Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome.
Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.
Croes
Evi
E
Laboratory of Food Chemistry and Biochemistry, Department of Microbial and Molecular systems, Katholieke Universiteit Leuven, Leuven, Belgium.
Gebruers
Kurt
K
Robben
Johan
J
Noben
Jean-Paul
JP
Samyn
Bart
B
Debyser
Griet
G
Van Beeumen
Jozef
J
Delcour
Jan A
JA
Courtin
Christophe M
CM
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000494:pharmacology*; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006031:Glycosylation; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000494:pharmacology*; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000737:chemistry*
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D010940:Plant Proteins; D020543:Proteome; C422246:TAXI I protein, Triticum aestivum; C485063:XIP-I protein, Triticum aestivum; D043364:Endo-1,4-beta Xylanases