Arthrobacter endo-beta-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C

Abstract

Asparagine-linked glycosylation is a major form of posttranslational modifications, which plays important roles in protein folding, intracellular signaling, and a number of other biological recognition events ^[1]. Glycoproteins are often characterized by their structural micro-heterogeneity where different glycoforms have the same polypeptide backbone but differ in the pendant oligosaccharides. Of particular interest are the findings that subtle difference in the attached glycans can have a significant impact on the biological functions of a given glycoprotein ^{[2, 3]}. The urgent need of pure glycoforms for functional studies and biomedical applications has stimulated a great interest in exploring new methods for making homogeneous glycoproteins ^[4]. Major advances include the application of native chemical ligation and expressed protein ligation for constructing full-size glycoproteins ^[–7], chemoselective ligation to introduce homogeneous glycans ^[8], and the engineering of yeast glycosylation pathways to produce single glycoforms ^[9]. Yet another interesting advance in the field is the endoglycosidase-catalyzed transglycosylation for glycosylation engineering and glycoprotein synthesis ^[–16].

Figure 1

Conformational comparison of Man₃GlcNAc-thiazoline (green) and CT-oxazoline (yellow) docked into substrate-binding site of Endo-A (macromolecule displayed in ribbon).

Figure 2

SDS-PAGE analysis of the one-pot transglycosylation reaction and the product. lane 0: marker; lane 1: RNase B; lane 2: an aliquot of the one-pot reaction mixture at 5min; lane 3: an aliquot of the one-pot reaction mixture at 3 h; lane 4: purified sialylated RNase C; lane 5: sialylated RNase C treated with Endo-M.

Figure 3

ESI-MS of the sialylated RNase C.

Scheme 1

Semi-synthesis of sialylated N-glycan oxazoline.

Scheme 2

Endo-A and EndoM-N175A act on sialylated N-glycan oxazoline for transglycosylation without product hydrolysis. Enzyme activity definition: 1 mU was defined as the amount of Endo-A or EndoM-N175A needed for transferring 1 nmol of Man₃GlcNAc-oxazoline to GlcNAc-PNP in 1 min at 23 °C.

Scheme 3

Endo-A and EndoM-N175A catalyzed one-pot conversion of RNase B to sialylated RNase C.