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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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Schematic representation of the putative endo-α-GalNAcases. Alignment of the putative endo-α-GalNAcases that were revealed by BLAST search against the protein sequence of EngBF and the identity results are shown. The gray shaded boxes correspond to the centrally located conserved region, while the black and patterned boxes indicate different types of SBDs. CBM_4_9, carbohydrate binding module (Pfam 02018); NPCBM, novel putative carbohydrate binding module (Pfam 08305); FIVAR, uncharacterized sugar-binding domain (Pfam 07554); F5-F8 type C, member of the galactose-binding domain-like superfamily (Pfam 00754). The protein accession numbers are B. longum (ABY836679), R. torques (ZP_01966813), B. capillosus (ZP_02035456), S. pneumoniae (YP_873926.1), E. faecalis (NP_815498.1), Janibacter sp. (ZP_00993766.1), A. aurescens (YP_947239.1), S. coelicolor (NP_630440.1), P. acnes (YP_056270.1), and C. perfrigens (YP_695137.1).
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Figure 1: Schematic representation of the putative endo-α-GalNAcases. Alignment of the putative endo-α-GalNAcases that were revealed by BLAST search against the protein sequence of EngBF and the identity results are shown. The gray shaded boxes correspond to the centrally located conserved region, while the black and patterned boxes indicate different types of SBDs. CBM_4_9, carbohydrate binding module (Pfam 02018); NPCBM, novel putative carbohydrate binding module (Pfam 08305); FIVAR, uncharacterized sugar-binding domain (Pfam 07554); F5-F8 type C, member of the galactose-binding domain-like superfamily (Pfam 00754). The protein accession numbers are B. longum (ABY836679), R. torques (ZP_01966813), B. capillosus (ZP_02035456), S. pneumoniae (YP_873926.1), E. faecalis (NP_815498.1), Janibacter sp. (ZP_00993766.1), A. aurescens (YP_947239.1), S. coelicolor (NP_630440.1), P. acnes (YP_056270.1), and C. perfrigens (YP_695137.1).

Mentions: In order to identify putative endo-α-GalNAcases, a BLAST (Altschul et al. 1997) search was run using the endo-α-GalNAcase protein sequence EngBF from B. longum JCM1217. The top nine hits were from bacteria and had 28% and higher overall sequence identity with EngBF (Figure 1). The predicted molecular mass of EngSP corresponds to that previously characterized from S. pneumoniae (Brooks and Savage 1997; Glasgow et al. 1977; Umemoto et al. 1977). All of the putative endo-α-GalNAcases had a centrally located conserved region of 700–800 amino acid residues (Figure 1). Using the Pfam database (Finn et al. 2006) to analyze the protein sequences, all sequences with the exception of EngEF from Enterococcus faecalis contained predicted domains that are presumably involved with the identification or binding of sugars (sugar binding domain (SBD)). To determine if the existence or the positioning of the SBDs could play a role on the substrate specificity of this family of enzymes, it was decided to clone and characterize four of these proteins each with a different organization. EngEF has no predicted SBD. EngPA from Propionibacterium acnes has an F5_F8 type C domain (a member of the galactose-binding domain-like superfamily) close to the N-terminus. EngAA from Arthrobacter aurescens has two novel putative carbohydrate binding module (NPCBM) domains at the C-terminus. EngCP from C. perfringens has a CBM 4_9 domain (carbohydrate binding module) inside the conserved region. All of the other potential endo-α-GalNacases as well as the already characterized enzymes EngBF and EngSP have SBDs at the C-terminus or inside the conserved region (Figure 1).


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

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

Schematic representation of the putative endo-α-GalNAcases. Alignment of the putative endo-α-GalNAcases that were revealed by BLAST search against the protein sequence of EngBF and the identity results are shown. The gray shaded boxes correspond to the centrally located conserved region, while the black and patterned boxes indicate different types of SBDs. CBM_4_9, carbohydrate binding module (Pfam 02018); NPCBM, novel putative carbohydrate binding module (Pfam 08305); FIVAR, uncharacterized sugar-binding domain (Pfam 07554); F5-F8 type C, member of the galactose-binding domain-like superfamily (Pfam 00754). The protein accession numbers are B. longum (ABY836679), R. torques (ZP_01966813), B. capillosus (ZP_02035456), S. pneumoniae (YP_873926.1), E. faecalis (NP_815498.1), Janibacter sp. (ZP_00993766.1), A. aurescens (YP_947239.1), S. coelicolor (NP_630440.1), P. acnes (YP_056270.1), and C. perfrigens (YP_695137.1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Schematic representation of the putative endo-α-GalNAcases. Alignment of the putative endo-α-GalNAcases that were revealed by BLAST search against the protein sequence of EngBF and the identity results are shown. The gray shaded boxes correspond to the centrally located conserved region, while the black and patterned boxes indicate different types of SBDs. CBM_4_9, carbohydrate binding module (Pfam 02018); NPCBM, novel putative carbohydrate binding module (Pfam 08305); FIVAR, uncharacterized sugar-binding domain (Pfam 07554); F5-F8 type C, member of the galactose-binding domain-like superfamily (Pfam 00754). The protein accession numbers are B. longum (ABY836679), R. torques (ZP_01966813), B. capillosus (ZP_02035456), S. pneumoniae (YP_873926.1), E. faecalis (NP_815498.1), Janibacter sp. (ZP_00993766.1), A. aurescens (YP_947239.1), S. coelicolor (NP_630440.1), P. acnes (YP_056270.1), and C. perfrigens (YP_695137.1).
Mentions: In order to identify putative endo-α-GalNAcases, a BLAST (Altschul et al. 1997) search was run using the endo-α-GalNAcase protein sequence EngBF from B. longum JCM1217. The top nine hits were from bacteria and had 28% and higher overall sequence identity with EngBF (Figure 1). The predicted molecular mass of EngSP corresponds to that previously characterized from S. pneumoniae (Brooks and Savage 1997; Glasgow et al. 1977; Umemoto et al. 1977). All of the putative endo-α-GalNAcases had a centrally located conserved region of 700–800 amino acid residues (Figure 1). Using the Pfam database (Finn et al. 2006) to analyze the protein sequences, all sequences with the exception of EngEF from Enterococcus faecalis contained predicted domains that are presumably involved with the identification or binding of sugars (sugar binding domain (SBD)). To determine if the existence or the positioning of the SBDs could play a role on the substrate specificity of this family of enzymes, it was decided to clone and characterize four of these proteins each with a different organization. EngEF has no predicted SBD. EngPA from Propionibacterium acnes has an F5_F8 type C domain (a member of the galactose-binding domain-like superfamily) close to the N-terminus. EngAA from Arthrobacter aurescens has two novel putative carbohydrate binding module (NPCBM) domains at the C-terminus. EngCP from C. perfringens has a CBM 4_9 domain (carbohydrate binding module) inside the conserved region. All of the other potential endo-α-GalNacases as well as the already characterized enzymes EngBF and EngSP have SBDs at the C-terminus or inside the conserved region (Figure 1).

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.

Show MeSH
Related in: MedlinePlus