functional properties of recombinant human mannan-binding lectin and its variants

Immunology Letters 123 (2009) 114–124

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New insights on the structural/functional properties of recombinant human mannan-binding lectin and its variants夽 Rema Rajagopalan a , Veena P. Salvi a , Jens Chr. Jensenius b , Nenoo Rawal a,∗ a b

Department of Biochemistry, University of Texas Health Science Center, 11937, US Highway 271, Tyler, TX 75708-3154, USA Department of Medical Microbiology and Immunology, University of Aarhus, Wilhelm Meyers Allé, 8000 Aarhus, DK, Denmark

a r t i c l e

i n f o

Article history: Received 10 October 2008 Received in revised form 13 February 2009 Accepted 24 February 2009 Available online 19 March 2009 Keywords: Recombinant mannan-binding lectin (MBL) Recombinant MBL structural variants Oligomer formation Complement lectin pathway

a b s t r a c t Inefficient activation of complement lectin pathway in individuals with variant mannan-binding lectin (MBL) genotypes has been attributed to poor formation of higher order oligomers by MBL. But recent studies have shown the presence of large oligomers of MBL (∼450 kDa) in serum of individuals with variant MBL alleles. The recombinant forms of MBL (rMBL) variants except MBL/B that assemble into higher order oligomers have not yet been reported. In the present study, structural/functional properties of recombinant forms of wild type MBL (rMBL/A) and its three structural variants, rMBL/B, C, and D generated in insect cells were examined. Western blot analysis indicated covalently linked monomers to hexamers while gel filtration chromatography exhibited non-covalently linked higher order oligomers in addition to prevalent low oligomeric forms. Mannan binding determined by ELISA showed rMBL/A but not the structural variants bind to mannan. Apparent avidity of monoclonal antibody used was found to be about 18- to 52-fold weaker for rMBL structural variants than rMBL/A. Complement activation varied with maximum impairment apparent in rMBL/C followed by rMBL/B, but rMBL/D was functional to the same extent as rMBL/A. Comparison of rMBL/A to MBL purified from plasma (pMBL/A) indicated 8- and 24-fold weaker binding to mannan by BIAcore analysis and ELISA and about 5-fold lesser efficiency in activating complement. The findings provide new insights on the structural/functional properties of rMBL variants and imply that lectin pathway activation may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D. © 2009 Published by Elsevier B.V.

1. Introduction Mannan-binding lectin (MBL) belongs to a family of carbohydrate-binding defense proteins called collectins [1–4]. Binding of MBL to the cell surface of microorganisms initiates activation of the lectin pathway of complement as part of the host’s innate immune response [5–12]. Recent studies have shown that the lectin pathway has the potential to generate four times more C3/C5 convertases than the classical pathway and hence the production of pro-inflammatory products C3a, C4a, and C5a required for mounting an effective innate immune response to infections

Abbreviations: MBL, mannan-binding lectin; pMBL/A, MBL purified from human plasma; rMBL, recombinant MBL; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; NBT/BCIP, nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate for alkaline phosphatase; mAb, monoclonal antibody; MASP, MBLassociated serine protease. 夽 This research was supported by National Institutes of Health Research Grant HL-073804 (N.R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ∗ Corresponding author. Tel.: +1 903 877 5840; fax: +1 903 877 5882. E-mail address: [email protected] (N. Rawal). 0165-2478/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.imlet.2009.02.013

[13]. MBL circulates in blood as oligomers of the basic structural subunit (75 kDa) made of 3 identical polypeptide chains (25 kDa each) [14–17]. Several studies have suggested that the oligomeric forms of MBL are responsible for complement activation [18–20]. Four structural allelic forms of MBL have been described [21–26]. The A allele is the wild type, where as alleles B, C, and D are the variant forms due to point mutations in codons 54, 57, and 52, respectively, of the MBL-2 gene. Single nucleotide mutations in the promoter and 5 -untranslated region of the MBL-2 gene resulting in seven haplotypes, have also been reported [27–29]. Hospital-based studies have associated the presence of MBL alleles with mutations in the structural gene as well as in the promoter region with increased susceptibility to recurrent infections in children and adults [7,21,23,26,29]. Individuals with certain MBL alleles are reported to be more sensitive than those with normal MBL to various health problems including HIV infections [30–32], and rheumatoid arthritis [33]. In addition, carriers of MBL alleles suffer increased morbidity in autoimmune disorders, such as systemic lupus erythematosus [34–36] and cystic fibrosis [37,38] and have a shorter life expectancy in diseases such as cystic fibrosis [39] because of a compromised immune system that renders them prone to infections. But studies have also suggested that MBL alleles may confer protection against parasitic infections [4,10,11].

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The low biological activities of the structural variants of MBL such as binding to mannan and complement activation have been attributed to poor formation of higher order oligomers. Several studies examining MBL in sera of individuals with MBL variants have reported different results on oligomer formation. Earlier studies examining oligomer formation of MBL in plasma have mostly reported poor formation of higher order oligomers by MBL structural variants [22,40]. The studies of Garred et al. [28] and Minchinton et al. [41] reported the presence of both higher and lower oligomer forms of MBL in sera of individuals heterozygous for MBL variants and predominantly lower oligomer forms in sera from homozygous and compound carriers of MBL alleles. But more recent studies by Terai et al. [42] and Frederiksen et al. [43] using gel filtration chromatography and new improved methods for detecting MBL structural variants have reported the presence of large oligomers of MBL with apparent molecular size of about 450 kDa in plasma from donors homozygous for MBL alleles. Studies expressing the recombinant forms of MBL (rMBL) structural variants have also mostly reported poor formation of higher order oligomers [19,44,45] except for rMBL/B [17]. The study of Larsen et al. expressing MBL in CHO cells [45] showed MBL structural variants did not form higher order oligomers but the same study reported loss of oligomer formation for rMBL/B upon purification. The study of Super et al. employing mouse hybridoma cells to express rMBL/B has reported formation of higher order oligomers [17]. Initial attempts to express wild type MBL/A in SF 9 insect cells also resulted in the generation of rMBL/A that did not oligomerize like plasma MBL/A (pMBL/A) [46] but later several groups successfully produced oligomeric forms of rMBL/A, with complete disulfide bridges in different cell lines [47–50]. Generation of recombinant form of MBL (rMBL) variants that assemble into higher order oligomers would be useful for studying functional defects associated with MBL variants and would also provide the biochemical support for hospital-based studies that have linked MBL alleles with mutations in the structural gene with increased susceptibility to recurrent infections [7,21,23,26,29]. Since, formation of multiple covalently linked (via disulfide) oligomeric forms have been reported for rMBL mutants that were expressed in insect cells and generated by deleting GXY triplets in the collagen region of the molecule [48], in the present study, MBL/A and its three structural variants, MBL/B, C, and D that arise due to single point mutations in the collagen region of the molecule, were generated using the insect cell system. Analysis of the structural/functional properties show that although rMBL structural variants generated in insect cells form covalently linked as well as non-covalently linked higher order oligomers (monomers to hexamers), rMBL/B and rMBL/C but not rMBL/D exhibit weaker and/or ineffective physiologic functions such as complement activation when compared to rMBL/A. The data also show that the lower biological activity of rMBL/A when compared to pMBL/A may be attributed to lower order oligomers being more prevalent in rMBL/A than pMBL/A. These findings on rMBL structural variants imply that activation of the lectin pathway and hence production of proinflammatory products C3a, C4a, and C5a that are required to mount an effective innate immune response may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D.

