Immunophurnmcology, 24 ( 1992) 91-99 0 1992 Elsevier Science Publishers B.V. All rights rcscrvcd 0162-J 109/92/$05.00 IMPHAR
91
0062 I
Mannan-binding
protein, a colrilplement activating a S teffen Thiel
Institute
uf Medical
Microbiology.
Bartholinbuildit~g.
Aarhm
tJniwr.city, Aarhus.
Denwork
Mannan-binding protein is an animal serum lectin (i.e. a molecule with the ability to bind specific&+ ir) certain Abstract: carbohydrate structures). The relevant carbohydrate ligands are found on many pathogenic microorganisms. After binding to suitable carbohydrate ligands, mannan-binding protein is found to be an activator of the classical pathway of complement via an activation ofthe Clr,Cls, complex, i.e. antibody and Clq independent. The molecular organization of MBP resembles that of Clq with a distinct division of collagen-like and globular amino acid sequences. This molecular similarity seems to be the basis for the common functional activity of the IWO proteins. MBP may play an important protective role, especially at early stages of infection prior to the generation of the specific humoral and cellular dcfcnce system. The paper explores the structure and the physiologicai functions of mannan-binding protein. Key words:
Lectin; Mannan-binding
protein:
Mannose-binding
Introduction
protein; Compfement
tion of the proteins
Corresponde?~ce to: S. Thiel, Institute of Medical Microhiology, Bartholinbuilding, Aarhus University, 8000 Aarhus, DK - Denmark. Abbreviations: ClqR, Clq receptor; CRD, carbohydrate recognition domain; CRP, C-reactive protein; GatNAc. N-acetyl-D-Galactosamine; GlcNAc. N-Acetyl-D-Glucosamine; HIV-l, human immunodeficiency virus type 1; LPS, lipopolysaccharide; ManNAc, N-Acetyl-D-Mannoscamine; MBP. mannan-binding protein; RaRF. Ra-reactive factor; SAA. Serum amyloid A protein; SDS-PAGE, sodium dodecyI sulphate polyacrylamide gel electrophorcsis; EM, electron microscopy.
Opsonic defect; Opsonin
according
to similarity
of their
In a number of cases, tire ability to bind carbohydrates can be assigned to a portion of the lectin, termed the carbohydrate recognition domain (CRD) (Drickamer, 1988). One such CRD, the so called C-type CRD, is defined on the basis of the requirement of calcium ions for binding. Comparison of the amino acid sequence of 22 lectins with a C-type CRD revealed within a 120 a.a. domain structure 14 absolutely conserved amino acids, with an additional 18 conserved in character (Weis et al., 1991). The C-type CRD has been identified in both vertebrates (primates, rodents, ruminants and birds) and invertebrates {flies and sea urchins). It is found connected to several other different structural domains such as the epidermal growth factor domain, the short consensus repeat of complement regulatory proteins, and collagen-like regions, suggesting that the C-type CRD originated early in evolution and has beamino
Mammalian lectins (i.e. proteins with the ability to bind specificalIy to carbohydrate structures, without being enzymes working on carbohydrate substrates, or without being anti-carbohydrate antibodies (Barondes, 1988)) are being recognized in ever-increasing numbers. Structural characterization form the basis for the classifica-
activation:
acid
sequence.
92 come associated with other domains through exon shuffling. Some of the humoral mammalian lectins have an unusual mosaic structure with a region of collagen-like amino acid sequences connected to a C-type CRD. Collagen regions are rod-like structures: built of three polypeptide chains twisted in a triple helix. The chains have a highly characteristic amino acid sequence in which every third position is occupied by a glycine residue; only this residue can be accommodated along the axis of the helix. In the -Gly-Xaa-Yaasequences, the Xaa and Yaa amino acids are frequently hydroxylated forms of proline or lysine which are found only in a few other proteins. Three plasma proteins, mannan-binding protein (MBP), conglutinin and complement component Clq and two lung proteins, pulmonary surfactant associated protein A and D (SP-A and SF-D), are now known to contain collagen-like regions (reviewed in Reid and Thiel, 1992). The name collectins has been proposed for the lectins belonging to this group of proteins (Malhotra et al., 1991).
Mannan-binding
Protein
With regard to nomenclature, MBP has been given varying names by different research groups; mannan-binding protein (Kawasaki et al., 1978; Lu et al., 1990; Super et al., 1989), mannosebinding protein (Drickamer, 1988; Ezekowitz and Stahl, 1988), core-specific lectin (Brownell et al., 1984) and Ra-reactive factor (RaRF) (Matsushita et al., 1992). Mannan-binding protein was, however, the original name used for the molecule and it seems appropriate to retain this nomenclature, which further distinguishes it from other lectins, such as the macrophage mannose specific lectin, and since none of the other names are especially correct concerning the sugar specificity (see below). Mannan, the ligand giving the name to the protein, is a carbohydrate rich constituent of yeast cell walls. MBP is synthesized by hepatocytes and bjr
liver cell lines (Mori et al., 1983; Brownell et al., 1984). In man and mice the presence of MBP mRNA seems to be restricted to the liver (Ezekowitz et al., 1988; Sastry et al., 1991). By immunohistochemical analysis MBP was found only in diseased or inflamed liver (Ryley et al., 1991). The synthesis in vitro of MBP is downregulated by interleukin 1 and tumour necrotic factor whereas y-interferon seems to induce synthesis (Ezekowitz and Stahl, 1988). The cDNAs for MBP in rat and man have been cloned and sequenced and parts of the polypeptide of the mature MBP has been sequenced by direct amino acid sequencing {Drickamer et al., 1986; Ezekowitz et al., 1988). In man, the mature MBP consists of four regions: (a) an NH,-terminal segment of 21 amino acids with 3 cysteines likely to be involved in the formation of interchain disulphide bonds stabilizing the multimeric forms of the protein; (b) a collagen-like domain; (c) a “neck)) region; and (d) a COOH-terminal C-type CRD (Fig. 1). In man the MBP gene is located on chromosome 10 (Sastry et al., 1989). It is split in 4 exons (Taylor et al., 1989; Sastry et al., 1989) each one coding for one of the four structural regions (Fig. 1). The polypeptide chains of MBP appear to be linked together by disulphide bonds. When purified MBP is analyzed by SDS-PAGE it is seen to migrate in unreduced form as a large (> 200 kDa) molecule and reduced as a 3 1 kDa polypeptide chain (Lu et al., 1990). E.lectron microscopy (EM), combined with size permeation chromatography and SDSPAGE analysis, has revealed that purified MBP is composed of a mixture of trimers, tetramers, pentamers and hexamers of an approximate 90 kDa structural unit, this unit being composed of 3 polypeptide chains (Thiel and Reid, 1989; Lu et al., 1990). Size permeation chromatography of serum followed by analysis for MBP showed MBP only in fractions corresponding to the pentamer and hexamers of approximately 750 kDa (Thiel, unpublished observation). The hexameric form of MBP is very similar in structure to Clq with both molecules composed of six
93
Polypept
Ide cham Gly-Yad.Yad
Fig. 1. Schematic drawing of the macromolecular organization, the polypeptide chain and the gene for MEP. The box (m) denotes a signal peptide. The hexameric form of MW consists of I8 identical chains and the organization of the gene encoding the hr:man MBP polypeptide chain is shown at the bottom of the figure. Three CRD regions are associated in each of the six globular heads seen in the schematic representation of MBP shown at the top of the figure.
globular heads each joined by connecting collagen strands to a central stem structure (Fig. 1). Several regulatory consensus sequences have been found in the 5’ flanking region of the gene: a region similar to the Drosophila heat shock promoter consensus sequence, three regions similar to the consensus sequences for glucocorticoidresponsive elements, and a sequence with a high degree of homology to a cytokine responsive element found in front of the gene for serum amyfoid A protein {SAA), an acute phase protein (Taylor et al., 1989; Sastry et al., 1989). The heat shock consensus sequence is also present in the 5’ flanking region of the human gene for C-reactive protein (CRP), another acute phase protein (Woo et al., 1985). The acute phase response is a systemic reaction to inflammatory stimuli. It is characterized by a multitude of physiological changes including alterations in
hormone and electrolyte levels, and alterations in the concentrations of a range of serum proteins, which have thus been classified as acute phase reactants. One may speculate that the glucocorticoid responsive elements, the heat shock pmmoter region and the cytokine responsive elements in the 5’ flanking promoter region of the MBP gene serve to regulate the expression of MBP during the acute phase response. In accordance with this, Sastry et al_ (1991) found the mRNA for mouse MBP-A to be upregulated following intraperitoneal injection of thioglycollate or azocasein. Human MBP has been described as an acute phase protein on the basis of a report by Ezekowitz et al. (19SS) that hepatic MBP mRNA concentrations were few in one presumed normal liver (from a patient with Hodgkin’s disease) md were increased in a human liver from a person who had been ex-
94 posed IO extreme physical stress in the form of fatal ii;juries as the fesuit of a trafik accident.
Seqilentia? serum and plasma samples from patients undergoing major surgery and from individwls during and after malarial attacks have been etamined, and the concentrations of MBP were found to increase two- to four-fold during the aciite phase response in these patients {Thiel et al., in press).
Binding
Specificity
of MBP
The most potent monosaccharide inhibitors of the binding of MBP to mannan are ManNAc and mannosc, while GlcNAc can also inhibit, although at slightly higher concentrations. Other monosaccharides like mannosamine, glucosamine, galactose, GalNAc, mannose-Qphosphate, glucose-6-phosphate and N-accetylneuraminic acid are either very poor inhibitors or do not inhibit the binding at ail (Kawasaki et al., 1978; Townsend and Stahl, 1981; Mizuno et at., 1981). Dissociation constants of 2.3 x 10e9 M have been measured in the binding of MBP to mannan and ~-mannusidase (a glycoprotein expressing high-mannose ol~gosacch~des} and 2.1 x lo-’ M to mannose (Kozutsumi et al., 198 1). Oligosaccharide recognition by MBP has been investigated using a series of structurally characterized neoglycolipids as probes in thin layer chromato~aphy (Childs et al., 1989). When the binding to N-linked biantennary complextype oiigosaccharides were investigated, MBP showed preferential reactivities with those containing two peripheral GlcNAc residues. Substitution with galactose masked n;activity, i.e. the presence of a terminal galactose residue on one of the peripheral GlcNAc residues, rn~kedl3f decreases binding and, when galactose residues are present on both GlcNAc, the binding is abolished. Similarly, mannose residues have to be exposed (i.e. at the non-reducing termini) to be effective in binding. Fucose, when bound Ql-4 or xl-3 to GlcNAc at the ends of complex-type oligosaccharides, is also a ligand for MBP. MBP
thus clearly has the potential to react with several glycocoujugates, but a major determining factor is the accessibility of the ol~gosacch~dcs which may be influenced by the folding of the protein chain (Lcfeless et al., 1989; Kawasaki et al., 1985). High-mannose oligosaccharides are present on several microorganisms (Hakomori, 1981; Olden et al., 1985). The human immunode~cjency virus type one {HIV-l) has high-mannose as well as complex and hybrid type oligosaccharides attached to the envelope proteins (Geyer et al., 1988; Mizuochi et al., 1990; Hansen et al., 1990). Purified human MBP has been found to bind to HIV-l infected CD4+ H9 cells and to HIV I infected ceils from the monocytic cell line U937 (Ezekowitz et al., 1989). It was found that preit+ cubation of HIV-l with MBP inhibits subsequent infection of H9 lymphoblasts. Another approach to the study of mannosebinding proteins and their ability to interact with virus is presented in a study on the &inhibitors of influenza virus A jAnders et al., 1990). These inhibitors are identified as heat-labile components of normal sera that have virus-neutralizing as well as hema~lutination-inhibiting activity. Investigation into the nature of the interaction of p-inhibitors with influenza virus revealed that these are mannose-binding lectins (i.e. the hemagglutination-inhibiting activity is inhibited by mannose). Further studies will reveal if these inhibitors are identical to any of the known mannose-bindjng serum proteins, MBP or congiutinin. MBP has been shown to bind to several species of bacteria (Kuhlmann et al., 1989; van Emmerik et al., 1992). Apparently encapsulation hampers the binding of MBP (van Emmerik et al., 1992). Scatchard plot analysis of MBP binding to rough strains of E. co/i showed that approx. 3 x lo4 MBP molecules could be bound per bacteria with a dissociation constant of 6 x IOm9 M (Kawasaki et al., 1989). Recently a component of human RaRF has been shown to be identical to MBP (Matsushita et al., 1992). RaRF was identified by its binding to rough
95
strains of Saimone& and to rough R2 strains ot E. co& (Kawskami et al., 1982; Ihara et al., 1988). The used strains have a Ra chemotypa rough core LPS in common and the determinants to which RaRF binds are probably the GlcNAc, glucose and L-glycero-D-mannoheptosyl residues present in the nonreducing termini of the polysaccharide chains (Ihara et ai., 1982; Ihara et al., 1991).
Interaction with the Compfement System The overall macromolecular org~iz~tion of MBP is in many ways similar to the complement component C iq. This suggests that MBP may be able to mimic the role that Clq plays in the activation of the Clr,Cls, complex, The function of Clq is to bind to IgG and IgM in immune compfexes and then, through an interaction with the Clr,Cls, complex, to activate the classical activation pathway of the complement system. The residues primarily involved in the interaction between Clq and IgG is found in the CH2 domain of the IgG (the residues involved being probably Glut, 1sp Lys(320) =d Lys(322,) ami in the globular COOH-terminal “heads” of Clq (Duncan and Winter, 1988). The binding to IgM involves the CH3 domain of the IgM heavy chain. Ikeda et al. (1987) described that purified MBP from man, rabbit and rat in the presence of serum could mediate a dose-dependent lysis of sheep erythrocytes coated with mannan. The lysis was dependent on the presence of C4 and it could be effectively inhibited by reievant monosaccha~d~s. Further studies by the Japanese group indicated that Cls binds to MBP only when Clr is also present (Ohta et al., 1990). In another functional study the ligand for MBP was zymosan, and the activation of the complement system was followed by measurement of the a~tiva~on of the proenzyme C 1s in the C lr,C I s2 complex (Lu et al., 1990). It was shown that only the pentamer/hexamer forms of MBP (supported by electron microscopy studies of the relevant
fractions), and not smaller forms of the molecule, could activate Cis. Another approach to the study of complement activation was taken by Super et al. (1990). Using microtiter plates coated with mannan as !+id for MBP, sera was analyzed for their ability to deposit campiement factors onto the surface. Analysis of 176 heahhy blood donors revealed that the levels of complement factors bound to the mannan coated surface showed strong correlation with the amount of MBP in the sera. Strikingly, no snch correlation was found to the levels of anti-mannan antibodies in the sera. MBP, when bound to bacteria, is abie to mediate a complement-de~nd~~ bactericidal activity. Thus. if rough strain E. cd or S&nonella has been sensitized with MBP and complement is added, a killing occurs (Kawasaki et al, 1989; Ji et al., 1988; lhara et ai., 1991). Eased on the presented data it is possiWe to reformulate the initiating steps in the activation of the complement system as summarised schematically in Fig, 2. With regard to the evolution of the activation pathways indicated, the alternative pathway is thought to be the oldest, as complement-like factors have been found in invertebrates, although no conclusive evidence has been provided for the existence of a complement cascade (Koch and Nielsen, 1984). Among the vertebrates, even the most primitive forms have at least some ~om~nents of the complement cascade - the lamprey, for example, possesses a C3b-like protein, probably activated via the alternative pathway. I3 cells, and therefore the ability to produce antibodies to trigger the classical pathway of complement via Clq, are found only in vertebrates_ The lectin pathway, dependent on the presence of complement factors, but not on the presence of antibodies, could have evolved simultaneously with or after the appearance of complement-like factors, but before a specific immune system had evolved. One might also envisage the lectins to be part of the p~mordial defense systems with complement evolving subsequently as a amplifying mechanism.
