Uridine Diphosphate N-acetylgalactosamine Research Articles

GALNT12 promotes fibrosarcoma growth by accelerating YAP1 nuclear localization.

Fibrosarcoma is a highly malignant type of soft tissue sarcoma that currently lacks effective treatment options. Polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) belongs to the uridine diphosphate N-acetylgalactosamine gene family, which is involved in numerous biological processes of diseases, such as tumor progression. Its upregulated expression is closely associated with the development of colorectal cancer. However, research on the role of GALNT12 in fibrosarcoma is currently limited. The present study aimed to assess the expression and biological function of GALNT12 in fibrosarcoma. Patient data and tissue samples were collected and public datasets were obtained from the Gene Expression Omnibus (GSE24369 and GSE21124). Immunofluorescence assays were performed to observe the cellular localization of GALNT12. GALNT12 expression was measured using reverse transcription-quantitative PCR, western blotting and immunohistochemistry. Small interfering RNAs were constructed to knock down GALNT12 expression in HT-1080 cells. Cell Counting Kit-8 and EdU assays were used to assess fibrosarcoma cell proliferation. Wound healing and Transwell assays were used to detect migration. Gene set enrichment analysis was performed to identify key pathways. Paired and unpaired Student's t-test, Fisher's exact test and one-way ANOVA (followed by Tukey's Honest Significant Difference test) were used to analyze the data. It was demonstrated that GALNT12 expression was upregulated in both fibrosarcoma cell lines and tissue samples and predicted poor patient prognosis. In vitro experiments demonstrated that high GALNT12 expression levels significantly increased HT-1080 cell proliferation and migration. Furthermore, it was demonstrated that high GALNT12 expression levels were closely associated with the yes1 associated transcriptional regulator (YAP1) signaling pathway. Knockdown of GALNT12 inhibited YAP1 nuclear translocation, which affected activation of key downstream genes including AMOTL2, BIRC5 and CYR61. Therefore, the present study demonstrated that GALNT12 promoted fibrosarcoma progression. GALNT12 could be a potential biomarker for this disease and may potentially provide new ideas for targeted therapy of fibrosarcoma in the future.

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs.

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

Structure-Based Prediction of Polypeptide Substrate Specificities of Glycosyltransferases

Glycans are some of the most abundant and diverse biopolymers, important in a number of biological processes including cell adhesion, receptor activation, molecular trafficking, endocytosis, and signal transduction. Protein glycosylation is primarily mediated by enzymes known as oligosaccharyltransferases (OSTs) and glycosyltransferases (GTs). Polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) are higher eukaryotic retaining GTs that transfer a GalNAc moiety from uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) onto Ser/Thr residues. This enzyme family initiates the most common type of O-glycosylation and thus affects the structure, stability, and function of many proteins. Structure-based prediction tools have the potential to advance our understanding of glycan-related phenomena as well as enable the design of novel glycoconjugates. Here, we present new computational protocols that allow rapid computational screening and scoring of polypeptide substrates of GTs such as GalNAcT2 and human-OGT. Our Rosetta-based algorithm docks a fully flexible glycosylated polypeptide substrate to the catalytic site of the GT, exhaustively sampling polypeptide and glycan conformational space, generating thousands of decoys for each docked polypeptide followed by ranking of decoys using the Rosetta score function. Using this protocol, we are able to predict relative specificities for polypeptide glycosylation for all possible amino acid permutations at substrate positions X-1 and X+1 for GalNAc-T2 and human-OGT. We compared predictions to experimental data (Kightlinger, W. et al., Nat. Chem. Biol.14, 627-635 (2018)) for all 400 polypeptide substrates per enzyme. Using a receiver operating curve analysis, the predictions were found to have an area-under-curve above 0.80, indicating that the method can often distinguish true and false positives. To the best of our knowledge, our protocol is the first example of a structure-based computational protocol for predicting polypeptide substrate specificities of GTs.

Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using (13)C6-glucose and (13)C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with (13)C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a (13)C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using (13)C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.

Isolation of a porcine UDP‐GalNAc transferase cDNA mapping to the region of the blood group EAA locus on pig chromosome 1

In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an alpha(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl-alpha(1,2)galactopyranoside; the enzyme did not use phenyl-beta-D-galactopyranoside (phenyl-beta-D-Gal) as an acceptor. Because the alpha(1,3)GalNAc transferase gene product requires an alpha(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the alpha(1,2)fucosyltransferase gene product is necessary for the functioning of the alpha(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen. CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fucalpha(1,2)Gal: fucosyl-alpha(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Galalpha(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl-beta-D-Gal: phenyl-beta-D-galactopyranoside; R: Galbeta1-4Glcbeta1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine

Chick neural retina N-acetylgalactosaminyltransferase/acceptor complex: catalysis involves the 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.