2. Materials and methods 2.1. Media, reagents and antibodies Restriction enzymes, T4 DNA ligase, calf intestinal alkaline phosphatase and DNA molecular weight markers were from New England Biolabs (USA). Qiagen Plasmid Mini kit and Qiaquick PCR

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purification kit were from Qiagen Sciences (USA) while GeneTailor Site-Directed Mutagenesis kit, Cellfectin, blasticidin, pIB/V5-His vector, pure link genomic DNA purification kit, Platinum PCR Super Mix High Fidelity, DH5␣ cells, High Five insect cells, and cell culture medium Sf-900 II SFM were from Invitrogen (USA). Gentamycin was from Life Technologies (USA) while pre-stained dual color protein molecular weight markers, SDS gels, MOPS buffer, polyvinylidene difluoride (PVDF) membrane and alkaline phosphatase substrate kit were from Bio-Rad (USA). Monoclonal antibodies (mAb) to human MBL: mAb 131-01 was from AntibodyShop (Denmark) and mAb 93C was from NatImmune A/S, Copenhagen, Denmark. Human MBL (pMBL/A) was purified from plasma by modifying the method of Tan et al. [51] as described [52]. Polyclonal antiserum (G5132) was raised to pMBL/A in goat at Bethyl Laboratories (USA).

2.2. Construction of wild type rMBL/A expression vector The vector, pUC19 containing cDNA for wild type human MBL (MBL/A) was a kind gift from Dr. R.A.B. Ezekowitz (Massachusetts General Hospital for Children, Boston, MA). For expression of MBL in insect cells, constructs were prepared by ligating a 1.4 kb MBL cDNA insert containing the coding region for MBL into pIB/V5-His vector and was transformed into DH5␣ cells. The MBL cDNA of ampicillin resistant clones with the right insert size were sequenced and only then used for transfection into insect cells to generate rMBL/A. The pIB/V5-His + MBL/A cDNA construct was used as the parent plasmid to generate MBL variant constructs as described below.

2.3. Construction of rMBL variant (rMBL/B, rMBL/C or rMBL/D) expression vector The standard GeneTailor Site-Directed Mutagenesis System was used for constructing the three variants of MBL; MBL/D (Arg52Cys), MBL/B (Gly54Asp), and MBL/C (Gly57Glu). The parent plasmid (pIB/V5-His + 1.4 kb MBL/A cDNA) was first methylated and then amplified with two overlapping primers, one of which contained the mutation in codon 52 for MBL/D, codon 54 for MBL/B, and codon 57 for MBL/C. The primers used for MBL/D were FWD 5 -TTCCCAGGCAAAGATGGGTGTGATGGCACC-3 and REV 5 -CCCATCTTTGCCTGGGAAGCCGTTGATG-3 . The primers for MBL/B were FWD 5 -AGGCAAAGATGGGCGTGATGACACCAAGGGAGAA-3 and REV 5 -CATCACGCCCATCTTTGCCTGGGAAGCCGTTG-3 . For MBL C the primers were FWD 5 -TGGGCGTGATGGCACCAAGGAAGAAAAGGGGGAA-3 and REV 5 -CCTTGGTGCCATCACGCCCATCTTTGCCTGGGAA-3 . PCR was done on a Rapid Cycler (Idaho Technology). The 5 kb PCR products obtained were transformed in DH5␣ cells. Plasmid DNA from ampicillin resistant clones, were sequenced to verify the mutation in each case.

2.4. Transfection of High Five cells with pIB/V5-His + MBL cDNA constructs The pIB/V5-His constructs containing cDNA for MBL/A, B, C, or D were purified from DH5␣ cells and transfected in to High Five insect cells using Cellfectin as per manufacturer’s instructions. Transfected cells were allowed to grow to confluence for 3 days after which stable transformants were selected in medium containing 50 ␮g/ml blasticidin. Controls were High Five insect cells without pIB/V5-His vector and with pIB/V5-His vector but no MBL cDNA. The stable transfected insect cells were grown in Sf-900 II serum free medium (SFM) with gentamycin and blasticidin (10 ␮g/ml of each) and supplemented with 0.3 mM l-ascorbic acid for hydroxylation of lysine and proline residues [47,48]. Cell cultures were amplified to large volumes of suspension cultures.

