MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation.
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 6.4mg/l (range: 2-12.9mg/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.


CONCLUSIONS
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.
Skjoedt
Mikkel-Ole
MO
Laboratory of Molecular Medicine, Department of Clinical Immunology, Sect 7631, Rigshospitalet, Faculty of Health Sciences, University Hospital of Copenhagen, Denmark. moskjoedt@health.sdu.dk
Palarasah
Yaseelan
Y
Munthe-Fog
Lea
L
Jie Ma
Ying
Y
Weiss
Gudrun
G
Skjodt
Karsten
K
Koch
Claus
C
Garred
Peter
P
D000818:Animals; D003181:Complement C4 / Q000276:immunology*; D037582:Complement Pathway, Mannose-Binding Lectin / Q000276:immunology*; D006224:Cricetinae; D006023:Glycoproteins / Q000097:blood / Q000276:immunology*; D006801:Humans; D037102:Lectins / Q000097:blood / Q000276:immunology*; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000032:analysis / Q000276:immunology*; D051379:Mice
D003181:Complement C4; C109662:FCN3 protein, human; D006023:Glycoproteins; D037102:Lectins; C084067:MASP1 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases