Structure of the Threonine-Rich Extensin from Zea mays (original) (raw)

Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete posttranslational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the 'P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains altemate with high regularity. The THRGP is not in the polyproline-11 conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HFsolvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot. In 1987, we reported (15) the first isolation and partial characterization of a THRGP2 and HRGP from a gramina-'Supported by the U.S. Department of Agriculture grant No. 88-37261-3682 and the U.S. Department of Energy contract No. DE-AC02-76ERO-1 338. 2 Abbreviations: THRGP, threonine-hydroxyproline-rich glycoprotein; HRGP, hydroxyproline-rich glycoprotein; dTHRGP, deglycosylated threonine-hydroxyproline-rich glycoproteins; dw, dry weight; P1, glycosylated tomato extensin type 1; dPI, deglycosylated tomato extensin type P1; P2, glycosylated tomato extensin type 2; dP2, deglycosylated tomato extensin type 2; HHRGP, histidinehydroxyproline-rich glycoprotein; dHHRGP, deglycosylated histidine-hydroxyproline-rich glycoprotein; HF, anhydrous hydrogen fluoride; OPA, orthophthalaldehyde; HFBA, hepta-fluorobutyric acid; AGP, arabinogalactan protein; AP, alkaline phosphatase. CD, circular dichroism; ABTS, 2,2'-Azino-bis(3-ethylbenzthiazinoline-6-sulfonic acid).