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2.5. Detection of rMBL expression in insect cell medium by Western blot analysis Cells from third day growth cultures were harvested by centrifugation and the supernatant was concentrated using Centricon 30 concentrator (Millipore) before electrophoresis on SDS PAGE using Criterion XT precast 10% acrylamide gels and XT MOPS buffer (Bio-Rad, USA). Proteins were electrophoretically transferred to PVDF membrane. The primary antibody used was various antiMBL antibodies (diluted 1/500 or 1/1000) as mentioned. The relevant secondary antibodies conjugated to alkaline phosphatase were commercial products and diluted as indicated. Bands were visualized by developing the blot with the substrate, nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as described by the manufacturer using the alkaline phosphatase conjugate substrate kit (Bio-Rad, USA). The images of Western blots were acquired in a GEL DOC 2000 machine (Bio-Rad, USA) and analyzed using ‘Quantity One’ software. 2.6. Detection of rMBL expression in insect cell medium by capture on anti-MBL followed by Western blot analysis Expression of rMBL molecules in the growth media of insect cells was determined by capture on anti-MBL. The microtiter wells were coated with goat IgG purified from MBL antiserum (G5132). The unoccupied sites were blocked with 200 ␮l 3% BSA in PBS containing 0.02% sodium azide. One hundred microliters of 10-fold concentrated medium was added to 12–24 consecutive wells. Between the various steps the wells were washed with PBS containing 0.05% Tween 20. The first well of each sample was incubated with 200 ␮l XT MOPS running buffer from BioRad, to release bound MBL and then the sample was transferred to the second well. The procedure was repeated through 12–24 consecutive wells so as to concentrate the captured MBL. The concentrated MBL sample (15–30 ␮l) was examined by Western blot analysis using mouse mAb 131-01 as the primary antibody and an anti-mouse IgG conjugated to alkaline phosphatase as the secondary antibody. The blot was developed using the alkaline phosphatase conjugate substrate kit (NBT/BCIP) from BioRad. 2.7. Purification of rMBL molecules by affinity chromatography After three days of growth, 10 mM EDTA was added to suspension cultures and cells removed by centrifugation for 10 min at 10,000 × g. Cell free medium (700 ml) was extensively dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.8 (TBS buffer A) containing 20 mM CaCl2 , applied on a mannan–Sepharose column equilibrated in the same buffer and bound MBL eluted with TBS buffer A containing 30 mM EDTA. Fractions containing MBL, detected by Western blot analysis, were pooled and concentrated. Mannan–Sepharose was prepared by coupling 8 mg mannan/ml Sepharose using the cyanogen bromide method [53]. To avoid cross contamination, separate columns were used for each rMBL protein. 2.8. Determination of MBL concentration Protein concentration of MBL purified from plasma (pMBL/A) was determined spectrophotometrically using 7.2 as the value for E1% /280 [54]. Analysis of rMBL molecules including rMBL/A by SDSPAGE indicated that the protein bands observed up on staining the gel did not correlate with the concentration determined by A280 when compared to MBL purified from plasma (pMBL/A). Therefore, concentration of rMBL molecules was measured by densitometric analysis of purified rMBL molecules electrophoresed on SDS-PAGE and calculated from a standard graph generated by densitometric

analysis of different concentrations of purified pMBL/A (0.5–3 ␮g) electrophoresed on the same SDS-PAGE gel.

2.9. Binding of rMBL molecules to mannan as determined by ELISA Binding of rMBL and pMBL/A to mannan was determined by modifying the procedure of Holmskov et al. [55]. Briefly, polypropylene flat bottom micro-plate wells were coated with 50 ␮l of mannan (20 ␮g/ml) in 50 mM carbonate–bicarbonate buffer pH 9.6 and incubated overnight at 4 ◦ C. The wells were washed three times with 10 mM Tris, 150 mM NaCl, pH 7.4 (TBS buffer B), nonspecific binding blocked with BSA (1%) in TBS buffer B for 1 h at 22 ◦ C, washed with TBSTCa++ buffer B (TBS buffer B containing 0.05% Tween 20, 20 mM CaCl2 ), incubated with varying concentrations of purified rMBL variants or pMBL/A for 1 h at 22 ◦ C and followed by washing with TBSTCa++ buffer B. The wells were incubated with IgG purified from anti-human MBL serum (goat) diluted 1/5000 in TBS buffer B as the primary antibody for 1 h at 22 ◦ C, washed with TBSTCa++ buffer B and incubated for 1 h at 22 ◦ C with commercial anti-goat antibody conjugated with alkaline phosphatase, diluted 1/1500 in TBS buffer B. The wells were washed with TBSTCa++ buffer B and binding to mannan detected by adding 6 mM of the substrate, p-nitrophenyl phosphate in 10% diethanolamine, pH 9.8 and the yellow color developed was measured at 405 nm.

2.10. Binding of rMBL molecules to mannan as determined by surface plasmon resonance using BIAcore Mannan was reductively aminated using ammonium chloride and sodium cyanoborohydride by the method of Osmond et al. [56]. The NH2 -(aminated) mannan was immobilized on a CM4 sensor chip (BIAcore AB) via amine groups using standard N-ethyl-N -(diethylaminopropyl) carbodiimide/N-hydroxy succinimide coupling according to the manufacturer’s protocol to give approximately 278 response units (RU) of NH2 -mannan bound to the chip. Binding experiments were conducted by passing MBL samples simultaneously over both the flow cells at 25 ◦ C in 20 mM Tris–HCl buffer pH 7.4 containing 0.15 M NaCl, 0.05% BSA, and 0.005% surfactant p-20 supplemented with 1 mM CaCl2 at a flow rate of 5 ␮l/min. Regeneration of the surfaces was achieved by injecting 10 ␮l running buffer containing 3 mM EDTA but no Ca++ .

2.11. Analysis of binding data Sensograms were analyzed using BIAevaluation 4.1 software by globally fitting the association and dissociation phases of the overlay plots simultaneously over a range of concentrations for pMBL/A and rMBL/A (Fig. 5A and B) to a 1:1 Langmuir binding model using BIAevaluation 4.1 software. The kinetic analysis of interaction of recombinant MBL variants with the immobilized amino mannan was performed as explained by Terada et al. [57] using the rate equation, dR/dt = ka CRmax − (ka C + kd )Rt , where dR/dt is the rate of change of the SPR signal (resonance units) due to each protein interaction with immobilized mannan at time t seconds, ka and kd are the association- and dissociation-rate constants, respectively, C is the concentration of each protein used and Rmax is the maximum MBL-binding capacity to mannan in resonance units. The apparent equilibrium dissociation constants (KD ) were calculated from the ratio of the dissociation and association rates (kd /ka ). The chi2 (2 ) value, a standard statistical measure of the fit was less than 2 in all the cases.

