Mannan-binding Protein Research Articles

Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein.

Mannan-binding protein (MBP) is a serum lectin participating in the innate immune defense by opsonizing various microorganisms for phagocytosis. Opsonization defect due to MBP deficiency and low levels of the protein can partially be explained by the dominant effect of three different mutations in the structural part of the MBP gene. Large interracial differences in the frequencies of these variants have previously been described, but they cannot explain the large interindividual variation in MBP serum concentration. We describe the existence of additional polymorphisms at positions -550 (H/L variants) and -221 (X/Y variants) in the promoter region of the gene. The promoter haplotypes, HY, LY, and LX, show associations with high, medium, and low levels of MBP serum concentrations, respectively. Moreover, this represents a genetic system with additive effect of haplotypes in which a low producing LX haplotype in the homozygous state down-regulates the basal expression of MBP as effectively as a single structural variant. Populations of pure Eskimos, Caucasoids, and black Africans show marked interethnic differences in the frequencies of promoter haplotypes regulating the expression of the normal peptide, with the HY haplotype frequency varying from 0.83 in Eskimos via 0.33 in Caucasoids to 0.08 in Africans. The LY haplotype frequency varies from 0.04 in Eskimos via 0.39 in Caucasoids to 0.23 in Africans. The LX haplotype frequency varies from 0.03 in Eskimos via 0.24 in Caucasoids to 0.23 in Africans. The effect of the promoter variants can explain almost all of the ethnic differences not explainable by the structural variants alone.

Association between mannan binding protein deficiency and recurrent miscarriage.

The distribution of mannan binding protein (MBP) in blood donor sera was determined by enzyme-linked immunosorbent assay to establish normal concentrations. Abnormally low MBP concentrations were found in 16% (21 out of 135) of female partners and 14% (15 out of 108) of male partners of couples experiencing recurrent miscarriage, compared with < 5% of obstetrically normal controls (P < 0.005). This relationship was even stronger (9.5 versus 1.0%) and more significant (P < 0.002) when only subjects presumed to be homozygous for the mutant allele responsible for MBP deficiency were considered. By immunohistochemistry, MBP could be demonstrated in first trimester placenta. We suggest that low concentrations of MBP within the feto-placental unit increase susceptibility to fetal loss, possibly via an infection-induced placental cytokine imbalance.

Mannan‐Binding Protein Forms Complexes with α‐2‐Macroglobulin. A Proposed Model for the Interaction

We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose. The occurrence of alpha 2M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-alpha 2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed alpha 2M subunits (94 and 180 kDa) and pMBP subunits (28kDa) in SDS-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing conditions. This band was also seen in eluates from the anti-alpha 2M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP-28 with alpha 2M.

Ontogeny of human mannan‐binding protein, a lectin of the innate immune system

Mannan-binding protein (MBP) is a member of the collectins. These lectins are composed of polypeptide chains which contains a collagen-like region and a calcium dependent carbohydrate recognition domain (named a C-type CRD). MBP binds via the CRD to carbohydrate structures on microorganisms. MBP can activate the complement system when bound to carbohydrate. MBP is thus thought to play a role in the defence against microorganisms. The present report describes the ontogeny of MBP. The level of MBP increase during the first 3 months of life, at which time a stable level is reached, comparable to that seen in adults. On average, the level of MBP at term was 37% of the post 3-month level. Measurements on infants born prematurely showed a 3-fold increase in MBP concentration from the time of birth at around 30 weeks from conception to the 10th week of life.

Mannan-binding protein and complement dependent opsonization in alcoholic cirrhosis.

