Galactosyltransferases: Physical, Chemical, and Biological Aspect

Ram, B. P.; Munjal, D.

doi:10.3109/10409238509113606

Cited by 49 publications

(17 citation statements)

References 283 publications

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“…For example, manganese needs to be delivered to the Golgi apparatus where the metal activates a variety of sugar transferases involved in protein processing (43)(44)(45)(46). In yeast, a useful marker for Golgi manganese is invertase, an enzyme that is modified by N-glycosylation via manganese-dependent mannosyltransferases (45)(46)(47)(48).…”

Section: Resultsmentioning

confidence: 99%

Manganese Superoxide Dismutase in Saccharomyces cerevisiae Acquires Its Metal Co-factor through a Pathway Involving the Nramp Metal Transporter, Smf2p

Luk¹,

Culotta²

2001

Journal of Biological Chemistry

134

166

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Eukaryotes express both copper/zinc (SOD1)-and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage. Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2. We demonstrate here that in Saccharomyces cerevisiae cells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p. SOD2 activity is greatly diminished in smf2⌬ mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria. Treating smf2⌬ cells with manganese supplements corrected the SOD2 defect, as did elevating intracellular manganese through mutations in PMR1. Hence, manganese appears to be inaccessible to mitochondrial SOD2 in smf2 mutants. Cells lacking SMF2 also exhibited defects in manganese-dependent steps in protein glycosylation and showed an overall decrease in steady-state levels of accumulated manganese. By comparison, mutations in the cell surface Nramp transporter, Smf1p, had very little impact on manganese accumulation and trafficking. Smf2p resides in intracellular vesicles and shows no evidence of plasma membrane localization, even in an end4 mutant blocked for endocytosis. We propose a model in which Smf2p-containing vesicles play a central role in manganese trafficking to the mitochondria and other cellular sites as well. Superoxide dismutases (SOD)1 are critical anti-oxidant defense enzymes that catalyze the disproportionation of superoxide anion to oxygen and hydrogen peroxide. Eukaryotic cells possess two evolutionary distinct forms of SOD: a copper-and zinc-containing SOD (SOD1) found mainly in the cytosol (1) and a manganese-containing SOD (SOD2) that localizes strictly to the mitochondrial matrix (2). Because both forms of SOD require a transition metal co-factor, enzyme activity in cells may be controlled through availability of the cognate metal ion. Indeed this is the case for the copper-requiring SOD1, where the enzyme relies on components of a defined copper trafficking pathway for acquisition of its metal ion (for reviews see Refs. 3-6). However, nothing is known regarding the mechanism by which SOD2 receives its manganese cofactor in vivo.Encoded in the nucleus, SOD2 is predicted to acquire its manganese after import into the mitochondrial matrix. The manganese-binding residues of SOD2 occur at distant regions of the polypeptide (7), and the metal binding site is not likely to remain intact during the protein-unfolding process needed for mitochondrial membrane translocation. Therefore, an intricate manganese trafficking pathway must warrant the accurate delivery of the metal from the cell surface to the mitochondria. To date, none of the factors that participate in this pathway have been identified.The bakers' yeast Saccharomyces cerevisiae represents an ideal eukaryotic system in which to unravel metal trafficking pathways. This organism has been extremely powerful for elucidating the copper acquisition pathw...

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Section: Resultsmentioning

confidence: 99%

Manganese Superoxide Dismutase in Saccharomyces cerevisiae Acquires Its Metal Co-factor through a Pathway Involving the Nramp Metal Transporter, Smf2p

Luk¹,

Culotta²

2001

Journal of Biological Chemistry

134

166

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“…The reaction, catalyzed by β-1,4-galactosyltransferase (EC 2.4.1.22), is the transfer of galactose from UDP-α-Dgalactose to terminal N-acetylglucosamine in glycoproteins and glycolipids with β-linkage (Ram and Munjal, 1985). The cDNAs that encode β-1,4-galactosyltransferase have been isolated from human (Appert et al, 1986;Masri et al, 1988), bovine (Narimatsu et al, 1986;D'Agostaro et al 1989), and murine sources (Nakazawa et al, 1988;Shaper et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Expression and Characterization of β-1,4-Galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae

Park¹,

Lee²,

Il³

et al. 2002

BMB Reports

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The lgtB genes that encode β-1,4-galactosyltransferases from Neisseria meningitidis ATCC 13102 and gonorrhoeae ATCC 31151 were isolated by a polymerase chain reaction using the pfu DNA polymerase. They were expressed under the control of lac and T7 promoters in Escherichia coli M15 and BL21 (DE3). Although the genes were efficiently expressed in E. coli M15 at 37 o C (33 kDa), most of the β-1,4-galactosyltransferases that were produced were insoluble and proteolysed into enzymatically inactive polypeptides that lacked C-terminal residues (29.5 kDa and 28 kDa) during the purification steps. When the temperature of the cell growth was lowered to 25 o C, however, the solubility of the β-1,4-galactosyltransferases increased substantially. A stable N-terminal his-tagged recombinant enzyme preparation could be achieved with E. coli BL21 (DE3) that expressed lgtB. Therefore, the cloned β-1,4-galactosyltransferases were expressed under the control of the T7 promoter in E. coli BL21 (DE3), mostly to the soluble form at 25 o C. The proteins were easily purified to homogeneity by column chromatography using Ni-NTA resin, and were found to be active. The galactosyltransferases exhibited pH optimum at 6.5-7.0, and had an essential requirement for the Mn +2 ions for its action. The Mg +2 and Ca +2 ions showed about half of the galactosyltransferase activities with the Mn +2 ion. In the presence of the Fe +2 ion, partial activation was observed with the β-1,4-galactosyltransferase from N. meningitidis (64% of the enzyme activity with the Mn +2 ion), but not from N. gonorrhoeae. ions, the Mn +2 ion could not activate the enzyme activities. Also, the β-1,4-galactosyltransferase activity was 1.5-fold stimulated with the non-ionic detergent Triton X-100 (0.1-5%).

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“…), and in the synthesis of cell wall polysaccharides in prokaryotes. This enzyme catalyzes the transfer of galactose from UDP-galactose to glycoproteins or glycolipids containing β-linkage (Ram and Munjal, 1985;Paulson and Colley, 1989). cDNAs encoding galactosyltransferase genes have been isolated from human (Appert et al, 1986;Marsi et al, 1988), bovine (Narimatsu et al, 1986), and murine sources (Shaper et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

A Mutagenic Study of β-1,4-Galactosyltransferases from Neisseria meningitidis

Park

Do²,

Lee³

et al. 2004

BMB Reports

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N-terminal His-tagged recombinant β-1,4-galactosyltransferase from Neisseria meningitidis was expressed and purified to homogeneity by column chromatography using Ni-NTA resin. Mutations were introduced to investigate the roles of, Ser68, His69, Glu88, Asp90, and Tyr156, which are components of a highly conserved region in recombinant β-1,4 galactosyltransferase. Also, the functions of three other cysteine residues, Cys65, Cys139, and Cys205, were investigated using site-directed mutagenesis to determine the location of the disulfide bond and the role of the sulfhydryl groups. Purified mutant galactosyltransferases, His69Phe, Glu88Gln and Asp90Asn completely shut down wild-type galactosyltransferase activity (1-3%). Also, Ser68Ala showed much lower activity than wild-type galactosyltransferase (19%). However, only the substitution of Tyr156Phe resulted in a slight reduction in galactosyltransferase activity (90%). The enzyme was found to remain active when the cysteine residues at positions 139 and 205 were replaced separately with serine. However, enzyme reactivity was found to be markedly reduced when Cys65 was replaced with serine (27%). These results indicate that conserved amino acids such as Cys65, Ser68, His69, Glu88, and Asp90 may be involved in the binding of substrates or in the catalysis of the galactosyltransferase reaction.

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Galactosyltransferases: Physical, Chemical, and Biological Aspect

Cited by 49 publications

References 283 publications

Manganese Superoxide Dismutase in Saccharomyces cerevisiae Acquires Its Metal Co-factor through a Pathway Involving the Nramp Metal Transporter, Smf2p

Manganese Superoxide Dismutase in Saccharomyces cerevisiae Acquires Its Metal Co-factor through a Pathway Involving the Nramp Metal Transporter, Smf2p

Expression and Characterization of β-1,4-Galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae

A Mutagenic Study of β-1,4-Galactosyltransferases from Neisseria meningitidis

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