked oligosaccharides, from Saccharomyces cerevisiae mnn1 mnn9 mutant mannoprotein extracted from the cells in hot citrate buffer, were separated by ion exchange into a monophosphate diester, a monophosphate monoester, a diphosphate diester, and a diphosphate monoester diester. The structures of the major components with diesterified phosphate were assigned as follows (where M = mannose), according to a recently revised oligosaccharide structure for the mnn mutants (Hernandez, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). formula; see text The monoester derivatives were mixtures of the possible isomers produced by removal of one or the other phosphoglycosyl-linked mannose units, and they were shown to arise by chemical degradation during isolation. The mnn1 mnn2 mnn10 acidic oligosaccharide fraction contained a mono- and a diphosphate ester. The monophosphate consisted predominantly of a single isomer with a mannosyl phosphate unit located at the end of the outer chain in an oligosaccharide with the following structure, where x may range from 2 to 12. The diphosphate had a mannosyl phosphate in this formula; see text position as well as one on the terminal alpha 1----6-linked mannose in the core. The presence in the mnn1 mnn9 or mnn1 mnn2 mnn10 background of the mnn4 or mnn6 mutations, which are known to regulate phosphorylation in yeast, reduced phosphorylation by 90% but did not eliminate it. AI-12522     

A single Caenorhabditis elegans Golgi apparatus-type transporter of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine

The genome of Caenorhabditis elegans encodes for 18 putative nucleotide sugar transporters even though its glycome only contains 7 different monosaccharides. To understand the biological significance of this phenomenon, we have begun a systematic substrate characterization of the above putative transporters and have determined that the gene ZK896.9 encodes a Golgi apparatus transporter for UDP-glucose, UDP-galactose, UDP- N-acetylglucosamine, and UDP- N-acetylgalactosamine. This is the first tetrasubstrate nucleotide sugar transporter characterized for any organism and is also the first nonplant transporter for UDP-glucose. Evidence for the above substrate specificity and substrate transport saturation kinetics was obtained by expression of ZK896.9 in Saccharomyces cerevisiae followed by Golgi enriched vesicle isolation and assays in vitro. Further evidence for UDP-glucose transport was obtained by expression of ZK 896.9 in Giardia lamblia, an organism recently characterized as having endogenous transport activity for only UDP- N-acetylglucosamine. Expression of ZK896.9 was also able to correct the phenotype of a mutant Chinese ovary cell line specifically defective in the transport of UDP-galactose into the Golgi apparatus and of a mutant of the yeast Kluyveromyces lactis specifically defective in the transport of UDP- N-acetylglucosamine into its Golgi apparatus. Because up to now all three other characterized nucleotide sugar transporters of C. elegans have been found to transport two or three substrates, the substrate specificity of ZK896.9 raises questions as to the evolutionary ancestry of this group of proteins in this nematode.     

An N-acetylglucosaminyltransferase of the Golgi apparatus of the yeast Saccharomyces cerevisiae that can modify N-linked glycans

The yeast Saccharomyces cerevisiae is widely regarded as being only capable of producing N-linked glycans with high-mannose structures. To investigate the glycan structures made in different mutant strains, we made use of a reporter protein consisting of a version of hen egg lysozyme that contains a single site for N-linked glycosylation. Mass spectrometry analysis of the attached glycans revealed that a large proportion contained an unexpected extra mass corresponding to a single N-acetylhexosamine residue. In addition, the glycosylated lysozyme was recognized by an N-acetylglucosamine specific lectin. The genome of S. cerevisiae contains an uncharacterized open reading frame, YOR320c, that is related to a known N-acetylglucosaminyltransferase. Deletion of this ORF resulted in the disappearance of the extra mass on the N-linked glycans and loss of lectin binding. We show that the protein encoded by YOR320c (which we term Gnt1p) is localized to the Golgi apparatus and has GlcNAc-transferase activity in vitro. The physiological role of Gnt1p is unclear because mutants lacking the protein show no obvious growth or cell wall defects. Nonetheless, these results indicate that heterologous glycoproteins expressed in yeast can receive N-glycans with structures other than high mannose. In addition, they indicate that the lumen of the yeast Golgi contains UDP-GlcNAc, which may facilitate reconstitution of higher eukaryotic N-glycan processing.     

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides

We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648-13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. (see structure below)     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

Localization of alpha 1----3-linked mannoses in the N-linked oligosaccharides of Saccharomyces cerevisiae mnn mutants

Neutral and phosphorylated N-linked oligosaccharides were isolated from Saccharomyces cerevisiae mnn9 and mnn9 gls1 mutant mannoproteins and separated into homologues that differed in the number of terminal alpha 1----3-linked mannoses. In each type of oligosaccharide, the addition of such mannose was shown to occur in an ordered rather than a random fashion. The results confirm and extend an earlier report that dealt with the N-linked oligosaccharides from yeast invertase [Trimble, R.B., & Atkinson, P.H. (1986) J. Biol. Chem. 261, 9815-9824], and they suggest that the postulated processing pathway can be generalized to include phosphorylated and glucose-containing N-linked oligomannosides. We conclude that this processing pathway is identical for the analogous oligosaccharides from the mnn9 and wild-type strains of S. cerevisiae. Analysis of the mnn2 mnn10 mannoprotein revealed that a similar modification occurred at the branched terminus of the outer chain as well as in the core in this mutant.     

