MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

TY - JOUR

T1 - MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

AU - Skjoedt, Mikkel-ole

AU - Palarasah, Yaseelan

AU - Munthe-fog, Lea

AU - Jie Ma, Ying

AU - Weiss, Gudrun

AU - Skjodt, Karsten

AU - Koch, Claus

AU - Garred, Peter

PY - 2010/11/1

Y1 - 2010/11/1

N2 - Background: The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and little is known about its biological characteristics. For this reason we expressed recombinant MASP-3 and generated specific monoclonal antibodies to establish biochemical characteristics and to determine the serum levels, the interactions with the LCP recognition molecules and the influence on complement activation of MASP-3. Methods: We expressed rMASP-3 in CHO-DG44 cells and used SDS-PAGE and Western blotting for biochemical characterization. We generated monoclonal antibodies against MASP-3 and developed a quantitative MASP-3 assay to establish the serum levels in 100 Danish blood donors. In addition we assessed the association levels between MASP-3 and Ficolin-2, -3 and MBL using both ELISA and immunoprecipitation techniques. Moreover, we assessed the influence on complement factor C4 deposition. Results: We found the mean serum MASP-3 concentration to be 6.4. mg/l (range: 2-12.9. mg/l) and that MASP-3 in serum is primarily found in complex with Ficolin-3. In contrast to this the MASP-3 association with Ficolin-2 and especially with MBL seems to be less evident. rMASP-3 significantly inhibited Ficolin-3 mediated C4 deposition, while the opposite was the case for rMASP-1. Conclusion: Our results show that MASP-3 is present in relatively high serum concentrations. Moreover, Ficolin-3 is the primary acceptor molecule of MASP-3 among the LCP activator molecules, but MASP-3 appears to down-regulate Ficolin-3 mediated complement activation through the lectin pathway.

AB - Background: The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and little is known about its biological characteristics. For this reason we expressed recombinant MASP-3 and generated specific monoclonal antibodies to establish biochemical characteristics and to determine the serum levels, the interactions with the LCP recognition molecules and the influence on complement activation of MASP-3. Methods: We expressed rMASP-3 in CHO-DG44 cells and used SDS-PAGE and Western blotting for biochemical characterization. We generated monoclonal antibodies against MASP-3 and developed a quantitative MASP-3 assay to establish the serum levels in 100 Danish blood donors. In addition we assessed the association levels between MASP-3 and Ficolin-2, -3 and MBL using both ELISA and immunoprecipitation techniques. Moreover, we assessed the influence on complement factor C4 deposition. Results: We found the mean serum MASP-3 concentration to be 6.4. mg/l (range: 2-12.9. mg/l) and that MASP-3 in serum is primarily found in complex with Ficolin-3. In contrast to this the MASP-3 association with Ficolin-2 and especially with MBL seems to be less evident. rMASP-3 significantly inhibited Ficolin-3 mediated C4 deposition, while the opposite was the case for rMASP-1. Conclusion: Our results show that MASP-3 is present in relatively high serum concentrations. Moreover, Ficolin-3 is the primary acceptor molecule of MASP-3 among the LCP activator molecules, but MASP-3 appears to down-regulate Ficolin-3 mediated complement activation through the lectin pathway.

U2 - 10.1016/j.imbio.2009.10.006

DO - 10.1016/j.imbio.2009.10.006

M3 - Journal article

SN - 0171-2985

VL - 215

SP - 921

EP - 931

JO - Immunobiology

JF - Immunobiology

IS - 11

ER -