Relationship Between Alg1 Activity And Lipid Structure Of Lipid Linked Oligosaccharide

Asparagine?N?-glycosylation is crucial post-translational modification of protein.In the endoplasmic reticulum?ER?,N-glycosylation begin with the stepwise assembly of the dolichol-linked-oligosaccharide?DLO?precursor Dol-PP-Gn2M9Glc3 on ER membrane,catalyzing by a series of glycosyltransferases encoded by asparagine-linked glycosylation?ALG?genes.Recently study indicated that the activity of Alg protein is closely related to the structure of its DLO substrate,especially the lipid chain.In the preliminary work of our laboratory,the human glycosyltransferases HsAlg1 was expressed in Escherichia coli.Using PPGn2(C₂₀,phytanyl-pyrophosphate-chitobiose)as substrate,but the HsAlg1 can't recognize it to synthesis the product PPGn2-Man.Therefore,we speculate the human-derived Alg proteins have high specificity for the structure of lipid chain in substrate.The LLO In eucaryon is dolichol-linked oligosaccharide,which lipid chain have 85-105 carbon.However,the detailed mechanism of protein-substrate recognition is still unclear,due to the diversity of LLO structures in the ER and difficult to obtain LLO substrates.In this study,we chemosynthesis the DPGn2?dolichyl-pyrophosphate-chitobiose?which is the nature substrate of Alg1,and its analogue.HsAlg1 protein was expressed in prokaryotic expression system,and explore the relationship between the HsAlg1 protein and the structure of lipid chain in substrate.The main research results are follows:?1?Extract and purified the polyprenol 10 from Ginkgo leaves,and through asymmetric hydrogenation to obtain dolichol 11.Use octaacetylchitobiose and dolichol as substrate,via six steps to get DPGn2 29(C₉₅),meanwhile chemosynthesis the lipid-linked chitobiose?LPGn2?with different lipid chain,solanesyl-pyrophosphate-chitobiose(C₄₅,SPGn2),?S?-solanesyl-pyrophosphate-chitobiose(C₄₅,SSPGn2),polyprenyl-pyrophosphate-chitobiose(C₉₅,PPPGn2).?2?The HsAlg1 was successfully overexpressed in Escherichia coli.The structure analysis of human Alg1 protein showed the prediction of a hydrophobic N-terminal transmembrane domain.Thus,truncated HsAlg1 lacking the first 22 amino acid was designed and successfully overexpressed in Escherichia coli.Compare with the HsAlg1 and HsAlg_123-464 activity,shows HsAlg_123-464 have no activity,which meanings maybe the transmembrane domain plays a important role in substrate recognization.?3?Explore the effect of lipid chain structure on HsAlg1 activity: Use LPGn2 with different lipid chain as stubstrate to detect the activity of HsAlg1,the result showed that HsAlg1 have higher activity on nature substrate than unnature substrate,revealed HsAlg1 have high specificity of the lipid chain length of substrate.Then detect the the substrate specificity of yeast glycosyltransferases Sc Alg1.Use LPGn2 with different lipid chain as stubstrate to detect the activity of Sc Alg1,the result showed that Sc Alg1 have high activity for most substrate,obviously,the substrate specificity is lower than HsAlg1.?4?Express five Alg1 protein related to ALG1-CDG,and use DPGn2 as stubstrate to detect the activity.The result showed that enzyme activity can be applied to determine the severity of ALG1-CDG patients.enzyme activity can be applied to determine the severity of ALG1-CDG patients.