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.     

Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class. Support for the stop signal proposed for regulation of outer chain elongation

Total cell mannoprotein was isolated from Saccharomyces cerevisiae X2180 mutants that have defects in elongation of the outer chain attached to the N-linked core oligosaccharides (mnn7, mnn8, mnn9, and mnn10) (Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem. 255, 5986-5991). Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9 mutation eliminates all but two mannoses of the outer chain, whereas the mnn8 and mnn10 strains produce outer chains of variable but similar lengths. The isolate designated mnn7 was found to be allelic with mnn8. Haploid mutants of the type mnn8 mnn9 or mnn9 mnn10 had the mnn9 phenotype, which established that the mnn9 defect is dominant and presumably acts at a processing step prior to the steps affected by mnn8 and mnn10. Analysis of the mnn1 mnn2 mnn10 oligosaccharides revealed that the heterogeneous outer chain contained 6-16 alpha 1----6-linked mannose units and each was terminated by a single alpha 1----2-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9 oligosaccharide. The results are consistent with and support the hypothesis (Gopal, P. K., and Ballou, C. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 84, 8824-8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wild-type strain.     

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.      

Oligosaccharide structures of the major exoglucanase secreted by Saccharomyces cerevisiae.

We have determined the structures of the N-linked carbohydrate chains, released by endo H, of exoglucanase II that are secreted by wild-type Saccharomyces cerevisiae and by the mnn1 mnn9 and mnn1 glycosylation mutants. The mnn9 mutation does not significantly affect N-linked oligosaccharides of exoglucanase II since we found almost identical structures in both mutant strains consisting of a slightly enlarged core with the basic structure shown in A (where M = mannose). Most of the molecules (77%) were phosphorylated on one of the starred mannoses (34%) or on both (43%) with a diesterified (alpha M-->P-->) or monoesterified phosphate group. In addition, some of the molecules apparently escape normal processing and retain the alpha-(1-->2)-linked mannose (italicized) and/or the three glucoses that are characteristic of the lipid-linked precursor (structure B). In the wild type, we found the same basic structure but more [formula; see text] than 90% of the molecules were modified with one to four alpha-(1-->3)-linked mannoses, which were absent in the strains bearing the mnn1 mutation (structure C). The proportion of acidic components was similar to that found in the mutants (78%), although, in this case, the monophosphorylated forms were more abundant (50%) than the diphosphorylated ones (28%). Most of the phosphate groups (69%) were diesterified by a disaccharide (alpha M-->3 alpha M-->P-->) instead of the single mannose found when the mnn1 mutation was present. In both mnn1 and wild type 10-15% of the oligosaccharides had an extra alpha-(1-->6)-linked mannose in the outer chain, a structure described in the recently isolated vrg1 mutant [Ballou, L., Hitzeman, R.A., Lewis, M. S., & Ballou, C. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3209-3212].     

Control of glycoprotein synthesis. Purification of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver

A new affinity chromatography adsorbant, in which UDP-GlcNAc has been linked to thiopropyl-Sepharose at the 5 position of the uracil via a 5-mercuri mercaptide bond, was utilized to purify UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II 60,000-fold from rat liver. After extraction of rat liver membranes with Triton X-100, the enzyme was found to exist in two molecular weight forms of markedly differing size, separable on Sephadex G-200. The low Mr form was separated from the high Mr form on columns of CM-Sephadex and hydroxylapatite, and was further purified by sequential elutions with NaCl, UDP-GlcNAc, and EDTA from the 5-mercuri-UDP-GlcNAc affinity adsorbant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified low Mr form under reducing conditions revealed two protein bands of Mr 48,000 and 43,000. The purified enzyme catalyzes the transfer of N-acetylglucosamine from UDP-GlcNAc to the compound: (Formula: see text) The high Mr form of the enzyme, which eluted in the void volume of Sephadex G-200, was resistant to a number of treatments in attempts to reduce its molecular weight. These results suggest that the high Mr form of the enzyme may represent either a complex which normally exists in Golgi membranes as a result of strong protein-protein interactions or a protein with one or more "anchor" segments.     

Overexpression of UDP-GlcNAc transporter partially corrects galactosylation defect caused by UDP-Gal transporter mutation

Nucleotide sugar transporters deliver substrates for glycosyltransferases into the endoplasmic reticulum and the Golgi apparatus. We demonstrated that overexpression of UDP-GlcNAc transporter (NGT) in MDCK-RCA(r) and CHO-Lec8 mutant cells defective in UDP-Gal transporter (UGT) restored galactosylation of N-glycans. NGT overexpression resulted in decreased transport of UDP-GlcNAc into the Golgi vesicles. This effect resembled the phenotype of mutant cells corrected by UGT1 overexpression. The transport of UDP-Gal was not significantly changed. Our data suggest that the biological function of UGT and NGT in galactosylation of macromolecules may be coupled.     

Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin.

The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.