Biological variations of MASP-3 and MAp44, two splice products of the MASP1 gene involved in regulation of the complement system

The lectin pathway of complement is part of the innate immune system. The complement-activating pattern-recognition molecules (for which we suggest the abbreviation CAPREMs) mannan-binding lectin (MBL) and the three ficolins (H-, L- and M-ficolin) circulate in complexes with MBL-associated serine proteases (MASP-1, -2 and -3) and two additional proteins (MAp19 and MAp44, also termed sMAP and MAP-1, respectively). When MBL or ficolins recognize a microorganism or altered self components, activation of the MASPs ensues, leading to the activation of the complement system. MASP-1, MASP-3 and MAp44 are all three encoded by the MASP1 gene. MASP-1 and -3 share five domains (constituting the so-called A-chain), but have unique protease domains (B-chains). MAp44 shares the first four domains with MASP-1 and MASP-3, followed by 17 unique C-terminal amino acid residues. Thus, assays for the protease domain of MASP-3 and for the 17 C-terminal amino acids of MAp44 are required to measure these proteins specifically and here we present such assays for MASP-3 and MAp44. MASP-3 was captured with a monoclonal antibody (5F5) reacting with a common domain of the three proteins (CCP1) and the assay was developed with a monoclonal antibody (38.12.3) specific for the C-terminal part of the MASP-3 protease domain. MAp44 was captured with a monoclonal antibody (2D5) reacting with the C-terminus of MAp44 followed by assay development with a monoclonal anti-CCP1 antibody (4H2). Using Superose 6 gel permeation chromatography of serum, MASP-3 and MAp44 were found in complexes, which eluted in positions corresponding to 600–800kDa and 500–700kDa, respectively. The level of MASP-3 in donor sera (N=200) was log-normally distributed with a median value of 5.0μg/ml (range: 1.8–10.6μg/ml), and the corresponding value for MAp44, also log-normally distributed, was 1.7μg/ml (range: 0.8–3.2μg/ml). For MASP-3, the inter-assay coefficients of variation of low, intermediate and high level internal controls were 4.9%, 6.9% and 3.9% (N=12). For MAp44, the corresponding inter-assay CVs were 7.6%, 6.2%, and 7.0% (N=12). MASP-3 levels were low at birth and reached adult levels within the first 6months, whereas MAp44 levels fell slightly during the first 6months. Concomitant with the acute phase response in patients undergoing major surgery, levels of both proteins fell slightly over 1–2days, but whereas MASP-3 recovered to baseline values over another 2days, MAp44 only reached baseline values at aroun