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2.12. Complement activation Complement activation of rMBL molecules was measured by modifying the method of Ikeda et al. [58] as described. Instead of using MBL depleted guinea pig serum, human MBL depleted (MBL-Dpl) serum was prepared by passing 10 ml pooled normal human serum on a mannose–Sepahrose column (20 ml) in tandem with a mannan–Sepharose column (20 ml). Depletion of MBL was confirmed by Western blot analysis. Serum concentration of MASP-2 is reported to be low (0.5 mg/ml) and there is no free MASP-2 in human serum, i.e., is bound to MBL and ficolins [59]. Depletion of MBL from serum only depletes MBL but not ficolin associated MASPs. Western blot analysis using mono-specific polyclonal antibody developed to a 19 amino acid long peptide sequence corresponding to the C-terminal region of MASP-2 confirmed that sufficient amount of MASP-2 (0.5 mg/ml) was present in human MBL-Dpl serum generated when compared to normal human serum. Sheep erythrocytes were coated with mannan (EMan ) and complement activation measured by hemolytic assays in which EMan cells were incubated together with rMBL and MBL-Dpl serum in the same reaction mixture instead of sensitizing EMan cells with MBL prior to the assay as described by Ikeda et al. [58]. Assay mixtures contained 2.5 ␮l EMan (2 × 109 /ml), 1 ␮l MBL-Dpl serum and varying concentrations of rMBL in a final volume of 25 ␮l of GVB++ buffer (gelatin veronal-buffered saline containing 0.5 mM MgCl2 and 0.15 mM CaCl2 ). After 8 h incubation at 37 ◦ C, assay tubes were transferred to an ice bath and 225 ␮l of cold GVB++ was added and the tubes centrifuged. The amount of hemolysis was determined by reading 200 ␮l of the supernatant at 415 nm. Controls containing EMan and MBL-Dpl serum were subtracted as background. EMan alone was also kept as a control. Lysis of EMan in the presence of MBL-Dpl serum in distilled water was considered 100%. 2.13. Gel filtration chromatography Recombinant MBL molecules that were purified by affinity chromatography on mannan–Sepharose columns were fractionated on a BioSep-Sec S 4000 column (600 mm × 7.8 mm, Phenomenex, USA) equilibrated in 20 mM Tris–HCl buffer containing 150 mM NaCl and 5 mM EDTA, pH 7.4 at a flow rate of 0.5 ml/min. The column was calibrated with blue dextran (2000 kDa), apoferritin (443 kDa) and gel filtration BioRad standards containing thyroglobulin (669 kDa), human IgG (158 kDa), ovalbumin (44 kDa), myoglobulin, (17 kDa), and Vitamin B12 (1.35 kDa). 3. Results 3.1. Detection of rMBL expression in insect cell medium by antibody capture followed by Western blot analysis Expression of rMBL molecules by insect cells was determined by Western blot analysis of the growth medium using various commercially available monoclonal antibodies to MBL. However, detection of the structural variants rMBL/B, C, and D but not rMBL/A (wild type) was weak even when the cell culture supernatant was concentrated 10-fold (data not shown). This could be due to low expression of rMBL variants by insect cells and/or weak avidity of the MBL antibody for MBL structural variants. The rMBL was therefore captured from 10-fold concentrated insect cell medium on to microtiter wells coated with IgG purified from MBL antiserum (G5132). Western blot analysis of the captured rMBL molecules using mAb 131-01 as the detecting antibody showed that the medium of insect cells transfected with DNA for MBL/A, B, C, and D (Fig. 1, lanes 2–5) expressed a protein (indicated by an arrow) that had the same molecular size (32 kDa) as pMBL/A purified from plasma (lane 7) and purified

Fig. 1. Detection of rMBL expression in insect cell medium by antibody capture followed by Western blot under reduced conditions. Recombinant MBL molecules in 10-fold concentrated third day culture media of insect cells were captured on microtiter wells coated with goat IgG purified from MBL antiserum (G5132) and examined by Western blot analysis using mAb 131-01 (1:500) as the primary detecting antibody. Lanes 2–5 represent MBL captured from the medium of insect cells expressing rMBL/A, rMBL/B, rMBL/C, and rMBL/D, respectively. Controls were culture medium of insect cells transfected with empty vector (lane 1), purified pMBL/A captured the same way as the rMBL molecules (lane 6), purified pMBL/A that was not captured (lane 7, 10 ng), and goat IgG purified from the MBL antiserum (lane 8, 300 ng and lane 9, 1500 ng).

pMBL/A captured by antibody (lane 6). The intensities of the bands for the structural variants rMBL/C (lane 4) and to a lesser extent rMBL/D (lane 5) were weak when compared to rMBL/A (lane 2) and rMBL/B (lane 3) and may be due to low expression levels and/or due to poor reaction of the mAb with the mutated rMBL molecules. The medium of insect cells transfected with empty vector (lane 1) did not show the 32 kDa band but two other bands having a molecular size of ∼50 and 25 kDa were seen. These two bands were also observed in lanes representing the medium of insect cells transfected with MBL cDNA (lanes 2–5) and in pMBL/A that was captured by antibody (lane 6) but not in the conventionally purified pMBL/A (lane 7). Controls with purified IgG from the MBL antiserum that was not captured (lane 8 and 9) showed identical 50 and 25 kDa bands indicating that these bands represent IgG eluted from the capture wells up on the addition of SDS buffer. In addition to the 32 kDa band of MBL, a 64 kDa band was observed for rMBL/A and to a lesser extent for rMBL/B. This represents the dimer of the MBL polypeptide, and was also observed for pMBL/A (lanes 6 and 7). The data presented in Fig. 1 show that insect cells transfected with MBL cDNA express rMBL that has the same polypeptide size as purified pMBL/A. 3.2. Oligomer formation of rMBL molecules determined by Western blot analysis The rMBL molecules purified by affinity chromatography on mannan–Sepharose columns were examined for oligomer formation by Western blot analysis using mAb 131-01 as the detecting antibody. As seen in Fig. 2A, rMBL/A, B, C, and D (lanes 2, 4, 6, 8) under reducing conditions exhibited a major band having an apparent molecular size of about 32 kDa similar to that observed for pMBL/A (lane 10). In the current analysis, the 32 kDa band migrated as a 25 ± 1 kDa band under non-reducing conditions (lanes 1, 3, 5, 7, and 9, Fig. 2A), which has been reported to be its true molecular size as determined by sequence and chemical composition [15]. The 25 kDa polypeptide of rMBL/A and its three structural

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Fig. 3. SDS-PAGE analysis and quantification of purified rMBL molecules. Four different concentrations of purified pMBL/A (0.5, 1.0, 2.0, and 3.0 ␮g, lanes 1–4) as determined by A280 were analyzed under reducing conditions by SDS-PAGE along with affinity purified rMBL proteins on mannan–Sepharose columns (lanes 5–8). After electrophoresis, the gel was stained with Coomassie brilliant blue R-250 (BioRad, USA). Concentration of purified recombinant molecules, rMBL/A (1.9 ␮g, lane 5), rMBL/B (0.47 ␮g, lane 6), rMBL/C (0.5 ␮g, lane 7), and rMBL/D (0.68 ␮g, lane 8) was determined from the standard graph generated with the indicated concentrations of pMBL/A by densitometric analysis of the bands using Quantity One software in a GEL DOC 2000 machine (Bio-Rad, USA).

Fig. 2. Oligomer formation of affinity purified rMBL molecules on mannan–Sepharose columns determined by Western blot analysis. (A) Odd numbered lanes represent non-reduced samples and even numbered lanes represent reduced samples of purified rMBL and pMBL/A. Purified pMBL/A was 30 ng (lane 9) and 10 ng (lane 10). Recombinant MBL molecules present in 15–30 ␮l correspond to the amount of rMBLs purified from 26, 60, 45, and 60 ml of the third day growth cultures of insect cells for rMBL/A, B, C and D, respectively. The analysis was performed using mAb 131-01 as the primary detecting antibody. (B) The analysis was performed under non-reduced conditions using mAb 93C as the primary detecting antibody. Concentrations of purified MBL preparations were pMBL/A (30 ng), rMBL/A (45 ng), rMBL/B (465 ng), rMBL/C (480 ng), and rMBL/D (800 ng) as determined by SDS-PAGE analysis in Fig. 3.

variants were observed to form a dimer (50 kDa) and a trimer (75 kDa) (lanes 1, 3, 5, and 7, Fig. 2A). The trimer of the 25 kDa polypeptide chain of MBL, which forms the basic monomeric structural subunit of MBL (75 kDa) was observed to form higher order oligomers for rMBL/A (lane 1) as well as for the structural variants, rMBL/B (lane 3), rMBL/C (lane 5), and rMBL/D (lane 7). This was indicated by the presence of high molecular size bands ranging from 75 kDa to above the 250-kDa molecular size marker used in the analysis. The pattern of higher order oligomers was observed to be similar to that of pMBL/A (lane 9). Employing another mAb 93C, which like 131-01 recognizes an epitope in the lectin region of MBL but weakly recognizes reduced MBL, the structural variants, rMBL/B, rMBL/C, and rMBL/D were observed to form disulfide linked higher order oligomers comprised of monomers to hexamers under non-reducing conditions similar to that observed with rMBL/A and pMBL/A (Fig. 2B). The pattern of oligomer formation obtained with mAb 93C (Fig. 2B) was similar to that obtained with mAb 131-01 (Fig. 2A). Together, the findings show that the structural variants, rMBL/B, rMBL/C, and rMBL/D form disulfide linked

(covalent) higher order oligomers comprised of monomers to hexamers. The data in Fig. 2B also show that mAb 93C detected higher order oliogomers of pMBL/A and rMBL/A with as little as 30–45 ng of the purified proteins. In contrast, about 16–27 times more of the structural variants of MBL, rMBL/B (465 ng), rMBL/C (480 ng), and rMBL/D (800 ng, lane 5) were required for detection (Fig. 2B). These results show that the avidity of mAb 93C is about 16- to 27-fold weaker for the structural variants rMBL/B, C, and D, respectively than for pMBL/A while the avidity for rMBL/A is nearly similar to that for pMBL/A. The avidity of the mAb 131-01 was also determined but under reducing conditions. The results obtained indicated that the avidity of mAb 131-01 for the structural variants, rMBL/B, C, and D was respectively 31-, 52-, and 18-fold weaker than for pMBL/A while the avidity for rMBL/A was nearly similar to that for pMBL/A. Together, the findings show that mAb 131-01 and 93C have a weaker avidity for rMBL structural variants, rMBL/B, rMBL/C, and rMBL/D than rMBL/A and pMBL/A. 3.3. SDS-PAGE analysis of purified rMBLs As seen in Fig. 3, rMBL/A (lane 5), rMBL/C (lane 7), and rMBL/D (lane 8) purified by affinity chromatography on mannan–Sepharose columns exhibit a single major protein band on SDS-PAGE analysis having a molecular size of ∼32 kDa similar to that observed for purified pMBL/A (lanes 1–4) whereas for rMBL/B (lane 6) in addition to the 32 kDa band the dimer (64 kDa) of the MBL monomer peptide was also observed. The dimer (64 kDa) has also been observed by some studies for pMBL/A [18,49]. Low expression levels of rMBL variants by insect cells precluded additional experiments that were needed to determine stability, which in the case of rMBL/B may have resulted in the dimer form observed on SDS PAGE analysis in Fig. 3. 3.4. Mannan-binding determined by ELISA The mannan-binding capacity of rMBL/A and its structural variants purified on mannan–Sepharose columns (Fig. 3) was examined by ELISA using mAb 131-01 as the detecting antibody. As seen in Fig. 4A (inset), pMBL/A bound to mannan efficiently as only 0.002–0.032 ␮g/ml corresponding to 0.0044–0.071 nM (assuming

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Fig. 4. Binding of pMBL/A and rMBL molecules to mannan determined by ELISA. Mannan-binding capacity of purified rMBLs was determined by ELISA using mAb 131-01 as the detecting antibody. Results of one representative experiment performed in triplicate are shown. (A) Concentrations used in the assays ranged from 0.002 to 0.032 ␮g/ml for pMBL/A (, inset) and 0.2–1.2 ␮g/ml for rMBL/A (). (B). Concentrations of rMBL variants used in the assays ranged from 0.2 to 1.2 ␮g/ml, rMBL/A ( ), rMBL/B (), rMBL/C (), rMBL/D ().

