title: 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.
authors.affiliation: Department of Plant Biology, University of Murcia, 30100, Murcia, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Gabaldón
authors.forename: Laura V
authors.initials: LV
authors.lastname: Gómez-Ros
authors.forename: María J López
authors.initials: MJ
authors.lastname: Núñez-Flores
authors.forename: Alberto
authors.initials: A
authors.lastname: Esteban-Carrasco
authors.forename: Alfonso Ros
authors.initials: AR
authors.lastname: Barceló
mesh_terms: D000595:Amino Acid Sequence; D019659:Asteraceae / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity; D040681:Structural Homology, Protein; D013438:Sulfhydryl Compounds / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D011134:Polysaccharides; D013438:Sulfhydryl Compounds; D009195:Peroxidase