Marek Šebela1, Radka Končitíková1, René Lenobel2, Vojtěch Franc1, Pavel Řehulka3, Martina Tylichová1, David Kopečný1


1Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic;

2Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany AS CR, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic;

3Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, Třebešská 1575, CZ-500 01 Hradec Králové, Czech Republic


Cytokinin oxidase/dehydrogenase (CKO; EC catalyzes the metabolic degradation of the plant hormones cytokinins. The presence of a glycosylation in maize CKO isoenzyme 1 (ZmCKO1) was originally deduced from its binding to lectin affinity columns [1]. This was further confirmed by electrophoretic migration, which provided significantly higher molecular mass estimates than was a theoretical calculation. The amino acid sequence of ZmCKO1 comprises eight potential N-glycosylation sites [2]. The published crystal structures have shown that at least six of them may be occupied by glycans attached at the residues N63, N89, N134, N294, N323 and N338 [3,4]. However, the composition and size of the carbohydrate chains have not yet been investigated as well as their contribution to the activity and stability of the enzyme. In this work, a recombinant ZmCKO1 expressed in Yarrowia lipolytica was treated by endoglycosidase H, which resulted in a complete deglysosylation (except for an expected preservation of one N-acetyl-D-glucosamine per each glycosylation site). The released N-glycans were purified by reversed phase chromatography on graphitized carbon and subjected to MALDI-TOF MS using 6-azathiothymine as a matrix. Sodium and potassium adduct peaks were observed in the positive ion reflectron mode. The sodium adduct peaks appeared in a ladder starting at m/z 1216, which continued gradually by adding a mass difference of 162 units at least to m/z 2676. By means of a simple calculation and search in a library of known structures (, the peaks could be assigned to high-mannose type N-glycans from Man6GlcNAc to Man15GlcNAc. On a Q-TOF instrument with electrospray ionization (ESI), the same N-glycans were detected predominantly as doubly charged ions [M+H+K]2+. Several structures were confirmed by collision-induced tandem mass spectrometry (CID-MS/MS). A tryptic digest of ZmCKO1 was then prepared to analyze N-glycopeptides directly by liquid chromatography coupled to mass spectrometry. The digest was separated on a reversed phase column with gradient elution and detected by Q-TOF ESI-MS. In CID-MS/MS, a double charged ion with m/z 1267.53 ([M+2H]2+) yielded a peptide fragmentation pattern corresponding to the sequence 133-INVSADGR-140 binding two N-acetyl-D-glucosamines plus 8 mannoses. The final proof of the presence of high-mannose type N-glycans in recombinant ZmCKO1 was obtained by MALDI-TOF MS of the tryptic digest using ferulic acid as a matrix. A series of glycopeptides was observed with m/z values spanning the interval of 7986-12360 and differing from each other by 162 mass units. Calculations allowed assigning of the measured glycopeptides to the tryptic peptide 269-LTAPRPGGGGASFGPMSYVEGSVFVNQSLATDLANTGFFTDADVAR-313 bearing large N-glycan chains from Man20GlcNAc2 to Man45GlcNAc2 (indicating a hyperglycosylation.) The enzyme deglycosylated under non-denaturing conditions showed decreased activity as well as thermostability indicating the physiological importance of the carbohydrate chains in ZmCKO1.


[1] J.M. Chatfield & D.J. Armstrong, Plant Physiol. 88 (1988), 245-247.

[2] N. Houba-Hérin, C. Pethe, J. d'Alayer, M. Laloue, Plant J. 17 (1999) 615-626.

[3] E. Malito, A. Coda, K.D. Bilyeu, M.W. Fraaije, A. Mattevi, J. Mol. Biol. 341 (2004) 1237-1249.

[4] D. Kopečný, P. Briozzo, H. Popelková, M. Šebela, R. Končitíková, L. Spíchal, J. Nisler, C. Madzak, I. Frébort, M. Laloue, N. Houba-Hérin, Biochimie 92 (2010) 1052-1062.


This work was supported by grant 522/08/0555 from the Czech Science Foundation.