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

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Abstract

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.

Introduction

The recognition of microorganisms within the body is of immense importance and a number of recognition molecules, both membrane-bound and soluble, are involved in the recognition of such non-self structures. Within the innate immune system a family of soluble proteins that recognize microbial agents is formed by mannan-binding lectin (MBL) and the three ficolins (H-ficolin, L-ficolin, and M-ficolin). These proteins are recognition molecules within the complement system. Three different pathways (the classical, the alternative and the lectin pathway) may lead to the activation of the complement system. The lectin pathway of complement activation is initiated when MBL or one of the ficolins in complex with the MBL-associated serine proteases (MASPs) binds to natural or artificial targets (Matsushita et al., 2000a, Holmskov et al., 2003, Frederiksen et al., 2005, Liu et al., 2005). Targets for MBL display patterns of carbohydrates with adequately spaced terminal carbohydrates with horizontal 3- and 4-OH groups whereas targets for the ficolins may be carbohydrates with N-acetyl groups or indeed other compounds with a suitable pattern of acetyl groups. Thus, the term “lectin pathway” is somewhat of a misnomer, since the ficolins strictly speaking are not lectins, i.e., they do react with acetylated carbohydrates but they also react with other acetylated molecules (Krarup et al., 2004). Three MASPs (MASP-1, MASP-2 and MASP-3) have been described to be associated with MBL and ficolins, and in addition two non-enzymatic proteins, MAp19 (Stover et al., 1999, Takahashi et al., 1999) and MAp44 (Degn et al., 2009, Skjoedt et al., 2010) (also known as sMAP and MAP-1, respectively), form complexes with all four proteins.

The five MBL- and ficolin-associated proteins are encoded by two genes. The MASP1 gene encodes a primary transcript which can be spliced to three different mRNAs coding for MASP-1, MASP-3 and MAp44 (Degn et al., 2009). The MASP2 gene gives rise to a primary transcript, which can be spliced to two mRNAs, one coding for MASP-2 and another for MAp19 (Stover et al., 1999). The exact composition of the MBL/MASP or ficolin/MASP complexes remains unsolved, but it is generally agreed that MASP-2 is both required and sufficient for the generation of the C3 convertase, C4bC2b (Thiel et al., 1997, Rossi et al., 2001). While the role of MASP-2 appears reasonably well established, the roles of MASP-1 and MASP-3 are still debated (Gál et al., 2007, Thiel, 2007). Results indicate that MASP-1 will accelerate while MASP-3 will inhibit the generation of the C3 convertase (Moller-Kristensen et al., 2007). There are reports indicating a role of MASP-1 in coagulation (Krarup et al., 2008, Takahashi et al., 2010a) and in activating endothelial cells through cleavage of protease-activated receptor 4 (Megyeri et al., 2009) and recently it was reported that MASP-1 is the enzyme responsible for cleavage of pro-factor D (Takahashi et al., 2010b).

The role of MBL was discovered through the study of patients with unexplained susceptibility to infections and opsonic deficiency. After many years of investigation these patients were found to be deficient in MBL (Super et al., 1989). It seems plausible that elucidating the role(s) of the remaining MASPs as well as those of MAp19 and MAp44 may also benefit from epidemiological investigations on selected patient populations. We thus decided to construct assays for these components. The development of such assays was hampered by the difficulty in raising selective monoclonal antibodies (mAbs) due to the extensive sharing of domains between the proteins. We have now developed specific anti-MASP-3 and anti-MAp44 antibodies and present sandwich-type assays for these proteins, yielding biological parameters, which serve as a basis for future clinical investigations.

Section snippets

Blood samples

Serum and plasma samples were obtained from the following three cohorts after informed consent and according to the Declaration of Helsinki:

  • a.

    Adult Danish blood donors. The levels of MBL and MASP-2 were previously determined in these samples (Ytting et al., 2007).

  • b.

    Infants aged 0–12 months. Blood samples were obtained from the umbilical cords of 15 infants delivered at term, as well as sequentially at 3, 6, 8, 9 and/or 12 months after birth (Thiel et al., 1995).

  • c.

    Patients undergoing surgery. Blood

Monoclonal antibodies

The specificities of the mAbs were examined by application to blots of pMBL/MASP complexes separated by SDS-PAGE. On the reduced Western blot shown in Fig. 1A, the mAb raised against the C-terminal of MASP-3 (38.12.3) is seen to react with protein bands at positions corresponding to the mobility of the B-chain of activated MASP-3 (Mr ~ 40 kDa), but not with material at the positions of MASP-1, MASP-2, MAp19 or MAp44. The mAb derived from immunization with the C-terminal of MAp44 (2D5) reacted

Discussion

The sandwich assay described for MASP-3 makes use of a mAb against the A-chain common to MASP-1, MASP-3, and MAp44 for capture of these analytes from serum or plasma, followed by assay development with a mAb raised against a peptide representing the unique C-terminal of the B-chain of MASP-3, thus ensuring specificity for MASP-3. Terai et al. (1997) reported results on the concentration of MASP-1 based on an assay using a similar anti-A-chain antibody (mAb 2B11) for capture. However, they

Acknowledgements

The work presented here was supported by the Lundbeck Foundation and the Danish Graduate School of Immunology (to S.E.D.), the Novo Nordisk Foundation and the Danish Medical Research Council. We are indebted to professor Mac Turner for his critical reading of the manuscript and valuable suggestions.

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