Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

Molecular cloning and expression of Galbeta1,3GalNAc alpha2, 3-sialyltransferase from human fetal liver.

Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding Galbeta1, 3GalNAc alpha2,3-sialyltransferase (SIATFL) has been isolated from human fetal liver. Expression analysis of the gene has been performed with various carcinoma cell lines, fetal tissues, fetal and adult liver and both hepatoma and the surrounding tissue from the same liver. The SIATFL gene was expressed poorly in fetal liver and in adult liver, slightly in hepatoma and highly in the surrounding tissue of hepatoma. The cDNA encoding the putative active domain was expressed in COS-1, Escherichia coli, and Pichia pastoris. The recombinant protein expressed in COS-1 could catalyse the transfer of NeuAc from CMP-NeuAc to asialo-fetuin. No enzyme activity was detected with a 32-kDa protein in E. coli and both 32-kDa and 41-kDa proteins in P. pastoris. These results suggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.     

Cloning and expression of human core 1 beta1,3-galactosyltransferase.

The common core 1 O-glycan structure Galbeta1--> 3GalNAc-R is the precursor for many extended mucin-type O-glycan structures in animal cell surface and secreted glycoproteins. Core 1 is synthesized by the transfer of Gal from UDP-Gal to GalNAcalpha1-R by core 1 beta3-galactosyltransferase (core 1 beta3-Gal-T). Amino acid sequences from purified rat core 1 beta3-Gal-T (Ju, T., Cummings, R. D., and Canfield, W. M. (2002) J. Biol. Chem. 277, 169-177) were used to identify the core 1 beta3-Gal-T sequences in the human expressed sequence tag data bases. A 1794-bp human core 1 beta3-Gal-T cDNA sequence was determined by sequencing the expressed sequence tag and performing 5'-rapid amplification of cDNA ends. The core 1 beta3-Gal-T predicts a 363-amino acid type II transmembrane protein. Expression of both the full-length and epitope-tagged soluble forms of the putative enzyme in human 293T cells generated core 1 beta3-Gal-T activity that transferred galactose from UDP-Gal to GalNAcalpha1-O-phenyl, and a synthetic glycopeptide with Thr-linked GalNAc and the product was shown to have the core 1 structure. Northern analysis demonstrated widespread expression of core 1 beta3-Gal-T in tissues with a predominance in kidney, heart, placenta, and liver. Highly homologous cDNAs were identified and cloned from rat, mouse, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that the enzyme is widely distributed in metazoans. The core 1 beta3-Gal-T sequence has minimal homology with conserved sequences found in previously described beta3-galactosyltransferases, suggesting this enzyme is only distantly related to the known beta3-galactosyltransferase family.     

GalNAc beta 1----3 terminated glycosphingolipids of human erythrocytes

Nonacid glycosphingolipids with 4 to 10 sugar residues isolated from pooled erythrocytes of blood group O donors have been efficiently separated as peracetylated derivatives on silicic acid. This procedure enabled a quantitative estimate of individual compounds and also revealed several GalNAc beta 1----3 terminated structures. The structural characterization of these glycolipids with 1H-NMR spectroscopy, direct inlet mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry identified the compounds as GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl sphingosine and GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl phytosphingosine, GalNAc beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 ceramide, and GalNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.     

Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme.

cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.     

Mucin biosynthesis revisited. The enzymatic transfer of Gal in beta 1,3 linkage to the GalNAc moiety of the core structure R1-GlcNAc beta 1,6GalNAc alpha-O-R2.

