Tomato extensin precursors P1 and P2 are highly periodic structures

Abstract

Intact cultured tomato cells eluted with dilute salt solutions yielded two soluble precursors (P1 and P2) to the bound extensin network. HF-deglycosylation of P1 and P2 (with methanol as scavenger) followed by tryptic degradation and HPLC on Hamilton PRP-1, gave unique tryptic peptide maps. The P1 map consisted predominantly of the deca- and hexadecapeptides, H5 and H20: (P1-H5) Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, and (H20) Ser-Hyp-Hyp-Hyp-Hyp-Val-Lys-Pro-Tyr-His-Pro-Thr-Hyp-Val-Tyr-Lys; the P2 map consisted almost entirely of the di- and octapeptides, H3 and H4: (P2-H3) Tyr-Lys; (H4) Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys, and small amounts of a closely related decapeptide containing intramolecularly-linked isodityrosine: (H11) Ser-Hyp-Hyp-Hyp-Hyp-Val- 1 2 IDT-Lys- 1 2 IDT-Lys. Both P1 and P2 are therefore highly periodic structures: P1 consists to a considerable extent of repeated H5 and H20 peptide blocks, while P2 may consist entirely of a single repeating decapeptide Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys-Tyr-Lys with occasional isoleucine for valine substitutions, and varying only in the extent of intramolecular IDT formation, which could stiffen the molecular rod. In P1, single tyrosine residues occur within the hexapeptide Val-Lys-Pro-Tyr-His-Pro region ofH20. This nonglycosylated region is sterically unhindered. Assuming a polyproline II conformation, all the basic residues of H20 lie in the same plane (available for pectic binding), while the hexapeptide tyrosine residue lies out of that plane and is therefore a prime candidate for the postulated intermolecular isodityrosine crosslink. Our sequences also reveal fundamental tetrapeptide and tripeptide periodicities of tomato extensin precursors: the contiguous decamers and hexadecamers consist of hydroxyproline tetrapeptides separated by a relatively few tripeptide sequences, (notably Tyr-Lys-Ser, Val-Tyr-Lys, Thr-Hyp-Val, Val-Lys-Pro, and Tyr-His-Pro). Furthermore, the presence of the pentapeptide Ser-Hyp-Hyp-Hyp-Hyp in all the repeat peptides shows that extensin, like many synthetic block copolymers, consists of relatively rigid domains (glycosylated Ser-Hyp-Hyp-Hyp-Hyp) separated by intervening (non-glycosylated) flexible spacers. Thus extensin is well-designed for its suggested role in mechanically coupling the cellulosic load-bearing polymers of the primary cell wall through formation of a network of defined porosity.