Limits...
Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A.

Wang T, Cai ZP, Gu XQ, Ma HY, Du YM, Huang K, Voglmeir J, Liu L - Biosci. Rep. (2014)

Bottom Line: Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins.In addition, PNGase H+ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A.The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.

View Article: PubMed Central - PubMed

Affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.

ABSTRACT
Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H+. The gene of PNGase H+ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H+ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H+ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.

Show MeSH

Related in: MedlinePlus

Substrate specificity tests of recombinant PNGase H+ using RNase B and lactoferrin(A) SDS/PAGE analysis of heat denatured RNase B with (+) and without (−) PNGase H+ and F. (B) UPLC chromatographs of N-glycans released from RNase B. PNGase F was chosen as control. Heat denatured RNAse B and pepsin digested RNase B were used as substrates. (C) SDS/PAGE analysis of heat denatured lactoferrin with (+) and without (−) PNGase H+ and F. (D) UPLC chromatographs of N-glycans released from lactoferrin. PNGase F was chosen as control. Heat denatured lactoferrin and pepsin digested lactoferrin were used as the substrates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231336&req=5

Figure 4: Substrate specificity tests of recombinant PNGase H+ using RNase B and lactoferrin(A) SDS/PAGE analysis of heat denatured RNase B with (+) and without (−) PNGase H+ and F. (B) UPLC chromatographs of N-glycans released from RNase B. PNGase F was chosen as control. Heat denatured RNAse B and pepsin digested RNase B were used as substrates. (C) SDS/PAGE analysis of heat denatured lactoferrin with (+) and without (−) PNGase H+ and F. (D) UPLC chromatographs of N-glycans released from lactoferrin. PNGase F was chosen as control. Heat denatured lactoferrin and pepsin digested lactoferrin were used as the substrates.

Mentions: HRP, RNase B and lactoferrin were used for the substrate specificity test of the recombinant PNGase H+. SDS/PAGE was applied to monitor the deglycosylation capabilities of the recombinant PNGase H+ and PNGase F on heat-denatured glycoproteins without the addition of any denaturants. Assay mixtures without enzymes were used as negative controls. Heat-denatured RNase B was only partly deglycosylated by PNGase F, while it was almost completely deglycosylated by PNGase H+, as evidenced by the migration of the primary protein band from approximately 19 to 16 kDa (Figure 4A). In a similar manner, overnight incubations of heat-denatured lactoferrin with either PNGase F or PNGase H+ resulted in a complete shift of the primary protein band on the SDS/PAGE to a lower molecular mass compared with the negative control (Figure 4C).


Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A.

Wang T, Cai ZP, Gu XQ, Ma HY, Du YM, Huang K, Voglmeir J, Liu L - Biosci. Rep. (2014)

Substrate specificity tests of recombinant PNGase H+ using RNase B and lactoferrin(A) SDS/PAGE analysis of heat denatured RNase B with (+) and without (−) PNGase H+ and F. (B) UPLC chromatographs of N-glycans released from RNase B. PNGase F was chosen as control. Heat denatured RNAse B and pepsin digested RNase B were used as substrates. (C) SDS/PAGE analysis of heat denatured lactoferrin with (+) and without (−) PNGase H+ and F. (D) UPLC chromatographs of N-glycans released from lactoferrin. PNGase F was chosen as control. Heat denatured lactoferrin and pepsin digested lactoferrin were used as the substrates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231336&req=5

Figure 4: Substrate specificity tests of recombinant PNGase H+ using RNase B and lactoferrin(A) SDS/PAGE analysis of heat denatured RNase B with (+) and without (−) PNGase H+ and F. (B) UPLC chromatographs of N-glycans released from RNase B. PNGase F was chosen as control. Heat denatured RNAse B and pepsin digested RNase B were used as substrates. (C) SDS/PAGE analysis of heat denatured lactoferrin with (+) and without (−) PNGase H+ and F. (D) UPLC chromatographs of N-glycans released from lactoferrin. PNGase F was chosen as control. Heat denatured lactoferrin and pepsin digested lactoferrin were used as the substrates.
Mentions: HRP, RNase B and lactoferrin were used for the substrate specificity test of the recombinant PNGase H+. SDS/PAGE was applied to monitor the deglycosylation capabilities of the recombinant PNGase H+ and PNGase F on heat-denatured glycoproteins without the addition of any denaturants. Assay mixtures without enzymes were used as negative controls. Heat-denatured RNase B was only partly deglycosylated by PNGase F, while it was almost completely deglycosylated by PNGase H+, as evidenced by the migration of the primary protein band from approximately 19 to 16 kDa (Figure 4A). In a similar manner, overnight incubations of heat-denatured lactoferrin with either PNGase F or PNGase H+ resulted in a complete shift of the primary protein band on the SDS/PAGE to a lower molecular mass compared with the negative control (Figure 4C).

Bottom Line: Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins.In addition, PNGase H+ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A.The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.

View Article: PubMed Central - PubMed

Affiliation: *Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.

ABSTRACT
Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H+. The gene of PNGase H+ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H+ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H+ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.

Show MeSH
Related in: MedlinePlus