α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates

Joziasse, David H.; Shaper, Nancy L.; Salyer, Linda S.; Eijnden, Dirk H. van den; Spoel, Aarnoud C. van der; Shaper, Joël H.

doi:10.1111/j.1432-1033.1990.tb19095.x

Cited by 67 publications

(33 citation statements)

References 34 publications

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“…5=-Untranslated regions (5=UTRs), ORFs, 3=UTRs, and poly(A) tails present in a complete transcript of eukaryote DNA were found in the sequences of four ␤-glucanase genes (Table 1). This finding indicates that four cDNAs were transcribed from different ␤-glucanase loci and did not arise from alternative splicing of mRNA transcribed from one ␤-glucanase gene (19).…”

Section: Screening Of ␤-Glucanase Genesmentioning

confidence: 81%

Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

Hung

Chen

Liu

et al. 2012

Appl Environ Microbiol

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ABSTRACTFour types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designatedbglA13,bglA16,bglA51, andbglM2, were found in the cDNA library ofNeocallimastix patriciarumJ11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias toStreptococcus equinus. The presence of expansion and several predicted secondary structures in the 3′ untranslated regions (3′UTRs) ofbglA16andbglM2suggest that these two genes were duplicated recently, whereasbglA13andbglA16, which contain very short 3′UTRs, were replicated earlier. These findings indicate that the β-glucanase genes fromN. patriciarumJ11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes ofStreptococcus equinus,Ruminococcus albus7, andN. patriciarumJ11 were cloned and expressed byEscherichia coli. The recombinant β-glucanases cloned fromS. equinus,R. albus7, andN. patriciarumJ11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higherkcatvalues than others. These results support the notion that duplicated β-glucanase genes, namely,bglA16andbglM2, increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms inN. patriciarumJ11.

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Section: Screening Of ␤-Glucanase Genesmentioning

confidence: 81%

Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

Hung

Chen

Liu

et al. 2012

Appl Environ Microbiol

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“…Interestingly, it has been found previously that the Galfl(1M)GlcNAcfll-R ~(2-6)-sialyltransferase whether isolated from rat liver [10,21] or bovine colostrum [22] also can act on GalNAcfl(1-4)GlcNAcfll-R with high efficiency. In view of these results it will be of interest to investigate whether other Galfl(1M)GlcNAcfll-R specific glycosyltransferases such as a(2 3)-sialyltransferase [23], a3-galactosyltransferase [24] and fl3-N-acetylglucosaminyltransferase [25] are similarly capable of acting on GalNAcfl(1-4)GlcNAcfll-R.…”

Section: Discussionmentioning

confidence: 99%

Conversion of GalNAcβ(1–4)GlcNAcβ‐OMe into GalNAcβ(1–4)‐[Fucα(1–3)]GlcNAcβ‐OMe using human milk α3/4‐fucosyltransferase synthesis of a novel terminal element in glycoprotein glycans

et al. 1993

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Incubation of GalNAcβ(1–4)GlcNAcβ‐OMe with GDP‐Fuc in the presence of human milk α3/4‐fucosyltransferase resulted in the formation of GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe. Under conditions that led to complete α3‐fucosylation of Galβ(1–4)GlcNAcβ‐OEt, GalNAcβ(1–4)GlcNAcβ‐OMe was fucosylated for more than 85%. For the identification of the isolated fucosylated products one‐ and two‐ dimensional 1H‐NMR spectroscopy was applied. In vacuo molecular dynamics simulations of Galβ(1–4)[Fucα(1–3)]GlcNAcβ‐OEt and GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe using the CHARMm based force field CHEAT, demonstrated only small differences between the conformations of these compounds. This illustrates the minor conformational influence of the substituent at C‐2′, i.e. a hydroxyl function versus a N‐acetyl group.

