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Novel endo-alpha-N-acetylgalactosaminidases with broader substrate specificity.

Koutsioulis D, Landry D, Guthrie EP - Glycobiology (2008)

Bottom Line: EngEF exhibited the highest k(cat) for this substrate.EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAc beta 1,3GalNAc alpha 1pNP.Interestingly, there were no significant differences in transglycosylation activities when Gal beta 1,3GalNAc alpha 1pNP or GlcNAc beta 1,3GalNAc alpha 1pNP was incubated with various 1-alkanols in the presence of the endo-alpha-GalNAcases tested in this work.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc., Ipswich, MA 01938-2723, USA.

ABSTRACT
In an effort to identify novel endo-alpha-N-acetylgalactosaminidases (endo-alpha-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-alpha-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Gal beta 1,3GalNAc alpha 1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest k(cat) for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAc beta 1,3GalNAc alpha 1pNP. This is the first report of endo-alpha-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Gal beta 1,3GalNAc alpha 1pNP or GlcNAc beta 1,3GalNAc alpha 1pNP was incubated with various 1-alkanols in the presence of the endo-alpha-GalNAcases tested in this work.

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TLC analysis of the transglycosylation reactions. Transglycosylation of disaccharide Galβ1,3GalNAcα1pNP (A, B) or GlcNAcβ1,3GalNAcα1pNP (C) to various 1-alkanols by endo-α-GalNAcases. Lane 1, methanol; lane 2, ethanol; lane 3, 1-propanol; lane 4, 1-butanol; lane 5, 1-pentanol; lane 6, 1-hexanol; lane 7, 1-heptanol; lane 8, 1-octanol; lane 9, 1-nonalol; and lane S, Galβ1,3GalNAcα1pNP.
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Figure 4: TLC analysis of the transglycosylation reactions. Transglycosylation of disaccharide Galβ1,3GalNAcα1pNP (A, B) or GlcNAcβ1,3GalNAcα1pNP (C) to various 1-alkanols by endo-α-GalNAcases. Lane 1, methanol; lane 2, ethanol; lane 3, 1-propanol; lane 4, 1-butanol; lane 5, 1-pentanol; lane 6, 1-hexanol; lane 7, 1-heptanol; lane 8, 1-octanol; lane 9, 1-nonalol; and lane S, Galβ1,3GalNAcα1pNP.

Mentions: Several endoglycosidases have transglycosylation activity in addition to hydrolysis activity. To test for this activity, Galβ1,3GalNAcα1pNP was incubated with various 1-alkanols in the presence of EngCP, EngEF, EngPA, EngSP, and EngAL. Reaction products were analyzed on TLC plates (Figure 4). All of the enzymes tested exhibited similar transglycosylation activity. The longest 1-alkanol successfully incorporated in these transglycosylation reactions was 1-pentanol though the level of product was very low (Figure 4A and B). EngEF, EngPA, and EngSP were also tested for transglycosylation activity using the disaccharide GlcNAcβ1,3GalNAcα1pNP as the donor and 1-alkanols again as the acceptors. Since only EngEF and EngPA were capable of fully hydrolyzing GlcNAcβ1,3GalNAcα1pNP compared to the rest of the endo-α-GalNAcases which had very low activity on this substrate (Table II), it was surprising to find no significant difference in the amount of transglycosylation products produced by all three enzymes when observed on a TLC (Figure 4C).


Novel endo-alpha-N-acetylgalactosaminidases with broader substrate specificity.

Koutsioulis D, Landry D, Guthrie EP - Glycobiology (2008)

TLC analysis of the transglycosylation reactions. Transglycosylation of disaccharide Galβ1,3GalNAcα1pNP (A, B) or GlcNAcβ1,3GalNAcα1pNP (C) to various 1-alkanols by endo-α-GalNAcases. Lane 1, methanol; lane 2, ethanol; lane 3, 1-propanol; lane 4, 1-butanol; lane 5, 1-pentanol; lane 6, 1-hexanol; lane 7, 1-heptanol; lane 8, 1-octanol; lane 9, 1-nonalol; and lane S, Galβ1,3GalNAcα1pNP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553423&req=5

Figure 4: TLC analysis of the transglycosylation reactions. Transglycosylation of disaccharide Galβ1,3GalNAcα1pNP (A, B) or GlcNAcβ1,3GalNAcα1pNP (C) to various 1-alkanols by endo-α-GalNAcases. Lane 1, methanol; lane 2, ethanol; lane 3, 1-propanol; lane 4, 1-butanol; lane 5, 1-pentanol; lane 6, 1-hexanol; lane 7, 1-heptanol; lane 8, 1-octanol; lane 9, 1-nonalol; and lane S, Galβ1,3GalNAcα1pNP.
Mentions: Several endoglycosidases have transglycosylation activity in addition to hydrolysis activity. To test for this activity, Galβ1,3GalNAcα1pNP was incubated with various 1-alkanols in the presence of EngCP, EngEF, EngPA, EngSP, and EngAL. Reaction products were analyzed on TLC plates (Figure 4). All of the enzymes tested exhibited similar transglycosylation activity. The longest 1-alkanol successfully incorporated in these transglycosylation reactions was 1-pentanol though the level of product was very low (Figure 4A and B). EngEF, EngPA, and EngSP were also tested for transglycosylation activity using the disaccharide GlcNAcβ1,3GalNAcα1pNP as the donor and 1-alkanols again as the acceptors. Since only EngEF and EngPA were capable of fully hydrolyzing GlcNAcβ1,3GalNAcα1pNP compared to the rest of the endo-α-GalNAcases which had very low activity on this substrate (Table II), it was surprising to find no significant difference in the amount of transglycosylation products produced by all three enzymes when observed on a TLC (Figure 4C).

Bottom Line: EngEF exhibited the highest k(cat) for this substrate.EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAc beta 1,3GalNAc alpha 1pNP.Interestingly, there were no significant differences in transglycosylation activities when Gal beta 1,3GalNAc alpha 1pNP or GlcNAc beta 1,3GalNAc alpha 1pNP was incubated with various 1-alkanols in the presence of the endo-alpha-GalNAcases tested in this work.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc., Ipswich, MA 01938-2723, USA.

ABSTRACT
In an effort to identify novel endo-alpha-N-acetylgalactosaminidases (endo-alpha-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-alpha-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Gal beta 1,3GalNAc alpha 1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest k(cat) for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAc beta 1,3GalNAc alpha 1pNP. This is the first report of endo-alpha-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Gal beta 1,3GalNAc alpha 1pNP or GlcNAc beta 1,3GalNAc alpha 1pNP was incubated with various 1-alkanols in the presence of the endo-alpha-GalNAcases tested in this work.

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Related in: MedlinePlus