Chemo-enzymatic Synthesis Of Human Complex-type N-glycan

N-glycosylation is one of the most important post-modifications of protein,in which the glycans are covalently linked to the newly formed polypeptide.Expect for the effect on the protein folding,N-glycans also help to maintain the structure and the activity of proteins,as well as induce the signal transductions between cell to cell.In human,almost all of the cell surface and secreted glycoproteins are modified with complex-type N-glycan.Thus,a method to obtain the complex-type N-glycans is essential for the comprehensive analyses of the glycan structure and properties of glycoproteins.On the other hand,since homogeneous certain complex-type N-glycan structure is critical for some therapeutic glycoproteins,preparation of the exact glycan would guarantee the quality of these glycoproteins.This work focuses on the synthesis of complex-type N-glycan,using a bottom-up chemo-enzymatic method.The chemical synthesized Gn2-P-P is the substrate.Several glycosyltransferases were expressed in E.coli,which were used to elongate the glycan chain,resulting in the target N-glycans.Among those glycosyltransferases,GnT-I and GnT-II are characterized.First,various glycosyltransferases were expressed in E.coli.The yeast Alg1 and Alg2 were co-expressed and the membrane of E.coli was used for the reaction.On the other hand,four Golgi glycosyltransferases,namely hGnT-I,hGnT-II and h?4GalT1 were expressed and purified.LC-MS and MALDI-TOF mass were used to monitor the reaction and verify the products,which showed the synthesis of complex-type N-glycan Gal2Gn2Man3Gn2 was completed after five-step reactions,at the milligram level.In addition,recombinant human GnT-I and GnT-II were purified and applied to protein characterization.GnT-I,the key enzyme to trimming the N-glycans from high-mannose type to hybrid-and complex-type,showed relatively low substrate specificity,while GnT-II,the enzyme to process the complex-type glycan synthesis,showed more strict substrate specificity,requiring the GlcNAc on the left branch of the substrate.GnT-II also required the metal ion co-factor,such as Mn²⁺and Co²⁺.The Km value of hGnT-II was calculated as 55?M,which was similar as the protein expressed in insect cells.In this work,a bottom-up chemo-enzymatic method is established to synthesis complex-type N-glycan.The characterization of key enzymes hGnT-I and hGnT-II helps to improve the conversion rata and productivity of glycans.The successful preparation of complex-type N-glycans provides substrate for glycosyltransferase assay,and leads to the production of homogenous glycoprotein with complex-type N-glycan.