The major basic peroxidase (ZePrx) from Zinnia elegans suspension cell cultures was purified and cloned. The purification resolved ZePrxs in two isoforms (ZePrx33.44 and ZePrx34.70), whose co-translational and post-translational modifications are characterized. Based on the N-terminal sequence obtained by Edman degradation of mature ZePxs, it may be expected that the immature polypeptides of ZePrxs contain a signal peptide (N-terminal pro-peptide) of 30 amino acids, which directs the polypeptide chains to the ER membrane. These immature polypeptides are co-translationally processed by proteolytic cleavage, and modeling studies of digestions suggested that the processing of the N-terminal pro-peptide of ZePrxs is performed by a peptidase from the SB clan (S8 family, subfamily A) of serine-type proteases. When the post-translational modifications of ZePrxs were characterized by trypsin digestion, and tryptic peptides were analyzed by reverse phase nano liquid chromatography (RP-nanoLC) coupled to MALDI-TOF MS, it was seen that, despite the presence in the primary structure of the protein of several (disulphide bridges, N-glycosylation, phosphorylation and N-myristoylation) potential post-translational modification sites, ZePrxs are only post-translationated modified by the formation of N-terminal pyroglutamate residues, disulphide bridges and N-glycosylation. Glycans of ZePrxs belong to three main types and conduce to the existence of at least ten different molecular isoforms. The first glycans belong to both low and high mannose-type glycans, with the growing structure Man3–9(GlcNAc)2. Low mannose-type glycans, Man3–4(GlcNAc)2, coexist with the truncated (paucimannosidic-type) glycan, Man3Xyl1Fuc1(GlcNAc)2, in the G3 and G4 sub-isoforms of ZePrx33.44. In ZePrx34.70, on the other hand, the complex-type biantennary glycan, Man3Xyl1Fuc3(GlcNAc)5, and the truncated (paucimannosidic-type) glycan, Man3Xyl1Fuc1(GlcNAc)2, appear to fill the two putative sites for N-glycosylation. Since the two N-glycosylation sites in ZePrxs are located in an immediately upstream loop region of helix F′′ (close to the proximal histidine) and in helix F′′ itself, and are flanked by positive-charged amino acids that produce an unusual positive-net surface electrostatic charge pattern, it may be expected that glycans not only affect reaction dynamics but may well participate in protein/cell wall interactions. These results emphasize the complexity of the ZePrx proteome and the difficulties involved in establishing any fine structure-function relationship.
Plant Molecular Biology – Springer Journals
Published: Jun 22, 2007
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