Topological Analysis Of N-glycosylation Related Proteins And Research On GPI Remodeling Related Genes On The ER

On the cell surface of higher organism,there are various glycoconjugates,which are closely related to different physiological processes.Therefore,it is important to research the molecular mechanism of proteins or genes involved in glycoconjugates synthesis in the glycosylation pathways.The N-glycan or GPI anchor modification of proteins,which starts on the endoplasmic reticulum(ER),is ubiquitous and highly conserved.During N-glycosylation,the biosynthesis of DLO(Dolichol-Linked Oligosaccharide)begins on the cytoplasmic side of the endoplasmic reticulum and ends in the lumen,resulting in Glc3Man9GlcNAc2-PP-Dol consists of 14 single sugars.Two functionally distinguished heteromeric glycosyltransferase(GTase)complexes are responsible for the cytosolic DLO assembly.Alg1,a ?-1,4 mannosyltransferase(MTase)physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate.Despite the fact that Alg1 plays a central role in formation of the multi-MTase has been confirmed,the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood.Using a combination of bioinformatics and biological approaches,we have undertaken a structural and functional study on Alg1 protein,in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr-ALG1 yeast strain,and further confirmed by a LC-MS-based in vitro activity assay.Computational evidences confirmed that Alg1 and Alg13/14 complex have structural similarities and can be defined as membraneassociated GT-B glycosyltransferase.We provided clear evidence that the N-terminal transmembrane domain(TMD)including the following positively charged amino acids and an N-terminal amphiphilic-like ? helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane.The detailed membrane topology of Alg1 further reveals its biological significance for coordinating DLO synthesis on the cytoplasmic face of the ER.In S.cerevisiae,the topology of Alg2(ScAlg2p)was resolved ten years ago,however,the transmembrane prediction of human Alg2(hAlg2p)is completely different from that of ScAlg2 p.Therefore,we aimed to analyze the topological structure of hAlg2 p.In the preliminary experiments,we realized that hAlg2 p is mainly localized to the ER in HEK293 cells detected by immunoblotting and immunofluorescence.The transmembrane prediction and helical projection analysis of hAlg2 p showed that hAlg2 p possesses only one hydrophobic region which locates between amino acids 85-104 and it shows as amphiphilic-tendency ? helix.In this paper,molecular biological methods were used to further analyze the membrane topology of hAlg2 p,and it was proved that the N-terminus and C-terminus of hAlg2 p both face to the cytoplasmic side of the ER;the amphiphilic-like ? helix of hAlg2 p is the unique ER membrane-binding site.Compared with ScAlg2 p,hAlg2p exhibited higher in vitro activity which was independent of lipid,due to its simpler membrane topology.Additionally,the established activity assay of TrxA-hAlg2 can be used to identify the in vitro activity of ALG2-CDG related mutants.In S.cerevisiae,the synthesis of GPI anchor protein starts from the ER.Before further transport to the Golgi,the lipid part of GPI anchor protein undergoes remodeling.It is reported that the proteins encoded by PER1,GUP1 and CWH43 genes participate in GPI lipid remodeling.Here,we focused on the putative GPI remodeling pathway in the methylotrophic yeast Ogataea minuta.We found that the O.minuta homologs of PER1,GUP1,and CWH43 are functionally compatible with those of S.cerevisiae.Disruption of GUP1 or CWH43 in O.minuta caused a growth defect under non-permissive conditions.The O.minuta per1(35)mutant exhibited a more fragile phenotype than the gup1(35)or cwh43(35)mutants.To address the role of GPI modification in O.minuta,we assessed the effect of these mutations on the processing and localization of the O.minuta homologs of the Gas1 protein;in S.cerevisiae,Gas1 p is an abundant and well-characterized GPI-anchored protein.We found that O.minuta possesses two copies of the GAS1 gene,which we designate GAS1 A and GAS1 B.Microscopy and western blotting analysis showed mislocalization and/or lower retention of Gas1 Ap and Gas1 Bp within the membrane fraction in per1(35)or gup1(35)mutant cells,suggesting the significance of lipid remodeling for GPI-anchored proteins in O.minuta.Our data reveals,for the first time,the existence of genes related to GPI anchor remodeling in O.minuta cells.