Studies on the glycosylation of wild-type and mutant forms of Aspergillus niger pectin methylesterase.
Pectin methylesterase (PME) is one of a number of enzymes released by the fungus Aspergillus niger that are involved in the degradation of specific plant cell-wall structures. PME is a glycoprotein with three potential sites for N-linked glycosylation. The glycosylation may affect the hydrolytic activity or the substrate specificity of PME. In this work, we investigate first the structures and the attachment sites of the glycans present on recombinant wild-type PME. Further, a series of PME mutants was created in which the three potential N-linked glycosylation sites were eliminated in all possible combinations. The glycosylation of the mutants and their activities were then studied. Mass spectrometric techniques tailored for carbohydrate analysis were applied to both characterize the glycan structures and to determine the specific sites of attachment. High mannose structures with variable numbers of mannose were found on the wild-type, as well as the mutant forms. Studies using the mutants suggest that glycosylation does not strongly influence the activity. Whether it may affect the substrate specify of the enzyme is unknown, and that aspect will be explored in future work.
Warren
Maria E
ME
Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road, Athens 30602-4712, USA.
Kester
Harry
H
Benen
Jacques
J
Colangelo
Jennifer
J
Visser
Jaap
J
Bergmann
Carl
C
Orlando
Ron
R
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D008358:Mannose / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011134:Polysaccharides / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011134:Polysaccharides; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D008358:Mannose