Purification and characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase specific for glycosylation of threonine residues.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from porcine submaxillary glands was purified to electrophoretic homogeneity. IgG prepared from antisera against the pure enzyme immunoprecipitated the transferase in Triton X-100 extracts of submaxillary glands. The submaxillary transferase is a membrane-bound enzyme in contrast to the pure bovine colostrum enzyme, which is soluble in the absence of detergents. Both transferases have similar properties but also differ significantly. Examination of the acceptor substrate specificity of the submaxillary gland transferase showed that it specifically transferred N-acetylgalactosamine from UDP-GalNAc to the hydroxyl group of threonine and was devoid of transferase activity toward serine-containing peptides. These results imply that more than one transferase is involved in forming the GalNAc-threonine and the GalNAc-serine linkages found in O-linked oligosaccharides in glycoproteins. The amino acid sequence adjacent to glycosylated threonine residues may influence the rate of glycosylation by the pure transferase. For example, the second threonine residue in the sequence, Thr-Thr, appears to be glycosylated about twice as fast as the first and more rapidly than single, isolated threonine residues. However, no unique consensus sequence for glycosylation of threonine residues is evident, and any accessible threonine residue appears to be a potential acceptor substrate.     

UDP-GalNAc : GalNAc-mucin α-N-acetylgalactosamine transferase activity in human intestinal cancerous tissues

GalNAc transferase activities of 6 human intestinal cancerous tissues were examined using bovine submaxillary gland mucin and its desialylated derivative, asialomucin, as acceptors. A Triton X-100 extract of these tissues was used as an enzyme source. All the tissues examined had GalNAc transferase that catalyzes the transfer of GalNAc from UDP-GalNAc to serine or threonine residues of the polypeptide chain. One of 6 specimens showed in addition UDP-GalNAc:GalNAc-mucin α-GalNAc transferase activity, synthesizing a disaccharide unit, GalNAcα→ GalNAc, when asialomucin was used as an acceptor. This carbohydrate structure was deduced on the basis of results of gel filtration, exoglycosidase digestion, and high-voltage paper electrophoresis.GalNAc transferaseHuman intestinal cancerous tissueBovine submaxillary gland mucin O-Glycosidically linked sugar chain     

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.     

Identification of the chick neural retina cell surface N-acetylgalactosaminyltransferase using monoclonal antibodies

Intact embryonic chick neural retina cells have at their surface an N-acetylgalactosaminyltransferase which catalyzes the incorporation of N-acetylgalactosamine from UDP-N-acetylgalactosamine into endogenous macromolecular acceptors. The enzyme along with its endogenous acceptors can be isolated as a particulate complex following treatment of membrane-enriched fractions with Triton X-100. In this paper we report on two separate fusions generating monoclonal antibodies: one using as immunogen the particulate complex and the second using as immunogen a soluble N-acetylgalactosaminyltransferase found in tissue-culture-conditioned medium which lacks endogenous acceptor activity. Antibodies from both fusions recognize an antigen which is tightly associated with the particulate transferase/acceptor complex and a soluble antigen having N-acetylgalactosaminyltransferase activity toward exogenously added acceptors. The antibodies recognize a component of ca Mr 220,000, which shows N-acetylgalactosaminyltransferase activity after SDS-gel electrophoresis and transfer to nitrocellulose. This component comigrates on two-dimensional gel electrophoresis with an iodinatable cell surface component whose presence at the cell surface correlates with endogenous transferase activity. We conclude that the antibodies recognize the transferase enzyme itself. Immunohistochemical analysis shows that the enzyme is initially localized throughout the embryonic neural retina in a pattern indicative of a cell surface disposition but becomes restricted to the outer plexiform layer and to outer segments in the adult.     

PARTICULATE AND SOLUBILIZED FUCOSYL TRANSFERASES FROM MOUSE BRAIN

The transfer of [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to endogenous and exogenous acceptors by particulate and solubilized preparations from mouse brain is described. Suspensions of brain microsomes incorporated [¹⁴C]fucose into a heterogenous group of glycoprotein products, which have a distribution on gel electrophoresis similar to those synthesized in vivo. Fucosyl transferase, extracted from brain microsomes by Triton X-100, transferred [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to terminal galactose residues exposed by mild acid hydrolysis of porcine plasma glycoprotein. Comparison of the specific activities of the solubilized fucosyl transferase from a number of organs showed that, in the presence of the exogenous acceptor which was used, the transferase of brain was more active than the transferases from all other organs tested, with the exception of kidney. Examination of subcellular fractions of brain, with endogenous and exogenous acceptors, showed that activity was limited to fractions containing microsomal membranes, whereas synaptosomal and other fractions were virtually inactive.     

Purification, properties and partial amino acid sequence of the blood-group-A-gene-associated alpha-3-N-acetylgalactosaminyltransferase from human gut mucosal tissue.

An alpha-3-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-N-acetylgalactosamine to H-active structures to form A determinants was purified to homogeneity from human gut mucosal tissue of blood-group-A subjects. The mucosa was homogenized, then treated with Triton X-100, and the solubilized enzyme was purified by affinity chromatography on UDP-hexanolamine-agarose and octyl-Sepharose CL-4B. Enzyme activity was recovered in 44% yield with a specific activity of approx. 7 mumol/min per mg. The only effective acceptor substrates for the transferase were those containing a subterminal beta-galactosyl residue substituted at the O-2 position with L-fucose. The purified enzyme had a weak capacity to transfer D-galactose from UDP-D-galactose to similar acceptors to make blood-group-B determinants. H.p.l.c. and SDS/PAGE analysis indicated an Mr of 40,000 for the purified enzyme. For the first time a partial amino acid sequence Xaa-Ser-Leu-Pro-Arg-Met-Val-Tyr-Pro-Gln-Ile-Ser?-Val-Leu was obtained for the N-terminal region of the soluble alpha-3-N-acetylgalactosaminyltransferase.     

Purification and properties of two enzymes catalyzing galactose transfer to GM2 ganglioside from rat liver Golgi

An enzyme activity which catalyzed the transfer of galactose from UDP-galactose to GM2 ganglioside was demonstrated in rat liver homogenate and enriched 38-fold in specific activity by preparation of Golgi membranes. This activity could be solubilized from Golgi membranes by sonication and extraction with 1% Triton X-100. The solubilized activity catalyzed the formation of GM1 ganglioside and was completely dependent upon the addition of acceptor. The rate of galactose incorporation was constant for up to 5 h at 30 degrees C. This enzyme activity was further purified by gel filtration on Sepharose CL-6B and ion exchange chromatography on DEAE-Sepharose. The elution position on gel filtration corresponded to a molecular weight for the enzyme of 38,000 which was in good agreement with that obtained by sedimentation velocity studies. Ion exchange chromatography resolved GM2 ganglioside galactosyltransferase into two species. The more basic enzyme (I) comprising 28% of the recovered activity was not retarded by the column, whereas enzyme II was eluted from the resin following the application of a salt gradient. Net purification was 120- to 140-fold for each enzyme with a total recovery of 42%. Unlike the activity in the Golgi extract, the purified enzymes I and II were labile to freezing and could be stored at -20 degrees C only in the presence of 50% glycerol. Both enzymes I and II had similar molecular weights and Michaelis constants and both had a strict requirement for Mn2+. Properties which distinguish the two enzymes included pH optima (enzyme I 7.0, enzyme II 6.0) and surfactant requirements. Neither enzyme was active following removal of Triton X-100 from the preparation. Among a series of glycolipids tested for ability to serve as substrates for galactose transfer only GM2 and asialo-GM2 ganglioside served as acceptors.     

Solubilization, purification, and characterization of succinate dehydrogenase from membranes of Mycobacterium phlei.

Succinate dehydrogenase (SDH) was solubilized from membranes of Mycobacterium phlei by Triton X-100 with a recovery of about 90%. The solubilized SDH was purified about 90-fold by Sephacryl S-300, DEAE-cellulose, hydroxylapatite, and isoelectric focusing in the presence of Triton X-100 with a 20% recovery. SDH was homogeneous, as determined by polyacrylamide gel electrophoresis in nondenaturing gels containing Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme revealed two subunits with molecular weights of 62,000 and 26,000. SDH is a flavoprotein containing 1 mol of flavin adenine dinucleotide, 7 to 8 mol of nonheme iron, and 7 to 8 mol of acid-labile sulfide per mol of protein. Using phenazine methosulfate and 2,6-dichloroindophenol as electron acceptors, the enzyme had an apparent Km of 0.12 mM succinate. SDH exhibited a sigmoidal relationship of rate to succinate concentration, indicating cooperativity. The enzyme was competitively inhibited by fumarate with a Ki of 0.15 mM. In the absence of Triton X-100, the enzyme aggregated, retained 50% of the activity, and could be resolubilized with Triton X-100 with full restoration of activity. Cardiolipin had no effect on the enzyme activity in the absence of Triton X-100, but it stimulated the activity by about 30% in the presence of 0.1% Triton X-100 in the assay mixture. Menaquinone-9(2H), isolated from M. phlei, had no effect on the enzyme activity either in the presence or absence of Triton X-100.     

The influence of flanking sequence on the O-glycosylation of threonine in vitro.

To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist.     

Biosynthesis of wall teichoic acids in Staphylococcus aureus H, Micrococcus varians and Bacillus subtilis W23. Involvement of lipid intermediates containing the disaccharide N-acetylmannosaminyl N-acetylglucosamine

The precursors for linkage unit (LU) synthesis in Staphylococcus aureus H were UDP-GlcNAc, UDP-N-acetylmannosamine (ManNAc) and CDP-glycerol and synthesis was stimulated by ATP. Moraprenol-PP-GlcNAc-ManNAc-(glycerol phosphate)1-3 was formed from chemically synthesised moraprenol-PP-GlcNAc, UDP-ManNAc and CDP-glycerol in the presence of Triton X-100. LU intermediates formed under both conditions served as acceptors for ribitol phosphate residues, from CDP-ribitol, which comprise the main chain. The initial transfer of GlcNAc-1-phosphate from UDP-GlcNAc was very sensitive to tunicamycin whereas the subsequent transfer of ManNAc from UDP-ManNAc was not. Poly(GlcNAc-1-phosphate) and LU synthesis in Micrococcus varians, with endogenous lipid acceptor, UDP-GlcNAc and CDP-glycerol, was stimulated by UDP-ManNAc. Synthesis of LU on exogenous moraprenol-PP-GlcNAc, with Triton X-100, was dependent on UDP-ManNAc and CDP-glycerol and the intermediates formed served as substrates for polymer synthesis. Membranes from Bacillus subtilis W23 had much lower levels of LU synthesis, but UDP-ManNAc was again required for optimal synthesis in the presence of UDP-GlcNAc and CDP-glycerol. Conditions for LU synthesis on exogenous moraprenol-PP-GlcNAc were not found in this organism. LU synthesis on endogenous acceptor in the absence of UDP-ManNAc was explained by contamination of membranes with UDP-GlcNAc 2-epimerase. Under appropriate conditions, low levels of this enzyme were sufficient to convert UDP-GlcNAc into a mixture of UDP-Glc-NAc and UDP-ManNAc and account for LU synthesis. The results indicate the formation of prenol-PP-GlcNAc-ManNAc-(glycerol phosphate)1-3 which is involved in the synthesis of wall teichoic acids in S. aureus H, M. varians and B. subtilis W23 and their attachment to peptidoglycan.     

Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1

All UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases cloned to date contain a lectin domain at the C-terminus, consisting of three tandem repeat sequences (alpha,beta, and gamma). We previously reported that the alpha repeat of one of the most ubiquitous isozymes, GalNAc-T1, is a functional lectin that recognizes O-linked GalNAc residues on the acceptor polypeptides with multiple acceptor sites; the domain appears not to be involved in the glycosylation of acceptors with a single acceptor site. In this report, we studied the function of the beta and gamma repeats in the GalNAc-T1 lectin domain, by site-directed mutagenesis and analysis of the catalytic properties of mutant enzymes. We found that the beta repeat recognizes GalNAc and is involved in glycosylation of acceptors with multiple glycosylation sites. The gamma repeat, on the other hand, showed no significant GalNAc-binding activity. These results indicate that the lectin domain of GalNAc-T1 has at least two functional repeats, allowing the possibility of multivalent interactions with GalNAc residues on the acceptor polypeptide during glycosylation.     

Characterization of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

The phosphorylation of two purified vaccinia virus proteins (Acceptors I and II) by a protein kinase isolated from vaccinia virus cores has been studied. Phosphorylation of viral acceptor proteins by the purified enzyme was dependent on the presence of ATP, Mg2+, and protamine or other basic proteins, and was maximal at alkaline pH values. Cyclic mononucleotides did not stimulate the vaccinia protein kinase under a variety of conditions. Protamine, however, was shown to function as an enzyme activator. In its presence, the purified vaccinia protein kinase phosphorylated mainly serine residues in Acceptor I, and predominantly threonine residues in Acceptor II. Phosphorylation of protamine accounted for less than 1% of the total 23P incorporation. Tryptic peptide maps prepared from 32P-labeled Acceptors I and II demonstrated that they contained different labeled peptide sequences and were, therefore, distinct protein species. From additional studies on both purified and virus-associated protein kinase it was concluded that various proteins affected the protein kinase reaction in one of three ways. One class of proteins served as phosphate acceptors, but only when another activator protein was present. A second class consisted of proteins that were strong activators but poor phosphate acceptors. The third class contained proteins that were fair phosphate acceptors, but which also activated the phosphorylation of other acceptor proteins.     

Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.     

In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain

MEKK2 and MEKK3 are mitogen-activated protein kinase kinase kinases (MAP3 kinases) of 70 and 71 kDa respectively that are markedly homologous (94%) in their kinase domains. Both MEKK2 and MEKK3 are able to activate the Jun kinase pathway in vivo. However, following routine immunoprecipitation in Triton X-100, MEKK2 but not MEKK3 is able to effectively phosphorylate both SEK-1 and MEK-1 and to undergo autophosphorylation. Unexpectedly, both MEKK2 and MEKK3 are functional in an in vitro kinase assay when cells are solubilized with the closely related detergent, NP-40. Given the high homology between these kinases, we set out to relate this differential sensitivity to Triton X-100 to differences in primary structure. A set of chimeric molecules were generated and the loss of activity in Triton X-100 mapped to kinase domain II/III and specifically to serine 390 of MEKK3 and valine 384 of MEKK2, residues immediately N-terminal to the active site lysine. Mutation of serine 390 of MEKK3 to a valine (as is found in MEKK2) conferred catalytic activity to MEKK3 in Triton X-100 whereas the reciprocal alteration of valine 384 of MEKK2 to a serine conferred lack of activity in Triton X-100 to MEKK2. Search of the protein database identified only three kinases, MEKK3, Pbs2p and Dd-PKI, with a serine or threonine at this site. The presence of a serine or threonine adjacent to the active site lysine in protein kinases is rare and, in MEKK3, results in detergent instability.     

Purification and Characterization of UDP-N-Acetylgalactosamine: κ-Casein Polypeptide N-Acetylgalactosaminyltransferase from Mammary Gland of Lactating Cow

UDP-N-acetyl-d-galactosamine: κ-casein polypeptide N-acetylgalactosaminyltransferase was purified from a crude Golgi apparatus of lactating bovine mammary gland after solubilization with Triton X-100. Through chromatography on DEAE-Sephadex A-50, apomucin-Sepharose 4B, FPLC mono S, and Sephacryl S-200, and then electrofocusing, the enzyme was purified up to 7500-fold from the homogenate.

