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Statements

Subject Item
n2:RIV%2F61989592%3A15310%2F12%3A33142674%21RIV13-MSM-15310___
rdf:type
skos:Concept n9:Vysledek
dcterms:description
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. 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.
dcterms:title
Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry
skos:prefLabel
Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry
skos:notation
RIV/61989592:15310/12:33142674!RIV13-MSM-15310___
n9:predkladatel
n10:orjk%3A15310
n3:aktivita
n15:I n15:P
n3:aktivity
I, P(ED0007/01/01), P(GA522/08/0555)
n3:cisloPeriodika
13
n3:dodaniDat
n6:2013
n3:domaciTvurceVysledku
n5:7905785 n5:5685303 n5:4945603 n5:9896821 n5:9708790
n3:druhVysledku
n11:J
n3:duvernostUdaju
n7:S
n3:entitaPredkladatele
n20:predkladatel
n3:idSjednocenehoVysledku
122184
n3:idVysledku
RIV/61989592:15310/12:33142674
n3:jazykVysledku
n16:eng
n3:klicovaSlova
Yarrowia lipolytica; mass spectrometry; N-glycosylation; glycan; endoglycosidase H; Cytokinin oxidase/dehydrogenase
n3:klicoveSlovo
n8:Yarrowia%20lipolytica n8:Cytokinin%20oxidase%2Fdehydrogenase n8:mass%20spectrometry n8:glycan n8:N-glycosylation n8:endoglycosidase%20H
n3:kodStatuVydavatele
NL - Nizozemsko
n3:kontrolniKodProRIV
[2BEEDD732D35]
n3:nazevZdroje
Journal of Proteomics
n3:obor
n18:CE
n3:pocetDomacichTvurcuVysledku
5
n3:pocetTvurcuVysledku
7
n3:projekt
n13:GA522%2F08%2F0555 n13:ED0007%2F01%2F01
n3:rokUplatneniVysledku
n6:2012
n3:svazekPeriodika
75
n3:tvurceVysledku
Končitíková, Radka Řehulka, Pavel Šebela, Marek Madzak, Catherine Franc, Vojtěch Lenobel, René Kopečný, David
n3:wos
000307025100019
s:issn
1874-3919
s:numberOfPages
11
n4:doi
10.1016/j.jprot.2012.05.013
n19:organizacniJednotka
15310