title: Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase.
abstract: Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.
authors.affiliation: Friedrich Miescher-Institute, Basel, Switzerland.
authors.forename: C W
authors.initials: CW
authors.lastname: Basse
authors.forename: K
authors.initials: K
authors.lastname: Bock
authors.forename: T
authors.initials: T
authors.lastname: Boller
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D010944:Plants; D011134:Polysaccharides / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D043324:beta-Fructofuranosidase
chemical_list: D006020:Glycopeptides; D011134:Polysaccharides; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D002918:Chymotrypsin; D008358:Mannose