Efficient production and characterization of soluble active human ß-1,2-N-acetylglucosaminyltransferase II in bacteria.

In humans, almost all the cell surface and secreted glycoproteins are modified with complex-type N-glycans. Thus, it is essential to obtain complex-type N-glycans to fully understand the biological properties of glycoproteins. Here, human ß-1,2-N-acetylglucosaminyltransferase II (hGnT-II), a Golgi-localized enzyme integral to complex-type N-glycan biosynthesis, was cloned as a truncated transmembrane form (GnT-II-ΔTM) and heterologously overexpressed in Escherichia coli. Our results showed that hGnT-II could be overexpressed in its soluble form by fusing the truncated enzyme with a thioredoxin (Trx)-tag in the Rosetta-Gami 2 strain. Using the optimized induction conditions, the expression level of recombinant protein was enhanced to yield approximately 4 mg per liter culture after affinity purification. The enzyme exhibited appropriate glycosyltransferase activity, and the calculated Km value was 52.4 µM, similar to the protein expressed in mammalian cells. Furthermore, the effect of MGAT2-CDG mutations on enzyme activity was also measured. These results suggested that the E. coli expression system was capable of the large-scale production of bioactive hGnT-II, which can be used for functional study and effective synthesis of complex-type N-glycans.

Alg mannosyltransferases: From functional and structural analyses to the lipid-linked oligosaccharide pathway reconstitution.

BACKGROUND: N-glycosylation is initiated from the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER), which is catalyzed by a series of Alg (asparagine-linked glycosylation) proteins. SCOPE OF REVIEW: This review summarizes our recent studies on the enzymology of Alg mannosyltransferases (MTases). We also discuss the membrane topology and physiological importance of several ER cytosolic Alg proteins. MAJOR CONCLUSIONS: Utilizing an efficient prokaryotic protein expression system and a new LC-MS quantitative activity assay, we overexpressed all Alg MTases and performed enzymology studies. Moreover, by reconstituting the LLO pathway, the high-yield chemoenzymatic synthesis of high-mannose-type N-glycans was accomplished using recombinant Alg MTases. GENERAL SIGNIFICANCE: The analysis of the enzymology and topology of Alg MTases has provided valuable biochemical information in the LLO biosynthesis pathway. In addition, an efficient chemoenzymatic strategy that could prepare various oligomannose-type N-glycans in sufficient amounts was established for further biological assays.

Topological and enzymatic analysis of human Alg2 mannosyltransferase reveals its role in lipid-linked oligosaccharide biosynthetic pathway.

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

Recent Progress in Chemo-Enzymatic Methods for the Synthesis of N-Glycans.

Asparagine (N)-linked glycosylation is one of the most common co- and post-translational modifications of both intra- and extracellularly distributing proteins, which directly affects their biological functions, such as protein folding, stability and intercellular traffic. Production of the structural well-defined homogeneous N-glycans contributes to comprehensive investigation of their biological roles and molecular basis. Among the various methods, chemo-enzymatic approach serves as an alternative to chemical synthesis, providing high stereoselectivity and economic efficiency. This review summarizes some recent advances in the chemo-enzymatic methods for the production of N-glycans, including the preparation of substrates and sugar donors, and the progress in the glycosyltransferases characterization which leads to the diversity of N-glycan synthesis. We discuss the bottle-neck and new opportunities in exploiting the chemo-enzymatic synthesis of N-glycans based on our research experiences. In addition, downstream applications of the constructed N-glycans, such as automation devices and homogeneous glycoproteins synthesis are also described.