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Production and purification of recombinants of mouse MASP-2 and sMAP
1Department of Biochemistry, Fukushima Medical University, Fukushima and CREST, Japan Science and Technology Agency, Japan.
Abstract:
Small mannose-binding lectin (MBL)-associated protein (sMAP) is a component of the complex consisting of MBL and MBL-associated serine proteases (MASPs) in the lectin complement pathway. sMAP is a truncated form of MASP-2, which is generated by an alternative splicing from a single structural MASP-2 gene. Upon activation of the MBL-MASPs complex, MASP-2 cleaves the complement C4, but the role of sMAP which lacks the serine protease domain is not clear. To clarify the role of sMAP in activation of the lectin pathway, we have generated sMAP-gene deficient mice which are also deficient for MASP-2. In this study, we generated and purified mouse recombinant sMAP (rsMAP) and rMASP-2 using the Drosophila expression system for the reconstitution assay of the deficient mice. In preliminary experiments, these purified recombinants were able to reconstitute the MBL-MASPs-sMAP complexes and the addition of rMASP-2 to deficient serum restored the C4 cleavage activity of the MBL-MASPs complex. From these data, rsMAP and rMASP-2 generated in this study seem to be useful in analysis of the deficient mice.
Insights
Researchers generated recombinant mouse small mannose-binding lectin (MBL)-associated protein (sMAP) and MBL-associated serine protease-2 (MASP-2). These tools aid in studying the lectin complement pathway using gene-deficient mice.
Area of Science:
- Immunology
- Complement System
- Molecular Biology
Background:
- Small mannose-binding lectin (MBL)-associated protein (sMAP) is part of the MBL-MASP complex in the lectin complement pathway.
- sMAP is a truncated form of MASP-2, derived from alternative splicing of the MASP-2 gene.
- The precise function of sMAP, lacking a serine protease domain, in complement activation remains unclear.
Purpose of the Study:
- To investigate the role of sMAP in the activation of the lectin complement pathway.
- To generate tools for studying sMAP function in gene-deficient mouse models.
Main Methods:
- Generation of sMAP-gene and MASP-2 deficient mice.
- Production and purification of mouse recombinant sMAP (rsMAP) and recombinant MASP-2 (rMASP-2) using the Drosophila expression system.
- Reconstitution assays using purified recombinants and deficient mouse serum.
Main Results:
- Purified rsMAP and rMASP-2 successfully reconstituted MBL-MASPs-sMAP complexes in vitro.
- Addition of rMASP-2 to deficient serum restored C4 cleavage activity.
- The generated recombinant proteins are suitable for analyzing the function of sMAP and MASP-2 in deficient mice.
Conclusions:
- The study provides valuable recombinant tools (rsMAP and rMASP-2) for investigating the lectin complement pathway.
- These tools will facilitate further research into the specific roles of sMAP and MASP-2 in complement activation using genetically modified mouse models.
