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 600–800 kDa and 500–700 kDa, respectively. The level of MASP-3 in donor sera (N = 200) was log-normally distributed with a median value of 5.0 μg/ml (range: 1.8–10.6 μg/ml), and the corresponding value for MAp44, also log-normally distributed, was 1.7 μg/ml (range: 0.8–3.2 μg/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 6 months, whereas MAp44 levels fell slightly during the first 6 months. Concomitant with the acute phase response in patients undergoing major surgery, levels of both proteins fell slightly over 1–2 days, but whereas MASP-3 recovered to baseline values over another 2 days, MAp44 only reached baseline values at around day 30. Thus, neither of the two proteins behaves as a classical acute phase protein.

Research Highlights
► MASP-3 and MAp44 are found in serum in high molecular weight complexes.
► MASP-3 levels are log-normally distributed with a median of 5.0 μg/ml (range: 1.8-10.6 μg/ml, N=200).
► MAp44 levels are log-normally distributed with a median of 1.7 μg/ml (range: 0.8-3.2 μg/ml).
► MASP-3 levels are low at birth and reach normal levels within the first 6 months.
► MAp44 levels drop slightly during the first 6 months after birth.
► Neither MAp44 nor MASP-3 behaves as a classical acute phase protein.