Natural antibodies (nAbs) are immunoglobulins present in circulation or tissues in the absence of pathological conditions or deliberate immunization. The decrease in nAb levels in germ- and antigen-free mice (Coutinho et al., 1995, Haury et al., 1997) suggests that their production is driven, at least in part, by self-antigens. NAbs belong to the IgM, IgG, or IgA subclasses (Holodick et al., 2017, Palma et al., 2018) and are encoded by germline variable (V) genes without extensive mutations. The major source of natural IgM in mice is long-lived peritoneal B1 cells; however, the production of IgM nAbs has also been observed in B1 cells in the spleen (Baumgarth, 2011, Baumgarth, 2016) and marginal zone B cells (Martin and Kearney, 2001).

NAbs play several functional roles in the immune system. For example, some nAbs provide immediate protection against infection, whereas the adaptive arm of the immune system mounts a specific and long-term response, and they can cause direct neutralization of circulating bacteria or viruses (Maddur et al., 2019, Pulendran and Maddur, 2015). IgM nAbs are the first line of defense linked to the complement system, as antibody/antigen complexes can be efficiently removed by connecting to the complement receptor (CR) CR3 or CR4 on phagocytic innate immune cells or transported to the spleen by CR1 as part of TI antibody responses (Matter and Ochsenbein, 2008, Ochsenbein and Zinkernagel, 2000). Beyond immediate protection from infection, nAbs also play important roles in the clearance of apoptotic cells and debris, as the absence of IgM nAbs significantly reduces apoptotic cell clearance (Quartier et al., 2005). nAbs promote the engulfment of apoptotic cells by the apoptotic cell clearing machinery (Ogden et al., 2005) and downregulate inflammatory signals released by macrophages and activated dendritic cells (Chen et al., 2009a, Chen et al., 2009b).

Recent research has shown that nAbs are also important in regulating B cell responses, selecting the B cell repertoire, and regulating B cell development (Baumgarth, 2016). These functions of nAbs are facilitated by their antigen reactivity, which is typically broad, cross-reactive, and recognizes evolutionarily fixed epitopes shared between foreign and self-antigens. Furthermore, nAbs have unique characteristics that contribute to their functional roles and distinguish them from antigen-specific Abs (Palma et al., 2018).

Additional roles for nAbs have been revealed by studies that have demonstrated that certain nAb subsets can recognize neo-epitopes revealed in injured tissues and cells and can catalyze complement activation and inflammation (Fleming, 2006, Ochsenbein and Zinkernagel, 2000, Quartier et al., 2005). For example, a hybridoma producing a B4-IgM monoclonal antibody (mAb) was obtained from the fusion of splenocytes from unmanipulated mice. Moreover, B4-IgM induced intestinal ischemia-reperfusion injury (IRI) in RAG-/- mice and bound to mouse annexin-4 (mAn4) protein on the surface of apoptotic cells (Kulik et al., 2009).

Subsequently, B4-IgM was found to be highly pathogenic that induced IRI and tissue injury in many mouse models. Furthermore, B4-IgM caused hepatic injury in RAG-/- mice (Marshall et al., 2018), catalyzed post-transplant cardiac IRI (Atkinson et al., 2015), and propagated cerebral injury following ischemic stroke (Elvington et al., 2012, Narang et al., 2017). Injection of recombinant mAn4 blocked IRI (Elvington et al., 2012, Kulik et al., 2009). In addition, serum from patients undergoing liver transplantation demonstrated greatly reduced nAb levels recognizing human An4 (hAn4), pointing to the likely importance of An4 reactivity in the post-transplantation immunological status (Marshall et al., 2018). Another study showed that the epitope recognized by B4-IgM is expressed on murine cardiac allografts and that nAb-induced IRI can be blocked by adding a single-chain construct based on the B4-IgM Fab domain antibody (Atkinson et al., 2015), suggesting a dominant role for this epitope. Similar data were obtained in liver IRI, where the single chain construct based on the B4-IgM Fab domain blocked B4-IgM-induced liver injury in mice, pointing to the broad expression of the epitope in different tissues and the possibility of using an inhibitory reagent to block the epitope and prevent IRI (Marshall et al., 2018). In this study, we further investigated the epitope recognized by nAb B4-IgM in humans.