The molecular weight of the enzyme was estimated at 200,000 from gel filtration. The pI value of the enzyme was 6.4 on electrofocusing. The purified enzyme transferred GalNAc from UDP-GalNAc, not to the carbohydrate chains but to the polypeptide chains of the substrates, κ-casein and mucin. The enzyme required Mn²⁺, DTT, and Triton X-100 for maximal activity. The Km value for UDP-GalNAc was 16.2μm. Km values for K-subcomponents 1 and 7, and apomucin were 1.15, 5.10, and 0.192mg/ml, and V_max values were 254, 259, and 581 nmol/hr/mg, respectively. Thermal stability and the effects of pH, milk components, lectins, and nucleotides were examined.

α_s1-Casein strongly inhibited GalNAc transfer to κ-casein. The inhibitory effect of α_s1-casein was canceled by the addition of Ca²⁺, which causes casein micelle formation. This means that the glycosylation of κ-casein occurs after casein micelle formation triggered by the accumulation of Ca²⁺ in vivo.     

Characterization and partial purification of beta-N-acetylgalactosaminyltransferase from urine of Sd(a+) individuals

Urine from Sd(a+) individuals was found to contain a beta-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to 3'-sialyllactose and glycoproteins carrying the terminal NeuAc alpha-3Gal beta group. This enzyme has been purified 174-fold by affinity chromatography on Blue Sepharose and DEAE-Sephacel chromatography in a yield of 33%. Neither endogenous incorporation nor sugar nucleotide degrading enzymes were found in the purified preparation. The transferase had a pH optimum of pH 7.5 and a requirement for Mn2+ but not for detergents. The Km for UDP-GalNAc was 66 X 10(-6) M, using fetuin as an acceptor. Like beta-GalNAc-transferase from other sources the urinary enzyme had a strict requirement for sialylated acceptors. On the basis of enzymatic and chemical treatment of the product obtained by the transfer of [3H]GalNAc to 3'-sialyllactose, we propose that the enzyme attaches GalNAc in beta-anomeric configuration to O-4 of the galactose residue that is substituted at O-3 by sialic acid. A preparation of Tamm-Horsfall glycoprotein from a Sd(a-) donor lacking beta-GalNAc was found to be the best acceptor among the glycoproteins tested. Studies on the transferase activity toward fetuin, human chorionic gonadotropin, and glycophorin A indicated that the enzyme preferentially adds the sugar to the sialylated terminal end of N-linked oligosaccharides. Unlike the beta-GalNAc-transferase bound to human kidney microsomes (F. Piller et al. (1986) Carbohydr. Res. 149, 171-184) the urinary transferase is able to transfer beta-GalNAc to the NeuAc alpha-3Gal beta-3(NeuAc alpha-6)GalNAc chains bound to the native glycophorin.     

Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 and T2 derived from oriented random peptide substrates

A large family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAc Ts) catalyzes the first step of mucin-type protein O-glycosylation by transferring GalNAc to serine and threonine residues of acceptor polypeptides. The acceptor peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly characterized and poorly understood, despite the fact that mutations in two individual isoforms are deleterious to man and the fly. In this work a series of oriented random peptide substrate libraries, based on the GAGAXXXTXXXAGAGK sequence motif (where X = randomized positions), have been used to obtain the first comprehensive determination of the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 isoforms. ppGalNAc T-glycosylated random peptides were isolated by lectin affinity chromatography, and transferase amino acid preferences were determined by Edman amino acid sequencing. The results reveal common and unique position-sensitive features for both transferases, consistent with previous reports of the preferences of ppGalNAc T1 and T2. The random peptide substrates also reveal additional specific features that have never been described before that are consistent with the x-ray crystal structures of the two transferases and furthermore are reflected in a data base analysis of in vivo O-glycosylation sites. By using the transferase-specific preferences, optimum and selective acceptor peptide substrates have been generated for each transferase. This approach represents a relatively complete, facile, and reproducible method for obtaining ppGalNAc T peptide substrate specificity. Such information will be invaluable for identifying isoform-specific peptide acceptors, creating isoform-specific substrates, and predicting O-glycosylation sites.     

Some biochemical properties of human breast tumor sialyltransferase

Sialyltransferase activity in normal human breast tissue and tumors was investigated with lactose, desialylated fetuin, and bovine submaxillary mucin as the acceptors. While microsomal preparations from the normal tissue showed little or no sialyltransferase activity toward these acceptors, tumors showed elevated enzymic activities. Tween-20 at 0.5% concentrations stimulated sialic acid transfer to all three acceptors. Another nonionic detergent, Triton X-100, stimulated asialo fetuin sialyltransferase activity while inhibiting activity toward asialo BSM and lactose. Interestingly, lysolecithin, a normal cellular constituent which possesses detergent properties also had an effect similar to that of Triton X-100. Thermal denaturation curves of enzymic activity toward asialo BSM, however, resembled those seen with asialo fetuin as the acceptor. Kinetic studies showed that at acceptor concentrations of 500 micrograms each, sialyl transfers to asialo fetuin, asialo BSM, and lactose showed apparent Km values of 50, 60, and 300 microM, respectively. At CMP-sialic acid concentrations of 300 microM, the Km values for the above acceptors were 25, 15, and 5000 microM.     

Occurrence of ribitol-containing lipoteichoic acid in Staphylococcus aureus H and its glycosylation

A ribitol-containing lipoteichoic acid was obtained from the 20,000 x g supernatant fraction of Staphylococcus aureus H by extraction with Triton X-100 followed by fractionation on Sepharose 6B and DEAE-cellulose columns. The purified lipoteichoic acid was composed of phosphate, glycerol, glucose, glucosamine, ribitol, and fatty acids in a molar ratio of 1 : 0.9 : 0.06 : 0.03 : 0.09 : 0.07. Based on the structural analysis of fragments from alkali and HF hydrolysis, the lipoteichoic acid appears to consist of three moieties, namely a ribitol phosphate oligomer, poly(glycerol phosphate) which has about 30 glycerol phosphate units, and beta-glucosyl-beta-glucosyl(1 leads to 1)diacylglycerol. N-Acetylglucosamine was linked to the ribitol residues. The lipoteichoic acid serves as an acceptor of glycosyl moieties from UDP-glucose and UDP-N-acetylglucosamine in the enzyme reaction catalyzed by the membrane preparation. The rate of enzymatic glycosylation was increased by prior treatment of the lipoteichoic acid with N-acetyl-beta-D-glucosaminidase. The glycosylation seems to occur at the ribitol residues of the lipoteichoic acid.     

Collagen glucosyl- and galactosyltransferases of cultured human fetal lung fibroblasts

Collagen-galactosyltransferase and collagen-glucosyltransferase activities have been studied in cultured human fetal lung WI-38 and IMR-90 diploid fibroblasts. These enzymes functioned in concert to synthesize glucosylgalactosylhydroxylysine units as found naturally in collagens, basement membranes, and certain serum glycoproteins. The transferases used UDP-Gal and UDP-Glc as glycose donors, collagens and collagen-derived peptides or glycopeptides as glycose acceptors, and worked best in the presence of manganese as a required divalent cation. Two pH optima, between pH 6 and 6.5 and between pH 7.5 and 8, were noted for each type of transferase, and these optima, particularly in the case of glucosyltransferase, were evident regardless of size of acceptor employed in the assay. About 35% of the total activity of each enzyme was found in the soluble fractions of cell homogenates, and, of the particulate fraction activities, about 50% could be released by mild sonication or by treatment with Triton X-100. Assessment of transferase activities as a function of cellular aging in culture revealed that significant decreases in enzyme levels occurred as the cell approached senescence (late Phase II), and these effects were reversed when cells attained senescence (Phase III). Addition of ascorbic acid to young cultures, under conditions known to increase endogenous collagenpeptide hydroxylation, caused no effects on the activities of the glycosyltransferases toward exogenous acceptors. These results suggested that the activities of collagen-hydroxylases and glycosyltransferase might not be coordinately regulated, and that, regardless of the hydroxylation events, glycosylation of the peptide might be limited to a specific fraction of hydroxylysine residues during the post-translational modification of collagen.