Study On Complex Formation Of Alg3,9,12 Mannosyltransferases And Quantitative Analysis For Enzymatic Activity Of The ALG3-CDG Related Proteins

Glycosylation is one of the important protein modification methods,not only in eukaryotic cells,but also in some archaea and bacteria.N-linked glycosylation is a main form of glycosylation of proteins that beginning in the endoplasmic reticulum(ER).The N-glycosylation process in the ER includes the assembly of dolichol-linked oligosaccharides(DLO)and the transfer of oligosaccharides to the asparagine residues of nascent polypeptides in ER lumen.The assembly of DLO occurs on the ER membrane and is catalyzed by specific glycosyltransferases(Alg proteins)whose process is relatively conserved.Abnormal Alg proteins in the human body will lead to congenital disorders of glycosylation: CDG).Previous studies have shown that the Alg proteins on the cytosolic side of ER exhibit as two enzyme complexes,namely Alg7,Alg13 and Alg14 complex and Alg1,Alg2 and Alg11 complex,indicates glycosyltransferases in the DLO pathway tend to exist in enzyme complex.On the other hand,Alg3,Alg9 and Alg12 share the same sugar donor,there is a possibility that Alg3,Alg9 and Alg12 exhibit as enzyme complexs.This study explored the interactions between the Alg3,Alg9 and Alg12 on the luminal side of the ER,and demonstrated that Alg3,Alg9 and Alg12 are also exist as complexes;in addition,ALG3-CDG caused by mutation of Alg3 protein was studied by quantitative analysis of activity in vitro.The main research includes:1)Three yeast strains which co-express Alg3 and Alg9,Alg3 and Alg12,Alg9 and Alg12 were constructed.Afterwards yeast membrane proteins were extracted by lysis buffer.Then Anti-FLAG Agrose beads coprecipitate protein,finally using western blot analysis.Similarly,the co-immunoprecipitation assay and western blot analysis showed that there were interactions between the two of the Alg3,Alg9 and Alg12 three mannosyltransferase.Yeast Mistic-Alg3,Alg9 and Alg12 were co-expressed in Escherichia coli(E.coli),and the membrane fractions of E.coli were extracted.After expression of Mistic-Alg3,Alg9 and Alg12 mannosyltransferase,co-immunoprecipitation experiments and western blot analysis were found that Mistic-Alg3,Alg9 and Alg12 exist in the form of a multi-enzyme complex.This study is the first time to show the interactions between the two of Alg3,Alg9 and Alg12 three mannosyltransferase.2)The activity of co-expressed enzymes were analyzed in vitro: PPGn2-M5 was used as substrate,the E.coli membrane containing Mistic-Alg3,Alg9 and Alg12 catalyzes the synthesis of PPGn2-M9.In comparison with the catalytic reaction which expressing Mistic-Alg3,Alg9 and Alg12 individually,the co-expressed enzymes showed higher catalytic activity.3)Human ALG3-CDG conserved yeast recombinant Mistic-Alg3 mutants were expressed in E.coli(mutations: I70 T,Y72R,G98 R,L157K,R171 Q and M221T).The enzymatic activity of these six mutant proteins were quantitative analyzed by LC-MS.It turned out that the mutants showed decreased activities,whose degrees are related to the severity of Alg3-CDG disease and the lifetime of patients.