Expression of yeast invertase in oocytes from Xenopus laevis. Secretion of active enzyme differing in glycosylation.
In an effort to understand factors that control glycosylation of proteins and processing of carbohydrate chains, invertase from Saccharomyces cerevisiae was expressed in a heterologous system. Microinjection of invertase-specific in vitro transcripts into oocytes from Xenopus laevis resulted in synthesis, glycosylation and secretion of enzymatically active invertase. It was found that although the number of carbohydrate chains acquired is the same as in yeast, the carbohydrate processing is different. This is consistent with the notion that the usage of a glycosylation site is determined by the protein part, whereas subsequent processing occurs in a host-dependent manner. Both, high-mannose and complex type glycans, most likely tri- and tetra-antennary structures, were synthesized in oocytes. The data obtained suggests that in this system the core chains of yeast invertase remain high-mannose type, whereas the more extensively processed polymannose chains are modified to complex oligosaccharides. In the presence of the glycosylation inhibitor, tunicamycin, and the glucosidase processing inhibitor, methyldeoxynojirimycin, secretion of invertase is significantly decreased, whereas in the presence of the mannosidase inhibitor, deoxymannojirimycin, no influence of secretion is seen. This may suggest that glycosylation of invertase is important for early secretion events. Expression of invertase lacking the leader sequence results in loss of glycosylation and secretion in oocytes. This indicates that yeast signals for secretion are functional in this higher eukaryote.
Roitsch
T
T
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Federal Republic of Germany.
Lehle
L
L
D000818:Animals; D005260:Female; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D066298:In Vitro Techniques; D009865:Oocytes / Q000378:metabolism* / Q000382:microbiology; D010957:Plasmids; D011134:Polysaccharides / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D014982:Xenopus laevis / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D006023:Glycoproteins; D011134:Polysaccharides; D012333:RNA, Messenger; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase