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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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Purified endo-α-GalNAcases. SDS–PAGE analysis using 10–20% polyacrylamide gel and stained by Coomassie Brilliant Blue R-250. Lane M, molecular mass standards; lane 1, EngCP; lane 2, EngEF; and lane 3, EngPA.
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Figure 2: Purified endo-α-GalNAcases. SDS–PAGE analysis using 10–20% polyacrylamide gel and stained by Coomassie Brilliant Blue R-250. Lane M, molecular mass standards; lane 1, EngCP; lane 2, EngEF; and lane 3, EngPA.

Mentions: Oligonucliotide primers (Table I) were designed to amplify the genes engEF, engCP, and engAA from the genomic DNA of E. faecalis ATCC 700802, C. perfringens ATCC 13124, and A. aurescens TCI. Following polymerase chain reaction (PCR), the amplified genes were cloned in frame into the pET21a expression vector. Unlike engEF and engCP, expression of engAA was very low. The very weak endo-α-GalNAcase activity of EngAA could be detected using Galβ1,3GalNAcα1pNP as a substrate but only after an extended incubation. For this reason, EngAA was dropped from this study. The gene coding the putative endo-α-GalNAcase protein engPA from P. acnes could not be amplified by PCR from the genomic DNA of P. acnes ATCC25746. To clone the ORF coding for protein EngPA, it was necessary to chemically synthesize the gene using codons optimized for gene expression in Escherichia coli. Once synthesized the gene was cloned into the expression vector pNEB206A. All three recombinant proteins were purified to apparent homogeneity by the chromatographic steps described in the experimental procedures section. The purified proteins migrated as single bands and the apparent molecular weights were in agreement with the predicted molecular masses (188,000 Da for EngCP, 147,000 Da for EngEF, 142,000 Da for EngPA) (Figure 2).Fig. 2


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

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

Purified endo-α-GalNAcases. SDS–PAGE analysis using 10–20% polyacrylamide gel and stained by Coomassie Brilliant Blue R-250. Lane M, molecular mass standards; lane 1, EngCP; lane 2, EngEF; and lane 3, EngPA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Purified endo-α-GalNAcases. SDS–PAGE analysis using 10–20% polyacrylamide gel and stained by Coomassie Brilliant Blue R-250. Lane M, molecular mass standards; lane 1, EngCP; lane 2, EngEF; and lane 3, EngPA.
Mentions: Oligonucliotide primers (Table I) were designed to amplify the genes engEF, engCP, and engAA from the genomic DNA of E. faecalis ATCC 700802, C. perfringens ATCC 13124, and A. aurescens TCI. Following polymerase chain reaction (PCR), the amplified genes were cloned in frame into the pET21a expression vector. Unlike engEF and engCP, expression of engAA was very low. The very weak endo-α-GalNAcase activity of EngAA could be detected using Galβ1,3GalNAcα1pNP as a substrate but only after an extended incubation. For this reason, EngAA was dropped from this study. The gene coding the putative endo-α-GalNAcase protein engPA from P. acnes could not be amplified by PCR from the genomic DNA of P. acnes ATCC25746. To clone the ORF coding for protein EngPA, it was necessary to chemically synthesize the gene using codons optimized for gene expression in Escherichia coli. Once synthesized the gene was cloned into the expression vector pNEB206A. All three recombinant proteins were purified to apparent homogeneity by the chromatographic steps described in the experimental procedures section. The purified proteins migrated as single bands and the apparent molecular weights were in agreement with the predicted molecular masses (188,000 Da for EngCP, 147,000 Da for EngEF, 142,000 Da for EngPA) (Figure 2).Fig. 2

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