Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
22634084
DOI
10.1016/j.jprot.2012.05.013
PII: S1874-3919(12)00301-6
Knihovny.cz E-resources
- MeSH
- Glycosylation MeSH
- Cloning, Molecular MeSH
- Zea mays enzymology MeSH
- Molecular Sequence Data MeSH
- Oxidoreductases metabolism MeSH
- Polysaccharides analysis MeSH
- Recombinant Proteins isolation & purification MeSH
- Amino Acid Sequence MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Enzyme Stability MeSH
- Tandem Mass Spectrometry MeSH
- Yarrowia enzymology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- cytokinin oxidase MeSH Browser
- Oxidoreductases MeSH
- Polysaccharides MeSH
- Recombinant Proteins MeSH
Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.
References provided by Crossref.org
Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce
Characterization of a long-chain α-galactosidase from Papiliotrema flavescens