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“…Recombinant forms of bovine and other ␣3GTs have been previously expressed (18,19); the cytosolic and transmembrane domains together with a 67-residue stem can be deleted to produce a fully active soluble enzyme (18). We have described the use of a bacterially expressed soluble form of the catalytic domain of bovine ␣3GT (residues 80 -367; ␣3GTcd) for the enzymatic production of substantial amounts of ␣-Gal-containing oligosaccharides (20).…”

mentioning

confidence: 99%

Specificity and Mechanism of Metal Ion Activation in UDP-galactose:β-Galactoside-α-1,3-galactosyltransferase

Zhang¹,

Wang²,

Brew³

2001

Journal of Biological Chemistry

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UDP-galactose:␤-galactosyl-␣1,3-galactosyltransferase (␣3GT) catalyzes the synthesis of galactosyl-␣-1,3-␤-galactosyl structures in mammalian glycoconjugates. In humans the gene for ␣3GT is inactivated, and its product, the ␣-Gal epitope, is the target of a large fraction of natural antibodies. ␣3GT is a member of a family of metal-dependent-retaining glycosyltransferases that includes the histo blood group A and B enzymes. Mn 2؉activates the catalytic domain of ␣3GT (␣3GTcd), but the affinity reported for this ion is very low relative to physiological levels. Enzyme activity over a wide range of metal ion concentrations indicates a dependence on Mn 2؉ binding to two sites. At physiological metal ion concentrations, Zn 2؉ gives higher levels of activity and may be the natural cofactor. To determine the role of the cation, metal activation was perturbed by substituting Co 2؉ and Zn 2؉ for Mn 2؉ and by mutagenesis of a conserved D 149 VD 151 sequence motif that is considered to act in cation binding in many glycosyltransferases. The aspartates of this motif were found to be essential for activity, and the kinetic properties of a Val 150 to Ala mutant with reduced activity were determined. The results indicate that the cofactor is involved in binding UDP-galactose and has a crucial influence on catalytic efficiency for galactose transfer and for the low endogenous UDP-galactose hydrolase activity. It may therefore interact with one or more phosphates of UDP-galactose in the Michaelis complex and in the transition state for cleavage of the UDP to galactose bond. The DXD motif conserved in many glycosyltransferases appears to have a key role in metal-mediated donor substrate binding and phosphate-sugar bond cleavage.a Golgi membrane-bound enzyme, catalyzes the synthesis of galactosyl ␣-1-3-galactosyl ␤-OR structures in glycoconjugates (Refs. 1 and 2; see Fig. 1). ␣3GTcd and the products of its action are found in most mammals but not in humans and their closest relatives, the old world monkeys and apes (3,4). In these species the gene for ␣3GT is mutationally inactivated (4, 5), and the absence of active enzyme allows the production of antibodies against the product of ␣3GT action, the ␣-Gal epitope (Fig. 1). Primates lacking this enzyme have natural antibodies (1-3% of circulating IgG, designated anti-Gal) that bind ␣-Gal (5) and facilitate immune defenses against pathogens but also present a barrier to the xenotransplantation of organs from mammalian species that produce active ␣3GT (6).Like other glycoprotein glycosyltransferases, ␣3GTs are type-2 membrane proteins with a short N-terminal cytosolic domain, a transmembrane helix, a stem, and a C-terminal catalytic domain (7-10). Only a few subgroups of glycosyltransferases that function in the processing of glycoproteins and glycolipids show global homology in primary structure. At present, a 2.4-Å structure of the catalytic domain of ␤-1,4-galactosyltransferase I is the only reported three-dimensional structure of an enzyme of this type (11). ␣1,3-GT is homologous...

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α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates

Cited by 67 publications

References 34 publications

Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

Conversion of GalNAcβ(1–4)GlcNAcβ‐OMe into GalNAcβ(1–4)‐[Fucα(1–3)]GlcNAcβ‐OMe using human milk α3/4‐fucosyltransferase synthesis of a novel terminal element in glycoprotein glycans

Specificity and Mechanism of Metal Ion Activation in UDP-galactose:β-Galactoside-α-1,3-galactosyltransferase

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