In Vitro Assay For The ER UDP-N-acetylglucosamine Transferase Complex And Its Application

N-linked glycosylation is one of the most important posttranslational modifications,directly affects a multitude of essential biological processes.Protein N-glycosylation begins with the assembly of dolichol-linked oligosaccharide（DLO）precursor whose synthesis starts on the cytosolic side of the endoplasmic reticulum（ER）membrane and finishes in its lumen side.Alg（Asparagine-linked glycosylation）glycosyltransferases are responsible for the sequential addition of sugars to dolichol pyrophosphate（Dolichol-P）at the ER membrane,resulting a DLO containing fourteen sugars.The second step of DLO synthesis transfers aβ-1,4-linked N-acetylglucosamine（Glc NAc）to Dolichyl-pyrophosphate-Glc NAc（DPGn）,which requires two enzymes Alg13 and Alg14.These two Alg proteins perform UDP-N-acetylglucosamine transferase（Gn Tase）activity only when they form a heterodimeric complex.There are at least four ALG13 isoforms generated by alternative splicing in human cells.However,the biological relevance of these isoforms is still unknown.In addition,mutations in human Alg13 and Alg14 proteins cause congenital disorders of glycosylation（CDG）.Noteworthy,most ALG13-CDG and ALG14-CDG variants show a normal N-glycosylation pattern on transferrin,a common biomarker for CDG.The direct evidence linking Alg13/14 Gn Tase activity to the CDG phenotypes remains elusive.An in vitro quantitative assay for studying the enzymatic properties of Alg13/14 Gn Tase is required for addressing the above questions.Because of its complex structure and the difficulty for preparing its DLO substrate,there are still no suitable assay system established for Alg13/14 Gn Tase.In this study,using a chemically synthesized DLO substrate,we succeeded in establishing a LC-MS based in vitro quantitative assay to detect the Gn Tase activity,and further applied it for enzymatic studies on Alg13/14 complex and its variants.The main results of this study are as follows:（1）Establishment of a quantitative in vitro assay for Alg13/14 transferase activity.A series of Lipid-pyrophosphate Glc NAc substrates with different length(C₂₀,C₄₅ and C₉₅)and structure of lipid chains were prepared and used for screening the suitable DLO substrate of Alg13/14 Gn Tase.Meanwhile,we successfully co-expressed human Alg13 and Alg14（HsAlg13 and HsAlg14）in E.coli cells,and purified an active HsAlg13/14 complex from the membrane fraction.Using this recombinant complex,we were able to identify the Dolichyl-PP-Glc NAc（C95）as the best substrate for HsAlg13/14 complex and further developed a LC-MS based quantitative assay for studying its Gn Tase activity.In addition,K_mvalue of HsAlg13/14 Gn Tase was determined as 94.1μmol·L^-1.（2）Studying the Gn Tase activity on HsAlg13 isoforms.In gene evolution,ALG13 fused with other genes and form different isoforms.Among multiple HsAlg13 isoforms,only isoform1（HsAlg13-iso1）and isoform2（HsAlg13-iso2）contain the catalytic domain of Alg13/14 Gn Tase.It has been reported that HsAlg13-iso2forms an active Gn Tase complex with HsAlg14,while the Gn Tase activity of HsAlg13-iso1has not been assessed yet.In this study,using our in vitro assay,we confirmed that HsAlg13-iso1 fails to form complex with HsAlg14 and is not directly involved in HsAlg13/14Gn Tase activity.（3）Discriminating the Gn Tase activity of ALG13-and ALG14-CDG mutants.To confirm the pathogenicity of ALG13-CDG and ALG14-CDG,the recombinant HsAlg13-iso2-CDG and HsAlg14-CDG variants proteins were tested utilizing our quantitative Gn Tase assay system.We demonstrated that both ALG13-CDG and ALG14-CDG mutations reduced both the Gn Tase activity and the protein stability of Alg13/14 complex,indicating the diminished the Gn Tase activity as the cause of ALG13/14-CDG.