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

Abstract

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 Man(3-9)(GlcNAc)(2). Low mannose-type glycans, Man(3-4)(GlcNAc)(2), coexist with the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(GlcNAc)(2), in the G(3) and G(4 )sub-isoforms of ZePrx33.44. In ZePrx34.70, on the other hand, the complex-type biantennary glycan, Man(3)Xyl(1)Fuc(3)(GlcNAc)(5), and the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(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.

Specific functions of individual class III peroxidase genes

“…Beside, because of different post-translational modifications, often more than one protein form (isoform) originates from a particular gene (e.g. Gabaldon et al, 2007;Laugesen et al, 2007).…”

“…In the case of the cationic peanut peroxidase, site-directed removal of each of the three N-linked complex glycans revealed that the N-60 and N-144 glycans influence the peroxidase catalytic activity, whereas the N-185 glycan is important for the thermostability of the enzyme (Lige et al, 2001). It has been postulated that glycans could affect substrate access because of their large size (Douroupi et al, 2005;Gabaldon et al, 2007), and could affect reaction dynamics due to a dampening of backbone motion (Nielsen et al, 2001).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Deduced structures (see text) (Gabaldon, et al, 2007) Peroxidase Hordeum vulgare (Barley) Trypsin, R-TOF, Glycopeptides…”

“…As an alternative to working with the intact glycopeptide, the mass of a glycan can be determined by comparing the masses of a glycopeptide before and after deglycosylation. As an example of the latter technique, glycan masses and constituent monosaccharide compositions from peroxidise extracted from the plant Zinnia elegans have been deduced from measurements of tryptic glycopeptides before and after deglycosylation with trifluoromethanesulfonic acid (TFMS) (Gabaldon et al, 2007). By the application of rules such as the conserved nature of the constituent monosaccharides and the known presence of the trimannosylchitobiose core, together with information on known structures from plants, the authors were able to propose the presence of low-mannose (Man 3-4 GlcNAc 2 ) and paucimannosidic structures.…”

Hormonal regulation of the basic peroxidase isoenzyme from Zinnia elegans

“…Based on the N-terminal sequence obtained by Edman degradation, it has been concluded that the immature polypeptide of ZePrx contains a signal peptide (N-terminal pro-peptide) of 30 amino acids, which directs the polypeptide chain to the ER membrane and later to the default pathway (Gabaldón et al 2007). The immature polypeptide of ZePrx is co-translationally processed by proteolytic cleavage, and modeling studies of digestions suggest that the processing of the N-terminal pro-peptide of ZePrx is performed by a peptidase from the SB clan (S8 family, subfamily A) of serine-type proteases (Gabaldón et al 2007).…”