Dextranase (alpha-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation.
Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.
Betancourt
L H
LH
Center for Genetic Engineering and Biotechnology, Havana, Cuba.
García
R
R
González
J
J
Montesino
R
R
Quintero
O
O
Takao
T
T
Shimonishi
Y
Y
Cremata
J A
JA
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003910:Dextranase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003910:Dextranase