Mass spectrometric characterization of extensin cross-linkages in arabidopsis tissue culture cell walls

Abstract

In this work we characterize and identify tyrosine-tyrosine cross-linkages occurring in the Arabidopsis extensin scaffold. Cross-linked extensin peptides were solubilized out of Arabidopsis tissue culture cell walls using chemical and enzymatic methods adapted from Qi et al. The resulting peptides were analyzed by liquid chromatography mass spectrometry (LC-MS/MS). Peptides derived from four hydroxyproline-rich extensins and one leucine-rich extensin were identified. Four different types of tyrosine cross-linkages were identified and characterized. One intramolecular linkage was identified: isodityrosine. This cross-linkage occurred at the amino acid sequence yXy (where y is a crosslinked tyrosine, and X is any amino acid). Three intermolecular linkages between two peptides were also identified: dityrosine, pulcherosine, and di-isodityrosine made up of two, three, and four tyrosines respectively. This is the first identification and characterization of cross-linkages formed in the extensin scaffold in vivo. These tyrosine containing cross-linkages corroborate earlier suggestions that the extensin scaffold is a highly cross-linked network in muro.