Secretion of glycosylated alpha-lactalbumin in yeast Pichia pastoris.
The secretion of N-linked glycosylated alpha-lactalbumin was much higher in the expression system of yeast Pichia pastoris carrying goat alpha-lactalbumin cDNA than in mammalian milk. This is possibly because of the presence of N-linked glycosylation signal sequences, Asn(45)-Asp(46)-Ser(47) and Asn(74)-Ile(75)-Ser(76), in wild-type alpha-lactalbumin. Attempts to elucidate the mechanism of the higher secretion of glycosylated alpha-lactalbumin in P. pastoris were made. Mutant N45D that deleted the N-linked glycosylation signal sequence at position 45 predominantly secreted nonglycosylated protein. On the other hand, mutant D46N with another N-glycosylation signal site at position 46 only secreted N-linked glycosylated alpha-lactalbumin, i.e. not the nonglycosylated protein. The total secreted amount of mutant N45D was greatly enhanced, while the secreted amounts of the wild-type and mutant D46N were very low, suggesting that the increase in the number of glycosylation sites greatly reduced the secretion of alpha-lactalbumin. It seems likely that the glycosylated alpha-lactalbumin may be degraded by the quality control system.
Saito
Akira
A
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan.
Usui
Masakatsu
M
Song
Youtao
Y
Azakami
Hiroyuki
H
Kato
Akio
A
D002241:Carbohydrates / Q000737:chemistry; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D007768:Lactalbumin / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D010843:Pichia / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D002241:Carbohydrates; D017931:DNA Primers; D011994:Recombinant Proteins; D007768:Lactalbumin