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1-Deoxymannojirimycin inhibits Golgi-mediated processing of glycoprotein in Xenopus oocytes
M S Fabbrini1, M Zoppè, R Bollini
1Istituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, Milano, Italy.
Abstract:
We prepared in vitro an mRNA transcript coding for the erythroagglutinating subunit of the kidney bean glycoprotein phytohemagglutinin, E-PHA. The mRNA, injected into Xenopus oocytes, synthesized E-PHA carrying two Asn-linked carbohydrate chains, one of which was processed and acquired resistance to endo-beta-N-acetylglucosaminidase H, as occurs in the native bean cells. When the mannose analog 1-deoxymannojirimycin, an inhibitor of mammalian Golgi mannosidase I, was included in the oocyte culture medium, the acquisition of endo-beta-N-acetylglucosaminidase H resistance was abolished, indicating that also in an amphibian cell the inhibitor blocks a key reaction in Golgi-mediated processing.
Insights
Researchers synthesized kidney bean phytohemagglutinin (E-PHA) in frog eggs using mRNA. An inhibitor blocked sugar chain processing, showing conserved Golgi mannosidase I function in amphibians.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Phytohemagglutinin (PHA) is a glycoprotein from kidney beans with erythroagglutinating properties.
- PHA contains N-linked carbohydrate chains crucial for its function and processing.
- Understanding glycoprotein processing in different organisms can reveal conserved biological mechanisms.
Purpose of the Study:
- To investigate the in vitro synthesis and post-translational modification of the erythroagglutinating subunit of kidney bean phytohemagglutinin (E-PHA).
- To determine if Xenopus oocytes can correctly process E-PHA N-linked glycans.
- To assess the effect of a mannosidase inhibitor on E-PHA glycan processing in oocytes.
Main Methods:
- In vitro synthesis of mRNA encoding E-PHA.
- Injection of E-PHA mRNA into Xenopus oocytes.
- Culture of oocytes with and without 1-deoxymannojirimycin (an inhibitor of Golgi mannosidase I).
- Analysis of N-linked carbohydrate chain processing using endo-beta-N-acetylglucosaminidase H.
Main Results:
- Xenopus oocytes synthesized E-PHA with two Asn-linked carbohydrate chains.
- One carbohydrate chain acquired resistance to endo-beta-N-acetylglucosaminidase H, indicating processing.
- Inclusion of 1-deoxymannojirimycin abolished this resistance, preventing glycan processing.
Conclusions:
- Xenopus oocytes can perform key steps in Golgi-mediated N-linked glycan processing.
- The inhibitor 1-deoxymannojirimycin effectively blocks Golgi mannosidase I activity in amphibian cells.
- This study demonstrates conserved mechanisms of glycoprotein processing across species.