title: Dissociation and re-association studies on the interaction domains of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, provide evidence for heterodimer formation.
abstract: Activation of the lectin pathway of complement begins with the activation of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, which are bound to the recognition molecules, MBL and ficolins. MASPs are Ca(2+)-dependent dimers. Dimerization and Ca(2+)-dependent association with the recognition molecules occurs via the first 3 domains, the CUB1-EGF-CUB2 region. The CUB1-EGF-CUB2 (D1-3) regions of MASP-1 and MASP-2, and also their tagged versions, were expressed in E. coli, refolded and purified. The first three domains of MASP-1 are identical with the respective regions of MASP-3 and MAp44, which are also associated with MBL and ficolins. The functionality of the fragments was checked by inhibition of C3 deposition from human serum. Time-course of the dissociation and re-association was examined by size exclusion chromatography. Both refolded proteins are tight Ca(2+)-dependent dimers, as expected. In buffer containing EDTA MASP-1_D1-3 dissociated to monomers, however it took about 1h to reach an equilibrium. Upon re-calcification dimers were re-formed, but this process was even slower; only after overnight incubation was the dimerization completed. MASP-2_D1-3 showed a somewhat different behavior: dissociation by EDTA was even slower, less complete, and higher MW aggregates also appeared. Heterodimer formation was detected by native PAGE. As modeled by the D1-3 fragments, MASP-1 and MASP-2 can readily form heterodimers after dissociation and re-association, however, in the presence of Ca(2+) exchange of subunits is slow between the homodimers. MASP-1:MASP-3 heterodimer formation was modeled by the tagged and untagged D1-3 fragments, and data indicate that subunits of these proteins are readily exchanged even in the presence of Ca(2+). The existence of heterodimers influences the current view on the composition of lectin pathway complexes and their activation.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 29 Karolina Street, H-1113 Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Paréj
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 29 Karolina Street, H-1113 Budapest, Hungary; Faculty of Information Technology, Pázmány Péter Catholic University, 50/A Práter Street, H-1083 Budapest, Hungary.
authors.forename: Agnes
authors.initials: A
authors.lastname: Hermann
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 29 Karolina Street, H-1113 Budapest, Hungary.
authors.forename: Nóra
authors.initials: N
authors.lastname: Donáth
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 29 Karolina Street, H-1113 Budapest, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Závodszky
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 29 Karolina Street, H-1113 Budapest, Hungary. Electronic address: gal.peter@ttk.mta.hu.
authors.forename: Péter
authors.initials: P
authors.lastname: Gál
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 29 Karolina Street, H-1113 Budapest, Hungary. Electronic address: dobo.jozsef@ttk.mta.hu.
authors.forename: József
authors.initials: J
authors.lastname: Dobó
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003176:Complement C3 / Q000378:metabolism; D037582:Complement Pathway, Mannose-Binding Lectin; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D037601:Mannose-Binding Lectin / Q000737:chemistry* / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000097604:Ficolins
chemical_list: D002118:Calcium; D003176:Complement C3; D037102:Lectins; D037601:Mannose-Binding Lectin; D050606:Mannose-Binding Protein-Associated Serine Proteases; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000097604:Ficolins; C084067:MASP1 protein, human; C105296:MASP2 protein, human
keywords: Ca(2+)-dependent dimer; Complement; Dissociation; Heterodimer; Lectin pathway; MBL-associated serine proteases