Synthetic glycosides containing the core, -Glc-NAc beta 1,6GalNAc alpha-, acted as acceptors for beta-galactosyltransferase of human ovarian tumor. A significant amount of Gal was transferred from UDP-Gal (100 nmol) to the alpha-benzylglycoside of LacNAc beta 1,6GalNAc (LGBn) (25.1 nmol of Gal) and the alpha-ortho-nitrophenylglycosides of LacNAc beta 1,6GalNAc (22.0 nmol of Gal), GlcNAc beta 1,6GalNAc (15.5 nmol of Gal), and Fuc alpha 1,3GlcNAc beta 1,6GalNAc (25.9 nmol of Gal); LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn (where Bn is benzyl) was almost inactive (only 1.2 nmol of Gal), indicating the Gal transfer to the alpha-GalNAc moiety. The product from LGBn was isolated in microgram quantities and identified by fast atom bombardment mass spectrometry as LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. The alpha GalNAc:beta 1,3Gal transferase was present in high concentration in ovarian tumor tissue (ovarian cancer serum----1.4; ascitic fluid----0.9; tumor----17.4). Asialo Cowper's gland mucin (ACGM) at 5 mg/ml reaction mixture inhibited the transfer of Gal to LGBn (25.2 and 53.4% respectively for 2 and 18 h incubation at 37 degrees C); inhibition by LGBn was 13.4 and 24.5%, respectively. In contrast to the inhibition by ACGM (25.2-31.6%), there was substantial increase (13.4-35.7%) in the inhibition by LGBn, when the incubation for 2 h at 37 degrees C was continued for 40 h at 4 degrees C, indicating the high affinity of LGBn for the enzyme at lower temp. Km for LGBn in presence of ACGM was 7.6 mM and in absence, 2.7 mM; Km for ACGM (M(r) 200,000) in presence of LGBn was 16.1 microM and Ki for ACGM (as the inhibitor) was 41.7 microM. In comparison with two normal ovarian tissues, the enzyme was found to be low (55-67%) in three ovarian tumors and high (146-260%) in two ovarian and one uterus tumors, as measured with ACGM; the synthetic acceptors showed similar activities. The enzyme had nearly the same extent of activity in the pH range 6-8. Fuc alpha 1,3GlcNAc beta 1,6GalNAc alpha-O-ONP had the highest affinity for the enzyme. The present study demonstrates the feasibility of beta 1,3Gal attachment on alpha GalNAc, which has already been substituted by beta 1,6GlcNAc, then elongated by beta 1,4Gal and also terminated by alpha 1,3Fuc.     

UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme.

All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability.     

Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme.

An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate. The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK). The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103810 Da). The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid. It had a molecular mass of 105 kDa and a pI value of 5.0. The maximum activity was observed at pH 8 and 55 degrees C in Tris-HCl buffer. The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum. The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx. 30% identity for both. High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes.     

Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme.

The pyruvate oxidase structural gene (poxB) of Escherichia coli was cloned into derivatives of plasmid pBR322. The gene was first cloned into a cosmid vector by selection for the tetracycline resistance determinant of a closely linked Tn10 insertion (no direct selection for the gene was available). Subsequent subcloning resulted in localization of the gene to a 3.1-kilobase-pair DNA segment. Two of the smaller poxB plasmids were shown to cause the overproduction of oxidase activity (by six- to eightfold), and one of these plasmids was shown to encode a protein having the size and antigenic determinants of pyruvate oxidase. Introduction of poxB plasmids into strains (aceEF) lacking pyruvate dehydrogenase activity relieved the aerobic growth requirement of the strains for exogenous acetate.     

Stable expression of a cDNA encoding a human beta 1 --> 3galactosyltransferase responsible for lacto-series type 1 core chain synthesis in non-expressing cells: variation in the nature of cell surface antigens expressed.

Transient expression of a human colonic adenocarcinoma Colo 205 cell derived cDNA in cell lines which ordinarily express only neolacto-series glycolipids has resulted in the expression of a beta 1 --> 3galactosyltransferase gene responsible for synthesis of glycolipids based upon the lacto-series type 1 core chain. Calcium phosphate transfected cells were panned on anti-IgM coated plates after initial treatment with a combination of monoclonal antibodies specific for type 1 chain terminal structures (TE-3) and a very broadly specific antibody reactive with multiple type 1 chain derivatives (TE-2). Adherent cells after panning were capable of efficiently transferring Gal in beta 1 --> 3-linkage to the acceptor glycolipid Lc3. Using these reagents, clones of stably transfected human colonic adenocarcinoma HCT-15 cells were produced and isolated. Parental HCT-15 cells do not express type 1 chain based antigens. The nature of the type 1 chain based antigens produced in each of these clones was analyzed by solid phase antibody binding assays. Three types of behavior were observed. Formation of type 1 terminal structures that were either exclusively sialylated or fucosylated, or a mixture of sialylated and fucosylated determinants occurred. In contrast, no difference in type 2 antigen expression between any clone and the parental cells was observed. These data suggest that coordination of subsequent reactions capable of modifying type 1 chain structures is not the same in all clones. The relationship of these results to aspects of cellular regulation of carbohydrate biosynthesis is discussed.