An4 belongs to a family of annexins, which are Ca2+- and phospholipid-binding proteins (Morgan et al., 2004, Rescher and Gerke, 2004). The structure of annexins consists of a conserved Ca2+- and membrane-binding core of four annexin repeats (eight for An6), and variable N-terminal regions (Gerke et al., 2005). Annexins are soluble cytosolic proteins that are expressed without obvious signal sequences and are unable to enter the classical secretory pathway. However, they have also been identified in extracellular fluids or associated with the external cell surface through poorly understood binding sites and mechanisms (Kundranda et al., 2004, Satoh et al., 1996, Yeatman et al., 1993). An4 is predominantly detected in epithelial cells, and has been found to be present throughout the cytoplasm (Massey-Harroche et al., 1998, Massey et al., 1991, Mayran et al., 1996). An4 inhibits the epithelial calcium-activated chloride ion conductance (Piljic and Schultz, 2006), plays a role in the formation of pronephric tubules (Seville et al., 2002), and regulates the passive membrane permeability to water and protons (Hill et al., 2003). An4 has been identified in both zebrafish and mouse endoderm, and while it is broadly produced in the developing liver and pancreas, its expression later becomes restricted to the hepatopancreatic ducts and pancreatic islets, including insulin-producing β-cells (Zhang et al., 2014).

In vitro studies have suggested that An4 exhibits an anti-apoptotic effect in response to cytotoxic stress (Han et al., 2000, Kim et al., 2010) by activating NF-κB transcriptional activity (Jeon et al., 2010, Sohma et al., 2003). An4 overexpression has been reported in various tumors, such as lung, gastric, colorectal, renal, pancreatic, ovarian, and prostate cancer, and is associated with tumor invasiveness, metastasis, and chemoresistance (Wei et al., 2015, Yao et al., 2016a, Yao et al., 2016b, Zimmermann et al., 2004). An4 and An6 were identified as proteins involved in plasma membrane repair (Boye et al., 2017, Boye and Nylandsted, 2016), and this phenomenon may explain, in part, why upregulation of An4 in cancers results in resistance to treatment. Recent studies have demonstrated that annexin is involved in the regulation of immunological tolerance and activation (Iwasa et al., 2012, Krammer and Weyd, 2017, Weyd, 2016, Weyd et al., 2013). Finally, membrane expression of An4 has also been recognized as an early marker for apoptotic cell death (Herzog et al., 2004) and possibly serves as a marker for apoptosis by binding to phosphatidylserine (Rosenbaum et al., 2011) and other members of the annexin family. An1 has been proposed to promote apoptotic cell clearance and the resolution of inflammation (Arur et al., 2003, Everts-Graber et al., 2019, Scannell et al., 2007).

The biosynthesis of polypeptide chains on ribosomes does not immediately produce a fully functional protein. The newly formed polypeptide chain must undergo modifications outside of the ribosome, which include the cleavage of certain peptide bonds, resulting in the removal of some of the formed polypeptide fragments, as well as side chain modifications of amino acid residues that result in differential function or localization of proteins (Karve and Cheema, 2011, Knorre et al., 2009). These chemical modifications are commonly referred to as post-translational modifications (PTMs), as they occur after protein biosynthesis. PTMs are of major interest in biology, as many intra-and extracellular events depend on the occurrence of a specific chemical alteration that drives changes in both protein structure and function (Basle et al., 2010).

Protein acetylation is a major post-translational modification in which the acetyl group from acetyl coenzyme A (Ac-CoA) is transferred to a specific site on a polypeptide chain (Drazic et al., 2016). Two distinct acetylation activities are performed by two distinct enzymes: lysine acetyltransferases (KATs), that modify the lysine side chain amino group and N-terminal acetyltransferases (NATs), that acetylate the N-terminal amino groups of polypeptides (Ree et al., 2018). Two co-translational processes, cleavage of N-terminal methionine residues and N-terminal acetylation, are by far the most common modifications that occur in the vast majority of eukaryotic proteins.

Six NATs, named sequentially NatA to NatF, were identified and classified based on their substrate preferences. NATs are mono- or multi-subunit enzymes consisting of a unique catalytic subunit, a unique ribosomal anchor, and up to two auxiliary subunits. The major auxiliary subunit modulates the activity and substrate specificity of the catalytic subunit (Arnesen, 2011, Liszczak et al., 2013). Approximately 70% of mature proteins do not retain N-terminal methionine (Bradshaw et al., 1998, Reddi et al., 2016) and approximately 90% of newly synthesized cytosolic proteins are acetylated (Arnesen et al., 2009, Drazic et al., 2016, Reddi et al., 2016). By exploring the proteins recognized by the highly pathogenic IgM-B4 nAb, we found that the antibody binds to a common epitope composed of the post-translation acetylation of N-terminal methionine, followed by glutamic or aspartic acid as the second amino acid. The epitope is detected on mAn4, as well as other proteins of human and mouse origin. We also found that the B4-IgM antibody can bind to the epitope in intracellular proteins through the plasma membrane of necrotic cells, thus presuming that the aims of nAbs are to remove not only apoptotic cells but also necrotic cell debris, thus helping the body maintain immunogenic homeostasis under properly controlled self-antigen responses.