Two Arabidopsis thaliana Golgi alpha-mannosidase I enzymes are responsible for plant N-glycan maturation.
N-Glycosylation is an important post-translational modification that occurs in many secreted and membrane proteins in eukaryotic cells. Golgi alpha-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 alpha-mannosidase I and act on Man(8)GlcNAc(2) and Man(9)GlcNAc(2) structures to yield Man(5)GlcNAc(2). 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 Man(8)GlcNAc(2) 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 alpha-mannosidase I that produces the Man(5)GlcNAc(2) N-glycan structure necessary for complex N-glycan synthesis.
Kajiura
Hiroyuki
H
International Center for Biotechnology, Osaka University, Suita-shi, Osaka 565-0871, Japan.
Koiwa
Hisashi
H
Nakazawa
Yoshihisa
Y
Okazawa
Atsushi
A
Kobayashi
Akio
A
Seki
Tatsuji
T
Fujiyama
Kazuhito
K
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D006056:Golgi Apparatus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013696:Temperature
D011134:Polysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase