The mannan-binding lectin pathway of complement activation: biology and disease association

Molecular Immunology 38 (2001) 133– 149 www.elsevier.com/locate/molimm

Review

The mannan-binding lectin pathway of complement activation: biology and disease association Steen Vang Petersen *, Steffen Thiel, Jens Christian Jensenius Department of Medical Microbiology and Immunology, The Bartholin Building, Uni6ersity of Aarhus, DK-8000 Aarhus, Denmark

Abstract Mannan-binding lectin (MBL) is a plasma protein found in association with several serine proteases (MASPs) forming the MBL complex. MBL recognises carbohydrate structures arranged in a particular geometry, such as those found on the surface of micro-organisms. When bound to e.g. bacteria the MBL complex will initiate the activation of the complement cascade. Mounting evidence supports the importance of the MBL pathway of complement activation in innate immunity. In this review, we focus on the structure and function of the proteins within the MBL pathway and address the properties of the pathway as an initiator of the host response against potential pathogenic micro-organisms. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Plasma protein; MBL, Complement; MASP, Infectious disease

1. Introduction The innate immune system is considered as the first line of host defence against infectious agents, which have penetrated the mechanical barriers. It is comprised of soluble and membrane bound proteins with a predefined specificity, in many cases involving carbohydrate moieties. The adaptive immune system subsequently plays an important role in generating specific responses towards the infectious agents. Although considered as separate systems, components of the innate immune system interact with the adaptive system, thus conferring an instructive role of innate immunity on the adaptive immune defence (Fearon and Locksley, 1996). Mounting evidence supports the importance of the mannan-binding lectin (MBL) pathway of complement activation in innate immunity. The initiator of the pathway consists of complexes of the plasma protein MBL and MBL-associated serine proteases (MASPs). MBL binds to carbohydrate structures presented by a wide range of pathogenic bacteria, viruses, fungi, and parasites. The involvement of the MBL pathway in first

line host defence is indicated by the finding that the frequency of MBL deficiency in individuals with severe and repeated infections is significantly elevated as compared to a control population. The presence of promoter polymorphisms and mutations in exon one of the gene encoding MBL results in pronounced inter-individual variation in the level of circulating MBL, and studies have shown that low MBL level to be the most frequent immuno-deficiency. Although extensive studies have yielded detailed information on the structure of MBL and on the activity of the associated serine proteases our understanding of the structure and functions of the MBL complex is still incomplete. In this review, we will focus on the structure and function of the proteins within the MBL pathway and address the properties of the pathway as an initiator of the host response against potential pathogenic micro-organisms.

2. Mannan-binding lectin, a pattern recognition molecule

2.1. Structure and function * Corresponding author. Tel.: +45-89-42-1778; fax: + 45-86-196128. E-mail address: [email protected] (S.V. Petersen).

Collectins constitute a family of proteins containing a collagenous region and a carbohydrate-binding lectin

0161-5890/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0161-5890(01)00038-4

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domain (Holmskov et al., 1994). In humans four members of this group of proteins are known: the plasma protein MBL, the lung surfactant proteins SP-A and SP-D, and CL-L1, which is localised to the cytosol of hepatocytes (Hakansson and Reid, 2000). Two additional collectins, conglutinin and CL-43, are found in the bovidae. Proteins of the collectin family are composed of subunits made up of three identical polypeptide chains each containing a short N-terminal cross-linking region containing two or three cysteines followed by a collagen-like region of variable length, a neck region and a C-terminal lectin domain or carbohy-

drate recognition domain (CRD) (Fig. 1). The collagenlike region of human MBL contains eight potential hydroxylation sites and four potential O-glycosylation sites. By formation of an a-helical coiled-coil structure the neck-region initiates the trimerisation of the polypeptides to form the structural subunit presenting a collagen region and three C-terminal lectin domains (Fig. 1). The subunit is stabilised by hydrophobic interactions and interchain disulfide bonds in the N-terminal cross-linking region (Wallis and Drickamer, 1999). In the circulation, human MBL is found as dimers to hexamers of the structural subunit giving MBL a serti-

Fig. 1. The structure of the mature MBL polypeptide chain. The structural elements are shown with the Gly-X-Y interruption of the collagen-like region indicated. Three identical polypeptide chains associate and generate the structural subunit. This subunit is stabilised through dislufide bonds in the cross-linking region (not shown). The interruption in the collagen-like region gives rise to a bend in the structural subunit. The structural subunit is found in the circulation as oligomers. The hexameric structure of MBL (shown here) presents an array of 18 CRD, allowing for avid binding to micro-organisms displaying appropriate carbohydrate ligands.

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Fig. 2. Three mutations in exon 1 of the MBL gene generate three amino acid substitutions in the collagen-like region. As shown, two of these substitutions disrupt the Gly-X-Y repeats by exchanging a glycine residue with aspartic acid (Variant B) or with glutamine (Variant C). A third substitutes a cysteine for an argenine (Variant D). As indicated these amino acid substitutions disrupt the assembly of the MBL molecule (see text for details). The natural occurring interruption of the collagen-like region is underlined.

form appearance when viewed by electron microscopy (Lu et al., 1990). Studies have shown that in recombinant rat MBL-A and in mouse serum MBL-A, the structural subunits are covalently joined via inter-subunit disulfide bonds in the N-terminal cross-linking region (Wallis and Drickamer, 1999; Liu et al., 2001), while the intermolecular forces joining the subunits in mouse MBL-C to a large extend are non-covalent only (Liu et al., 2001). MBL is a C-type lectin as it requires calcium for binding to a carbohydrate ligand. The crystal structure of recombinant lectin-domains from rat MBL-A complexed with an oligosaccharide clearly shows the direct participation of calcium in the binding by formation of coordination bonds to the 3- and 4-OH groups of the bound sugar (Weis et al., 1992). In addition, the structure also revealed the requirement of the 3- and 4-OH groups of the complexed sugar to be in the equatorial plane of the hexose ring-structure in order to allow for hydrogen bonding to amino acid side chains. Thus, MBL binds to several monosaccharides, e.g. N-acetylglucosamine, mannose, N-acetylmannoseamine, L-fucose, and glucose, whereas galactose is not bound (Holmskov et al., 1994). The dissociation constant of the interaction between one CRD and carbohydrate is very weak (10 − 3 M) (Iobst et al., 1994). But the multiple CRDs being presented by the quaternary structure of the MBL molecule allows for high avidity binding to repetitive carbohydrate ligands or to patches with high concentration of the relevant carbohydrates. Indeed, the X-ray structure of trimeric MBL shows that the CRDs are spaced 45 A, apart, thus dictating a repetitive nature of the ligand to achieve avid binding (Sheriff et al., 1994). Using different approaches the KD for human MBL binding to mannan or glycosylated

bovine serum albumin was estimated to be in the order of 10 − 9 M (Kawasaki et al., 1983; Lee et al., 1992). This type of binding characteristics has recently been referred to as micro-pattern and macro-pattern recognition, respectively (Hoffmann et al. 1999). MBL has accordingly been shown to bind to a wide range of clinically relevant mirco-organisms (including bacteria, viruses, fungi and parasites) displaying repetitive carbohydrate structures on their surfaces (Holmskov et al., 1994; van Emmerik et al., 1994; Neth et al., 2000). MBL does not appear to bind to self-surfaces under normal physiological conditions as a result of the prevalent termination of the glycans with sialic acid and maybe due to the lack of repetitive carbohydrate structures on the surface of animal cells.

2.2. Structural mutations within the mannan-binding lectin gene The constitutional level of MBL in the circulation is very stable (Nielsen et al., 1995), whereas the level in different individuals varies from below 50 ng/ml to above 3 mg/ml. This very large variation resides in the occurrence of four allotypes due to mutations in exon 1 of the gene encoding the MBL polypeptide as well as several polymorphisms in the promoter region. This result in a number of different genotypes, some of which are associated with a decreased level of MBL. Single base mutations in codon 54 and 57 of exon 1 give rise to substitution of glycin with aspartic acid and glycine with glutamic acid at residue position 34 and 37 in the mature protein, respectively (Sumiya et al., 1991; Lipscombe et al., 1992) (Fig. 2). These mutations disrupt the Gly-X-Y repeats of the collagenous region. Studies conducted on recombinant rat MBL-A with

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mutations homologous to the human alleles introduced indicate that these mutations alter the interchain disulfide bonds within the N-terminal cross-linking region (Wallis and Cheng, 1999). Pulse chase experiments on CHO cells expressing these two mutant forms of MBL suggested that the low level of circulating MBL associated with these two alleles in humans is due to impaired secretion (Heise et al., 2000). Using different expression systems other studies show that recombinant human MBL carrying the codon 54 mutation and wild type MBL are secreted at similar rates (Super et al., 1992; Kurata et al., 1993; Ma et al. 1997). Naito et al. (1999a) suggest that the low level of circulating MBL in individuals carrying the codon 54 mutation could be due to a higher turnover rate in serum of the mutant form as compared to wild type MBL. A third single base mutation in exon 1 results in substitution of cysteine for an argenine at residue 32 of the mature protein (referred to as the codon 52 mutation). While this substitution does not interrupt the Gly-X-Y sequence, the presence of this additional cysteine residue has been suggested to disrupt oligomer formation by generation of additional disulfide bonds involving the introduced cysteine residue (Wallis and Cheng, 1999). The disulfide bridge pattern in the N-terminal region was found not to be affected by this substitution. The low amounts of MBL found in the circulation of individuals homozygous for any of these three mutations, or combinations hereof, was shown to be of lower molecular weight than wild type MBL. Heterozygotes produced primarily the high molecular weight wild type MBL but also small amounts of the lower molecular weight form (Lipscombe et al., 1995). The alleles containing the codon 52, 54, or 57 mutation are designated D, B and C, respectively, while the wild type allele is designated A (Madsen et al., 1994). The frequency of these mutations in a population varies between ethnic groups (reviewed by Turner and Hamvas, 2000), e.g., in Danish Caucasians, the B and C alleles are found in 13 and 2% of the population, respectively, whereas the frequencies in sub-Saharan Africans (Kenya) are 3 and 23%, respectively. The frequency of the D allele is 5% in both populations.

2.3. Promoter polymorphisms The identification of promotor variants showed that the level of MBL in plasma is also modulated at the transcriptional level (Madsen et al., 1995). Nucleotide substitutions at position-550 (G to C) and at position221 (G to C) gives rise to the H(G)/L(C) and Y(G)/ X(C) variants, respectively. Another substitution in the 5% untranslated region of exon 1 (position +4) gives rise to the P(C)/Q(T) variant (Fig. 3). Several other promoter variants have been described but found not

to influence the MBL levels (Madsen et al., 1998). The haplotypes HY, LY and LX were found to be associated with high, medium, and low plasma levels of MBL, respectively (Madsen et al., 1995). Because of linkage disequilibrium between some of the variants only seven MBL haplotypes (HYPA, LYQA, LYPA, LXPA, LYPB, LYQC and HYPD) have been established (Madsen et al., 1998). In addition, the HXPA haplotype has been reported as occurring in three systemic lupus erythematosus (SLE) patients (Sullivan et al., 1996).

2.4. From gene to plasma le6els The median level of MBL within a Danish Caucasian population was reported at 1.2 mg/ml plasma for individuals homozygous for the wild type allele (Garred et al., 1992a). However, the levels ranged from 0 to 5 mg MBL/ml plasma. In the same study, the median level for individuals heterozygous for the codon 54 mutation was found to be 0.2 mg MBL/ml (range 0–1.2 mg/ml). Similar median levels were also obtained in an earlier study (Lipscombe et al., 1992). This large variation between individuals with identical structural haplotypes can in part be ascribed to the presence of promotor variants. Interestingly, it has been shown that the LX haplotype has a dominant effect on the MBL level. Thus, individuals homozygous for the LXA haplotype show a median level of circulating MBL comparable to that of a genotype with two structural mutations (Madsen et al., 1995; Steffensen et al., 2000) although the first group did not find this in a later study (Garred et al., 1999a). One should note that even taking all known allotypes into account there is still considerable variation (up to 6-fold) between individuals with identical genotypes (Steffensen et al., 2000). This agrees with the up to 2.5-fold inter-individual difference between mice of the same inbred strain (Liu et al., 2001).

2.5. Acute-phase modulation of mannan-binding lectin le6els The promotor region of the MBL gene contains several acute phase response elements (Ezekowitz et al., 1988; Taylor et al., 1989). In agreement with this the circulating level of MBL was found to increase 1.5 to 3 fold in patients undergoing major surgery and in patients suffering an attack of malaria (Thiel et al., 1992). Also, the MBL levels in mice receiving an intraperitoneal injection of LPS or casein showed an increase in MBL plasma levels of up to 2-fold (Liu et al., 2001). In comparison with the classical acute phase reactant Creactive protein, the increase in MBL levels detected in both studies designates MBL as a weak acute phase reactant. It should be noted that the increase detected is

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small, when compared to the 1000-fold variation found between individuals. However, when analysing the MBL promoter using a luciferase assay system, Naito et al. (1999b) observed that an acute-phase agonist (a glucocorticoid) decreased the transcription of the MBL gene. This finding questions the understanding of MBL as an acute-phase reactant.

3. Mannan-binding lectin-associated proteins Initial analysis of affinity purified MBL from both human and mouse serum revealed the presence of an MASP (referred to as P100 in the mouse) (Matsushita and Fujita, 1992; Takada et al., 1993). Later another MBL-associated serine protease termed MASP-2 (Thiel et al., 1997) as well as a smaller protein of 19 kDa, termed MBL-associated protein of 19 kDa (MAp19) (Stover et al., 1999a) or small MBL-associated protein (sMAP) (Takahashi et al., 1999) were identified as components of the MBL complex. The complexity of the first component of the MBL pathway of complement activation was further underscored, when recently a third serine protease (MASP-3) was cloned (Dahl et al., 2001).

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3.1. Mannan-binding lectin-associated serine proteases The serine proteases of the MBL pathway are modular proteins with a domain composition identical to that of C1r and C1s of the classical pathway of complement activation (Takayama et al., 1994; Sato et al., 1994; Thiel et al., 1997) (Fig. 4). An N-terminal CUBdomain (CUB-I) is followed by an epidermal growth factor (EGF)-like domain, another CUB-domain (CUB-II) and two complement control protein (CCP)domains also known as short consensus repeats (SCRs). The C-terminal serine protease (SP) domains are homologous to serine proteases of the chymotrypsinogen family. Hence, the catalytic residues consisting of His, Asp and Ser are located at the interface between two six-stranded b-barrels. The amino acid located in the substrate specificity pocket is Asp thus dictating a trypsin-like substrate specificity. A short link segment separates the SP domain from the preceding five domains. This segment contains a conserved Arg-Ile peptide bond. Cleavage of this bond generates an active serine protease consisting of an A-chain composed of the first five domains and the link region and an enzymatic B-chain held together by a disulfide bond. Although homologous in domain architecture Endo et

Fig. 3. The organization of the MBL gene located at chromosome 10q21 (Sastry et al., 1989; Taylor et al., 1989). Two promotor polymorphisms at positions -550 and -221 are indicated. A third polymorphism is found at position + 4 in the 5% untranslated region. Exon 1 of the gene encodes the 5% untranslated region, a signal peptide of 20 amino acids, the N-terminal cross-linking region and the first part of the collagenous region harbouring the base mutations that results in the production of the MBL variants. The second exon encodes the remaining part of the collagenous region including the disruption of the Gly-X-Y repeat. A third exon encodes the neck region. The last exon encodes the carbohydrate recognition domain (CRD) and the 3% untranslated region. Because of linkage disequilibrium only the indicated haplotype has been described. Figure not to scale.

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al. (1998) suggested the presence of two different lineages of MASPs in vertebrates, the TCN-type and the AGYtype. The first group contains those MASPs, which have the active site serine encoded by a TCN codon (N denotes A, G, C or T), a serine protease domain encoded by a split exon structure, and a histidine loop (a structural feature of primitive serine proteases denoting an intramolecular disulfide bond between cysteines residues on each side of the active site histidine residue). Human MASP-1 belongs to the TCN-type. The other group has an active site serine encoded by an AGY codon (Y denotes the nucleotides T or C), a single exon encoding the serine protease domain, and lacks the cysteines involved in the formation of the histidine loop. This group encompasses human MASP-2 and -3 and also C1r and C1s of the classical pathway. Since the TCN lineage contains features also found in trypsin and chymotrypsin the AGY lineage was proposed to have diverged from the TCN lineage (Endo et al., 1998). Thus, it appears appropriate to apply the term MASP-like serine proteases for the family containing MASPs, C1r, and C1s (Gadjeva et al., 2001). cDNA encoding proteins of the MASP-like serine proteases have now been cloned from mouse and rat (Takada et al., 1993; Stover et al., 1999b), carp, shark, lamprey, and Xenopus (Endo et al., 1998) and the ascidian Halocynthia roretzi (Ji et al., 1997). The presence of MASPs in acidian indicates that the MBL pathway antedates the classical pathway of complement activation.

3.2. Proteolytic acti6ities of mannan-binding lectin-associated serine proteases Initially, it was shown that MBL purified from mouse serum (referred to as Ra-reactive factor (RaRF)) and from rat, rabbit and human serum was capable of consuming complement components C4 and C2 (Ikeda et al., 1987; Ji et al., 1988). Based on the structural similarity between C1q of the classical pathway and MBL it was hypothesised that MBL could bind and activate C1r2C1s2 of the classical pathway (Ikeda et al., 1987). Using different in vitro systems, it was shown that

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purified human MBL could bind and facilitate the activation of C1r2C12 (Lu et al., 1990; Ohta et al., 1990). However, further analysis of MBL preparations from human and mouse revealed the presence of a specific serine protease, which was believed to exert this C1s-like substrate specificity (Matsushita and Fujita, 1992; Ji et al., 1993). When analysing full serum, Thiel et al. (2000) found that MBL circulated in complex with its specific MASP proteases while C1r and C1s were found in association with C1q only. Studies by Ogata et al. (1995) showed that mouse MASP (P100) cleaved murine as well as human C4 (with a 20- to 100-fold higher specific activity than C1s) and to a lesser extent murine C3. Matsushita and Fujita (1995) reported that also human MASP could cleave C3. As only one MBL-associated serine protease was identified in 1995, it now seems likely that these experiments were conducted using a mixture of MASPs. With the identification of MASP-2 it was revealed that the C4 cleaving activity was a feature of MASP-2 and not MASP-1 (previously MASP) (Thiel et al., 1997). Further studies performed with human recombinant MASP-2 expressed in mammalian cells, clearly showed that MASP-2 cleaves C4 and C2, thereby generating the C3 convertase, C4b2b (Vorup-Jensen et al., 2000). A recent report used sequential affinity chromatography to isolate MASP-1 and -2 from human serum and confirmed that MASP-2 is a C4 cleaving protease, and further showed that both MASP-1 and -2 have the ability to cleave complement component C2 (Matsushita et al., 2000). It was also corroborated that MASP-1 cleaves C3. Using a different purification procedure, we also find that MASP-1 cleaves C3. This experiment is conducted using a 50-fold surplus of protease and an incubation time of 16 h at 37 °C. Takahashi et al. (2000) reported that the ability to deposit C3b on zymosan was impaired in serum from MASP-1 knockout mice at 0 °C in comparison with wild type animals. No difference was seen at 37 °C. This indicates a possible physiologic role of MASP-1 in C3b deposition although, the specific phenotype was only observed at 0 °C. However, it should be noted that, as the MASP-1 gene had been disrupted in the second exon, the MASP-1

Fig. 4. Schematic representation of the genes encoding human MBL associated proteins and the transcription products. The gene encoding MASP-1/3 (a) is located at chromosome 3q27 – q28 and encompasses 17 exons. The first exon encoding the 5% untranslated region of the mRNA is followed by 9 exons encoding a signal peptide and the five N-terminal domains of the proteins. As indicated, these exons for the A chain are combined identically in the mRNA encoding MASP-1 and -3. These exons are further combined with either a MASP-3 specific exon encoding the link region, the protease domain and the 3% untranslated region or with the remaining six exons encoding the homologous regions of MASP-1. Thus, as a result of the gene structure and alternative splicing the first five domains of mature MASP-1 and MASP-3 are identical. The gene encoding MAp19/MASP-2 (b) encompasses 12 exons and is located on chromosome 1 (1p36). The 5% untranslated region encoded by the first exon are combined with the following three exons encoding a signal peptide, a CUB domain and an EGF-like domain. As shown, the formation of a MAp19 specific mRNA is obtained by the addition of a fifth exon encoding the four MAp19 specific amino acids (EQSL) and the 3% untranslated region. MASP-2 mRNA is obtained by alternative splicing combining the first four exons with the remaining 7 exons thus removing the MAp19 specific exon. Hence, mature MAp19 and MASP-2 shares the first CUB domain and the EGF-like domain. The base-triplet encoding the active serine of the protease domain is indicated. The regions of the gene and mRNA that codes for the individual protein domains are shown and a key given. Introns above 3000 bp are indicated (/). Data were obtained from Endo et al. (1996, 1998), Takada et al. (1995), Stover et al. (1999c, 2001) and Dahl et al. (2001).

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knockouts would also be expected to be deficient in MASP-3 (Fig. 4, see below). Thus, it remains to be clarified whether this activity is of physiological importance. One could speculate that a low turnover rate of C3 by MASP-1 could initiate the alternative pathway by depositing small amounts of C3b on an activating surface. Others have not been able to detect any C3 cleaving activity of purified MBL/MASP complexes (Wong et al., 1999). Thus, while the proteolytic activity of MASP-2 has been established the physiologic activity of MASP-1 is still controversial. Recently, a third MBL-associated serine protease (MASP-3) was characterised (Dahl et al., 2001). MASP-3 shares domain organisation with the other members of the MASP-like family of serine proteases (Fig. 4). The protease domain contains the genetic and structural features of the AGY lineage. Interestingly, it was found that the exons encoding the A chain of MASP-3 were identical to the exons encoding the A chain of MASP-1. The region encoding the MASP-1/3 A chain is followed by a single exon encoding the protease domain of MASP-3 and further downstream by a split exon structure with 6 exons encoding the protease domain of MASP-1 (Fig. 4). Hence, MASP-1 and -3 are produced as a consequence of alternative splicing of primary mRNA transcripts. It has been suggested that this gene structure is an evolutionary intermediate between the TCN lineage and the AGY lineage of the MASP-like serine protease family (Nonaka, 2001). The physiological activity of MASP-3 remains to be determined.

3.3. Mannan-binding lectin-associated protein of 19 kDa When a MBL preparation was analysed by Western blotting using an antibody raised against a peptide representing 19 N-terminal amino acids of MASP-2 a band of 20 kDa was detected (Thiel et al., 1997). This was initially interpreted as a truncated form of MASP2. With the analysis of the gene encoding MASP-2 it became clear that the 20 kDa band observed by Thiel et al. (1997) was a protein encoded by a specific mRNA. This protein is now referred to as MAp19 (Stover et al., 1999a) or sMAP (Takahashi et al., 1999). MASP-2 and MAp19 are products of the same structural gene (Fig. 4). Because of alternative splicing the first amino acids of MASP-2 and MAp19 (comprising the CUB-I domain and the EGF-like domain) are identical. A separate exon encodes four unique C-terminal amino acids (EQSL) of MAp19. Hitherto no physiological role of MAp19 has been reported. In accordance with our own results, Takahashi et al. (1999) reported that MAp19/ sMAP co-purified with MASP-1 in the absence of calcium. In analogy with C1r and C1s, where the N-terminal CUB-I/EGF domains are involved in the

calcium-dependent formation of the C1r2C1s2 tetramer and also in the interaction with C1q, it was suggested that the CUB-I/EGF domain of sMAP/MAp19 might mediate a possible calcium-dependent interaction between MASP-1 and MBL (Takahashi et al., 1999). It was also suggested by that MAp19 could have a regulatory function in the MBL pathway.

4. Mannan-binding lectin/MBL-associated serine protease complexes Due to striking structural similarities it was initially believed that the quaternary structure of the complex initiating the MBL pathway of complement activation was highly homologous to that of the classical pathway. With the identification of three MASPs and a fourth MBL-associated protein with no protease domain, a picture of a more complex system than initially anticipated is emerging. Only little is known about the stoichiometric composition and the molecular interactions involved in the assembly of the MBL complexes. Using recombinant MASP-2 Vorup-Jensen et al. (2000) showed that recombinant MASP-2 alone was able to associate with purified MBL and that this complex could activate C4. These findings were supported by Wallis and Dodd (2000) who found that recombinant truncated forms of rat MASP-1 and -2 containing the three first domains of the A chain were able to interact, independently, with rat MBL. This interaction was found to be calcium dependent. These authors also found the first two N-terminal domains (CUB-I/EGF (Fig. 4.)) of MASP-1 and -2 to be sufficient for the formation of calcium independent homodimers. Using a baculovirus/insect cell expression system Thielens et al. (2001), showed that truncated human MASP-1 and -2 (containing the two first domains) could bind independently to immobilised MBL in a calcium dependent manner. However, the dissociation constant was significantly lower, when full length MASP-1 and -2 were used. Wallis and Dodd (2000) found that the CUB-I/ EGF fragment of MASP-2 and MAp19 formed homodimers in the presence of calcium. However, the CUB-I/EGF domain of MASP-1 seemed to form homodimers independent of calcium. It was found that MASP-1, -2 and MAp19 did not interact with each other. As mentioned above, several studies have indicated the presence of a complex of MASP-1 and MAp19 in serum. The reason for this discrepancy is not known, but clearly indicates the need for further studies on purified serum components. The interactions described above are in contrast to the classical pathway where C1r mediates the interaction between the C4/C2 cleaving protease C1s and C1q. The existence of a circulating complex composed of MBL and MASP-2 was supported with the analysis of MBL purified from plasma. By use of anion exchange chromatography or

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sucrose density gradient centrifugation Dahl et al. (2001) were able to separate circulating MBL/MASP complexes into two forms, MBL-I with a smaller molecular size than a second form, MBL-II. MASP-2 and -3 were found to be associated mainly with the MBL-II complex while MAp19 was present in the MBL-I complex. MASP-1 was found in both complexes but predominantly in the MBL-I complex. These findings suggest the presence of a complex composed of one subpopulation of MBL and MASP-2/MASP-3 and another composed of a second subpopulation of MBL and MASP-1/MAp19. The biological activities of these complexes are suggested to be C4/C2 activation of the MBL-II/MASP-2/MASP-3 complex and direct activation of C3 by the MBL-I/MASP-1/MAp19 complex. It should be noted that although the names suggest that the MASPs and MAp19 are only found in complex with MBL a large proportion of these proteins exists as non MBL-complexed in the circulation (Terai et al., 1997; Thiel et al., 2000; Dahl et al., 2001). This differs to the classical pathway where the C1 subcomponents in circulation are found in approximately equimolar ratios.

5. Complement activation via mannan-binding lectin

5.1. Implications of quaternary structure Super et al. (1989) showed that the capability of serum to deposit complement components C3b and C4b onto a mannan-coated surface was correlated to the levels of MBL in the circulation. This opsonic defect in MBL-deficient serum was subsequently linked to the codon 54 mutation (Sumiya et al., 1991). Using gel-permeation chromatography and sucrose density gradient centrifugation Lipscombe et al. (1995) showed that individuals homozygous for the structural mutations produced small amounts of MBL with a lower molecular weight than wild type MBL. Individuals with a heterozygous genotype produced primarily high molecular weight MBL but also some low molecular weight MBL. Several studies have indicated that the complement activating capacity of MBL is highly dependent on the oligomeric form(Ikeda et al., 1987; Lu et al., 1990; Yokota et al., 1995). Thus, high molecular weight MBL, i.e., tetramers to hexamers of structural subunits, were found to be capable of activating complement whereas low molecular weight MBL could not. When the MBL variants with codon 54 and 57 mutation were expressed in COS-1 cells, Kurata et al. (1993) found that the mutant proteins were secreted into the medium in levels similar to wild type MBL. However, they found that the amount of higher order oligomers produced by cells expressing mutant MBL was significantly reduced in favour of lower forms

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relative to cells expressing wild type MBL. The authors observed that this altered distribution of oligomers significantly reduces the capacity to activate complement. Although only one kind of MBL exists in human two different species, MBL-A and MBL-C are found in rats (Drickamer et al., 1986). Also mice (Sastry et al., 1991) and rhesus monkeys (Mogues et al., 1996) have both MBL-A and MBL-C. Originally, MBL-C was thought to be a low molecular weight liver protein whereas MBL-A was referred to as a high molecular weight serum protein (Oka et al., 1988). MBL-A has been referred to as the homologue of human MBL partly on this basis and partly because MBL-A, like human MBL, has three cysteines in the N-terminal cross-linking region and MBL-C only two. However, the overall amino acid sequence identity is 57 and 61% between human MBL and mouse MBL-A and -C, respectively. Fifty-three percent identity is seen between mouse MBL-A and -C. This perception of the existence of serum and liver MBL species has been questioned by the purification of both MBL proteins as high molecular weight forms from mouse serum (Hansen et al., 2000). Further, it has been found that MBL-C is present in mouse serum at about six times higher concentration than MBL-A (Liu et al., 2001). Studies performed on purified mouse MBL-A and -C (Hansen et al., 2000) and on recombinant rat MBL-A and -C (Wallis and Drickamer, 1999) have shown that the complement activating capacity of MBL-A is higher than that of MBL-C. Using rat MBL-A and -C chimeras expressed in CHO cells Wallis and Drickamer (1999) showed that the difference in complement activation between MBL-A and -C could be ascribed to the N-terminal cross-linking region and the first part of the collagenous region. Thus, MBL-C containing this cassette of MBL-A has as similar complement activating capacity when compared to MBL-A. Gel permeation chromatography indicated that this was due to the formation of higher oligomers. This supports the view that higher oligomeric structures of MBL are needed for complement activation. In contrast to this, mouse MBL-A and -C have in other studies been found to occur in circulation as high molecular weight forms with the MBL-C oligomers to a large extend held together by non-covalent interactions Hansen et al., 2000; Liu et al., 2001) Observations have indicated mouse MBL-C and especially rat MBL-C to be sensitive to proteolytic degradation during purification (Hansen et al., 2000). This might explain the previous contention that MBL-C was largely a liver protein. Future studies will address the complement activating capacity of these higher oligomeric structures of MBL-C.

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5.2. Implications of mannan-binding lectin allotypes When Super et al. (1992) expressed human MBL with the codon 54 mutation (B allotype) in a mouse hybridoma cell line they found that this mutant MBL could not mediate the activation of C4 and C3. A plausible explanation for this defect was presented when it was shown that purified MASPs could not associate with rMBL carrying the codon 54 mutation (Matsushita et al., 1995). These observations were interpreted to indicate that the MASP interaction site on MBL could be mapped to the fifth collagen repeat (containing amino acid 54) or that the mutation disrupts a distal interaction site. Alternatively, it simply reflects a failure to form stable oligomers as it was found that the rMBL B variant produced oligomers that were more easily dissociated than wild type rMBL. Wallis and Cheng (1999) introduced the three structural mutations of human MBL into recombinant rat MBL-A (Fig. 2). Analysis of the codon 52 mutation (Arg “Cys) revealed that oligomer formation was disrupted as a result of aberrant disulfide bonds generated as a consequence of the extra cysteine. Gel permeation chromatography (GPC) indicates that this mutant MBL mainly circulates as monomers of the structural unit. Thus, the loss of complement activating capacity of this mutant is probably due to failure to form higher oligomers. When the mutations in codons 54 and 57 were introduced into rat MBL-A GPC analysis revealed only a slight decrease in the higher oligomeric forms in favour of the lower molecular forms. Further analysis of this material indicated that the disulfide-bonding pattern in the N-terminal region was altered. It was speculated that the altered disulfide bonding pattern could disrupt the MASP interaction site which was suggested to be located within the N-terminal cysteine-rich region and the first half of the collagen domain (Wallis and Drickamer, 1999). A recent paper from the same group shows that recombinant fragments of rat MASP-1 and -2 binds to these mutants with a lower affinity than to wild type MBL-A (Wallis and Dodd, 2000). These findings are in line with the observation that human MBL with the codon 54 mutation is unable to associate with purified human MASP as discussed above (Matsushita et al., 1995). Taken together, the three structural mutations found in human MBL leads to defective complement activation as a consequence of both altered oligomer distribution and an inability to associate with the serine proteases comprising the catalytic unity of the complex. It has been hypothesised that the mutant forms of MBL might subserve functions associated with binding to MBL receptors (see below).

6. Receptor mediated activities of mannan-binding lectin

6.1. Direct opsonisation by mannan-binding lectin Besides opsonisation by the deposition of complement components on an activator via the MBL pathway, MBL has been reported to act directly as an opsonin (Kuhlman et al., 1989) Salmonella monte6ideo (displaying a mannose-rich O-polysaccharide) was thus found to be ingested by monocytes in an MBL-dependent manner using both human MBL (20 mg/ml) and recombinant MBL expressed in CHO cells (1 mg/ml). Another study showed that strains of influenza A virus displaying a high mannose oligosaccharide near the sialic acid bindingpocket of hemagglutinin could bind recombinant human MBL and thereby enhance the H2O2 production of human neutrophils (Hartshorn et al., 1993). In this study, a similar effect was seen using wild type rMBL and rMBL with the codon 54 mutation. Using recombinant human MBL produced in the same expression system Super et al. (1992)showed that both forms of rMBL had a similar potential of mediating the uptake of S. monte6ideo by human neutrophils. These studies indicate that both wild type MBL and mutant MBL interacts with a receptor present on the surfaces of monocytes and neutrophils. Several candidates of a putative MBL receptor have been presented in the literature (reviewed by Eggleton et al. (1998)). One receptor, cC1qR (‘c’ for collagen) was later found to be identical to the intracellular protein calreticulin. Another putative receptor, gC1qR (‘g’ for globular head), was subsequently found to be a mitochondrial protein. The cellular localisation of these putative receptors makes a physiologically relevant interaction with MBL questionable. A third receptor, C1qRp (‘p’ for phagocytic), has been identified as the foetal stem cell marker AA4, a protein involved in cell–cell adhesion. A recent publication has suggested that complement receptor 1 (CR1) besides acting as a receptor for C3b also acts as an MBL-receptor (Ghiran et al., 2000). It was shown that MBL even at a super-physiological concentration (20 mg MBL/ml) could not stimulate phagocytosis of S. monte6ideo by unactivated polymorphonuclear leukocytes (PMNs). If PMNs were activated by fibronectin treatment phagocytosis of S. monte6ideo could be detected when the bacteria were opsonised with MBL at a physiological concentration (1 mg/ml). Phagocytosis by unactivated PMNs was also supported when bacteria were opsonised with both MBL (1 mg/ml) and a sub-opsonizing concentration of rabbit anti-Salmonella IgG. These results are analogous to the findings of CR1 interaction with the opsonins C4b and C3b and thus support the physiological relevance of this interaction. The dissociation constant between recombinant soluble CR1 and MBL was estimated to be 5 nM (Ghiran et al., 2000).

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Recently Bajtay et al. (2000) analysed the interaction between MBL and C1q and various cells and cell lines. Using flow cytometry it was found that whereas C1q binds to isolated B and T lymphocytes MBL does not. This contrasts to the findings of Ghiran et al. (2000) as B cells are known to express CR1. The analysis of MBL and C1q binding to monocytes by flow cytometry showed that there is no competition between the ligands suggesting the presence of different receptors for C1q and MBL on the surface of monocytes (Bajtay et al., 2000). This also contrast the findings of Ghiran et al. (2000), where C1q and MBL were found to compete for binding sites on immobilised soluble CR1. Clearly, further studies are called for to illuminate the structure and importance of the putative MBL receptors.

6.2. Mannan-binding lectin-dependent cell-mediated cytotoxicity Fujita et al. reported in 1995 that several glioma cell lines could bind human MBL in a dose dependent manner. In only one of these cells lines (1321N1) the binding of MBL could be inhibited by mannose (40 mM) or EDTA (10 mM). However, it was shown that purified MBL complexes (containing MASPs) bound to 1321N1 cells could consume complement components C4 and C3. Interestingly, it was also found that MBL binding to a cell line, in an EDTA sensitive manner only, consumed C4. These data suggests that MBL bound to the surface of malignant cells can facilitate complement activation. Ma et al. (1999) substantially extended this finding through experiments on nude mice transplanted with an MBL-reactive human colorectal carcinoma cell line, SW1116. When vaccinia virus expressing wild type MBL was injected directly into such tumours regression of growth was observed. Inhibition of tumour growth, but not regression, was seen when the recombinant vaccinia virus was injected subcutaneously at a distant site. Surprisingly, a similar effect was observed using vaccinia virus expressing MBL variant B. As this mutant is known to have an impaired complement activating capacity, the authors suggested that the effect on the tumour was due to a cellular effector mechanism killing tumour cells that bind MBL as a consequence of an aberrant expression of carbohydrate structures on the cell surface. This activity was referred to as MBLdependent cell-mediated cytotoxicity, MDCC. Using an in vitro model Kawasaki et al. (2000) recently reported that PMNs binds to MBL. This binding results in an increased production from the leukocytes of superoxide, a highly reactive oxygen radical. It was also shown that mutant MBL could exert the same effect, thus supporting the results obtained using the in vivo model. These data suggest the presence of an MBL-receptor on

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the surface of PMN leukocytes in line with the reports discussed above.

7. The mannan-binding lectin pathway and disease Since the first report by Super et al. (1989) of linkage between recurrent infections and low levels of MBL, a large number of papers have been published on association between MBL deficiency and increased susceptibility to various infectious as well as to autoimmune diseases. This has been reviewed in detail by Turner (1998) and Turner and Hamvas (2000) and only selected topics shall be discussed below. It should be noted that 90% of MBL deficient individuals do not acquire repeated infections. This is likely due to the redundancy of the complement system. Hence, it could be speculated that the phenotypical manifestation of MBL deficiency is only observed, when combined with another humoral immunodeficiency being acquired or genetically determined. In support of this notion, Aittoniemi et al. (1998) found that MBL deficient children with recurrent infections had concomitant IgG subclass deficiency or transient IgG subclass deficiency. Until now the correlation between MBL deficiency and susceptibility to infection has been evaluated by selecting an arbitrary level for deficiency, usually the lower detection level of the assay used. There is no clinical data that supports this definition of MBL deficiency, and the MBL level that impose an increased susceptibility may differ between diseases. Other studies compare the frequency of variant MBL alleles between the study group and selected controls. This approach is rendered difficult by the large variation of MBL level found between individuals of identical genotypes (Steffensen et al., 2000).

7.1. Mannan-binding lectin deficiency and increased risk of infection In 1995, Garred et al. (1995) published a study on 228 patients with suspected immunodeficiency. These patients presented clinical symptoms such as recurrent lung infections, recurrent otitis media, diarrhoea and septicaemia. When the MBL genotype of these patients was analysed an increased frequency of individuals homozygous for the structural mutations was found (8.3% in patients versus 0.8% a random control group). There was no increase in the frequency of heterozygous individuals among the patients as compared to the control group. Summerfield et al. (1997) compared the MBL genotypes of 345 children admitted to hospital with infections to those of 272 children admitted for other reasons. They found a significant higher frequency of children heterozygous (133/345 versus 60/ 272) or homozygous (146/345 versus 64/272) for mutant

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MBL alleles in the infected group. These authors conclude that there is a significant increase in susceptibility to infectious diseases in children both heterozygous and homozygous for mutant MBL alleles. The importance of MBL mediated defence mechanisms in combination with an acquired immunodeficiency has recently been noted, when it was found that deficiency of MBL is associated with severe infections after chemotherapy (Peterslund et al., 2001).

7.2. Human immunodeficiency 6irus and mannan-binding lectin A possible correlation between HIV infection and MBL has been subject to a number of studies. It was initially showed in an in vitro model that the infection of CD4 + lymphocytes by HIV could be partially inhibited (25%) by MBL at physiological concentration (1 mg/ml) while 100% inhibition was seen at 50 mg MBL/ml (Ezekowitz et al., 1989). Using a plate binding assay these authors also showed that the HIV envelope protein, gp120, containing high mannose oligosaccharides, interacted with MBL. This interaction was confirmed by Haurum et al. (1993) who also showed that the MBL pathway could be activated by gp110 (HIV-2) and gp120 (HIV-1)-bound MBL. The implications of this finding is two faced; the deposition of complement on the HIV surface could promote the elimination of the virus, but might on the other hand also mediate an CD4-independent uptake of virus in cells expressing complement receptors. When Nielsen et al. (1995) determined the level of circulating MBL in 80 HIV infected individuals 10% (8/80) were found to have levels of MBL below the detection limit of the assay, a significantly higher frequency than in their normal control group (3/123; 2.4%). No correlation was seen between the level of MBL and the length of time from the detection of HIV antibodies and development of AIDS or from AIDS diagnosis to occurrence of death. This conclusion was supported in a later study on both MBL genotype and MBL level (McBride et al., 1998). In another study, the MBL genotypes were determined in 96 HIV infected men (Garred et al., 1997a). Eight percent (8/96) were found to be homozygous for the mutant MBL alleles. This was significantly higher than in controls. Whether this overrepresentation of mutant alleles in HIV infected individuals reflects an increased susceptibility to HIV infection or whether it predisposes to HIV infection as a consequence of other infections could not be concluded. An increased frequency of MBL deficiency in HIV-seropositive individuals was also found in sub-Saharan Africans (Garred et al., 1997b) and in Hungarians (Prohaszka et al., 1997). Others have failed to detect any signifi-

cant increase of MBL deficiency in HIV infected individuals (Senaldi et al., 1995). In contrast to the study by Nielsen et al. (1995) other publications conclude that HIV infected individuals with low MBL levels have a significantly reduced survival time after the diagnosis of AIDS (Garred et al., 1997a) or that the time from seroconversion to the development of AIDS and death is slightly increased (Maas et al., 1998; Amoroso et al., 1999). The latter findings were not supported by Hundt et al. (2000) who found that six long-term nonprogressors had normal levels of circulating MBL. The influence of MBL on the susceptibility to HIV infection and on disease development and progression remains controversial.

7.3. Autoimmune disease Several reports have presented evidence for the involvement of MBL in SLE. The frequency of the MBL B allele was found to be significantly increased in British (Davies et al., 1995) and in Hong Kong Chinese (Lau et al., 1996) SLE patients. In the latter study, a significant correlation between the serum level of MBL and the risk of acquiring SLE was also found. A similar increase in SLE susceptibility was detected when Sullivan et al. (1996) analysed MBL promoter and structural variants in 92 Black American SLE patients. The frequency of the B and C variant alleles and the LX promoter haplotype, associated with low levels of circulating MBL, was found to be significantly increased as compared to a control group. These findings were confirmed by Ip et al. (1998) who found the LX haplotype at a frequency of 0.259 in 112 Chinese SLE patients (58/224). This was significantly different from controls (0.164, 36/220). When Garred et al. (1999b) genotyped 91 Danish SLE patients a significant increase of individuals homozygous for the variant MBL alleles was observed, when compared to normal controls. An increase was not seen when comparing the frequency of heterozygotes in the two populations. These findings suggest that impairment of the MBL pathway due to production of dysfunctional MBL or due to a reduction in the amount of circulating MBL predispose to acquisition of SLE. As shown by Davies et al. (1997), the risk of SLE acquisition due to MBL pathway impairment can be further increased in the presence of C4B null alleles. This underscores the importance of dysfunctional complement activation cascades in the development of SLE. A recent study failed to shown any significant difference in the frequency of the B allele in 95 Japanese SLE patients when compared to healthy controls (Horiuchi et al., 2000).

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7.4. Mannan-binding lectin, a port of entry for intracellular pathogens? Because of the high frequency of variant MBL alleles in different populations it has been speculated whether MBL deficiency could protect against certain infectious diseases. When Garred et al. (1992b) analysed the MBL level in 56 Kenyan Africans, they found 10 to have an MBL level below the detection limit of the assay. Based on this high frequency of MBL deficiency the authors suggest that low circulating levels of MBL may protect against infections with intracellular micro-organisms that display MBL ligands on their surface. Several studies have shown that MBL bind parasites such as Leichmania major and L. mexicana (Garred et al., 1994) Trypanosoma cruzi (Kahn et al., 1996) Schistosoma mansoni (Klabunde et al., 2000), and to sonicates of both Mycobacterium leprae and M. tuberculosis (Garred et al., 1994). In addition, MBL was found to bind proteophosphoglycan secreted from the intracellular L. mexicana (Green et al., 1994). The presence of MBL on the surface of the parasites might allow for cellular uptake either directly via an MBL receptor or via receptors for complement fragments deposited on the parasite as a result of complement activation via the MBL pathway. In support of the protective effect of MBL deficiency, it was found that Ethiopians infected with M. leprae had a significant higher level of MBL than non-infected normal controls (Garred et al., 1994). Subsequently, it was also shown that individuals infected with tuberculosis had a significantly higher MBL level than controls (Garred et al., 1997b). Other studies have supported the hypothesis of a possible protective role of low MBL alleles and infection with intracellular micro-organisms (Bellamy et al., 1998; Hoal-Van Helden et al., 1999).

7.5. Ischemia/reperfusion injury Tissue and organs that are deprived of oxygen, as occurs at coronary artery obstruction or organ transplantation, are often severely damaged when revascularised. This phenomenon is known as ischemia/reperfusion (I/R) injury. The destruction of tissue and organs is in part due to the exposure of hypoxic (ischemic) tissue to oxygen when reperfused. The sudden increase in oxygen results in the formation of highly reactive oxygen radicals (Vermeiren et al., 2000). The activation of the complement cascade also appears to be important in I/R injury. This give rise to the formation of anaphylatoxins, which in combination with pro-inflammatory cytokines supports the recruitment of PMNs into the reperfused tissue (Vermeiren et al., 2000). Here the activated PMNs will

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release the contents of their granules leading to severe tissue destruction. The classical complement pathway has been implicated in initiating complement activation during I/R to a large extend through studies showing a significant reduction in tissue destruction, when experimental animals were pre-treated with C1 inhibitor before reperfusion or when C4-deficient animals were used (Caliezi et al., 2000). Collard et al. (1997) showed that complement was activated in a C2-dependent manner, when hypoxic human umbilical vein endothelial cells (HUVECs) were reoxygenated. The cause of complement activation has been speculated to be mediated through the classical pathway via natural antibodies (Dong et al., 1999). Since hypoxia is known to induce alterations in endothelial cell surface protein expression and glycosylation, it was also speculated that complement could be activated through the MBL pathway of complement activation (Collard et al., 1999). Studies conducted on HUVECs subjected to oxidative stress clearly shows that MBL binds to the surface of reoxygenated cells and mediates the deposition of complement component iC3b (Collard et al., 2000). This suggests an important role of the MBL pathway in the pathophysiology of I/R injury, which has inspired the development of inhibitors specific for the pathway (Collard et al., 2000; Petersen et al., 2000; Montalto et al., 2001; Lekowski et al., 2001).

8. Concluding remarks The understanding of the physiological properties and interactions of the proteins involved in the MBL pathway is rapidly increasing. The value of recombinant components has been great but natural, plasmaderived components are needed to support the conclusions. The generation of knock-out mice will be of great importance in addressing the physiological functions of the components. The recent discovery of a third MBL associated serine protease and the identification of distinct MBL complexes calls for a new perception of the MBL pathway of complement activation. Future studies will throw light on the activities of the different complexes. A large body of research shows that defect of MBL (and the MBL pathway) is involved in a number of infectious as well as autoimmune diseases. With MBL deficiency being the most widespread immune defect it seems reasonable to develop reconstitution therapy with plasma-derived MBL or preferably with fully biologically active recombinant MBL. The MBL pathway is a potent activator of inflammation and it thus seems pertinent to search for suitable MBL pathway inhibitors, as possible therapeutic agents in acute and chronic debilitating inflammatory.

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