In vivo glycosylation suppresses the aggregation of amyloidogenic hen egg white lysozymes expressed in yeast.
The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae. The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild-type protein. To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N-glycosylation signal sequence (Asn-X-Ser) by the substitution of glycine with asparagine at position 49. The secretion of these glycosylated mutant proteins is greatly increased in S. cerevisiae, compared with that of non-glycosylated type. Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37 degrees C whereas the non-glycosylated proteins eventually lost all activity under these conditions. These results suggest that the glycosylated chains could mask the beta-strand of amyloidogenic lysozymes from the intermolecular cross-beta-sheet association, thus improving the solubility of amyloidogenic lysozymes.
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan.
Y
Y
Song
H
H
Azakami
M
M
Hamasu
A
A
Kato
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D002645:Chickens; D004527:Egg Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D000682:Amyloid; D002241:Carbohydrates; D004527:Egg Proteins; D011994:Recombinant Proteins; D009113:Muramidase