STRUCTURAL AND FUNCTIONAL STUDY ON THE
GLYCOSYLATION IN MAIZE CYTOKININ OXIDASE/DEHYDROGENASE 1
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 220.127.116.11) 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 . 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 . 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 (http://www.expasy.ch/tools/glycomod), 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.
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This work was supported by grant 522/08/0555 from the Czech Science Foundation.