The isolation and characterization of a tobacco extensin precursor and two Arabidopsis hydroxyproline-rich glycoproteins.

摘要

Hydroxyproline rich glycoproteins (HRGPs) are structural proteins found in all plant species. The HRGP superfamily can be split into three different subgroups: proline-rich proteins, arabinogalactan proteins, and extensins. In the case of this research, one extensin precursor from tobacco NtP1 as well as an extensin precursor and an arabinogalactan protein from Arabidopsis were isolated and characterized. Extensins are components contributing to the primary cell wall architecture. They form porous covalently cross-linked networks in the wall during normal cell growth and in response to pathogens and stress. NtP1 was characterized as an abundant HRGP salt eluted from the cell surface of tobacco cell suspension cultures (Bright Yellow 2) and characterized by the glycosylation profiles, crosslinking analysis and HRGP peptide sequences from the insoluble wall network. NtP1's main saccharides are arabinose (Ara) and galactose (Gal). A hydroxyproline arabinoside (Hyp-Ara) profile revealed tobacco P1 to be mainly Hyp-Ara4, with lesser amounts of Hyp-Ara 3, Hyp-Ara2, Hyp-Ara1, and little nonglycosylated Hyp. Isodityrosine (IDT) assays and that the protein has the ability to form intermolecular crosslinks. Amino acid composition analysis revealed the protein consisted mainly of hydroxyproline, tyrosine, and lysine, with smaller amounts of threonine, serine, proline, valine, histidine, and alanine. Peptide mapping and sequence analysis of the major peptides yielded the following sequences: Ser-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys and Lys-Pro-Tyr-Tyr-Pro-Hyp-His-Thr-Hyp-Val-Tyr-Lys and identified the protein as a P1 type cross-linking extensin. AtHRGP and Ara P were salt eluted from the cell surface of an Arabidopsis suspension culture and purified by a combination of gel filtration chromatography on Superose 6 and reverse phase chromatography on a Hamilton PRP-1 column. Each of the proteins were characterized by amino acid composition analysis, glycosylation profiles, crosslinking analysis, and analysis of the insoluble walls for crosslinked peptides. AtHRGP coprecipitated with the Yariv reagent and had a glycosyl composition analysis rich in Gal, Ara and uronic acids consistent with an AGP. Ara P characterization resulted in its identification as an extensin via the sugar composition of mainly Ara and Gal, Hyp-Ara profile, and amino acid composition.