%0 Thesis %A Dahl, Mads Ronald %D 2014 %T Mannan-binding lectin {MBL) associated serine protease-3 (MASP-3) : complex formation in serum and plasma, conditions required for the conversion of the zymogen form into a two-chain serin protease, and a search for substrates using recombinant material produced by stable expression in eukaryotic cell lines %U https://figshare.le.ac.uk/articles/thesis/Mannan-binding_lectin_MBL_associated_serine_protease-3_MASP-3_complex_formation_in_serum_and_plasma_conditions_required_for_the_conversion_of_the_zymogen_form_into_a_two-chain_serin_protease_and_a_search_for_substrates_using_recombinant_mat/10138592 %2 https://figshare.le.ac.uk/ndownloader/files/18271832 %K IR content %X The complement system is part of the innate immune system and is crucial for identifying invading microorganisms. The lectin pathway of complement activation is initiated through multimeric macromolecules which recognise pathogen-associated patterns and translate binding through activation of associated serine proteases that start a cascade of proteolytic events leading to bactericidal, opsonising and proinflammatory responses. Mannan-binding lectin (MBL) is one of the macromolecules mediating binding to specific carbohydrate structures common to a range of microorganisms. Three different mannan-binding lectin associated serine proteases (MASP-1/-2/-3) and a non-enzymatic protein of 19 kDa (MApl9) have been described. MASP-2 appears to mediate all processes required for complement activation while little or no complement-related functional activity was found to be mediated by MASP-1, MASP-3 or MApl9. Functional and biophysical studies of MASP-3 relied on continuous and reliable supply of recombinant MASP-3. In this work production of recombinant MASP-3 in mammalian cells with subsequent affinity purification and characterisation of the recombinant MASP-3 was performed to obtain large quantities of homogeneous enzyme. The development of screening assays and assays for quantitative determination of MASP-3 levels were two other tools essential for the development of this thesis. As a result of this thesis MASP-3 levels in different body fluids were determined in healthy individuals using a quantitative assay. The assays were used to analyse the MASP-3 level in sample collections from patients suffering from Alzheimer disease and type-1 diabetes. The correlation of MASP-3 level and MBL genotype, H-/L-Ficolin concentration, age, BMI, acute phase and time of year were analysed. The enzymatic activity of MASP-3 was analysed on chromogenic substrates and the results permitted a study of MASP-3 inhibition. Attempts were made to affinity purify potential MASP-3 substrates and ligands using beads coupled with recombinant MASP-3 and anti- MASP-3 antibodies. The influence of calcium on MASP-3 complex formation, dissociation, activation and stability was analysed. %I University of Leicester