Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry

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.

Cartoon representation of ZmCKO1 molecule (PDB 3C0P). Six glycosylated Asn sites and FAD cofactor, both shown in spheres are colored in red and yellow, respectively. Black ellipse indicates the entrance to the active site.Figure optionsDownload high-quality image (141 K)Download as PowerPoint slideHighlights
► The N-glycosylation pattern of a recombinant maize cytokinin oxidase/dehydrogenase 1 (ZmCKO1) was studied.
► N-linked high-mannose glycans were released from the enzyme by endoglycosidase H treatment and their structure confirmed.
► N-glycopeptides were separated from tryptic digests by reversed-phase chromatography using a simple microgradient device.
► MALDI-TOF/TOF tandem mass spectrometry of N-glycopeptides demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338.
► The presence of large N-glycans at Asn338 (more than 25 mannoses) indicated a partial hyperglycosylation of the enzyme.