Fig. 2. A ~ia~~mati~
representation
of the different pathways
Interaction with the Clq Receptor (ClqR) A general property of the collectins is their direct opsonizing effect. MBP, bovine conglutinin and SPA all function as opsonins, Le. they enhance phagocytosis when bound to microorganisms (Wright et al., 1987; Kuhlman et al., 1989; Tenner et al., 1989; Friis-Christianser et al., 1990; ~~~ke~nke et all., 1992). it has been demonstrated that binding to the Clq receptor (ClqR) is a common feature of the collagen containing lectins (Malhotra et al., 1990). Since CtqR is known to bind to the collagenous region of Clq (Arvieux et al., 1984) it is likely that it binds to the collagenous segments of the collectins as well. CIqR should thus more correctly be referred to as a general receptor for humoral collagencontaining proteins, The existence of Clq receptors on several human cell types has been reported by numerous researchers (reviewed in Erdei and Reid, 1989). The binding of Clq to the ClqR has been reported to mediate a range of phenomena including phagocytosis, antibody-
leading to activation
of the complement
system.
dependent cell-mediated cytotoxicity, modulation of cytokine and immunoglobulin secretion and polymorph-endoth~~~um interaction (Ghebrehiwet, 1989; IX&a, 1989). Phagocytosis is thought of as the most primitive cellular defence system. The direct opsonic effect of the collectins indicates that proteins related to these are candidates for evolutionary early opsonins. The interaction with ClqR. together with the complement activating features, suggest that there are two ways in which MBP opsonizes microorganisms: (1) a direct opsonizing function by binding to the microorganism and mediating a phagocytosis via ClqR; and (2) an indirect opsonizing effect by binding to a mj~roorg~ism and activating the complement system, where the subsequently deposited complement factors (especially C3b and iC3b) will serve as opsonins. The latter function is likely to be the most important one, as the amplification achieved through the activation of the complement system is enormous.
97 Clinical Observations Failure of serum to opsonise baker’s yeast (Saccharomyces cemvjsiae), for phagocytosis by normal polymorphonuclear leukocytes, has been reported in about a quarter of children with frequent, unexplained infmtioms (Soothill and Harvey, 19?6), in association with chronic diarrhoea of infancy (Candy et al., 1980), and in association with otitis media in infants (Richardson et al_, 1983). The opsonic defect is surprisingly quite common (5-7%) in the general population (Soothill and Harvey, 1976; Levinsky et al., 1978; Turner et al., 1981). An association between the defect to opsonize yeast for phagocytosis and a reduced ability to deposit C3b/iC3b fragments on the yeast surface has been established (Turner et al., 1981; Turner et al., 1985). Association of low levels of MBP with the defect in opsonization has been estab~shed in a study where ten children, previously shown to suffer from the mentioned opsonic defect, all had undetectable or very low levels of MBP in their sera (Super et al., 1989). Furthermore, addition of purified MBP could correct the opsonic defect in the sera in a dos~~endent manner. Previous observations have shown that people with the opsonic defect deposit lower levels of C3b not only on S. cerevisiae and CaFldida a&cans, but also on Staphylococcus aureus and E. co& (Turner et al., 1976), su~esting that low levels of MBP may result in suboptimal responses to a wide range of common bacterial infections. It is clear that low levels of MBP does not invariably lead to clinical disease, as low levels of MBP are found in about 10% of normal healthy children. Sequence analysis of the MBP gene in three unrelated children with recurrent infections, opsonic deficiency and low serum MBP concentrations has been reported (Sumiya et al., 1391). No abnormalities in the MBP gene were observed except for a point mutation in exon 1 which caused a shift of codon 54 from GGC to CAC. This results in the replacement of a glycine by aspartic acid, thus disrupting the fifth Gly-XaaYaa repeating triplet within the collagen-like re-
gion of the MBP chain. The gene frequency of the GAC aiiele is found to be 0.17 (Lipscombe et al., 1992). A deficiency of MBP may especially become pathologically important in individuals where the other immunological responses toward polysaccharides is suboptimal. This is Iikely to be the case in infants aged 6-18 months, as these will have little residual maternal antibodies while their own immune system still has not reached a mature state. With the maturation of the humoral and the cellular immune system, the host will depend less oiz non-antigen-specific immune defence mechanisms, such as mediated by MBP, and the clinical relevance of low levels of MBP may thus be limited. In conclusion, MBP may play an important protective role. it is likely to represent, in evolutionary terms, an early defense mechanism. In higher animals, with both humors and cellular immune systems, MBP may be of the greatest importance at early stages of infection, prior to the generation of the specific humoral or cellular defense systems. The protein may also be important during infancy and early childhood, before the maturation of the immune system is completed. Acknowledgements I thank Dr. Jens Chr. Jensenius and Dr. Graham Leslie for careful reading and useful comments on this manuscript.
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defect
of
opsonisation.
Lancet
1989;
2:
Taylor ME, Brickell PM, Craig RK. Sumrneriield JA. Structure and evolutionary origin of the gene encoding a human serum m~nose-binding protein. B&hem J 1989; 262: 763-771. Tenner AJ, Robinson SL, Borchelt J, Wright JR. Human pulmonary surfactant protein (SP-A), a protein structuraily homologous to Clq, can enhance FcR- and CRl-mediated phagocytosis. J Biol Chem 1989; 264: 13923-13928. Thiel S, Reid KB. Structures and functions associated with the group of mammalian lectins containing collagen-like sequences. FEBS L&t 1989,250: 78-84. Townsend R, Stahl P. Isolation and characterization of a maunose/N-acetylgIucosamine/fucose-binding protein from rat liver. B&hem J 1981; 194: 209-214. Turner MW, Mowbray JF, Roberton DR. A study of C3b deposition on yeast surfaces by sera of known opsonic potential. Clm Exp Immunol 1981; 46~ 412-419. Turner MW, Seymour NI3, Kazatchkine MD, Mowbry JF. Suboptimal C3b/C3bi deposition and defective yeast opsonlzation. I. Evidence for the absence of essential cofactor activity. Clin Exp Immunol 1985; 62: 427-434. Turner MW, Grant C. Seymour ND, Harvey B, Lcvinsky RJ. Evaluation of C3blC3bi opsonization and chemiluminescence with selected yeasts and bacteria using sera of different opsonic potmntial. 1~~01 1976: 58: 111-115. van Emmcrik LC, ThieI S, Ktijper EJ, Fijen CAP. Dankert 3. Interaction of mannan-binding protein with common pathogens causing bacterial meningitis. Immunobiology 1992; 184: 423. Weis WI, Kahn R, Fourme R, Drickamer K, Hendrickson WA. Structure of the Cecil-dependent leetin domain from a rat m~nose-bong protein determined by MAR phasing, Science 19991;254: 1608-I615 Woo P, Korcnberg JR, Whibhead AS. Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component. J Biol Chem 1985: 260: 13384-1338s. Wright JR, Wager RE, Hawgood S. Dobbs I.,, elements JA. Surfactant apoprotein Mr = X,000-36,000 enhances uptake of liposomes by type II cells. 9 Biol Chem 1987; 262: 2888-2894.
91
0062 I
Mannan-binding
protein, a colrilplement activating a S teffen Thiel
Institute
uf Medical
Microbiology.
Bartholinbuildit~g.
Aarhm
tJniwr.city, Aarhus.
Denwork
Mannan-binding protein is an animal serum lectin (i.e. a molecule with the ability to bind specific&+ ir) certain Abstract: carbohydrate structures). The relevant carbohydrate ligands are found on many pathogenic microorganisms. After binding to suitable carbohydrate ligands, mannan-binding protein is found to be an activator of the classical pathway of complement via an activation ofthe Clr,Cls, complex, i.e. antibody and Clq independent. The molecular organization of MBP resembles that of Clq with a distinct division of collagen-like and globular amino acid sequences. This molecular similarity seems to be the basis for the common functional activity of the IWO proteins. MBP may play an important protective role, especially at early stages of infection prior to the generation of the specific humoral and cellular dcfcnce system. The paper explores the structure and the physiologicai functions of mannan-binding protein. Key words:
Lectin; Mannan-binding
protein:
Mannose-binding
Introduction
protein; Compfement
tion of the proteins
Corresponde?~ce to: S. Thiel, Institute of Medical Microhiology, Bartholinbuilding, Aarhus University, 8000 Aarhus, DK - Denmark. Abbreviations: ClqR, Clq receptor; CRD, carbohydrate recognition domain; CRP, C-reactive protein; GatNAc. N-acetyl-D-Galactosamine; GlcNAc. N-Acetyl-D-Glucosamine; HIV-l, human immunodeficiency virus type 1; LPS, lipopolysaccharide; ManNAc, N-Acetyl-D-Mannoscamine; MBP. mannan-binding protein; RaRF. Ra-reactive factor; SAA. Serum amyloid A protein; SDS-PAGE, sodium dodecyI sulphate polyacrylamide gel electrophorcsis; EM, electron microscopy.
Opsonic defect; Opsonin
according
to similarity
of their
In a number of cases, tire ability to bind carbohydrates can be assigned to a portion of the lectin, termed the carbohydrate recognition domain (CRD) (Drickamer, 1988). One such CRD, the so called C-type CRD, is defined on the basis of the requirement of calcium ions for binding. Comparison of the amino acid sequence of 22 lectins with a C-type CRD revealed within a 120 a.a. domain structure 14 absolutely conserved amino acids, with an additional 18 conserved in character (Weis et al., 1991). The C-type CRD has been identified in both vertebrates (primates, rodents, ruminants and birds) and invertebrates {flies and sea urchins). It is found connected to several other different structural domains such as the epidermal growth factor domain, the short consensus repeat of complement regulatory proteins, and collagen-like regions, suggesting that the C-type CRD originated early in evolution and has beamino
Mammalian lectins (i.e. proteins with the ability to bind specificalIy to carbohydrate structures, without being enzymes working on carbohydrate substrates, or without being anti-carbohydrate antibodies (Barondes, 1988)) are being recognized in ever-increasing numbers. Structural characterization form the basis for the classifica-
activation:
acid
sequence.
92 come associated with other domains through exon shuffling. Some of the humoral mammalian lectins have an unusual mosaic structure with a region of collagen-like amino acid sequences connected to a C-type CRD. Collagen regions are rod-like structures: built of three polypeptide chains twisted in a triple helix. The chains have a highly characteristic amino acid sequence in which every third position is occupied by a glycine residue; only this residue can be accommodated along the axis of the helix. In the -Gly-Xaa-Yaasequences, the Xaa and Yaa amino acids are frequently hydroxylated forms of proline or lysine which are found only in a few other proteins. Three plasma proteins, mannan-binding protein (MBP), conglutinin and complement component Clq and two lung proteins, pulmonary surfactant associated protein A and D (SP-A and SF-D), are now known to contain collagen-like regions (reviewed in Reid and Thiel, 1992). The name collectins has been proposed for the lectins belonging to this group of proteins (Malhotra et al., 1991).
Mannan-binding
Protein
With regard to nomenclature, MBP has been given varying names by different research groups; mannan-binding protein (Kawasaki et al., 1978; Lu et al., 1990; Super et al., 1989), mannosebinding protein (Drickamer, 1988; Ezekowitz and Stahl, 1988), core-specific lectin (Brownell et al., 1984) and Ra-reactive factor (RaRF) (Matsushita et al., 1992). Mannan-binding protein was, however, the original name used for the molecule and it seems appropriate to retain this nomenclature, which further distinguishes it from other lectins, such as the macrophage mannose specific lectin, and since none of the other names are especially correct concerning the sugar specificity (see below). Mannan, the ligand giving the name to the protein, is a carbohydrate rich constituent of yeast cell walls. MBP is synthesized by hepatocytes and bjr
liver cell lines (Mori et al., 1983; Brownell et al., 1984). In man and mice the presence of MBP mRNA seems to be restricted to the liver (Ezekowitz et al., 1988; Sastry et al., 1991). By immunohistochemical analysis MBP was found only in diseased or inflamed liver (Ryley et al., 1991). The synthesis in vitro of MBP is downregulated by interleukin 1 and tumour necrotic factor whereas y-interferon seems to induce synthesis (Ezekowitz and Stahl, 1988). The cDNAs for MBP in rat and man have been cloned and sequenced and parts of the polypeptide of the mature MBP has been sequenced by direct amino acid sequencing {Drickamer et al., 1986; Ezekowitz et al., 1988). In man, the mature MBP consists of four regions: (a) an NH,-terminal segment of 21 amino acids with 3 cysteines likely to be involved in the formation of interchain disulphide bonds stabilizing the multimeric forms of the protein; (b) a collagen-like domain; (c) a “neck)) region; and (d) a COOH-terminal C-type CRD (Fig. 1). In man the MBP gene is located on chromosome 10 (Sastry et al., 1989). It is split in 4 exons (Taylor et al., 1989; Sastry et al., 1989) each one coding for one of the four structural regions (Fig. 1). The polypeptide chains of MBP appear to be linked together by disulphide bonds. When purified MBP is analyzed by SDS-PAGE it is seen to migrate in unreduced form as a large (> 200 kDa) molecule and reduced as a 3 1 kDa polypeptide chain (Lu et al., 1990). E.lectron microscopy (EM), combined with size permeation chromatography and SDSPAGE analysis, has revealed that purified MBP is composed of a mixture of trimers, tetramers, pentamers and hexamers of an approximate 90 kDa structural unit, this unit being composed of 3 polypeptide chains (Thiel and Reid, 1989; Lu et al., 1990). Size permeation chromatography of serum followed by analysis for MBP showed MBP only in fractions corresponding to the pentamer and hexamers of approximately 750 kDa (Thiel, unpublished observation). The hexameric form of MBP is very similar in structure to Clq with both molecules composed of six
93
Polypept
Ide cham Gly-Yad.Yad
Fig. 1. Schematic drawing of the macromolecular organization, the polypeptide chain and the gene for MEP. The box (m) denotes a signal peptide. The hexameric form of MW consists of I8 identical chains and the organization of the gene encoding the hr:man MBP polypeptide chain is shown at the bottom of the figure. Three CRD regions are associated in each of the six globular heads seen in the schematic representation of MBP shown at the top of the figure.
globular heads each joined by connecting collagen strands to a central stem structure (Fig. 1). Several regulatory consensus sequences have been found in the 5’ flanking region of the gene: a region similar to the Drosophila heat shock promoter consensus sequence, three regions similar to the consensus sequences for glucocorticoidresponsive elements, and a sequence with a high degree of homology to a cytokine responsive element found in front of the gene for serum amyfoid A protein {SAA), an acute phase protein (Taylor et al., 1989; Sastry et al., 1989). The heat shock consensus sequence is also present in the 5’ flanking region of the human gene for C-reactive protein (CRP), another acute phase protein (Woo et al., 1985). The acute phase response is a systemic reaction to inflammatory stimuli. It is characterized by a multitude of physiological changes including alterations in
hormone and electrolyte levels, and alterations in the concentrations of a range of serum proteins, which have thus been classified as acute phase reactants. One may speculate that the glucocorticoid responsive elements, the heat shock pmmoter region and the cytokine responsive elements in the 5’ flanking promoter region of the MBP gene serve to regulate the expression of MBP during the acute phase response. In accordance with this, Sastry et al_ (1991) found the mRNA for mouse MBP-A to be upregulated following intraperitoneal injection of thioglycollate or azocasein. Human MBP has been described as an acute phase protein on the basis of a report by Ezekowitz et al. (19SS) that hepatic MBP mRNA concentrations were few in one presumed normal liver (from a patient with Hodgkin’s disease) md were increased in a human liver from a person who had been ex-
94 posed IO extreme physical stress in the form of fatal ii;juries as the fesuit of a trafik accident.
Seqilentia? serum and plasma samples from patients undergoing major surgery and from individwls during and after malarial attacks have been etamined, and the concentrations of MBP were found to increase two- to four-fold during the aciite phase response in these patients {Thiel et al., in press).
