Specificity of a soluble UDP-galactose: fucoside alpha1,3-galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium

Skp1 is an adaptor-like protein in E3(SCF)-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galalpha1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn(2+) and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K(m) and 35-fold higher V(max) compared with a simple alpha-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V(max) advantage, but still much less active than Skp1 itself because of a K(m) difference. These findings indicate that alpha-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.     

A non-Golgi alpha 1,2-fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.

Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose alpha1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDP-Fuc:Skp1 alpha 1,2-fucosyltransferase. Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known alpha 1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membrane-bound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus.     

Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of dictyostelium

Skp1 is a ubiquitous eukaryotic protein found in several cytoplasmic and nuclear protein complexes, including the SCF-type E3 ubiquitin ligase. In Dictyostelium, Skp1 is hydroxylated at proline 143, which is then modified by a pentasaccharide chain. The enzyme activity that attaches the first sugar, GlcNAc, was previously shown to copurify with the GnT51 polypeptide whose gene has now been cloned using a proteomics approach based on a quadrupole/time-of-flight hybrid mass spectrometer. When expressed in Escherichia coli, recombinant GnT51 exhibits UDP-GlcNAc:hydroxyproline Skp1 GlcNAc-transferase activity. Based on amino acid sequence alignments, GnT51 defines a new family of microbial polypeptide glycosyltransferases that appear to be distantly related to the catalytic domain of mucin-type UDP-GalNAc:Ser/Thr polypeptide alpha-GalNAc-transferases expressed in the Golgi compartment of animal cells. This relationship is supported by the effects of site-directed mutagenesis of GnT51 amino acids associated with its predicted DXD-like motif, DAH. In contrast, GnT51 lacks the N-terminal signal anchor sequence present in the Golgi enzymes, consistent with the cytoplasmic localization of the Skp1 acceptor substrate and the biochemical properties of the enzyme. The first glycosylation step of Dictyostelium Skp1 is concluded to be mechanistically similar to that of animal mucin type O-linked glycosylation, except that it occurs in the cytoplasm rather than the Golgi compartment of the cell.     

Purification and properties of rat liver globotriaosylceramide synthase, UDP-galactose:lactosylceramide alpha 1-4-galactosyltransferase

The enzyme which catalyzes the transfer of galactose from UDP-galactose to lactosylceramide (LacCer) was obtained in a 32,000-fold purified and apparently homogeneous form from rat liver by a procedure involving affinity chromatography on UDP-hexanolamine-Sepharose and LacCer-Sepharose. The enzyme is composed of two nonidentical subunits whose apparent molecular weights are 65,000 and 22,000. Methylation and hydrolysis of the product formed by incubation of the enzyme with UDP-galactose and [3H]LacCer yielded 2,3,6-tri-O-methyl-[3H]galactose, indicating that a galactose residue was introduced to position C-4 of the terminal galactose of the LacCer. The product also specifically reacted with monoclonal antibody directed to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). This indicates that the purified enzyme is exclusively alpha 1-4-galactosyltransferase. Studies on substrate specificity indicate that the purified enzyme is highly specific for the synthesis of GbOse3Cer and is clearly distinct from the enzymes responsible for the formation of iGbOse3Cer (Gal alpha 1-3Gal beta 1-4Glc-Cer) and blood group-B substance, which possess alpha 1-3 galactosidic linkages at the nonreducing termini. The enzyme is also distinct from the alpha 1-4-galactosyltransferase which catalyzes the formation of galabiaosylceramide (Gal alpha 1-4Gal beta 1-1Cer) and IV4Gal-nLacOse4 (P1 antigen). These studies represent the first report of the properties of a highly purified alpha-galactosyltransferase catalyzing the transfer of sugar residues to glycolipids.     

