Production and purification of recombinants of mouse MASP-2 and sMAP.
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.
Iwaki
Daisuke
D
Department of Biochemistry, Fukushima Medical University, Fukushima and CREST, Japan Science and Technology Agency, Japan.
Fujita
Teizo
T
D017398:Alternative Splicing; D000818:Animals; D003167:Complement Activation; D003181:Complement C4 / Q000378:metabolism; D015151:Immunoblotting; D037601:Mannose-Binding Lectin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D003181:Complement C4; D037601:Mannose-Binding Lectin; D011994:Recombinant Proteins; C491340:MASP-2 protein, mouse; D050606:Mannose-Binding Protein-Associated Serine Proteases; D012697:Serine Endopeptidases