Comprehensive glycan analysis of recombinant Aspergillus niger endo-polygalacturonase C.
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.
Woosley
Bryan
B
Complex Carbohydrate Research Center and Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
Xie
Min
M
Wells
Lance
L
Orlando
Ron
R
Garrison
Derek
D
King
Daniel
D
Bergmann
Carl
C
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D002853:Chromatography, Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D037102:Lectins / Q000032:analysis; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011096:Polygalacturonase / Q000737:chemistry* / Q000235:genetics; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D037102:Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins; D011096:Polygalacturonase; D004229:Dithiothreitol