450 kDa as the molecular size of MBL) of pMBL/A was required. rMBL/A also bound to mannan in the nanomolar concentration range (0.2–1.2 ␮g/ml corresponding to 0.44–2.67 nM) but as indicated by the dashed line in Fig. 4A about 24-fold more of rMBL/A (0.46 ␮g/ml or 1.022 nM) than pMBL/A (0.019 ␮g/ml or 0.042 nM) was required. In contrast, the structural variants exhibited very poor binding to mannan when similar concentrations as rMBL/A were used (Fig. 4B). 3.5. Mannan-binding determined by surface plasmon resonance using BIAcore The fact that the structural variants could be purified by affinity chromatography on a mannan–Sepharose (Fig. 3) indicated that the structural variants bind to sugars. However, quantification of rMBL binding to mannan in Fig. 4B as determined by ELISA using mAb 131-01 as the detecting antibody showed that the structural variants rMBL/B, rMBL/C, and rMBL/D bind to mannan very weakly. The poor binding of the structural variants is attributed to the weaker (18- to 52-fold) avidity of mAb 131-01 for the variants than for pMBL/A (see Section 3.2). Therefore, another method such as surface plasmon resonance that did not require an anti-MBL antibody for detection was used and rMBL binding to mannan was followed in real time. Fig. 5A shows an overlay plot of the binding response observed between immobilized mannan and various concentrations of pMBL/A ranging from 0.14 to 2.22 nM. Similar binding interactions were observed when rMBL/A concentrations ranging from 1.1 to 35.5 nM were used (Fig. 5B). Measurement of the rate constants by globally fitting the data simultaneously to

Fig. 5. Binding curves of pMBL/A and rMBL/A to NH2 -mannan immobilized on a CM4 sensor chip using surface plasmon resonance (BIAcore). (A) Concentrations of pMBL/A used were 0.14, 0.28, 0.56, 1.11, and 2.22 nM. Binding was measured in 20 mM Tris–HCl buffer pH 7.4 containing 0.15 M NaCl, 0.05% BSA, and 0.005% surfactant p20 supplemented with 1 mM CaCl2 at a flow rate of 5 ␮l/min at 25 ◦ C. After injecting 20 ␮l of protein the ligand surface was regenerated using 10 ␮l of 3 mM EDTA. Data were analyzed by globally fitting the association and dissociation phases of the overlay plots simultaneously to a 1:1 Langmuir binding model using BIAevaluation 4.1 software. Dark serrated lines are binding curves whereas thin smooth lines show the global fit. Residuals to the fits are shown in the respective panels below the figure. (B) Concentrations of rMBL/A used were 1.1, 2.2, 4.4, 8.9, 17.8, 35.5 nM. Binding was measured as described for pMBL/A in A.

a 1:1 Langmuir binding model for several concentrations using 450 kDa (hexamer) as the molecular size of MBL are shown in the lower panel of Fig. 5A and B. The apparent dissociation constant (KD ) obtained from the global fit shows that pMBL/A (KD = 1.56 nM, 2 = 1.11) binds mannan with high affinity and that rMBL/A also binds with high affinity (KD = 13.1 nM, 2 = 1.06) but the interaction is about 8-fold weaker than pMBL/A. Low expression levels by insect cells together with low yields obtained up on affinity chromatography (19–57 ␮g/l of cell culture medium) limited the amount of pure rMBL structural variants generated to determine mannan-binding by surface plasmon resonance. Analysis of single binding curve for each rMBL structural variant indicated that rMBL structural variants bind to mannan, but since an array of binding curves could not be generated, the KD values were not determined. Clearly, additional

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Fig. 6. Complement activation by pMBL/A and rMBL variants determined by hemolytic assays. Lysis of sheep erythrocytes coated with mannan (EMan ) was measured as the amount of hemoglobin released up on incubation with MBL-depleted serum and the indicated amount of purified pMBL/A ( ) or rMBL variants; rMBL/A (), rMBL/B (䊉), rMBL/C (), rMBL/D (♦). Values plotted are of three independent experiments ± S.D. (n = 3). Data presented are after subtracting background lysis of EMan and MBL-depleted serum, which was less than 20%. The dashed line indicates the normal concentration of pMBL/A in blood (median ∼1 ␮g/ml) [17].

experiments are required to confirm the binding of rMBL structural variants to mannan. 3.6. Complement activation Hemolytic assays containing EMan cells and MBL-depleted serum were used to analyze the ability of rMBL/A and its structural variants to activate complement. As seen in Fig. 6, different results on complement activation were obtained for the three structural variants of MBL. Based on the amount of rMBL required for 30% lysis of EMan cells via the lectin pathway, complement fixing activity of rMBL/A (0.0257 ± 0.0088 ␮g/ml) was about 5-fold less than for pMBL/A purified from plasma (0.0048 ± 0.0047 ␮g/ml). The structural variant rMBL/C was ineffective while rMBL/B activated complement only weakly as almost 100 times more of rMBL/B (2.43 ± 0.18 ␮g/ml) than rMBL/A was required for 30% lysis of EMan cells. In contrast, rMBL/D was fully functional (0.0321 ± 0.0133 ␮g/ml) and activated complement nearly to the same extent as rMBL/A. 3.7. Native PAGE analysis of rMBL The order of oligomeric structures under non-denaturing conditions was determined by native PAGE analysis of rMBL molecules that were purified on mannan–Sepharose columns and compared to that of pMBL/A. As seen in Fig. 7, purified pMBL/A primarily exhibited two protein bands that migrated in the upper region of the native gel (indicated by arrowheads in lane 1) suggesting the presence of high oligomeric forms. The recombinant forms of wild type MBL (rMBL/A, lanes 3 and 4) as well as the structural variants (rMBL/B, lanes 5 and 6; rMBL/C, lanes 7–10; rMBL/D, lanes 11 and 12) exhibited bands that migrated in the upper as well as the lower region of the native gel suggesting the presence of many forms of MBL; higher and lower oligomeric forms. The bands observed in the upper region of the native gel for structural variants rMBL/A, B, C, and D migrated to the same distance as those observed for pMBL/A. The intensity of the faster migrating band seen in the lower part of the native gel suggested that low oligomeric forms of MBL were more prevalent for the structural variants, rMBL/B, C, and D than for rMBL/A. The results indicate structural differences between pMBL/A and rMBL/A as well as between rMBL/A and the structural variants generated in insect cells.

Fig. 7. Native-PAGE analysis of pMBL/A and rMBL molecules. Various concentrations of MBL: lanes 1 and 2, pMBL/A (250 and 100 ng); lanes 3 and 4 rMBL/A (70 and 140 ng); lanes 5 and 6, rMBL/B (14 and 140 ng); lanes 7–10, rMBL/C (15 and 150 ng, 23 and 46 ng); lanes 11 and 12, rMBL/D (140 and 55 ng) were electrophoresed under native (non-denaturing) conditions using 4–15% gradient gel Tris–HCl (BioRad, USA). The protein bands were visualized by staining with the silver staining kit from Bio-Rad (USA).

3.8. Gel filtration chromatography The presence of higher order oligomeric structures was determined by gel filtration chromatography using the FPLC system. As seen in Fig. 8A, pMBL/A showed two peaks. The first peak (16 ml) corresponded to a higher order oligomer of ∼1000 kDa and was the major form while the second peak (25 ml) corresponded to the salt peak and exhibited a shoulder suggesting the presence of a lower molecular size form. In addition, the dimer form (150 kDa) was observed but in negligible amounts at 20 ml. Gel filtration analysis of rMBL/A showed 6 major peaks (Fig. 8A). The first form eluted in the void volume (12 ml) corresponding to an apparent molecular size of about ≥2000 kDa and suggested the presence of large complexes. The second form (peak 2) eluted at 17 ml corresponding to nonamers of ∼670 kDa and the third form (peak 3, 18 ml indicated by a dashed line) corresponded to hexamers of ∼450 kDa. The presence of the fourth (22 ml) and fifth (23 ml) peaks with a shoulder (21 ml) observed on the fourth peak indicated low oligomeric forms corresponding to ∼150–25 kDa. The sixth peak (25 ml) was the salt peak. These results show the presence of many oligomeric forms that include higher and predominant lower order oligomers for rMBL/A. Gel filtration analysis of the structural variants rMBL/B, C, and D showed 4 major peaks indicating many oligomeric forms but fewer than those observed for rMBL/A (Fig. 8A). The first form (peak 1) eluted in the void volume (12 ml) suggesting the presence of large complexes of about ≥2000 kDa, the second form (peak 2, indicated by a dashed line) eluted at 18 ml indicating hexamers of ∼450 kDa while the third peak (20–24 ml) indicated lower order oligomers of ∼150–25 kDa to be the major forms for the structural variants. The fourth peak was the salt peak. Distribution of various forms of oligomers in the rMBL molecules was determined by weighing the area of each peak and then calculating the percentage of the specific oligomer. The area encompassing 20–24 ml was considered as one peak for low oligomeric forms corresponding to ∼150–25 kDa. As seen in Table 1, for pMBL/A, 88.5% were higher order oligomers (∼1000 kDa) and 11.5% lower order oligomers while for rMBL/A, 12% were nonamers, 23.3% hexamers, and 44% low oligomeric forms (∼150–25 kDa). Since nonamers and hexamers comprised 35% of higher order oligomers, the ratio of 0.8 for higher to lower oligomers suggests that rMBL/A generated in insect cells was made up of slightly greater amounts (1.3-fold more) of lower than higher order oligomers. In contrast, the ratio of 7.7 indicates pMBL/A was made up of 7.7-fold more of

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Fig. 8. (A) Gel filtration chromatography of pMBL/A and rMBL molecules. Purified pMBL/A or rMBL molecules were diluted in 20 mM Tris–HCl buffer containing 150 mM NaCl and 5 mM EDTA, pH 7.4 and fractionated on a BioSep-SEC S 4000 column. The concentration of the rMBL molecules used was pMBL/A (4.5 ␮g), rMBL/A (4.5 ␮g), rMBL/B (4.0 ␮g), rMBL/C (3.5 ␮g), and rMBL/D (2.4 ␮g). pMBL/A was purified from human plasma while rMBL molecules were purified by affinity chromatography on mannan–Sepharose columns. The elution profile of the Bio-Rad molecular weight markers used is shown in the graph: blue dextran (void volume), thyroglobulin (669 kDa), apoferritin (443 kDa) IgG (158 kDa), ovalbumin (44 kDa), myoglobulin (17 kDa), and Vitamin B12 (1.35 kDa). (B) Western blot analysis of rMBL/A fractionated by gel chromatography. Specified fractions (30 ␮l) obtained from gel filtration chromatography profiles of rMBL/A described in A were analyzed by Western blot under non-reducing condition using goat IgG purified from MBL antiserum (G5132) as the primary antibody. Control used was pMBLA (50 ng). (C) Western blot analysis of rMBL/B fractionated by gel chromatography in A as described for rMBL/A in the legend of B.

Table 1 Oligomer distribution of rMBL variants using gel filtration chromatography.

pMBL/A

Peak #

Elution volume (ml)

Type of oligomer

1

16 20–24

Large oligomers Lower oligomersb

Percentage of oligomer (%)

Oligomer ratioa

≥1000 150–25

88.5 11.5

7.7

Apparent molecular size (kDa)

rMBL/A

1 2 3 4–5

12 17 18 20–24

Large complexes Nonamer Hexamer Lower oligomers

≥2000 670 450 150–25

20.7 12 23.3 44

0.80

rMBL/B

1 2 3

12 18 20–24

Large complexes Hexamer Lower oligomers

≥2000 450 150–25

27.6 16.9 55.5

0.30

rMBL/C

1 2 3

12 18 20–24

Large complexes Hexamer Lower oligomers

≥2000 450 150–25

15.4 9.4 75.2

0.13

1 2 3

12 18 20–24

Large complexes Hexamer Lower oligomers

≥2000 450 150–25

26.5 12.8 60.7

0.21

rMBL/D

The distribution of various forms of oligomers present in rMBL molecules was determined by weighing the area of each peak obtained up on gel filtration chromatography (Fig. 8A) and calculating the percentage of the specific oligomers. For low oligomeric forms of ∼150–25 kDa, the area encompassing 20–24 ml was considered as one peak. a The oligomer ratio was calculated by dividing the percent value of the higher oligomers (excluding the large complexes) by the percent value of the lower oligomers. b These peaks were very small compared to the major peaks, and are therefore not numbered in the chromatogram.