Binding
Specificity
of MBP
The most potent monosaccharide inhibitors of the binding of MBP to mannan are ManNAc and mannosc, while GlcNAc can also inhibit, although at slightly higher concentrations. Other monosaccharides like mannosamine, glucosamine, galactose, GalNAc, mannose-Qphosphate, glucose-6-phosphate and N-accetylneuraminic acid are either very poor inhibitors or do not inhibit the binding at ail (Kawasaki et al., 1978; Townsend and Stahl, 1981; Mizuno et at., 1981). Dissociation constants of 2.3 x 10e9 M have been measured in the binding of MBP to mannan and ~-mannusidase (a glycoprotein expressing high-mannose ol~gosacch~des} and 2.1 x lo-’ M to mannose (Kozutsumi et al., 198 1). Oligosaccharide recognition by MBP has been investigated using a series of structurally characterized neoglycolipids as probes in thin layer chromato~aphy (Childs et al., 1989). When the binding to N-linked biantennary complextype oiigosaccharides were investigated, MBP showed preferential reactivities with those containing two peripheral GlcNAc residues. Substitution with galactose masked n;activity, i.e. the presence of a terminal galactose residue on one of the peripheral GlcNAc residues, rn~kedl3f decreases binding and, when galactose residues are present on both GlcNAc, the binding is abolished. Similarly, mannose residues have to be exposed (i.e. at the non-reducing termini) to be effective in binding. Fucose, when bound Ql-4 or xl-3 to GlcNAc at the ends of complex-type oligosaccharides, is also a ligand for MBP. MBP
thus clearly has the potential to react with several glycocoujugates, but a major determining factor is the accessibility of the ol~gosacch~dcs which may be influenced by the folding of the protein chain (Lcfeless et al., 1989; Kawasaki et al., 1985). High-mannose oligosaccharides are present on several microorganisms (Hakomori, 1981; Olden et al., 1985). The human immunode~cjency virus type one {HIV-l) has high-mannose as well as complex and hybrid type oligosaccharides attached to the envelope proteins (Geyer et al., 1988; Mizuochi et al., 1990; Hansen et al., 1990). Purified human MBP has been found to bind to HIV-l infected CD4+ H9 cells and to HIV I infected ceils from the monocytic cell line U937 (Ezekowitz et al., 1989). It was found that preit+ cubation of HIV-l with MBP inhibits subsequent infection of H9 lymphoblasts. Another approach to the study of mannosebinding proteins and their ability to interact with virus is presented in a study on the &inhibitors of influenza virus A jAnders et al., 1990). These inhibitors are identified as heat-labile components of normal sera that have virus-neutralizing as well as hema~lutination-inhibiting activity. Investigation into the nature of the interaction of p-inhibitors with influenza virus revealed that these are mannose-binding lectins (i.e. the hemagglutination-inhibiting activity is inhibited by mannose). Further studies will reveal if these inhibitors are identical to any of the known mannose-bindjng serum proteins, MBP or congiutinin. MBP has been shown to bind to several species of bacteria (Kuhlmann et al., 1989; van Emmerik et al., 1992). Apparently encapsulation hampers the binding of MBP (van Emmerik et al., 1992). Scatchard plot analysis of MBP binding to rough strains of E. co/i showed that approx. 3 x lo4 MBP molecules could be bound per bacteria with a dissociation constant of 6 x IOm9 M (Kawasaki et al., 1989). Recently a component of human RaRF has been shown to be identical to MBP (Matsushita et al., 1992). RaRF was identified by its binding to rough
95
strains of Saimone& and to rough R2 strains ot E. co& (Kawskami et al., 1982; Ihara et al., 1988). The used strains have a Ra chemotypa rough core LPS in common and the determinants to which RaRF binds are probably the GlcNAc, glucose and L-glycero-D-mannoheptosyl residues present in the nonreducing termini of the polysaccharide chains (Ihara et ai., 1982; Ihara et al., 1991).
Interaction with the Compfement System The overall macromolecular org~iz~tion of MBP is in many ways similar to the complement component C iq. This suggests that MBP may be able to mimic the role that Clq plays in the activation of the Clr,Cls, complex, The function of Clq is to bind to IgG and IgM in immune compfexes and then, through an interaction with the Clr,Cls, complex, to activate the classical activation pathway of the complement system. The residues primarily involved in the interaction between Clq and IgG is found in the CH2 domain of the IgG (the residues involved being probably Glut, 1sp Lys(320) =d Lys(322,) ami in the globular COOH-terminal “heads” of Clq (Duncan and Winter, 1988). The binding to IgM involves the CH3 domain of the IgM heavy chain. Ikeda et al. (1987) described that purified MBP from man, rabbit and rat in the presence of serum could mediate a dose-dependent lysis of sheep erythrocytes coated with mannan. The lysis was dependent on the presence of C4 and it could be effectively inhibited by reievant monosaccha~d~s. Further studies by the Japanese group indicated that Cls binds to MBP only when Clr is also present (Ohta et al., 1990). In another functional study the ligand for MBP was zymosan, and the activation of the complement system was followed by measurement of the a~tiva~on of the proenzyme C 1s in the C lr,C I s2 complex (Lu et al., 1990). It was shown that only the pentamer/hexamer forms of MBP (supported by electron microscopy studies of the relevant
fractions), and not smaller forms of the molecule, could activate Cis. Another approach to the study of complement activation was taken by Super et al. (1990). Using microtiter plates coated with mannan as !+id for MBP, sera was analyzed for their ability to deposit campiement factors onto the surface. Analysis of 176 heahhy blood donors revealed that the levels of complement factors bound to the mannan coated surface showed strong correlation with the amount of MBP in the sera. Strikingly, no snch correlation was found to the levels of anti-mannan antibodies in the sera. MBP, when bound to bacteria, is abie to mediate a complement-de~nd~~ bactericidal activity. Thus. if rough strain E. cd or S&nonella has been sensitized with MBP and complement is added, a killing occurs (Kawasaki et al, 1989; Ji et al., 1988; lhara et ai., 1991). Eased on the presented data it is possiWe to reformulate the initiating steps in the activation of the complement system as summarised schematically in Fig, 2. With regard to the evolution of the activation pathways indicated, the alternative pathway is thought to be the oldest, as complement-like factors have been found in invertebrates, although no conclusive evidence has been provided for the existence of a complement cascade (Koch and Nielsen, 1984). Among the vertebrates, even the most primitive forms have at least some ~om~nents of the complement cascade - the lamprey, for example, possesses a C3b-like protein, probably activated via the alternative pathway. I3 cells, and therefore the ability to produce antibodies to trigger the classical pathway of complement via Clq, are found only in vertebrates_ The lectin pathway, dependent on the presence of complement factors, but not on the presence of antibodies, could have evolved simultaneously with or after the appearance of complement-like factors, but before a specific immune system had evolved. One might also envisage the lectins to be part of the p~mordial defense systems with complement evolving subsequently as a amplifying mechanism.
