Expression,purification And Activity Assay Of Yeast ?-1,2 Mannosyltransferase Alg11

N-glycosylation is one of the most abundant protein modifications in nature,the initial step the synthesis of dolichol-linked oligosaccharide?DLO?precursor,Glycosyltransferase Alg11,which is an important protein in N-glycosylation pathway,transfers the mannose moiety from GDP-Man to DPGn2M3?Dolichyl-pyrophosphate-GlcNAc2Mannose3?,forming DPGn₂M₄ and DPGn₂M₅ DLO precursors.Defects in human glycosyltransferase Alg11 can lead to the rare congenital glycosylated disease,but there is no method to establish the correlation between different mutations and the severity of the disease.In this study,the yeast-derived glycosyltransferase Alg11 was expressed in the prokaryotic expression system and subjected to soluble expression.A preliminary study of the characterization of the enzyme was performed in vitro.The structural analysis of Saccharomyces cerevisiae Alg11 showed the prediction of a hydrophobic N-terminal transmembrane domain.Thus,truncated Alg11 lacking the first 44amino acid was designed and successfully overexpressed in Escherichia coli.Using PPGn₂?Phytanyl-pyrophosphate-GlcNAc2?as substrate,the natural analogue acceptor substrate PPGn₂M₃ of Alg11 was synthesized rapidly and efficiently using the recombinant Alg1?TM and TRX-Alg2 enzymes in vitro.After the transferase activity assay,reaction mixture was applied to the liquid chromatography tandem mass spectrometry?LC-MS?,which revealed that Alg11_45-548 was able to generate PPGn₂M₅ from its acceptor substrate PPGn₂M₃.Structural analysis of Gn₂M₅ showed that two newly formed glycosidic bonds could be cleaved by?-1,2 mannosidase,meaning the two mannose moieties were attached to Gn2M3 by?-1,2 linkages.We first studied the enzymatic properties of glycosyltransferase Alg11 in vitro.The results showed that the optimal reaction temperature was 30°C,the most suitable metal ion was Ca²⁺,the most suitable sugar donor was GDP-Man,and the optimum reaction pH was 9.0.Substrate specificity assay indicated the recombinant Alg11 specifically recognized PPGn₂M₃rather than other LLOs,such as PPGn2 and PPGn2M1.Intermediates in N-glycosylation can not be used by this enzyme.Additionally,oligosaccharide Gn₂M₃ was not elongated by Alg11_45-548,suggesting that the lipid chain in the substrate PPGn₂M₃ was critical for the recognition.Two conserved motifs of G-rich and EX₇E in the glycosyltransferase Alg11 were found by homologous sequence comparisons.In vitro analysis proved that Alg1,Alg2 and Alg11 have a specific catalytic sequence.This property guarantees the ordered synthesis of DLO precursors in N-glycosylation.The human glycosyltransferase hAlg11 was expressed in the prokaryotic expression system.The activity of hAlg11 was detected by the above mentioned method and the relative activity of the mutation site in ALG11-CDG patients was analyzed for the first time,L86S and Q318P in vitro.In vitro relative activity of the mutants is consistent with the severity of the symptoms.To sum up,quantitative detection of enzyme activity by LC-MS can be applied to determine the severity of ALG11-CDG patients.