Cell-cycle dependence of a ganglioside glycosyltransferase activity and its inhibition by enkephalin in a neurotumor cell line.

Rat glioma X mouse neuroblastoma hybrid neurotumor cells (NG108-15), synchronized by amino acid deprivation, showed a cell-cycle-dependent peak of activity of a ganglioside N-acetylgalactosaminyl transferase 14-24 h following release from the cell cycle block (S/G2 phase). Maximal expression of two typical lysosomal hydrolases, N-acetyl-beta-hexosaminidase and beta-galactosidase, occurred between 18 and 21 h following release (S phase), declining to G1 phase levels during the peak of N-acetylgalactosamine (GalNAc) transferase activity. In addition, glycosyltransferase activity in G2 phase cells showed an increase in apparent Vmax (suggesting the presence of more enzyme/mg of cell protein) and apparent binding affinity for uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) (32 versus 14 microM) when compared to transferase activity in the G1 phase. However, the opioid peptide enkephalin [D-Ala2, D-Leu5], which inhibits ganglioside GalNAc transferase activity in unsynchronized NG108-15 cultures, was much more inhibitory in whole cells 8 h after release from the cell cycle block (G1 phase) than in cells 20 h after release (G2 phase), with 50% inhibition occurring at 2 X 10(-9) M and 2 X 10(-7) M, respectively. These results suggest that the GalNAc transferase activity is regulated in more than one way during the cell cycle, since both Vmax and Km changes are observed, and that the cyclic AMP-dependent mechanism by which opiates reduce transferase activity is receptor mediated and cell cycle dependent.

Preparation of Uridine Diphosphate- N -Acetylgalactosamine from Uridine Diphosphate- N -Acetylglucosamine by Using Microbial Enzymes

A method was developed for the large scale preparation of uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc) from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by means of microbial enzymes. With Bacillus subtilis cell-free extract as a source of UDP-GlcNAc 4-epimerase, about 35% of the UDP-GlcNAc added was converted to UDP-GalNAc. After the residual UDP-GlcNAc was degraded to uridine triphosphate and N-acetylglucosamine-1-phosphate with a protamine-treated extract of bakers' yeast as a source of UDP-GlcNAc pyrophosphorylase, UDP-GalNAc was separated by anion-exchange column chromatography. The nucleotide was recovered by adsorption on charcoal and elution with ammoniacal ethanol. The final yield was about 100 mumol.

The occurrence of uridine diphosphate N-acetylgalactosamine 6-sulfate in quail egg white and characteristic distribution of sulfated sugar nucleotides in different avian eggs

A sulfated sugar nucleotide has been isolated from quail egg white, and accounts for nearly 80% of the total sugar nucleotides found in the egg white. Evidence is presented that this nucleotide is uridine diphosphate N-acetyl-galactosamine 6-sulfate, an isomer of the 4-sulfated derivative of uridine diphosphate N-acetylgalactosamine previously found in chicken egg white. Further studies on the distribution of sulfated sugar nucleotides in egg white of various birds (chicken, quail, pheasant, peafowl, turkey, goose, and duck) demonstrate that each species has a characteristic composition, differing from one another regarding the relative amounts of 4-sulfated, 6-sulfated, and 4,6-bissulfated derivatives of uridine diphosphate N-acetylgalactosamine.

A common biochemical change in SV40 and polyoma virus transformed mouse cells coupled to control of cell growth in culture

In mouse cell lines after transformation with SV40 or polyoma virus there was shown to be a reduction in the content of higher ganglioside homologs and in the activity of an enzyme (uridine diphosphate N-acetylgalactosamine: hematoside 2 2 Abbreviations used: hematoside, G M3NAc or G M3NG, N-acetyl- or N-glycolyl- neuraminyl-galactosylglucosylceramide, respectively; G M2, N-acetylgalactosaminyl ( N-acetylneuraminyl) galactosylglucosylceramide; G M1, galactosyl- N-acetylgalactosaminyl ( N-acetylneuraminyl) galactosylglucosylceramide; G D1 a , N-acetylneuraminyl-galactosyl- N-acetylgalactosaminyl [ N-acetylneuraminyl] galactosylglucosylceramide; FUdR, 5-fluoro-2-deoxyuridine. N-acetylgalactosaminyltransferase) necessary for the synthesis of the higher gangliosides. These changes were found to be reversibly coupled to the changes in growth properties in tissue culture: in the flat derivatives of the virus transformed cell lines which reverted to normal phenotypic growth properties in culture there was a trend of reversion in ganglioside pattern and in enzymatic activity near to that of the parent 3T3 line, in spite of the continued presence of the viral DNA. Mouse cells productively infected with polyoma virus did not show decrease in the aminosugar transferase activity. Cocultivation of virally transformed cells with enzymatically active cells did not result in reduced enzyme activity in the enzymatically active cells, or in the restoration of enzyme activity in the virally transformed cells. Similarly, no change in enzyme activity occurred when admixing homogenates of the two type of cells. These biochemical findings are consistent with a similar repressor-like mechanism in both SV40 and polyoma virus induced cell transformation.

