title: Identification of the pI 4.6 extensin peroxidase from Lycopersicon esculentum using proteomics and reverse-genomics.
abstract: The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Lycopersicon esculentum), insolubilization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, we have identified tomato EP candidates using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP candidates, which contained a putative signal sequence and a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated (CG5). To test for EP crosslinking activity, we cloned into a bacterial expression vector the CG5 open-reading frame from tomato cDNA. The CG5 was expressed in Escherichia coli, fractionated from inclusion bodies, and folded in vitro. The peroxidase activity of CG5 was assayed and quantified by ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting our hypothesis that CG5 encodes a tomato EP.
authors.affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, United States. Electronic address: wd113009@ohio.edu.
authors.forename: Wen
authors.initials: W
authors.lastname: Dong
authors.affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, United States. Electronic address: kielisze@ohio.edu.
authors.forename: Marcia
authors.initials: M
authors.lastname: Kieliszewski
authors.affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, United States. Electronic address: held@ohio.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Held
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology; D006023:Glycoproteins / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism*; D017510:Protein Folding; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
chemical_list: D006023:Glycoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C005488:extensin protein, plant; D010544:Peroxidases
keywords: Extensin; Hydroxyproline-rich glycoproteins; Peroxidase; Plant cell wall; Protein crosslinking