Related Experiment Videos
Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase
S N Sarkar1, S Bandyopadhyay, A Ghosh
1Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Abstract:
P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2'-5'-linked oligoadenylate products containing up to 30 residues. 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.
Insights
Human P69 enzyme activity depends on glycosylation, not myristoylation. This study highlights insect cell expression for active P69 production, crucial for understanding 2’-5’ oligoadenylate synthetase function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- P69 is a human 2'-5' oligoadenylate synthetase isozyme.
- Understanding P69's enzymatic properties and structural requirements is essential.
Purpose of the Study:
- To investigate the enzymological and structural properties of recombinant P69.
- To determine the role of post-translational modifications in P69 activity.
- To establish an efficient expression system for P69 production.
Main Methods:
- Recombinant P69 expression in bacterial and insect cells.
- Site-directed mutagenesis to assess myristoylation and glycosylation roles.
- Affinity chromatography for protein purification.
- Enzymatic assays using various double-stranded RNA (dsRNA) constructs.
Main Results:
- Bacterial P69 was inactive; insect cell P69 was highly active, indicating post-translational modification is crucial.
- Glycosylation, but not myristoylation, was essential for P69 enzymatic activity.
- Purified P69 is a stable, dimeric glycoprotein dependent on dsRNA for activity.
- P69 synthesizes 2'-5'-linked oligoadenylates up to 30 residues non-processively.
- dsRNA length and 2'-O-methylation affect P69 activation.
Conclusions:
- Glycosylation is a critical post-translational modification for human P69 enzyme activity.
- Insect cell expression provides a viable system for producing active, modified P69 for further studies.
- The characterized properties of P69 offer insights into 2'-5' oligoadenylate synthetase mechanisms.