Reduced efficiency in the glycosylation of the first sequon of Saccharomyces cerevisiae exoglucanase leads to the synthesis and secretion of a new glycoform of the molecule.
In addition to exoglucanases (EXGs) I and II, old cultures of Saccharomyces cerevisiae secreted into the culture medium a new immunologically-related material that exhibited exoglucanase activity. The new exoglucanase (EXGII1/2) was purified from stationary-phase cultures. It turned out to be a glycoprotein whose protein portion was identical to that of the other two isoenzymes in terms of ionic properties, size, amino acid composition and NH2-terminal sequence (25 residues). Disruption of the structural gene encoding EXGs I and II resulted in a strain unable to secrete all three isoenzymes. EXGII1/2 was indistinguishable in terms of molecular weight from the single intermediate detected during the deglycosylation (mediated by endo H) of EXGII by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, the new isoenzyme contains only one of the two slightly elongated mannan inner cores present in enzyme II. Two intermediates were, however, detected when the deglycosylation of EXGII was monitored by ion-exchange chromatography (high-pressure liquid chromatography). Site-directed mutagenesis indicated that the major intermediate, which eluted at about the same position as enzyme II1/2, corresponded to protein molecules carrying the oligosaccharide attached to the Asn of the second sequon, whereas the minor one carried the oligosaccharide in the first potential glycosylation site. Several lines of evidence indicate that EXGII1/2 is a biosynthetic product resulting from an imbalance between the rate of protein synthesis and the glycosylation capabilities of the glycosylation machinery.
Basco
R D
RD
Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Muñoz
M D
MD
Hernández
L M
LM
Vazquez de Aldana
C
C
Larriba
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D006031:Glycosylation; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D007527:Isoenzymes; D001617:beta-Glucosidase