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

• Published in: Molecular immunology 2014-05, Vol.59 (1), p.1-9
• Main Authors: Parej, Katalin, Hermann, Agnes, Donath, Nora, Zavodszky, Peter, Gal, Peter, Dobo, Jozsef
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites - genetics; Ca2+-dependent dimer; Calcium - chemistry; Calcium - metabolism; Complement; Complement C3 - metabolism; Complement Pathway, Mannose-Binding Lectin; Dissociation; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Ficolins; Heterodimer; Humans; Kinetics; Lectin pathway; Lectins - chemistry; Lectins - metabolism; Mannose-Binding Lectin - chemistry; Mannose-Binding Lectin - metabolism; Mannose-Binding Protein-Associated Serine Proteases - chemistry; Mannose-Binding Protein-Associated Serine Proteases - genetics; Mannose-Binding Protein-Associated Serine Proteases - metabolism; MBL-associated serine proteases; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Binding; Protein Multimerization; Protein Refolding; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2013.12.003

•Serine proteases of the lectin pathway (MASPs) were thought to be homodimers.•Dissociation and subunit exchange properties of MASPs were characterized.•Heterodimer formation between MASP-1 and MASP-2 is structurally allowed.•Exchange of subunits between MASP-1 and -2 is slow, between MASP-1 and -3 is fast.•Existence of heterodimers influences the current view of lectin pathway activation. 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 Ca2+-dependent dimers. Dimerization and Ca2+-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 Ca2+-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 Ca2+ 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 Ca2+. The existence of heterodimers influences the current view on the composition of lectin pathway complexes and their activation.