Identification of a region of UDP-galactose:N-acetylglucosamine beta 4-galactosyltransferase involved in UDP-galactose binding by differential labeling

The location of regions in the primary structure of UDP-galactose:N-acetylglucosamine beta 4-galactosyl-transferase (GT) that are involved in binding UDP-galactose has been investigated by differential chemical modification with two different reagents in the presence and absence of UDP-galactose. Treatment with periodate-cleaved UDP and NaCNBH3 resulted in a loss of 80% of GT activity, which was largely prevented by UDP-galactose. Stoichiometry of labeling and peptide maps of the modified enzyme samples indicated partial labeling at many sites. A major site of reaction in the absence of UDP-galactose that was essentially unmodified in its presence was found to correspond to Lys341 in the cDNA sequence of GT. As a second approach, the reactivities of the amino groups of GT were compared in the presence and absence of saturating levels of UDP-galactose by trace acetylation with [3H]acetic anhydride. UDP-galactose binding was found to perturb the reactivities of a number of lysines in the C-terminal region of GT, the most pronounced effect being a reduction in the reactivity of Lys351. The two procedures thus identified a region between residues 341 and 351 as being associated with UDP-galactose binding. This region overlaps a small section in the sequence of GT that was previously noted to be similar to part of bovine alpha-1,3-galactosyltransferase (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Sequence comparisons indicate that extended regions at the C terminus of each enzyme encompassing this area may represent homologous UDP-galactose-binding domains.     

Molecular and biochemical characterization of the Skp2-Cks1 binding interface

SCF(Skp2) is a multisubunit E3 ubiquitin ligase responsible for ubiquitination of cell cycle inhibitor p27. Ubiquitination of p27 requires an adapter protein, Cks1, to be in direct association with Skp2. The exact interface between Skp2 and Cks1 has not been elucidated. Here we have reported the definition of the critical functional interface between Skp2 and Cks1. We have identified eight amino acid residues in two discrete regions of Skp2 that are engaged in Cks1 binding. Mutation of any of these eight residues alone or in combination results in the loss of Cks1 association and negates Skp2-dependent p27 ubiquitination. These eight amino acid residues map on the same side of the Skp2 structure and likely constitute a functional binding surface for Cks1. Four of the eight amino acid residues are located in the largely unstructured carboxyl-terminal tail region of Skp2. These results uncovered the specificity of the Skp2-Cks1 interaction and reveal a critical function for the structurally flexible carboxyl-terminal tail region of Skp2 in Cks1 recognition and substrate ubiquitination.     

Changing the donor cofactor of bovine alpha 1, 3-galactosyltransferase by fusion with UDP-galactose 4-epimerase. More efficient biocatalysis for synthesis of alpha-Gal epitopes

Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.     

Molecular characterization of a novel beta1,3-galactosyltransferase for capsular polysaccharide synthesis by Streptococcus agalactiae type Ib

A group B streptococcus, Streptococcus agalactiae type Ib, produces a high-molecular-weight polysaccharide consisting of the following pentasaccharide repeating unit: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. The type-specific capsular polysaccharide (CP) synthesis (cps) genes of this strain were cloned and analyzed. A cloned 10-kb DNA fragment contained cpsIbE to L and neu (neuraminic acid synthesis gene) B. Comparison of the gene products with those of S. agalactiae type Ia, which has a similar but distinct CP, showed that the translation products of cpsIa and cpsIb genes exhibited very high homology except for those of cpsJ and K. In the type Ia strain, cpsIaJ encodes beta1,4-galactosyltransferase, which catalyzes the transfer of galactose as the fourth monosaccharide of the sugar repeating unit. In the type Ib CP, this galactose forms a beta1,3-linkage to GlcNAc. The low homology between the type Ia and Ib CpsJs seems to reflect this difference. By enzymatic activity measurement, the cpsIbJ product was found to display beta1,3-galactosyltransferase activity. Furthermore, hydrophobic cluster analysis clarified the similarities and differences of the structures in N-terminal regions, including the DXD motif, between the galactosyltransferases.     

Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase from rat liver.

The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase (beta 3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, beta 4-galactosyltransferase, beta-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the beta 3-Gal-T. beta 3-Gal-T activity is sensitive to changes in the R-group of the GalNAc alpha-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAc alpha-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished beta 3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAc alpha-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAc alpha-benzyl, which inhibited Gal incorporation into GalNAc alpha-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAc beta 6[Gal beta 3] GalNAc alpha-) is formed from core 1 (Gal beta 3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.     

Detection and characterization of a novel factor that stimulates DNA polymerase alpha

A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition.