Misao Matsushita

The lectin-complement pathway – its role in innate immunity and evolution

Summary:  Innate immunity was formerly thought to be a non‐specific immune response characterized by phagocytosis. However, innate immunity has considerable specificity and is capable of discriminating between pathogens and self. Recognition of pathogens is mediated by a set of pattern recognition receptors, which recognize conserved pathogen‐associated molecular patterns (PAMPs) shared by broad classes of microorganisms, thereby successfully defending invertebrates and vertebrates against infection. Lectins, carbohydrate‐binding proteins, play an important role in innate immunity by recognizing a wide range of pathogens. Mannose‐binding lectin (MBL) and ficolin are lectins composed of a lectin domain attached to collagenous region. However, they use a different lectin domain: a carbohydrate recognition domain (CRD) is responsible for MBL and a fibrinogen‐like domain for ficolin. These two collagenous lectins are pattern recognition receptors, and upon recognition of the infectious agent, they trigger the activation of the lectin‐complement pathway through attached serine proteases, MBL‐associated serine proteases (MASPs). A similar lectin‐based complement system, consisting of the lectin–protease complex and C3, is present in ascidians, our closest invertebrate relatives, and functions in an opsonic manner. We isolated several lectins homologous to MBLs and ficolins and several MASPs in invertebrates and lower vertebrates, and herein we discuss the molecular evolution of these molecules. Based on these findings, it seems likely that the complement system played a pivotal role in innate immunity before the evolution of an acquired immune system in jawed vertebrates.

MASP-3 and Its Association with Distinct Complexes of the Mannan-Binding Lectin Complement Activation Pathway

The mannan-binding lectin (MBL) pathway of complement activation is part of the innate immune defense. The binding of MBL to microbial carbohydrates activates the MBL-associated serine proteases (MASPs), which recruit the complement factors, C4 and C2, to generate the C3 convertase or directly activate C3. We present a phylogenetically highly conserved member of the MBL complex, MASP-3, which is generated through alternative splicing of the MASP-1/3 gene. The designation of MASP-3 as a protease is based on homology to known MASPs. Different MBL oligomers were found to have distinct MASP composition and biological activities. MASP-1, MAp19, and direct C3-cleaving activity are associated with smaller oligomers whereas MASP-3 is found together with MASP-2 on larger oligomers. MASP-3 downregulate the C4 and C2 cleaving activity of MASP-2.

A Novel Human Serum Lectin with Collagen- and Fibrinogen-like Domains That Functions as an Opsonin

Collectins are C-type animal lectins with both collagenous and carbohydrate recognition domains and are involved in the first line host defense against pathogens. We report here a novel Ca2+-dependent and GlcNAc-binding lectin consisting of subunits of 35 kDa (P35) with a collagen-like sequence. When P35 is isolated from human serum, it forms a homopolymer by means of intermolecular disulfide bonding, as is the case with collectins. P35 cDNA was cloned from a human liver cDNA library, and the deduced amino acid sequence of 313 residues revealed that the mature form of P35 consists mainly of collagen- and fibrinogen-like domains. The latter contained two potential Ca2+-binding sites that may be involved in carbohydrate binding. The overall sequence of P35 was highly homologous to porcine ficolins α and β. Northern blots of various human tissues showed that the major product of the 1.3-kilobase-long P35 transcript is expressed in liver. P35 enhanced phagocytosis of Salmonella typhimurium by neutrophils, suggesting an opsonic effect via the collagen region. P35 was found to bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as well as to neoglycolipids containing complex-type oligosaccharides derived from glycoproteins, suggesting that P35 recognizes GlcNAc residues such as those found in microbial glycoconjugates and complex-type oligosaccharides. Therefore, P35 represents a new type of GlcNAc-binding lectin with structural and functional similarities to collectins involved in innate immunity.

Cutting Edge: Complement-Activating Complex of Ficolin and Mannose-Binding Lectin-Associated Serine Protease

Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different. A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL. On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19). We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP. MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3. The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement. These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.

Proteolytic Activities of Two Types of Mannose-Binding Lectin-Associated Serine Protease

Mannose (or mannan)-binding lectin (MBL) is an oligomeric serum lectin that plays a role in innate immunity by activating the complement system. In human, two types of MBL-associated serine protease (MASP-1 and MASP-2) and a truncated protein of MASP-2 (small MBL-associated protein; sMAP or MAp19) are complexed with MBL. To clarify the proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3, we isolated these two types of MASP in activated forms from human serum by sequential affinity chromatography. On an anti-MASP-1 column, MASP-2 passed through the column in the presence of EDTA and high salt concentration, whereas MASP-1 was retained. Isolated MASP-1 and MASP-2 exhibited proteolytic activities against C3 and C4, respectively. C2 was activated by both MASPs. C1 inhibitor (C1 INH), an inhibitor for C1r and C1s, formed equimolar complexes with MASP-1 and MASP-2 and inhibited their proteolytic activities.

L-Ficolin Specifically Binds to Lipoteichoic Acid, a Cell Wall Constituent of Gram-Positive Bacteria, and Activates the Lectin Pathway of Complement

The lectin pathway of complement is activated when a carbohydrate recognition complex and associated serine proteases binds to the surface of a pathogen. Three recognition subcomponents have been shown to form active initiation complexes: mannan-binding lectin (MBL), L-ficolin, and H-ficolin. The importance of MBL in antimicrobial host defense is well recognized, but the role of the ficolins remains largely undefined. This report shows that L-ficolin specifically binds to lipoteichoic acid (LTA), a cell wall component found in all Gram-positive bacteria. Immobilized LTA from Staphylococcus aureus binds L-ficolin complexes from sera, and these complexes initiate lectin pathway-dependent C4 turnover. C4 activation correlates with serum L-ficolin concentration, but not with serum MBL levels. L-ficolin binding and corresponding levels of C4 turnover were observed on LTA purified from other clinically important bacteria, including Streptococcus pyogenes and Streptococcus agalactiae. None of the LTA preparations bound MBL, H-ficolin, or the classical pathway recognition molecule, C1q.

Glomerular Activation of the Lectin Pathway of Complement in IgA Nephropathy Is Associated with More Severe Renal Disease

IgA nephropathy (IgAN) is characterized by glomerular co-deposition of IgA and complement components. Earlier studies showed that IgA activates the alternative pathway of complement, whereas more recent data also indicate activation of the lectin pathway. The lectin pathway can be activated by binding of mannose-binding lectin (MBL) and ficolins to carbohydrate ligands, followed by activation of MBL-associated serine proteases and C4. This study examined the potential role of the lectin pathway in IgAN. Renal biopsies of patients with IgAN (n=60) showed mesangial deposition of IgA1 but not IgA2. Glomerular deposition of MBL was observed in 15 (25%) of 60 cases with IgAN and showed a mesangial pattern. All MBL-positive case, but none of the MBL-negative cases showed glomerular co-deposition of L-ficolin, MBL-associated serine proteases, and C4d. Glomerular deposition of MBL and L-ficolin was associated with more pronounced histologic damage, as evidenced by increased mesangial proliferation, extracapillary proliferation, glomerular sclerosis, and interstitial infiltration, as well as with significantly more proteinuria. Patients who had IgAN with or without glomerular MBL deposition did not show significant differences in serum levels of MBL, L-ficolin, or IgA or in the size distribution of circulating IgA. Furthermore, in vitro experiments showed clear binding of MBL to polymeric but not monomeric patient IgA, without a significant difference between both groups. Together, these findings strongly point to a role for the lectin pathway of complement in glomerular complement activation in IgAN and suggest a contribution for both MBL and L-ficolin in the progression of the disease.

Human M-Ficolin Is a Secretory Protein That Activates the Lectin Complement Pathway

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a coimmunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.

Activation of the Lectin Complement Pathway by H-Ficolin (Hakata Antigen)

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.

Ficolins and the lectin complement pathway.

Ficolins, found in various tissues, are a group of proteins containing both a collagen‐like and a fibrinogen‐like domain. Recently, it was shown that ficolins present in serum are lectins with a common binding specificity for N‐acetylglucosamine (GlcNAc). The fibrinogen‐like domain is responsible for the carbohydrate binding. Mannose‐binding lectin (MBL) is also a collagenous lectin in serum that is specific for GlcNAc and mannose binding. Its domain organization is similar to that of ficolins, except that MBL has a carbohydrate‐recognition domain instead of a fibrinogen‐like domain. MBL plays a role in innate immunity by acting as an opsonin and activating complement in association with MBL‐associated serine protease (MASP) via the lectin pathway. Investigations of two types of human serum ficolins, ficolin/P35 and Hakata antigen, revealed that they are associated with MASPs and sMAP, a truncated protein of MASP‐2, and that they activate complement. These findings indicate that serum ficolins are structurally and functionally similar to MBL and have the capacity to activate the lectin pathway and thus have a role in innate immunity.