O-glycosylation of a recombinant carbohydrate-binding module mutant secreted by Pichia pastoris.
Carbohydrate-binding modules (CBMs; previously called cellulose-binding domains) make excellent fusion partners for the immobilization or purification of polypeptides. However, their use in eukaryotic hosts has been limited by glycosylation, which interferes with the ability of the CBM to bind to cellulose. We have engineered the C-terminal carbohydrate-binding module from Cellulomonas fimi xylanase 10A such that it lacks N-glycosylation sites. This variant, called CBM2aNgly-, was produced and secreted by the methylotrophic yeast Pichia pastoris and found to be O-glycosylated. The O-linked glycans were composed entirely of mannose in a ratio of 1 mol of mannose to 4 mol of protein. The overall distribution of mannose on the O-glycosylated CBM mutant ranged from 1 to 9 mannose residues with the oligosaccharide sizes ranging from Man(1) to Man(4). MALDI-TOF (all matrix-assisted-laser-desorption time of flight) mass spectrometry (MS) was used to map the O-glycosylation to three regions of the polypeptide, each region having a maximum of 4 mannose residues attached to each. Glycans chemically released from CBM2aNgly- and analyzed by fluorophore-assisted carbohydrate electrophoresis were found to contain alpha-1,2-, alpha-1,3-, and alpha-1,6-linkages. Significantly, the O-glycosylation did not influence binding, making CBM2aNgly- a suitable fusion partner for polypeptides produced in P. pastoris and other eukaryotic hosts.
Boraston
Alisdair B
AB
The Protein Engineering Network of Centres of Excellence, PENCE Inc, Edmonton, Canada. boraston@ysbl.york.ac.uk
Sandercock
Linda  E
L
Warren
R Anthony J
RA
Kilburn
Douglas G
DG
D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D040143:Cellulomonas / Q000201:enzymology*; D002482:Cellulose / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D002482:Cellulose; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase