title: Unusual N-glycan structures in alpha-mannosidase II/IIx double null embryos identified by a systematic glycomics approach based on two-dimensional LC mapping and matrix-dependent selective fragmentation method in MALDI-TOF/TOF mass spectrometry.
abstract: alpha-Mannosidase IIx (MX) is an enzyme closely related to alpha-mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid- to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T. O., Nakagawa, H., Wong, N. K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M. N. (2006) Essential and mutually compensatory roles of alpha-mannosidase II and alpha-mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983-8988). In the present study, we determined minor but unusual N-glycan structures found in MII/MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. (2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097-6101). Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls, and wild-type mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.
authors.affiliation: Laboratory of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Hato
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Kurogochi
authors.forename: Tomoya O
authors.initials: TO
authors.lastname: Akama
authors.forename: Jamey D
authors.initials: JD
authors.lastname: Marth
authors.forename: Michiko N
authors.initials: MN
authors.lastname: Fukuda
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D043323:alpha-Mannosidase / Q000235:genetics*
chemical_list: D011134:Polysaccharides; D043323:alpha-Mannosidase