Journal: FEBS letters

Article Title: Enterococcus faecalis α1-2-mannosidase (EfMan-I): an efficient catalyst for glycoprotein N-glycan modification

doi: 10.1002/1873-3468.13618

Figure Lengend Snippet: Substrate specificity studies of EfMan-I by thin-layer chromatography (TLC) analysis with reactions carried out at 37°C for (A) 30 min or (B) 24 h in the presence of EfMan-I (1.0 μg) (developing solvent used was n-PrOH:H2O:NH3·H2O = 5:2:1, by volume). For (C), reactions were carried out in the presence of EfMan-I (10 μg) at room temperature (for 10a and 12a) or at 37°C (for 10b and 12b) for 18 h (developing solvent used was n-PrOH:H2O:NH3·H2O = 3:2:1, by volume). Lanes: 1, mannose; 2, ManαpNP in the presence of EfMan-I; 3, ManαpNP; 4, α2-mannobiose in the presence of EfMan-I; 5, α2-mannobiose; 6, α3-mannobiose in the presence of EfMan-I; 7, α3-mannobiose; 8, α6-mannobiose in the presence of EfMan-I; 9, α6-mannobiose; 10, Man3 in the presence of EfMan-I; 11, Man3; 12, Man5 in the presence of EfMan-I; 13, Man5.

Article Snippet: T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison, WI, USA). para -Nitrophenyl-α-D-mannopyranoside (Manα p NP) was from Chem-Impex Int’L Inc. (Wood Dale, IL, USA). α2-Mannobiose, α3-mannobiose, and 3α,6α-mannopentaose (Man 5 ) were from Santa Cruz Biotechnology (Dallas, TX, USA). α6-Mannobiose, hexokinase from S. cerevisiae , phosphoglucose isomerase from baker’s yeast, and glucose-6-phosphate dehydrogenase from L. mesenteroides were from Sigma-Aldrich (Saint Louis, MO, USA).

Techniques: Thin Layer Chromatography, Solvent

Journal: FEBS letters

Article Title: Enterococcus faecalis α1-2-mannosidase (EfMan-I): an efficient catalyst for glycoprotein N-glycan modification

doi: 10.1002/1873-3468.13618

Figure Lengend Snippet: HPLC analysis of 2-AB-labeled high-mannose N-linked glycans from RNase B without (A) and with (B) EfMan-I treatment; and schematic illustration of the reaction catalyzed by EfMan-I (C) [48].

Article Snippet: T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison, WI, USA). para -Nitrophenyl-α-D-mannopyranoside (Manα p NP) was from Chem-Impex Int’L Inc. (Wood Dale, IL, USA). α2-Mannobiose, α3-mannobiose, and 3α,6α-mannopentaose (Man 5 ) were from Santa Cruz Biotechnology (Dallas, TX, USA). α6-Mannobiose, hexokinase from S. cerevisiae , phosphoglucose isomerase from baker’s yeast, and glucose-6-phosphate dehydrogenase from L. mesenteroides were from Sigma-Aldrich (Saint Louis, MO, USA).

Techniques: Labeling

Journal: FEBS letters

Article Title: Enterococcus faecalis α1-2-mannosidase (EfMan-I): an efficient catalyst for glycoprotein N-glycan modification

doi: 10.1002/1873-3468.13618

Figure Lengend Snippet: Superposition of EfMan-I (salmon color) and SpGH92 (slate blue) structures (A) and their active sites (B). They superimpose with an rmsd of 1.07 Å over 672 equivalent α-carbons. Calcium ions in the active site are shown as green spheres, and the 359 loop near the active site is highlighted in bright red color for EfMan-I. The side chains of residues that coordinate the calcium ion are shown along with ordered glycerol molecules found in both mannosidase structures. Note that in each structure a glycerol coordinates the calcium ion and in the EfMan-I structure, another glycerol binds in a similar location to mannose observed in SpGH92, which is 3.4 Å below the indole ring of Trp68. Glu494 and Asp604 are proposed to be the respective general acid and general base in catalysis.

Article Snippet: T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison, WI, USA). para -Nitrophenyl-α-D-mannopyranoside (Manα p NP) was from Chem-Impex Int’L Inc. (Wood Dale, IL, USA). α2-Mannobiose, α3-mannobiose, and 3α,6α-mannopentaose (Man 5 ) were from Santa Cruz Biotechnology (Dallas, TX, USA). α6-Mannobiose, hexokinase from S. cerevisiae , phosphoglucose isomerase from baker’s yeast, and glucose-6-phosphate dehydrogenase from L. mesenteroides were from Sigma-Aldrich (Saint Louis, MO, USA).

Techniques: