Heterogeneity of MBL–MASP complexes

Molecular Immunology 43 (2006) 1286–1292

Short communication

Heterogeneity of MBL–MASP complexes Karine R. Mayilyan a,b , Julia S. Presanis a , James N. Arnold a , Krishnan Hajela a,c , Robert B. Sim a,∗ a

MRC Immunochemistry Unit, Department of Biochemistry, Oxford University, South Parks Roads, Oxford OX1 3QU, UK b Institute of Molecular Biology, Armenian NAS, 7 Hasratyan St., Yerevan 375014, Armenia c School of Life Sciences, Devi Ahilya University, Indore 452001, India Received 24 June 2005; accepted 11 July 2005 Available online 15 August 2005

Abstract In order to study aspects of the stoichiometry and composition of human MBL–MASP complexes in the population, MBL–MASP complexes were bound from sera of 152 healthy individuals onto mannan-coated microtitre plates. Bound mannan-binding lectin (MBL) was measured by ELISA, and the enzyme activities of MBL-bound MASP-1 and MASP-2 were measured by an amidolytic assay and a C4 fixation assay, respectively. MASP-1 activity correlated with MBL concentration, as did MASP-2 activity (in both cases: p < 0.0001). This is expected since MASP-1 and MASP-2 are bound to the mannan via MBL. However, when MASP activities were normalised to MBL concentration (i.e. MASP-1 activity/[MBL], MASP-2 activity/[MBL]) MASP-1 activity was inversely correlated with MASP-2. This means on average that high MASP-1 correlates with low MASP-2 and vice-versa, and confirms the hypothesis that native MBL–MASP complexes on average do not have fixed MBL-(MASP-1)-(MASP-2) stoichiometry. The findings are consistent with separate populations of MBL–MASP-1 complexes and MBL–MASP-2 complexes, the concentrations of which show wide inter-individual variation. © 2005 Elsevier Ltd. All rights reserved. Keywords: Complement; MBL; Lectin pathway; MASP-1; MASP-2

1. Introduction Mannan-binding lectin (MBL) is the only member of the collectin family of proteins to activate the complement system via the lectin pathway which is distinct from the wellcharacterized classical and alternative pathways (Holmskov et al., 1994; Presanis et al., 2003). The lectin pathway initiator complex consists of MBL and MBL-associated serine proteases 1–3 (MASPs) (Petersen et al., 2001), and the smaller non-enzymatic component, MAp19 or sMAP (Stover et al., 1999; Takahashi et al., 1999). MBL binds to carbohydrate structures presented by a wide range of pathogens (bacteria, viruses, fungi, etc.) and mediates complement activation via activation of MASP-2. MASP-2 cleaves and activates the complement proteins C2 and C4, thereby generating the C3 ∗

Corresponding author. Tel.: +44 1865 275351; fax: +44 1865 275729. E-mail address: [email protected] (R.B. Sim).

0161-5890/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2005.07.011

convertase, C4b2a (Hajela et al., 2002; Vorup-Jensen et al., 2000). This protease complex cleaves and activates the most abundant complement protein, C3. MBL can also act directly as an opsonin, by binding to carbohydrates on pathogens, then interacting with MBL receptors on phagocytic cells to promote clearance by phagocytosis (Malhotra et al., 1990; Ogden et al., 2001; Presanis et al., 2003). The roles of MBL and MASP-2 are quite clearly defined, while MASP-1 and MASP-3 are of unknown function. Several reports have been published of the cleavage of C3 by MASP-1 (Matsushita and Fujita, 1992, 1995; Matsushita et al., 2000), but it appears that this cleavage is too slow to be of biological significance (Ambrus et al., 2003; Hajela et al., 2002; Petersen et al., 2001; Wong et al., 1999). Recent studies suggest MASP-1 cleaves the haemolytically inactive form of C3 [C3(H2 O)], but not “live” C3 (Hajela et al., 2002). Chen and Wallis (2004), working with recombinant rat MASPs suggested that MASP-1 cleaves C2 almost

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as efficiently as MASP-2 does, but it does not cleave C4. Thus, MASP-1 might augment MASP-2 complement activation but cannot initiate complement fixation in the absence of MASP-2. MASP-1 has a substrate reactivity profile similar to that of thrombin and cleaves fibrinogen and factor XIII (Hajela et al., 2002), but its physiological substrate has yet to be determined. Concerning MASP-3, there is a report of down-regulatory activity on MASP-2 C4 and C2 cleavage activity, but the data have not yet been confirmed. The structural subunit of MBL is a 75 kDa helical molecule consisting of three identical 25 kDa polypeptides, each with its own C-terminal Ca2+ -dependent carbohydraterecognition domain. The three polypeptides associate by forming a collagen triple helix. Disulphide bonds in the cysteine-rich N-terminal region stabilize the subunit (Jensen et al., 2005), and promote oligomerisation to form MBL molecules with two to six subunits. Three point mutations have been identified in codons 52, 54 and 57 in exon 1 of the human MBL structural gene in the region encoding collagen-like sequences. These mutations either disrupt the Gly-X-Y-repeating motif in the collagenous region (codons 54 (Gly → Asp at position 34) and 57 (Gly → Glu at position 37)) (Lipscombe et al., 1992; Sumiya et al., 1991), or disrupt the N-terminal disulphide bonds (codon 52, Arg → Cys at position 37) and impair the formation and stability of MBL oligomers (Madsen et al., 1994). These mutations are present in 40% of humans, and result in profound reductions in the levels of circulating functionally active MBL (Larsen et al., 2004; Mead et al., 1997; Turner and Hamvas, 2000). The mutations in codons 54 and 57 are thought to interfere not only with the maintenance of stable quaternary structure but to promote degradation in the circulation (Heise et al., 2000; Wallis and Cheng, 1999), and are associated with the failure to bind MASPs efficiently (Wallis et al., 2004). Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs (Turner and Hamvas, 2000). It is suggested that the binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical (Wallis et al., 2004). In addition to the three mutations in exon 1 of the human MBL gene, which lead to MBL functional deficiency, single nucleotide polymorphisms in the promoter region at position −550 (G to C), −221 (G to C) and untranslated region +4 (C to T), of the MBL structural gene also alter the serum level of MBL (Madsen et al., 1995; Petersen et al., 2001). The frequency of these mutations in a population varies between ethnic groups (Turner and Hamvas, 2000). In this study, we examine the composition of MBL–MASP complexes captured from human serum onto mannan-coated surfaces. The quantity of MBL bound to mannan was measured by ELISA, and the activities of MBL-associated MASP-1 and MASP-2 were measured by enzymic assay. When adjusted for MBL concentration, there was an inverse correlation between the relative quantities of MBL-bound MASP-1 and MASP-2. This is consistent with the presence of separate and distinct populations of MBL–MASP-2 com-

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plexes and MBL–MASP-1 complexes, both of which vary widely in concentration between individuals. The existence of several separate populations of MBL–MASP complexes is consistent with recent data of Teillet et al. (2005), who suggest that MBL oligomers associate predominantly with only one MASP homodimer.

2. Materials and methods 2.1. Subjects One hundred fifty two healthy volunteers randomly selected from two Caucasoid populations ((1) n = 101, mean age M ± S.D. = 34 ± 9 years; (2) n = 51, adults) participated in the study. All of them gave their informed consent to provide 5 ml of venous blood. The study was approved by local Ethics Committees. 2.2. Blood sampling Blood samples from the first population (Yerevan, Armenia) were obtained from the antecubital vein into 3 ml EDTAvacutainers (n = 82) or vacutainers without anti-coagulant (n = 19) at 08:30–09:30 h before breakfast. Serum (after coagulation) and EDTA–plasma samples were prepared by separation of cells after centrifugation and kept at −30 ◦ C. EDTA–plasma samples were re-calcified before use. Samples from the second population were obtained as whole blood aliquots (without anti-coagulant) from the National Blood Transfusion Service, Bristol, UK. Serum samples were prepared from whole blood aliquots and kept at −20 ◦ C before use. 2.3. MBL purification MBL was isolated from pooled human serum, for use as a quantitative assay standard, as described previously (Arnold et al., 2004; Tan et al., 1996). Briefly this method involves precipitation of MBL from serum with 7% polyethylene glycol (PEG 3350), binding of MBL–MASP complexes to mannan–agarose and elution with EDTA-containing buffer. MBL was separated from the MASPs by gel-filtration on Superose 6 (Pharmacia) at pH 5.0 in sodium acetate EDTAcontaining buffer. Traces of IgM were removed by passage through an anti-IgM agarose column (Sigma–Aldrich, A9935). The MBL was estimated to be >98% pure via SDSPAGE and its concentration was then established by amino acid analysis. 2.4. Amino-acid analysis Amino-acid analysis was done as described by Heinrikson and Meredith (1984). Samples were run on an ABI 420A derivatiser/analyser (PE Biosystems, Warrington, UK) following hydrolysis for 24 h at 110 ◦ C in 5.7N hydrochloric

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acid. The ABI 420A utilises pre-column derivatisation with phenylisothiocyanate to form phenylthiocarbamyl amino acids. These are then automatically sampled to a narrowbore HPLC system (Applied Biosystems 130A) for analysis. Data handling is performed using Dionex Chromeleon software (Version 6.40 from Dionex UK Ltd., Macclesfield, UK). 2.5. Quantitation of serum MBL by ELISA The quantity of MBL capable of binding to mannan was measured using an ELISA in which mannan-coated wells were used for capture of MBL (Arnold et al., 2004). Briefly, ELISA plates (Maxisorp, Nunc, Kamstrup, Denmark) were coated with 50 ␮l of 1 mg/ml mannan (Sigma–Aldrich, M7504) in 0.1 M Na2 CO3 , pH 9.6 (coating buffer). The wells were washed three times and blocked with PBS, 0.1% Tween20 (w/v) for 2 h at RT. The wells were incubated for 1 h at RT with serum diluted 1/10–1/100 (v/v) in 10 mM HEPES, 1 M NaCl, 5 mM CaCl2 , pH 7.4. Negative control samples were diluted in 10 mM HEPES, 1 M NaCl, 5 mM EDTA, pH 7.4 (to prevent MBL binding). The wells were washed and then incubated for 1 h at RT with 100 ␮l of a 1/250 (v/v) dilution of rabbit anti-MBL polyclonal antiserum, depleted of anti-mannan antibodies by passage through a mannan agarose column (Sigma–Aldrich, M9917) and depleted of rheumatoid factors by adsorbtion on non-immune human IgG Sepharose (Arnold et al., 2004). The wells were washed and incubated at RT with 100 ␮l of a 1/700 (v/v) dilution of goat anti-rabbit IgG antibody conjugated to alkaline phosphatase (Sigma–Aldrich, A-3812) for 1 h. The wells were washed and 100 ␮l of 1 mg/ml p-nitrophenyl phosphate substrate in 0.2 M Tris buffer pH 9.5 (Sigma–Aldrich, N-2770) was added and absorbance read at 405 nm after 10 min. MBL level was expressed in ␮g/ml of serum (␮g/ml). Each serum sample was assayed three times. 2.6. Assay of MASP-1 activity The enzymic activity of MASP-1 bound to MBL captured on the mannan-coated microtitre plate (MBL-boundMASP-1 activity) was determined by use of a fluorescent substrate Val-Pro-Arg-aminomethylcoumarin (Bachem, Bubendorf, Switzerland) (Presanis et al., 2004). Serum was diluted 1:1 (v/v) with 40 mM HEPES, 2 M NaCl, 10 mM CaCl2 , pH 7.4 and 100 ␮l of each serum dilution was incubated in a mannan-coated microtitre plate well for 1 h on ice. After washing twice with 20 mM HEPES, 1 M NaCl, 5 mM CaCl2 , 0.1% (v/v) Tween-20, pH 7.4 (high salt washing buffer) and three times with 20 mM HEPES, 140 mM NaCl, 5 mM CaCl2 , 0.1% (v/v) Tween-20, pH 7.4 (washing buffer) at 37 ◦ C, 200 ␮l of 0.1 mM VPR-AMC substrate, in 20 mM HEPES, 5 mM CaCl2 pH 8.5, was added. The samples were excited at 355 nm and emission read at 460 nm every 30 s for 1 h using a microtitre plate reader (Fluoroskan, ThermoLife Sciences, Basingstoke, UK).

MASP-1 activity was calculated from the initial slope of the activity curve and expressed as relative fluorescence emission units per min (RFU). 2.7. Assay of MASP-2 activity MBL-bound-MASP-2 activity was evaluated by C4 fixation assay as described by Presanis et al. (2004). MBL–MASP complexes were bound to mannan-coated microtitre wells as for the MASP-1 assay. After washing, purified human C4 (0.1 ␮g per well) in 20 mM HEPES, 140 mM NaCl, 5 mM CaCl2 , pH 7.4, was added to each well and incubated for 1 h at 37 ◦ C. Following three further washes with 20 mM HEPES, 140 mM NaCl, 20 mM EDTA, 0.1% Tween-20, pH 7.4, 100 ␮l alkaline-phosphatase conjugated human anti-C4 (Immunsystem, Uppsala, Sweden) diluted 500-fold in the same buffer was added and incubated for 1 h. Wells were washed three times with the same buffer and developed using 100 ␮l p-nitrophenyl phosphate in 0.2 M Tris buffer pH 9.5 (Sigma–Aldrich) for 1 h at 37 ◦ C. The absorbance was read at 405 nm using a microtitre plate reader (Multiskan, ThermoLife Sciences). MASP-2 mediated C4b fixation activity was expressed in absorbance units per ml of serum (U/ml). 2.8. Statistics The Shapiro–Wilk W-test was done to determine the normality of distribution of the data obtained. For correlation analysis of the data, Spearman’s rank correlation was applied. Statistics were performed using “Analyse-it” statistical software (Analyse-it Software Ltd., UK).

3. Results The MBL serum level of all subjects investigated ranged from undetectable (marked as 0.13: the sensitivity limit of the assay was 0.13 ␮g/ml) to 20 ␮g/ml. On Fig. 1 are shown

Fig. 1. Frequency distribution of individuals by serum MBL level: lower detection limit of the assay is 0.13 ␮g/ml.

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percentage frequency distributions of the investigated individuals according to their serum MBL levels. Although 20% of them had undetectable concentration of the protein, the mean MBL serum concentration on average was high at 2.4 ␮g/ml, and thus, reflects the finding that the Armenian population has mean MBL levels higher than the UK population (Mayilyan et al., 2005). The mean MBL-bound MASP-1 activity in all of the investigated individuals was 2.1 RFU (range 0–14.88). The MASP-1 distribution characteristics are shown in Fig. 2. The percentage of individuals with undetectable MASP-1 activity within the group was similar to that obtained for MBL concentration (20%). Moreover, there was a strong correlation between the data obtained on serum MBL level and MASP1 activity (Fig. 2B; Spearman’s rank correlation: rs = 0.73, p < 0.0001). This is expected, as MASP-1 is bound to mannan via MBL. Of the 30 individuals with undetectable MBLbound MASP-1 activity, most also had no detectable MBL. However, two individuals with undetectable MASP-1 activity had low but detectable MBL (0.13–0.14 ␮g/ml), one had 0.75 ␮g/ml MBL and one had high MBL (4.1 ␮g/ml). Each of these four individuals had MASP-2 activity. These four cases may indicate some form of MASP-1 deficiency, and further studies are being done to investigate this. The mean MASP-2 activity in the individuals investigated was 6.0 U/ml (range 0–10.83 U/ml). Only two individuals had

Fig. 2. MBL-bound MASP-1 activity: (A) frequency distribution of individuals by activity (lower detection limit of the assay is about 20 ng/ml); (B) correlation with serum MBL concentration.

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undetectable MASP-2 activity, and both of these also had no detectable MBL. All other individuals with undetectable MBL (<0.13 ␮g/ml) had detectable MASP-2 activity. This is a reflection of the greater sensitivity of the MASP-2 assay. The MASP-2 assay is about 12-fold more sensitive than the MBL assay (on a molar basis), while the MASP-1 assay is only slightly more sensitivity than the MBL assay, on a molar basis. MASP-2 activity distribution is shown in Fig. 3A. A good correlation was seen between data obtained for MBL concentration and MASP-2 activity (Fig. 3B; rs = 0.55, p < 0.0001). Again this is expected, as MASP-2 is bound to mannan via MBL. Results of statistical analyses of all the data obtained for the three different measurements are presented in Table 1. For each of the measured parameters, the median and mean of data obtained are very different from each other. Thus, the Shapiro–Wilk W-test showed non-normal distribution of the data obtained (for each of three cases: P < 0.0001). For calculation of MASP-1 and MASP-2 activity per ␮g of MBL in the bound MBL–MASP complexes from each individual, the ratio MASP activity/MBL concentration was expressed in arbitrary units (AU). The data on individuals with undetectable MBL serum concentration were not included in the calculation. Fig. 4 shows the correlation of the data on MASP-1 and MASP-2 activities of MBL–MASP

Fig. 3. MBL-bound MASP-2 activity: (A) frequency distribution of individuals by serum MASP-2 activity (lower detection limit of the assay is about 2 ng/ml); (B) correlation with serum MBL concentration. As noted in the text, the intercept on the y-axis is a feature of difference in sensitivity (maximum detection level) of the two assays.

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Table 1 Statistical data of all three parameters of MBL–MASP complexes in healthy individuals [MBL], ␮g/ml MASP-2, U/ml MASP-1, RFU

n

Mean

S.D.

S.E.

95% CI of mean

Median

IQR

95% CI of median

145 152 147

2.40 6.02 2.13

2.74 2.88 2.83

0.23 0.23 0.23

1.95–2.85 5.56–6.48 1.67–2.59

1.90 6.31 0.91

3.02 3.69 3.56

1.14–2.24 6.04–6.82 0.41–1.39

CI, confidence interval; IQR, interquartiles.

Fig. 4. The correlation of the data on MASP-1 and MASP-2 activities adjusted per unit of MBL concentration.

complexes adjusted for MBL concentration. There was an inverse correlation between MASP-1/[MBL] and MASP2/[MBL] activities (Fig. 4; rs = −0.36, confidence interval: CI −0.51 to −0.19, P < 0.0001). The distribution fits the equation y = 2.860/x. This shows that individuals with a low MASP-2 activity/[MBL] have in general a high MASP1 activity/[MBL] and vice-versa. This finding reflects the MBL–MASP-1/MASP-2 complex composition in serum. Thus, when the MBL–MASP complexes were captured on mannan-coated surfaces for assaying, the MASP-1: MASP2 ratio was not constant, but showed wide inter-individual variation. It is also clear from Fig. 4 that the degree of occupancy of MBL with MASP-1 and MASP-2 is variable. Data points at the upper right have much more MASP-1 + MASP-2 per unit MBL than do points at the lower left.

4. Discussion and conclusions Super et al. (1989) showed that the capacity of serum to deposit complement components C3b and C4b onto a mannan-coated surface correlated with the levels of MBL in the circulation. Our data also support this observation, as we found good correlation between MASP-2 C4 fixation activity and MBL concentration (P < 0.0001). MASP-1 activities of individuals also had strong correlation with MBL quantity (P < 0.0001). The MASPs assay used here measured MBLbound MASP activity, and so a strong correlation between MBL level and MASPs activities is to be expected. Moreover, unlike normal frequency distributions of total serum MASP1 (Terai et al., 1997), the observed non-normal distribution of MASP-1 activity in this study reflects the non-normal dis-

tribution of MBL protein quantity in the circulation of the subjects investigated. A striking result from these data is the finding that the quantities of MASP-1 and MASP-2 bound to MBL are inversely correlated (Fig. 4), i.e. high MASP-2 activity/[MBL] correlates with low MASP-1 activity/[MBL] and vice-versa. This finding confirms that MASP-1 and MASP-2 have no fixed stoichiometry. Earlier work on MBL structure demonstrated the presence of hexamers (six subunits, 18 polypeptide chains) as well as smaller oligomers (Lu et al., 1990). Since MBL was shown to be able to bind C1r2 s2 and to activate complement via these proteases (Lu et al., 1990), it appeared likely that the major functionally active form of MBL in plasma was a hexamer, bound to four protease monomers, similar to the fixed stoichiometry C1 complex which contains one hexameric C1q, 2 C1r monomers and 2 C1s monomers. The discovery of 2 MASPs (MASP-1 and MASP-2) indicated that MBL might form complexes very similar to C1, with a composition MBL + 2(MASP-1) + 2(MASP-2). However, the subsequent characterization of MASP-3 and MAp19 indicated a more complex situation. There is some disagreement regarding the degree of polymerization of MBL in human serum. Lu et al. (1990) showed the presence of range of oligomers, from one to six subunits. Recent data (Teillet et al., 2005) indicates that trimeric and tetrameric forms of MBL predominate in human serum (3 or 4 subunits, 9 or 12 polypeptide chains) and that both forms bind two molecules (one homodimer) of MASPs. This is consistent with the work of Wallis et al. (2004), who showed with rat recombinant proteins that one MASP homodimer will bind to an MBL dimer, trimer or tetramer. Thus, each MBL oligomer (with the exception of monomers) in the circulation is likely to bind only one MASP dimer. Since the MASPs have not been reported to form heterodimers, each MBL oligomer may bind only one type of MASP. Therefore MBL–(MASP-2)2 complexes, MBL–(MASP-1)2 complexes will occur, but not MBL oligomers complexed to more than one type of MASP. Our results are consistent with this and can be most simply explained by postulating that, among the MBL oligomers large enough to bind to mannan, each MBL oligomer is complexed with a MASP-2 dimer or a MASP-1 dimer. Additional MBL oligomers complexed to MAp19 dimers or MASP-3 dimers may also be present, but we did not measure these. Thus, each individual has a population of MBL–MASP-1 complexes and of MBL–MASP2 complexes, but the relative concentrations of these are inversely related and vary widely between individuals (Fig. 4).

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As observed by (Dahl et al. (2001), there may be preferential binding of MASP-1, -2 or -3 to MBL oligomers of different size. In addition mutations in the collagen region of MBL are reported to influence the degree of oligomerisation (Larsen et al., 2004) and also the affinity of the MBL–MASP interaction (Wallis et al., 2004). Individuals who are heterozygous for wild type plus any of the three sequence variant genotypes will form mixed MBL oligomers containing wild type and sequence-variant polypeptides. This will increase the heterogenity in oligomerisation (relative to wild type homozygotes) and may result not only in inter-individual differences in quantities of each oligomer, but also in occurrence of oligomers of the same size, e.g. trimers, which have different affinities for MASPs. Thus, the degree of occupancy or saturation of mannan-bound MBL with MASPs is also variable, as is observed in Fig. 4. Thus, the MBL–MASP complexes, though superficially similar to the C1q-r-s (C1) complex, are really much more diverse in structure. This arises from two features of MBL. The first is the disulphide bridging pattern of MBL, which unlike that in C1q is heterogeneous and causes variable early termination of polymerization (Jensen et al., 2005; Larsen et al., 2004). The second feature is the high frequency occurrence of structural variants of the MBL polypeptide. The structural variants are defective in oligomer stability and/or MASP binding. The occurrence in heterozygote individuals of oligomers containing a mixture of WT and mutant polypeptides introduces further complexity in oligomer size and affinity for MASPs. Our data on the highly variable composition of MBL–MASP complexes and the variable degree of occupancy of MBL by MASPs is very likely to have biological significance. Further studies of factors controlling MBL oligomer formation (Jensen et al., 2005) are required to understand heterogeneity of MBL–MASP complex formation.

Acknowledgments We thank Tony Willis for amino acid analysis of the MBL preparation. We acknowledge the contribution of EU Grant QLGI-2001-01039. K.R.M. thanks the Royal Society for an RS/NATO postdoctoral fellowship (16312/03B/LD). J.S.P. and J.N.A. thank the UK Medical Research Council for postgraduate studentships.

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