Biosynthesis of Chondroitin Sulfate: III. Formation of a Sulfated Glycosaminoglycan with a Microsomal Preparation from Chick Embryo Cartilage

Abstract A microsomal preparation from chick embryo epiphyseal cartilage previously has been shown to catalyze the formation of chondroitin and to catalyze the incorporation of sulfate-35S into endogenous microsomal chondroitin sulfate. This same preparation has now been shown to catalyze the new formation of chondroitin-14C sulfate. This formation requires the addition of 3'-phosphoadenosine 5'-phosphosulfate as well as uridine diphosphate N-acetylgalactosamine and uridine diphosphate glucuronic acid-14C to the reaction mixture. The final products, which appear to be a mixture of approximately 25% chondroitin sulfate and 75% chondroitin, remain bound to the microsomal preparation. It is suggested that the polysaccharide polymerization and sulfation to form chondroitin sulfate take place in close proximity in the cell.

Formation of chondroitin sulfate by a microsomal preparation from chick embryo epiphyseal cartilage

It has been shown that radioactively labeled glucuronic acid and N-acetylgalactosamine are incorporated into a glycosaminoglycan (mucopolysaccharide) when labeled uridine diphosphate glucuronic acid and uridine diphosphate N-acetylgalactosamine are incubated with a microsomal preparation from chick embryo epiphyseal cartilage ( Silbert, 1964; Perlman et al. , 1964 ). This glycosaminoglycan has been identified by sugar analysis as the nonsulfated compound chondroitin (Silbert, 1964). The same microsomal preparation has also been shown to contain particle-bound chondroitin sulfate, and to be able to catalyze the incorporation of labeled sulfate from PAPS into this particlebound chondroitin sulfate ( DeLuca and Silbert, 1968). This suggests that the synthesis of the polysaccharide chain of chondroitin and sulfation to form the complete chondroitin sulfate product might take place with the same microsomal preparation. The present report describes the use of this microsomal preparation from chick embryo epiphyseal cartilage to form chondroitin sulfate.

Enzymatic Transfer of Sulfate from 3‘-Phosphoadenosine 5‘-Phosphosulfate to Uridine Diphosphate N-Acetylgalactosamine 4-Sulfate

Abstract A new enzyme has been purified from a homogenate of the albumen-secreting region of hen oviduct. The enzyme catalyzes the transfer of sulfate from 3' -phosphoadenosine 5'-phosphosulfate to position 6 of the N-acetylgalactosamine moiety of uridine diphosphate N-acetylgalactosamine 4-sulfate.

The chemistry of insect haemolymph—IV. Acid-soluble phosphates

The acid-soluble phosphate compounds in the haemolymph plasma of several lepidopterous insects have been fractionated quantitatively by ion-exchange chromatography, and the principal components identified. In Hyalophora cecropia, total acid-soluble P ranged from 26 to 44 mM, and the main components were inorganic orthophosphate, α-glycerophosphate, phosphorylcholine, and phosphorylethanolamine. Present in smaller amounts were uridine diphosphate n-acetylgalactosamine and some other uridine diphosphate derivatives. The presence of a phosphagen was also indicated. Quantitatively, orthophosphate and the basic phosphates tended to be more abundant during diapause than in the active stages, while glycerophosphate changed in the reverse manner. Injected orthophosphate- 32P was incorporated into the various esters at all stages, though at reduced rates during diapause; but when cell-free haemolymph was incubated with 32P, incorporation was negligible. In Antheraea polyphemus, Samia cynthia, and Protoparce sexta, the distribution of phosphates was similar to that in H. cecropia. In Bombyx mori, however, there was little α-glycerophosphate whereas sorbitol-6-phosphate was abundant and glucose-6-phosphate was also found.