Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysis of its biological activity.
The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.
Saskiawan
Iwan
I
Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, Japan. iwan@lif.kyoto-u.ac.jp
Mizuno
Mamoru
M
Inazu
Toshiyuki
T
Haneda
Katsuji
K
Harashima
Satoshi
S
Kumagai
Hidehiko
H
Yamamoto
Kenji
K
D000117:Acetylglucosamine / Q000378:metabolism*; D000373:Agglutinins / Q000096:biosynthesis; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D000072235:Mating Factor; D009089:Mucor / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000373:Agglutinins; D010455:Peptides; D000072235:Mating Factor; D006026:Glycoside Hydrolases; D000117:Acetylglucosamine