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Articles by K. Fujiyama
Total Records ( 2 ) for K. Fujiyama
  H Kajiura , H Koiwa , Y Nakazawa , A Okazawa , A Kobayashi , T Seki and K. Fujiyama
 

N-Glycosylation is an important post-translational modification that occurs in many secreted and membrane proteins in eukaryotic cells. Golgi -mannosidase I hydrolases (MANI) are key enzymes that play a role in the early N-glycan modification pathway in the Golgi apparatus. In Arabidopsis thaliana, two putative MANI genes, AtMANIa (At3g21160) and AtMANIb (At1g51590), were identified. Biochemical analysis using bacterially produced recombinant AtMANI isoforms revealed that both AtMANI isoforms encode 1-deoxymannojirimycin-sensitive -mannosidase I and act on Man8GlcNAc2 and Man9GlcNAc2 structures to yield Man5GlcNAc2. Structures of hydrolytic intermediates accumulated in the AtMANI reactions indicate that AtMANIs employ hydrolytic pathways distinct from those of mammalian MANIs. In planta, AtMANI-GFP/DsRed fusion proteins were detected in the Golgi stacks. Arabidopsis mutant lines manIa-1, manIa-2, manIb-1, and manIb-2 showed N-glycan profiles similar to that of wild type. On the other hand, the manIa manIb double mutant lines produced Man8GlcNAc2 as the predominant N-glycan and lacked plant-specific complex and hybrid N-glycans. These data indicate that either AtMANIa or AtMANIb can function as the Golgi -mannosidase I that produces the Man5GlcNAc2 N-glycan structure necessary for complex N-glycan synthesis.

  H Kajiura , T Seki and K. Fujiyama
 

The core oligosaccharide Glc3Man9GlcNAc2 is assembled by a series of membrane-bound glycosyltransferases as the lipid carrier dolichylpyrophosphate-linked glycan in the endoplasmic reticulum (ER). The first step of this assembly pathway on the ER luminal side is mediated by ALG3 (asparagine-linked glycosylation 3), which is a highly conserved reaction among eukaryotic cells. Complementary genetics compared with Saccharomyces cerevisiae ALG gene families and bioinformatic approaches have enabled the identification of ALG3 from other species. In Arabidopsis thaliana, AtALG3 (At2g47760) was identified as 1,3-mannosyltransferase. Complementation analysis showed that AtALG3 rescued the temperature-sensitive phenotype, that lipid-linked oligosaccharide assemblies and that protein underglycosylation of S. cerevisiae ALG3-deficient mutant. In Arabidopsis ALG3 mutant, an immature lipid-linked oligosaccharide structure, M5ER, was synthesized, and used for protein N-glycosylation, resulting in the blockade of subsequent maturation with the concanavalin A affinoactive and Endo H-insensitive structure. N-Glycan profiling of total proteins from alg3 mutants exhibited a unique structural profile, alg3 has rare N-glycan structures including Man3GlcNAc2, M4ER, M5ER and GlcM5ER, which are not usually detected in Arabidopsis, and a much less amount of complex-type N-glycan than that in wild type. Interestingly, despite protein N-glycosylation differences compared with wild type, alg3 showed no obvious phenotype under normal and high temperature or salt/osmotic stress conditions. These results indicate that AtALG3 is a critical factor for mature N-glycosylation of proteins, but not essential for cell viability and growth in Arabidopsis.

 
 
 
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