Molecular cloning and characterization of Arabidopsis thaliana Golgi α‐mannosidase II, a key enzyme in the formation of complex N‐glycans in plants

• Published in: The Plant journal : for cell and molecular biology 2006-03, Vol.45 (5), p.789-803
• Main Authors: Strasser, Richard, Schoberer, Jennifer, Jin, Chunsheng, Glössl, Josef, Mach, Lukas, Steinkellner, Herta
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Amino Acid Sequence; Animals; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis thaliana; Biological and medical sciences; Classical and quantitative genetics. Population genetics. Molecular genetics; Fundamental and applied biological sciences. Psychology; Generalities. Genetics. Plant material; Genetics and breeding of economic plants; Glycopeptides; glycosyl hydrolase; Golgi Apparatus - metabolism; Golgi mannosidase II; Golgi targeting; Mannosidases - metabolism; Molecular genetics; Molecular Sequence Data; Mutagenesis, Insertional; N‐glycosylation; Oligosaccharides, Branched-Chain - metabolism; Sequence Analysis, DNA; Spodoptera - genetics; Substrate Specificity; Transfection
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2005.02648.x

Summary N‐glycosylation is one of the major post‐translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N‐glycan precursors leading to hybrid‐type and finally complex‐type N‐glycans are not fully understood in plants. To investigate the role of Golgi α‐mannosidase II (GMII) in the formation of complex N‐glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N‐glycans. Furthermore, a detailed N‐glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N‐glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N‐glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N‐glycans to complex N‐glycans exist in plants. Transient expression of N‐terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.