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Functional bioactive recombinant acylation stimulating protein is distinct from C3a anaphylatoxin
I Murray1, R A Parker, T G Kirchgessner
1Mike Rosenbloom Laboratory for Cardiovascular Research, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Journal of Lipid Research
|February 11, 1998
Summary
Recombinant acylation stimulating protein (rASP) effectively mimics plasma ASP's role in stimulating triglyceride synthesis and glucose transport in fat cells. This bioavailable rASP offers a valuable tool for studying ASP's structure-function relationships.
Area of Science:
- Biochemistry
- Molecular Biology
- Metabolic Research
Background:
- Acylation stimulating protein (ASP) is crucial for triglyceride synthesis and glucose transport in adipose tissue.
- Understanding ASP's function requires reliable methods to study its bioactivity.
Purpose of the Study:
- To produce recombinant ASP (rASP) in E. coli.
- To assess the bioactivity of purified rASP in stimulating triglyceride synthesis and glucose transport.
- To investigate the structure-function relationship of ASP.
Main Methods:
- Cloning and expression of the ASP-coding cDNA region from complement C3 in E. coli.
- Testing rASP bioactivity via triglyceride synthesis assays, glucose transport assays, and competition binding assays.
- Utilizing 3T3 preadipocytes and human differentiated adipocytes for cellular assays.
Main Results:
- Recombinant ASP (rASP) demonstrated comparable stimulation of triglyceride synthesis in 3T3 preadipocytes and human adipocytes versus plasma ASP (pASP).
- rASP significantly increased glucose transport in L6 myocytes and human adipocytes, mirroring pASP activity.
- rASP competitively displaced radiolabeled pASP from cell surface binding sites and immunoprecipitation abolished its stimulatory effect.
Conclusions:
- The produced rASP retains full biological activity, validating its utility as a research tool.
- Lipogenic and anaphylatoxic activities of ASP are mediated by distinct structural domains.
- rASP provides a means to further explore ASP's structure-function dynamics in metabolic processes.