ABSTRACT
Polygalacturonase inhibiting proteins (PGIPs) are members of the leucine rich repeat family of proteins, involved in plant defense against fungal pathogens. PGIPs exhibit a remarkable degree of specificity in terms of their ability to bind and inhibit their target molecules, the endopolygalacturonases (EPGs). This specificity has been attributed for certain EPG/PGIP combinations to differences in primary sequence, but this explanation is unable to account for the full range of binding and inhibitory activities observed. In this paper, we have fully characterized the glycosylation on the PGIP derived from Pyrus communis and demonstrated, using a combination of PNGaseF and PNGaseA in (18)O-water, that the Pyrus communis PGIP utilizes all seven potential sites of N-linked glycosylation. Further, we demonstrate that certain sites appear to be modified only by glycans bearing alpha3-linked core fucosylation, while others are occupied by a mixture of fucosylated and nonfucosylated glycans. Modeling of the carbohydrates onto a homologous structure of PGIP indicates potential roles for glycosylation in mediating the interactions of PGIPs with EPGs.
Subject(s)
Plant Proteins , Polygalacturonase/chemistry , Polygalacturonase/metabolism , Polysaccharides/analysis , Pyrus , Antifungal Agents/chemistry , Antifungal Agents/metabolism , Carbohydrate Conformation , Carbohydrate Sequence , Glycosylation , Models, Molecular , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/metabolism , Polygalacturonase/genetics , Protein Conformation , Pyrus/chemistry , Pyrus/enzymologyABSTRACT
The enzyme PGC is produced by the fungus Aspergillus niger during invasion of plant cell walls. The enzyme has been homologously overexpressed to provide sufficient quantities of purified enzyme for biological studies. We have characterized this enzyme in terms of its posttranslational modifications (PTMs) and found it to be both N- and O-glycosylated. The glycosyl moieties have also been characterized. This has involved a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), liquid chromatography (LC)-ion trap, and LC-electrospray ionization (ESI) mass spectrometries in conjunction with trypsin degradation and beta-elimination, followed by Michael addition with dithiothreitol (BEMAD). This is the first demonstration of the ability of BEMAD to map glycosylation sites other than O-GlcNAc sites. The complete characterization of all PTMs on PGC allows us to model them on the peptide backbone, revealing potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.
