In Vitro Reconstitution Of The Dolichol-linked Oligosaccharide Precursor Biosynthesis Pathway For N-linked Glycosylation

Asparagine ?N?-linked glycosylation is an important posttranslational modification of protein that directly affects both structural and biological function in eukaryotes,and,most of the secretory or membrane proteins in eukaryotic cells are N-glycosylated.N-glycan synthesis begins with the ordered,stepwise assembly of the common dolichol-linked oligosaccharide?DLO?precursor Dol-PP-GlcNAc₂Man₉Glc₃,on the endoplasmic reticulum?ER?membrane.A series of ER-resident glycosyltransferases encoded by asparagine-linked glycosylation?ALG?genes are responsible for the biosynthesis of DLO precursor oligosaccharides.Extensive effort in the past three decades has led to the identification all the ALG genes required for DLO biosynthesis,but the biochemical function analysis of these glycosyltransferases has been blocked for a long time.In humans,mutations in ALG lead to congenital disorders of glycosylation?CDG?,the CDG patients suffer from broad multisystem defects with varying degrees of clinical severity.As far,there was no method to assay ALG-CDG mutations activity which correlating to the clinical severity.On the other hand,as a class of N-linked modification,high-mannose oligosaccharides were served as important materials for biological studies including the glycan arrays,vaccines,and glycoprotein quality control.Nevertheless,currently,the chemical synthesis and isolation from natural sources of the high-mannose oligosaccharides are extremely labor-intensive and low-yield.In order to solve the above problem,this study reconstituted the ordered assembly DLO pathway by a tractable in vitro LC-MS quantitative assay to define the precise roles of these glycosyltransferases,and to measure the activity of CDG-related mutations;furthermore to chemo-enzymatically synthesize high-mannose oligosaccharides with extreme efficiency.The main results of this study are summarized as follows:?1?Quantitative study of yeast Alg1 beta-1,4 mannosyltransferase activity by LC-MS.Yeast ALG1 encodes ab-1,4 mannosyltransferase that adds the first mannose?Man?onto GlcNAc2-PP-Dolichol?DPGn2?to produce a core trisaccharide DPGn2-Man.Here,we report a new method to purify recombinant Alg1 in high yield,and a LC-MS approach for accurately measuring itsb-1,4 mannosyltransferase activity.Using purified Alg1,its biochemical characteristics were investigated.Furthermore,this assay was applied to analyze patient relevant missense mutations in Alg1 to judge their clinical severity.?2?Alternative routes for synthesis of N-linked glycans by Alg2.Alg2 catalyzes addition of both thea1,3-anda1,6-linked mannose onto DPGn2-Man to form the trimannosyl core DPGn2-Man3.Although this bifunctionality has been recognized for some time,conflicting reports about the order of these reactions led to a gap in our understanding of these early events in DLO synthesis.The current accepted model for the order of Man addition by Alg2 isa1,3-mannosylation followed bya1,6-mannosylation to yield the branched core pentasaccharide product.A problem with this model is it contradicts experiments conducted almost 30 years ago that suggested the DPGn2-Man2 tetrasaccharide intermediate is a mixture.In this study,we successfully developed an LC-MS-based quantitative assay for Alg2 activity and applied it to study the enzyme activity of Alg2.This assay allowed us to precisely identify Alg2 reaction intermediates,demonstrating addition of thea1,3-anda1,6-mannose can occur independently in either order but at differing rates.We propose a different model,in which alternative orders of Man addition are possible.Thea1,3-linked Man can be added first,followed bya1,6-linked Man or vice versa.On the other hand,the conserved C-terminal EX₇E motif,N-terminal cytosolic tail,and 3G-rich loop motifs in Alg2 play crucial roles for these activities,both in vitro and in vivo.?3?An LC-MS quantitative assay reveals the severity of ALG11-CDG.ALG11 encodes a dual function mannosyltransferase,catalyzing the fourth and fifth mannose linkage onto DPGn2-Man3 with formation of DPGn2-Man5.In human,deficiency of Alg11 leads to CDG designated CDG-Ip?ALG11-CDG?.This study established a quantitative assay using LC-MS to measure the activity of hAlg11.Application of quantitative assay to measure the activity of ALG11-CDG related mutations indicated that the changes were correlated with the severity of ALG11-CDG phenotypes.Moreover,we identified several conserved amino acids mutations in the G-rich and EX₇E motifs affecting activity of hAlg11.?4?Chemoenzymatic stepwise assembly of the lipid-linked high-mannose oligosaccharides.This study expressed a series of recombinant mannosyltransferase.Starting with chemically synthesized PPGn2 and sequential combinations of Alg2,we reconstituted the eukaryotic DLO pathway of stepwise assembly of high-mannose oligosaccharides with extreme efficiency in vitro.Furthermore,using various combinations of acceptors,we can synthesize the unusual high-mannose oligosaccharides.