Mannan-binding protein is synthesized by the liver and functions in first-line host defence by opsonizing mannose-rich microorganisms due to activation of the classical complement pathway independent of Clq, and by an intrinsic ability to opsonize and mediate phagocytosis. We have investigated whether the increased susceptibility to bacterial infections in patients with cirrhosis could be explained by low plasma concentrations of mannan-binding protein and impaired complement-dependent opsonization. We examined 51 patients with compensated alcoholic cirrhosis, 34 who were decompensated and 16 healthy controls. Irrespective of group, we found a significant correlation (p < 0.05) between plasma mannan-binding protein concentration and deposition of the complement opsonin C4 on mannan from baker's yeast. In contrast to what was expected, this kind of opsonization and plasma levels of mannan-binding protein were significantly increased in the patients with decompensated cirrhosis (p = 0.01 and p = 0.007, respectively). A significant correlation (0 < 0.05) was found between mannan-binding protein and erythrocyte sedimentation rate, fibrinogen and haptoglobin in these patients. Though the correlations were weak (rho = 0.49, rho = 0.48 and rho = 0.40, respectively), the elevated levels of mannan-binding protein in the patients with decompensated cirrhosis may reflect an acute phase reaction. It is concluded that plasma levels of mannan-binding protein are increased in patients with decompensated cirrhosis and that complement-dependent opsonization of mannan does not seem to be compromized in patients with alcoholic cirrhosis.

Preparation and use of immunoglobulin-binding affinity supports on Emphaze beads

The Emphaze support couples to nucleophiles with a leak-resistant linkage resulting from the reaction with intrinsic azlactone functionalities. Protein A, protein G, and protein A/G were coupled to the Emphaze support and to an agarose matrix at various concentrations of ligand. Mannan Binding Protein was similarly coupled to the Emphaze matrix and agarose. These supports were investigated as to their binding characteristics toward immunoglobulins, both in a gravity separation format and using a medium pressure chromatography system under high linear flow. In addition, the lectin jacalin was coupled both to Emphaze and agarose and compared for total binding capacity toward human IgA. There were significant differences in the binding characteristics of human IgG for the Emphaze and agarose supports. Mouse monoclonal IgG1 showed a binding optimum at pH 5 on Emphaze protein A, A/G and G as compared to an optimum of pH 8 for the agarose supports. Similar binding pH optimums were observed for human IgG. Slightly higher capacities were observed for the Emphaze IgG binding than for the corresponding agarose supports. Emphaze Mannan Binding Protein had an almost identical binding capacity for mouse IgM as did a similarly loaded agarose support.

Activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by CD14 antigen, and mannan binding protein inhibits TNF-alpha release.

The present work was initiated to define mechanisms that account for the binding on human monocytes of streptococcal cell wall polysaccharides formed by rhamnose glucose polymers (RGPs), and subsequent stimulatory activities. We show here that RGPs bind to and stimulate human monocytes to produce TNF-alpha in a dose-dependent manner. To detect cell surface RGPs binding proteins, intact monocytes were biotinylated before lysis with Nonidet P-40 and solubilized proteins were incubated with RGPs Affi-Prep beads. One major membrane protein of 55 kDa was specifically detected and identified as CD14 because it reacted with anti-CD14 mAbs. Furthermore, anti-CD14 mAbs were able to perform a dose-dependent inhibition of RGPs binding, and suppressed TNF-alpha release from RGPs-stimulated monocytes. Moreover, we demonstrated that RGPs also bind to CD11b; however, this binding is not implicated in synthesis of TNF-alpha. Interestingly, RGPs binding to monocytes was enhanced by human normal serum (HNS) whereas HNS inhibits the TNF-alpha-stimulating activity of RGPs. Western blotting analysis of HNS proteins purified on RGPs Affi-prep beads revealed three specific bands of 75, 55, and 32 kDa reactive with anti-C3 Abs, anti-CD14 mAbs (TUK4), and anti-human mannan binding protein (hMBP)-derived peptide IgG, respectively. These results suggest that C3, soluble CD14, and hMBP form complexes that are probably active in enhancing the binding of RGPs to monocytes. Additional studies have shown that hMBP that recognizes RGPs prevents, unlike the LPS binding protein, TNF-alpha release by inhibiting the binding of RGPs to CD14 Ag. By incubating cells with a constant amount of RGPs-hMBP complexes in the presence or absence of increasing concentrations of C1q, we also demonstrated that C1q receptor mediates the binding and probably the uptake of RGPs-hMBP complexes by human monocytes.

Collectin in a non-mammalian species: isolation and characterization of mannan-binding protein (MBP) from chicken serum.

A chicken serum lectin was isolated by affinity chromatography on TSK-75 beads derivatized with the monosaccharide N-acetyl-D-mannosamine (ManNAc). Serum was applied to the column in a Ca(2+)-containing buffer and proteins were eluted with EDTA. After recalcification, the eluate was passed through a new ManNAc-derivatized column. Bound proteins were eluted with 50 mM ManNAc. Anti-carbohydrate antibodies present in the eluate were removed by passage through a rabbit anti-chicken immunoglobulin derivatized column, and the lectin was further purified by ion-exchange chromatography and gel-permeation chromatography. The purified chicken lectin shows an overall structure similar to mammalian mannan-binding protein (MBP). SDS-PAGE revealed two polypeptides of M(r) 33 and 34 kDa (reduced) with identical sequence for the first 30 NH2-terminal residues. The NH2-terminal sequence shows 43% identity with the human MBP. Like mammalian MBP, the polypeptides of the chicken lectin are degraded by treatment with collagenase. Residues 26-30 (G-L-P(OH)-G-D) are likely to represent the beginning of the collagenous region. Mobilities on SDS-PAGE of the COOH-terminal collagenase-resistant fragment under reduced and non-reduced conditions indicate the presence of intrachain disulphide bonds, as are also found in mammalian MBP. Gel chromatography showed an intact mol. wt of 750 kDa. Binding of the chicken MBP to mannan was inhibited by monosaccharides in the following order of potency: ManNAc > L-fucose > mannose > N-acetylglucosamine. Other monosaccharides inhibited poorly or not at all. Chicken MBP, bound to mannan, activated the classical complement pathway in human serum. Electron micrographs show structures and dimensions resembling human MBP. Overall, the results show that the purified lectin is the chicken homologue to mammalian MBP and indicate the presence of a MBP-like clearance system outside mammals.

Association between mannan binding protein deficiency and recurrent miscarriage

The distribution of mannan binding protein (MBP) in blood donor sera was determined by enzyme-linked immunosorbent assay to establish normal concentrations. Abnormally low MBP concentrations were found in 16% (21 out of 135) of female partners and 14% (15 out of 108) of male partners of couples experiencing recurrent miscarriage, compared with < 5% of obstetrically normal controls (P < 0.005). This relationship was even stronger (9.5 versus 1.0%) and more significant (P < 0.002) when only subjects presumed to be homozygous for the mutant allele responsible for MBP deficiency were considered. By immunohistochemistry, MBP could be demonstrated in first trimester placenta. We suggest that low concentrations of MBP within the feto-placental unit increase susceptibility to fetal loss, possibly via an infection-induced placental cytokine imbalance.

Binding of human collectins (SP-A and MBP) to influenza virus

Collectins are a group of soluble proteins each of which has collagenous domains and non-collagenous globular domains, the latter containing the consensus residues found in C-type lectins. Members of the collectin family are the serum proteins mannan-binding protein (MBP), conglutinin, CL-43, and the lung-associated proteins surfactant protein A (SP-A) and surfactant protein D (SP-D). MBP and conglutinin have been shown previously to bind to influenza viruses and to inhibit the infectivity and haemagglutinating activity of influenza viruses. We report here that the lung protein SP-A, like MBP, can bind to influenza virus (strain A/X31) through its lectin domain and inhibit the virus-mediated agglutination of red cells. The binding of SP-A or MBP to influenza virus was saturable, concentration-dependent, and required the presence of Ca2+ ions. Ligand-blot analysis, using MBP as ligand, of the virus lysate indicated that MBP binds to a 68 kDa viral species. The 68 kDa species was isolated to homogeneity and was shown to be the viral neuraminidase. The purified 68 kDa species inhibited the binding of both MBP and SP-A to influenza virus.

Calreticulin Is Released from Activated Neutrophils and Binds to C1q and Mannan-Binding Protein

The Ca 2+ storage protein calreticulin is associated with the endoplasmic reticulum and shares a high degree of amino acid homology with the surface receptor C1q-R. In this study, flow cytometric analysis detected calreticulin on the neutrophil surface, which decreased during stimulation probably as a consequence of shedding, as calreticulin was found by ELISA in the cell supernatants of stimulated cells. Antibodies raised against C1q-R and calreticulin demonstrated a high degree of immunological cross-reactivity for purified calreticulin as determined by dot blot analysis. Western blots of neutrophil subcellular fractions located calreticulin in both the cytosol and cell membrane fractions; C1q-R was largely confined to the cell membrane. Calreticulin and C1q-R both bind to C1q and mannan-binding protein. Therefore, calreticulin may be shed on cell activation and may be associated with the cell membrane, where it can potentially interact with C1q and serum lectins. The implications of this are discussed.

Recognition of the major cell surface glycoconjugates of Leishmania parasites by the human serum mannan-binding protein

Activation of complement on the surface of parasitic protozoa of the genus Leishmania appears to be important for parasite infectivity in the mammalian host, as it allows these parasites to attach to and invade macrophages via their surface complement receptors. Serum mannan-binding protein (MBP) is a known activator of complement. Therefore, in the present study, we have investigated whether serum MBP binds to live Leishmania parasites, and to mannose-containing saccharides derived from the parasite cell surface. We have observed by fluorescence microscopy that biotinylated MBP binds to the surface of L. major and L. mexicana promastigotes. At this developmental stage the parasites are coated by a mannose-containing lipophosphoglycan (LPG). We have observed that radioiodinated MBP binds in a mannose-inhibitable manner to purified LPG which has been immobilized in plastic microwells, as well as to purified mannose-terminating di-, tri- and tetrasaccharide fragments (‘cap’ structures) which have been released by mild acid hydrolysis from the outer chains of the LPG, converted into neoglycolipids and resolved by thin-layer chromatography. 125I-MBP also binds in the chromatogram-binding assay to the mannose-containing glycoinositol-phospholipids that are expressed in high copy number on both the promastigote and the intracellular amastigote stages of most Leishmania species. These data suggest that MBP has the potential to opsonize the major developmental stages of Leishmania parasites, and provide a possible mechanism for the antibody-independent activation of complement on their surface.

Collectins — soluble proteins containing collagenous regions and lectin domains — and their roles in innate immunity

The collectins are a group of mammalian lectins containing collagen-like regions. They include mannan binding protein, bovine conglutinin, lung surfactant protein A, lung surfactant protein D, and a newly discovered bovine protein named collectin-43. These proteins share a very similar modular domain composition and overall 3-dimensional structure. They also appear to play similar biological roles in the preimmune defense against micro-organisms in both serum and lung surfactant. The close evolutionary relationship between the collectins is further emphasized by a common pattern of exons in their genomic structures and the presence of a gene cluster on chromosome 10 in humans that contains the genes known for the human collectins. Studies on the structure/function relationships within the collectins could provide insight into the properties of a growing number of proteins also containing collagenous regions such as C1q, the hibernation protein, the alpha- and beta-ficolins, as well as the membrane acetylcholinesterase and the macrophage scavenger receptor.

Complement-dependent cytotoxic activity of serum mannan-binding protein towards mammalian cells with surface-exposed high-mannose type glycans

Serum mannan-binding protein (S-MBP), a lectin specific for mannose and N-acetylglucosamine, activates complement through the classical pathway. With the help of complement, S-MBP lyses red blood cells which have been coated with yeast mannan and kills bacteria which have N-acetylglucosamine and/or L-glycero-D-manno-heptose on their core oligosaccharide. In this study, we examined whether mammalian cells, on which S-MBP could bound, are killed by a complement-dependent mechanism. When baby hamster kidney (BHK) cells were treated with an alpha-mannosidase inhibitor, 1-deoxymannojirimycin (dMM), most of the cellular oligosaccharides were transformed from the complex-type to the high mannose-type. S-MBP bound to the dMM-treated BHK cells in the presence of Ca2+, and this binding was eliminated by mannose. When dMM-treated cells, labelled with 51Cr, were incubated with complement, radioactivity was released in a dose-dependent manner by S-MBP and complement. This release was not observed with heat-inactivated complement. These observations suggest that S-MBP is able, with the help of complement, to kill not only exogenous microorganisms but also mammalian cells which have high mannose-type oligosaccharides exposed on their surfaces.

Mannan-Binding Protein-Like Activity in the Sera of Newborn Piglets

The mechanism of the antibody-independent bactericidal activity of the sera of newborn piglets deprived of colostrum was studied using rough strains of E. coli and S. typhimurium. Although all strains were invariably killed by all newborn piglet sera tested, two different mechanisms of killing were encountered. Using specific anti-pig C1q antiserum, strains S. typhimurium LT2Ml and E. coli K-12, strain Gal 23 were found to be killed by a C1q-dependent mechanism, while the killing of E. coli S 16, E. coli K-12, strain W 3100 and E. coli B 41 could not be inhibited by anti-C1q antiserum. In order to test whether a mannan-binding protein is responsible for the bactericidal effect in the latter group of strains, we examined the inhibitory activity of two types of mannans isolated from S. cerevisiae and H. capsulata, respectively. The use of a purified rabbit mannan-binding protein showed that only strains killed by the C1q-independent mechanism were sensitive to the MBP-dependent mechanism of killing, the inhibitory activities of both mannans were found to be equal. As expected, the inhibitory effect of mannan on the bactericidal activity of newborn piglet sera was also detected only in strains killed by the C1q-independent mechanism. Surprisingly, only the phosphomannan isolated from H. capsulata was found to be an effective inhibitor of the bactericdial activity of piglet sera against E. coli S 16 and E. coli K-12, strain W 3100, while the mannan isolated from S. cerevisiae was inactive. Hence the factor present in newborn piglet sera is either MBP with slightly different binding properties, or a completely different protein.

Surfactant protein D binding to alveolar macrophages.

Surfactant protein D (SP-D) is a lung-specific protein, synthesized and secreted by lung epithelial cells. It belongs to group III of the family of C-type lectins; each member of this group has an unusual overall structure consisting of multiple globular 'head' regions (which contain the C-type lectin domains) linked by triple-helical, collagen-like, strands. This group includes the surfactant protein A (SP-A) and the serum proteins mannan-binding protein, conglutinin and collectin-43, all of which have been shown to bind to the C1q receptor found on a wide variety of cells, including macrophages. Both SP-D and SP-A have been shown to enhance oxygen radical production by alveolar macrophages. Although this strongly suggests a direct interaction between SP-D and a specific receptor on alveolar macrophages, it is still unclear whether SP-D binds to the same receptor used by SP-A and/or C1q. Human SP-D was isolated from amniotic fluid and was radiolabelled using 125I. Alveolar macrophages were isolated from human bronchioalveolar lavage fluid, and also from bovine lung washings, by differential adhesion to 24-well tissue-culture plates. The study was carried out using EDTA-containing buffers, to eliminate Ca(2+)-dependent C-type lectin binding, and was also carried out at 4 degrees C to eliminate possible internalization by the cells. 125I-SP-D showed specific binding to alveolar macrophages in both a time- and concentration-saturable manner. The binding was inhibited, by approx. 90%, on addition of a 200-fold excess of unlabelled SP-D. The apparent dissociation constant (Kd) was (3.6 +/- 1.3) x 10(-11) M, based on the assumption that native SP-D is assembled as a dodecamer of 12 identical polypeptides of 43 kDa to yield a protein of 516 kDa. C1q was also shown to bind alveolar macrophages (Kd 3 x 10(-6) M), but addition of C1q did not show inhibition of the binding of 125I-SP-D to the macrophages. We conclude that SP-D binds specifically to alveolar macrophages and the receptor involved is different from that utilized by C1q.

Mannan‐Binding Protein and Bovine Conglutinin Mediate Enhancement of Herpes Simplex Virus Type 2 Infection in Mice

A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type 1 (HSV-1) in vitro. We decided to investigate the role of mammalian lectins in infection with herpes simplex virus. Two lectins, conglutinin and mannan-binding protein (also called mannose-binding protein, MBP), belonging to the collectin family of lectins, were examined. Four week-old BALB/c mice were injected subcutaneously with 100 micrograms bovine conglutinin or 50 micrograms human MBP 1 day before intravenous infection with 5 x 10(4) PFU of herpes simplex virus type 2 (HSV-2). A three-fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with conglutinin or MBP, whereas no effect was seen on days 1 and 5. In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre-incubated with differing concentrations of the collectins. The concentrations used were similar to those used by us in vivo, and by others in in vitro experiments showing inhibition of the infectivity of HSV-1 with plant lectins. In an ELISA with HSV-2 antigens captured on anti-HSV-2 antibodies, calcium-dependent and carbohydrate inhibitable binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions with an alternative port of entry into cells leading to infection enhancement.

