Post-translational modifications of the basic peroxidase isoenzyme from Zinnia elegans

Area of Science:

• Plant Biochemistry
• Proteomics
• Glycobiology

Background:

• Peroxidases play crucial roles in plant defense and cell wall metabolism.
• Understanding the post-translational modifications of plant peroxidases is essential for elucidating their functions.
• Zinnia elegans major basic peroxidase (ZePrx) is a key enzyme in plant systems.

Purpose of the Study:

• To purify and characterize the isoforms of Zinnia elegans major basic peroxidase (ZePrx).
• To investigate the co-translational and post-translational modifications of ZePrx, focusing on glycosylation.
• To explore the potential impact of these modifications on ZePrx function and interactions.

Main Methods:

• Purification of ZePrx isoforms from Zinnia elegans cell cultures.
• N-terminal sequencing by Edman degradation.
• Proteolytic digestion (trypsin) followed by RP-nanoLC-MALDI-TOF MS analysis.
• Bioinformatic modeling for proteolytic cleavage site prediction.

Main Results:

• Two ZePrx isoforms (ZePrx33.44 and ZePrx34.70) were purified.
• ZePrx undergoes co-translational processing of its N-terminal signal peptide by serine proteases.
• Post-translational modifications include N-terminal pyroglutamate formation, disulfide bridges, and complex N-glycosylation.
• Glycans exhibit diversity, leading to at least ten molecular isoforms with varying mannose-type and complex-type structures.
• N-glycosylation sites are located near the proximal histidine, potentially influencing enzyme activity and cell wall interactions.

Conclusions:

• ZePrx isoforms exhibit intricate post-translational modifications, particularly diverse N-glycosylation patterns.
• The identified modifications contribute to a complex ZePrx proteome, challenging direct structure-function relationship establishment.
• Glycosylation likely plays a significant role in modulating ZePrx's catalytic activity and its interactions within the cellular environment.