Fig. 2. A ~ia~~mati~
representation
of the different pathways
Interaction with the Clq Receptor (ClqR) A general property of the collectins is their direct opsonizing effect. MBP, bovine conglutinin and SPA all function as opsonins, Le. they enhance phagocytosis when bound to microorganisms (Wright et al., 1987; Kuhlman et al., 1989; Tenner et al., 1989; Friis-Christianser et al., 1990; ~~~ke~nke et all., 1992). it has been demonstrated that binding to the Clq receptor (ClqR) is a common feature of the collagen containing lectins (Malhotra et al., 1990). Since CtqR is known to bind to the collagenous region of Clq (Arvieux et al., 1984) it is likely that it binds to the collagenous segments of the collectins as well. CIqR should thus more correctly be referred to as a general receptor for humoral collagencontaining proteins, The existence of Clq receptors on several human cell types has been reported by numerous researchers (reviewed in Erdei and Reid, 1989). The binding of Clq to the ClqR has been reported to mediate a range of phenomena including phagocytosis, antibody-
leading to activation
of the complement
system.
dependent cell-mediated cytotoxicity, modulation of cytokine and immunoglobulin secretion and polymorph-endoth~~~um interaction (Ghebrehiwet, 1989; IX&a, 1989). Phagocytosis is thought of as the most primitive cellular defence system. The direct opsonic effect of the collectins indicates that proteins related to these are candidates for evolutionary early opsonins. The interaction with ClqR. together with the complement activating features, suggest that there are two ways in which MBP opsonizes microorganisms: (1) a direct opsonizing function by binding to the microorganism and mediating a phagocytosis via ClqR; and (2) an indirect opsonizing effect by binding to a mj~roorg~ism and activating the complement system, where the subsequently deposited complement factors (especially C3b and iC3b) will serve as opsonins. The latter function is likely to be the most important one, as the amplification achieved through the activation of the complement system is enormous.
97 Clinical Observations Failure of serum to opsonise baker’s yeast (Saccharomyces cemvjsiae), for phagocytosis by normal polymorphonuclear leukocytes, has been reported in about a quarter of children with frequent, unexplained infmtioms (Soothill and Harvey, 19?6), in association with chronic diarrhoea of infancy (Candy et al., 1980), and in association with otitis media in infants (Richardson et al_, 1983). The opsonic defect is surprisingly quite common (5-7%) in the general population (Soothill and Harvey, 1976; Levinsky et al., 1978; Turner et al., 1981). An association between the defect to opsonize yeast for phagocytosis and a reduced ability to deposit C3b/iC3b fragments on the yeast surface has been established (Turner et al., 1981; Turner et al., 1985). Association of low levels of MBP with the defect in opsonization has been estab~shed in a study where ten children, previously shown to suffer from the mentioned opsonic defect, all had undetectable or very low levels of MBP in their sera (Super et al., 1989). Furthermore, addition of purified MBP could correct the opsonic defect in the sera in a dos~~endent manner. Previous observations have shown that people with the opsonic defect deposit lower levels of C3b not only on S. cerevisiae and CaFldida a&cans, but also on Staphylococcus aureus and E. co& (Turner et al., 1976), su~esting that low levels of MBP may result in suboptimal responses to a wide range of common bacterial infections. It is clear that low levels of MBP does not invariably lead to clinical disease, as low levels of MBP are found in about 10% of normal healthy children. Sequence analysis of the MBP gene in three unrelated children with recurrent infections, opsonic deficiency and low serum MBP concentrations has been reported (Sumiya et al., 1391). No abnormalities in the MBP gene were observed except for a point mutation in exon 1 which caused a shift of codon 54 from GGC to CAC. This results in the replacement of a glycine by aspartic acid, thus disrupting the fifth Gly-XaaYaa repeating triplet within the collagen-like re-
gion of the MBP chain. The gene frequency of the GAC aiiele is found to be 0.17 (Lipscombe et al., 1992). A deficiency of MBP may especially become pathologically important in individuals where the other immunological responses toward polysaccharides is suboptimal. This is Iikely to be the case in infants aged 6-18 months, as these will have little residual maternal antibodies while their own immune system still has not reached a mature state. With the maturation of the humoral and the cellular immune system, the host will depend less oiz non-antigen-specific immune defence mechanisms, such as mediated by MBP, and the clinical relevance of low levels of MBP may thus be limited. In conclusion, MBP may play an important protective role. it is likely to represent, in evolutionary terms, an early defense mechanism. In higher animals, with both humors and cellular immune systems, MBP may be of the greatest importance at early stages of infection, prior to the generation of the specific humoral or cellular defense systems. The protein may also be important during infancy and early childhood, before the maturation of the immune system is completed. Acknowledgements I thank Dr. Jens Chr. Jensenius and Dr. Graham Leslie for careful reading and useful comments on this manuscript.
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