Dual role of mannan-binding protein in infections: another case of heterosis?

Human mannan-binding protein (MBP) is a serum lectin that participates in the immune defence by mediating phagocytosis and activation of complement. Variant MBP alleles causing dominant low-serum concentrations have high frequencies in all populations studied, and therefore, low MBP concentrations may confer selective advantages to those individuals carrying the variant alleles. Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular parasite dependent on phagocytosis to invade host cells. The serum concentrations of MBP in 36 Ethiopian patients (median: 1688 micrograms l-1) with lepromatous or borderline lepromatous leprosy were significantly (P < 0.001) higher than in 26 healthy Ethiopian blood donors (median: 368 micrograms l-1). Only 17% of the patients vs. 58% of the donors (P = 0.0019) had the relatively low MBP concentrations usually associated with variant alleles. Functional studies revealed that M. leprae and M. tuberculosis sonicates bind MBP as strongly as pure mannan. These observations suggest a role for mycobacteria as a selective force in the positive selection of alleles causing low levels of MBP and warrant genetic studies of patients infected with these bacteria.

Differential recognition by conglutinin and mannan-binding protein of N-glycans presented on neoglycolipids and glycoproteins with special reference to complement glycoprotein C3 and ribonuclease B

Conglutinin and mannan-binding protein are serum proteins that have similar carbohydrate binding specificities toward high mannose-type oligosaccharides, and yet only conglutinin binds the complement glycoprotein iC3b, which contains oligosaccharides of this type. In the present study, the interactions of conglutinin and mannan-binding protein were evaluated with the complement glycoprotein C3, including various physiologically derived fragments of this glycoprotein, and neoglycolipids prepared from oligosaccharides released from C3 and its isolated alpha and beta chains. Several conclusions can be drawn. First, the interaction of conglutinin is profoundly influenced by the state of the protein moiety of the alpha chain in the vicinity of the glycosylation site Asn-917. Second, the binding to the C3-derived glycoprotein iC3b appears to be exclusively mediated through the Man8 or Man9 oligosaccharide on the alpha chain; there is no evidence for other N-linked oligosaccharides on C3 that are uniquely bound by conglutinin. Third, although conglutinin shows a more restricted binding relative to mannan-binding protein toward the oligosaccharides free of protein, it has a broader binding pattern toward the oligosaccharides as presented on C3-derived glycoproteins. From these and additional observations with RNase B, which contains high mannose-type oligosaccharides at Asn-34, it is clear that the protein moieties of these glycoproteins markedly influence the presentation of the oligosaccharides such that biological specificity is mediated by the commonly occurring high mannose-type oligosaccharides in the context of specific carrier proteins.

Purification and Characterization of Mannan‐Binding Protein from Mouse Serum

Mouse mannan-binding protein (MBP) was identified in serum by its Ca(2+)-dependent binding to mannan. On gel permeation chromatography, the protein eluted corresponding to a molecular weight of approximately 750 kDa. Analysed on SDS-PAGE under reducing conditions, the polypeptide showed an apparent molecular weight of 28 kDa, while several high molecular weight bands were seen under non-reducing conditions. The presence of collagen-like domains within the molecule was indicated by a high glycine content (14.9%) and substantiated by sensitivity to collagenase. Rabbit anti-mouse MBP antisera were raised. The concentration of MBP in serum from normal mice was measured by rocket immunoelectrophoresis and found to be from below 1 microgram/ml to 100 micrograms/ml (average 50 micrograms/ml, n = 60). The binding of mouse MBP to mannan could be inhibited by mono- and disaccharides in the following order of potency: L-fucose > D-mannose > N-acetyl-D-glucosamine > maltose > D-mannoheptulose > D-glucose > N-acetyl-D-mannosamine >> lactose > D-galactose >> N-acetyl-D-galactosamine. Mouse MBP was shown to activate the classical complement cascade after binding to mannan. The sequence of 14 NH2-terminal amino acid residues of the molecule showed 93% identity to rat MBP-A and complete identity to the translated cDNA sequences for mouse MBP-A and mouse Ra-reactive factor component P28b (RaRF P28b) published previously. The amino acid composition of mouse MBP showed a high degree of homology to MBPs from other species and mouse RaRF P28b.