Levels of mannan-binding lectin-associated serine protease-2 in healthy individuals

Abstract

The lectin pathway is part of the innate immune system providing a first line of defence against infections. Mannan-binding lectin (MBL) and ficolins, in complex with MBL-associated serine proteases (MASPs), are capable of activating the complement system, thus mediating the destruction of infectious agents. MASP-2 cleaves C4 and C2 and is thus crucial for the activation of downstream complement components. We present an assay for quantifying total MASP-2 in plasma and serum samples. The assay is a sandwich type assay using a combination of two monoclonal anti-MASP-2 antibodies, one directed against the N-terminal part of MASP-2 and the other against its C-terminal part. Based on a population of Danish blood donors, the average MASP-2 concentration was estimated at 534 (S.D.±213) ng per ml of plasma. Characterization of the MASP-2 protein in serum showed high stability at 4 °C and at ambient temperature but a rapid decline at 37 °C. Gel permeation chromatography (GPC) indicated that all MASP-2 in serum is present in complexes with MBL and ficolins.

Introduction

The ability of lectins, i.e., carbohydrate-binding proteins, and other pattern recognition molecules to distinguish between self and non-self is one of the characteristics of innate immunity. Mannan-binding lectin (MBL) and L- and H-ficolin detect pathogen-associated molecular patterns (PAMPs) and promote destruction of pathogens by activating effector mechanisms of the complement system (Holmskov et al., 2003). MBL shows selectivity for carbohydrates presenting 3- and 4-OH groups in the equatorial plane of the ring structure (Weis et al., 1992), while the epitopes recognized by ficolins have not been defined, but are present in, e.g., N-acetyl-glucosamine (GlcNAc) (Matsushita et al., 1996).

For activating the complement system, MBL, L- and H-ficolin rely on associated serine proteases. These were first discovered in complex with MBL and are referred to as MBL-associated serine proteases (MASPs). MBL, L- and H-ficolin Matsushita et al., 2000a, Matsushita et al., 2002 are associated with three MASPs, termed MASP-1 (Matsushita and Fujita, 1992), MASP-2 (Thiel et al., 1997) and MASP-3 (Dahl et al., 2001) as well as with a non-enzymatic protein referred to as MBL-associated protein of 19 kDa (MAp19) (Stover et al., 1999) or small MBL-associated protein (sMAP) (Takahashi et al., 1999). MAp19 comprises the first two domains of MASP-2, followed by an extra sequence of four unique amino acids. MASPs and MAp19 have been reported to bind to MBL in a calcium-dependent manner (Wallis and Dodd, 2000). It appears that the EGF-like domain of MASP-2, assisted by the two surrounding CUB domains, is involved in the calcium-dependent complex formation (Feinberg et al., 2003). Since MAp19 does not contain the second CUB domain, this domain is likely not to be crucial for complex formation, but nevertheless contributes to a more stable interaction (Thielens et al., 2001).

Thiel et al. (1997) discovered MASP-2 by affinity chromatography of plasma on mannan- and N-acetylglucosamine-derivatized Sepharose beads, and MASP-2 was shown to activate C4. The activation of MASP-1 and -2 in the MBL complex was initially thought to resemble the sequential activation of C1r and C1s of the C1 complex. However, experiments with recombinant MASP-2 showed that complexes comprising only rMBL and rMASP-2 were sufficient for C4 and C2 activation (Vorup-Jensen et al., 2000). MASP-1 does not activate MASP-2 but shows a weak C3 as well as a C2 activating capacity (Matsushita et al., 2000b). Recently, a genetically determined deficiency of MASP-2 was described (Stengaard-Pedersen et al., 2003). The mutation of a single nucleotide leads to an ASP-Gly exchange in the CUB1 domain and renders MASP-2 incapable of binding to MBL.

In the present paper, we describe the development of a specific assay for MASP-2, and use this assay to establish normal levels of the protein in human serum and plasma.