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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 reaction products using pNP substrates and natural glycoproteins. Reaction mixtures were incubated with (lane +) and without (lane −) EngEF at 25°C for 16 h. Lane 1, Core 2 trisaccharide Galβ1,3(GlcNAcβ1,6)GalNAcα1pNP; lane 2, Core 3 disaccharide GlcNAcβ1,3GalNAcα1pNP; lane 3; Galβ1,3GlcNAcα1pNP disaccharide; lane 4, GalNAcα1pNP monosaccharide; lane 5, Core 1 disaccharide Galβ1,3GalNAcα1pNP; lane 6, Fetuin; lane 7, Fetuin + Neuraminidase; lane 8, Asialofetuin; lane 9, κ-casein glycopeptides; lane 10, κ-casein glycopeptides + Neuraminidase; lane 11, Glycophorin A; lane 12, Glycophorin A + Neuraminidase; lane 13, Mucin glycopeptides; and lane 14, Mucin glycopeptides + Neuraminidase.
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Figure 3: TLC analysis of the reaction products using pNP substrates and natural glycoproteins. Reaction mixtures were incubated with (lane +) and without (lane −) EngEF at 25°C for 16 h. Lane 1, Core 2 trisaccharide Galβ1,3(GlcNAcβ1,6)GalNAcα1pNP; lane 2, Core 3 disaccharide GlcNAcβ1,3GalNAcα1pNP; lane 3; Galβ1,3GlcNAcα1pNP disaccharide; lane 4, GalNAcα1pNP monosaccharide; lane 5, Core 1 disaccharide Galβ1,3GalNAcα1pNP; lane 6, Fetuin; lane 7, Fetuin + Neuraminidase; lane 8, Asialofetuin; lane 9, κ-casein glycopeptides; lane 10, κ-casein glycopeptides + Neuraminidase; lane 11, Glycophorin A; lane 12, Glycophorin A + Neuraminidase; lane 13, Mucin glycopeptides; and lane 14, Mucin glycopeptides + Neuraminidase.

Mentions: The substrate specificities of the purified enzymes EngCP, EngEF, and EngPA and the commercially available EngSP and EngAL were determined. Each enzyme was incubated with various synthetic substrates. The released sugars were detected by colorimetric assay and thin layer chromatography (TLC) analysis (Table II, Figure 3). Galβ1,3GalNAcα1pNP was the most rapidly hydrolyzed substrate by all the enzymes tested. After 16 h of incubation, only EngEF and EngPA were capable of fully hydrolyzing the Core 3 disaccharide (GlcNAcβ1, 3GalNAcα1pNP). EngAL could only partially hydrolyze the Core 3 disaccharide (27%) after 16 h of incubation (Table II). EngAL could also partially release GalNAc while the rest of the enzymes released only traces of the monosaccharide (Table II). None of the enzymes could act on Galβ1,3GlcNAcα1pNP and low or no activity was detected when Core 2 trisaccharide (Galβ1,3(GlcNAcβ1,6)GalNAcα1pNP) was used as a substrate (Table II).


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

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

TLC analysis of the reaction products using pNP substrates and natural glycoproteins. Reaction mixtures were incubated with (lane +) and without (lane −) EngEF at 25°C for 16 h. Lane 1, Core 2 trisaccharide Galβ1,3(GlcNAcβ1,6)GalNAcα1pNP; lane 2, Core 3 disaccharide GlcNAcβ1,3GalNAcα1pNP; lane 3; Galβ1,3GlcNAcα1pNP disaccharide; lane 4, GalNAcα1pNP monosaccharide; lane 5, Core 1 disaccharide Galβ1,3GalNAcα1pNP; lane 6, Fetuin; lane 7, Fetuin + Neuraminidase; lane 8, Asialofetuin; lane 9, κ-casein glycopeptides; lane 10, κ-casein glycopeptides + Neuraminidase; lane 11, Glycophorin A; lane 12, Glycophorin A + Neuraminidase; lane 13, Mucin glycopeptides; and lane 14, Mucin glycopeptides + Neuraminidase.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553423&req=5

Figure 3: TLC analysis of the reaction products using pNP substrates and natural glycoproteins. Reaction mixtures were incubated with (lane +) and without (lane −) EngEF at 25°C for 16 h. Lane 1, Core 2 trisaccharide Galβ1,3(GlcNAcβ1,6)GalNAcα1pNP; lane 2, Core 3 disaccharide GlcNAcβ1,3GalNAcα1pNP; lane 3; Galβ1,3GlcNAcα1pNP disaccharide; lane 4, GalNAcα1pNP monosaccharide; lane 5, Core 1 disaccharide Galβ1,3GalNAcα1pNP; lane 6, Fetuin; lane 7, Fetuin + Neuraminidase; lane 8, Asialofetuin; lane 9, κ-casein glycopeptides; lane 10, κ-casein glycopeptides + Neuraminidase; lane 11, Glycophorin A; lane 12, Glycophorin A + Neuraminidase; lane 13, Mucin glycopeptides; and lane 14, Mucin glycopeptides + Neuraminidase.
Mentions: The substrate specificities of the purified enzymes EngCP, EngEF, and EngPA and the commercially available EngSP and EngAL were determined. Each enzyme was incubated with various synthetic substrates. The released sugars were detected by colorimetric assay and thin layer chromatography (TLC) analysis (Table II, Figure 3). Galβ1,3GalNAcα1pNP was the most rapidly hydrolyzed substrate by all the enzymes tested. After 16 h of incubation, only EngEF and EngPA were capable of fully hydrolyzing the Core 3 disaccharide (GlcNAcβ1, 3GalNAcα1pNP). EngAL could only partially hydrolyze the Core 3 disaccharide (27%) after 16 h of incubation (Table II). EngAL could also partially release GalNAc while the rest of the enzymes released only traces of the monosaccharide (Table II). None of the enzymes could act on Galβ1,3GlcNAcα1pNP and low or no activity was detected when Core 2 trisaccharide (Galβ1,3(GlcNAcβ1,6)GalNAcα1pNP) was used as a substrate (Table II).

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