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higher than lower order oligomers. The ratio of 0.30, 0.13, and 0.21 obtained for rMBL/B, rMBL/C, and rMBL/D indicated that the lower oligomeric structures were about 3, 8, and 5 times more predominant than the respective hexamer form of the structural variant. Large complexes (≥2000 kDa) were also observed for the structural variants of MBL (15–28%) and rMBL/A (20.7%). The results show that low oligomeric structures are the prevalent form for the structural variants of MBL and also for rMBL/A though to a lesser extent but not for pMBL/A. Fractions obtained from gel filtration chromatography (Fig. 8A) were examined by Western blot analysis to determine the type of oligomer. As seen in Fig. 8B analysis of rMBL/A fractions showed several bands, the mobility of which increased from peak 1 (12 ml) to peak 5 (23 ml). This was observed to be consistent with the estimated apparent molecular sizes determined by gel filtration. Peak 1 (12–14 ml), which corresponded to ≥2000 kDa showed higher order oligomers indicated by a broad diffused band below the sample well. Peak 2 (17 ml), which corresponded to ∼670 kDa showed many bands that included oligomer forms above the 250 kDa molecular marker used. Peak 3 (18–19 ml) exhibited ∼450 kDa hexamers including predominantly low molecular weight species while the shoulder (21 ml) and peaks 4 and 5 (20–24 ml) showed oligomer forms of ∼150–25 kDa. In contrast, Western blot analysis of rMBL/B fractions (Fig. 8C) obtained from gel filtration chromatography (Fig. 8A) exhibited bands with very weak intensity even though the blot was overexposed. And although distribution of the hexamer form of rMBL/B was nearly similar to that of rMBL/A by gel filtration chromatography (Fig. 8A), Western blot analysis primarily detected the dimer but not the hexamer form of rMBL/B (Fig. 8C) while all forms of rMBL/A were detected (Fig. 8B). Western blot analysis of rMBL/C and rMBL/D fractions that exhibited higher order oligomers on gel filtration (Fig. 8A) showed very faint bands that too only for the low oligomeric forms corresponding to peak 3 (20–24 ml) (data not shown). These results show a weak reaction of mAb 131-01 for the structural variants rMBL/B, rMBL/C, and rMBL/D but not rMBL/A.

4. Discussion The present study, based on the biochemical analyses of rMBL structural variants generated in insect cells, provides new insights on the functional defects associated with MBL variant alleles that help resolve some of the differences published on their structural/functional properties. Examination of the structural/functional properties of rMBL structural variants (rMBL/A, B, C, and D) revealed that rMBL variants generated in insect cells not only form covalently linked (via disulfide) higher order oligomers (monomers to hexamers) as detected by Western blot analysis (Fig. 2) but also non-covalently linked higher order oligomers as determined by gel filtration chromatography (Fig. 8A) and native PAGE analysis (Fig. 7). Analysis of physiological functions such as binding to mannan and complement activation indicated weaker and/or impaired activity depending on the rMBL variant examined. The most significant effect was observed when the mutation occurred near the kink in the collagen region of the molecule with maximum impairment apparent in rMBL/C, followed by rMBL/B. In contrast, rMBL/D was fully functional to the same extent as rMBL/A (Fig. 6). Most assays quantifying MBL have used monoclonal antibodies for detection and individuals who are carriers for variant MBL alleles were initially thought to have low levels of MBL. The study of Garred et al., which employing rabbit anti-MBL showed higher levels of variant MBL in circulation [60] than previously reported [61]. The low levels of MBL were attributed to preferential affinity of the mAb for wild type MBL. The studies of Garred et al. [28], Terai et al. [42], and Frederiksen et al. [43] employing anti-MBL mAbs 131-

11, 3E7 and 93C, respectively, reported significant MBL level in the sera of individuals with variant MBL alleles, i.e., up to 500 ng/ml as compared to below 50 ng/ml determined with other commercially available assays. Our data show that about 18- to 52-fold greater concentration of purified rMBL/B, C, and D than of rMBL/A or pMBL/A was required for detection with mAb 131-01 by Western blot analysis under reduced conditions while under non-reduced conditions about 16- to 27-fold greater concentration was required for detection with mAb 93C (Fig. 2B). The findings explain why detection of the structural variants but not rMBL/A was observed to be weak even when the insect cell medium was concentrated ten times (Fig. 1) or when Western blot analysis (Fig. 8C) of fractions from gel filtration chromatography (Fig. 8A) primarily detected the dimer (150 kDa) but not the hexamer form (∼450 kDa) of rMBL/B even though the distribution of this form of rMBL/B (peak 2, Fig. 8A) was almost similar to that of rMBL/A (peak 3, Fig. 8A). These findings may explain why earlier studies examining oligomer formation of MBL have mostly reported poor formation of higher order oligomers by MBL structural variants [19,22,40,44,45]. Our results when considered together with the published reports highlight the problem of detecting and quantifying MBL variants, a subject matter that will benefit from development of new antibodies with high avidity for the structural variants of MBL and/or new improved detection methods that do not involve the use of antibodies. Several groups that have made rMBL/A in different expression systems have reported many oligomeric structures [17,47,50]. In the present study, gel filtration analysis of rMBL molecules generated in insect cells also showed many forms that included both higher (∼670 and/or 450 kDa indicated by a dotted line in Fig. 8A) and more prevalent lower oligomeric structures ranging from ∼150 to 25 kDa (20–24 ml, Fig. 8A). The higher order oligomers were predominant in pMBL/A but less prevalent in rMBL/A and still less in the three structural variants of MBL (Fig. 8 and Table 1). Nevertheless, total oligomers present as higher order oligomers were 9.4–16.9% for the three rMBL structural variants, 35% for rMBL/A and 88.5% for pMBL/A (Fig. 8A and Table 1). These findings indicate structural differences not only between the recombinant form of MBL/A and that purified from plasma (pMBL/A) but also between rMBL/A and its three structural variants generated in insect cells. Studies have shown the ratio of higher to lower oligomers to be dependent on the cell line employed for MBL expression [47]. Ma et al. reported a significant difference in the oligomer ratio for rMBL/A when expressed in three human hepatoma cell lines with the highest ratio when expressed in HLF cells, the lowest in Chang liver cells and an intermediate one in Hep2 liver cells [47]. The hexamer/pentamer has also been reported by Super et al. to be the predominant oligomer for rMBL/A and rMBL/B generated in a mouse hybridoma cell line [17]. Our findings when considered together with the published reports stress the importance of exploring other expression systems to determine whether lower order oligomers are the major form of MBL structural variants or are a result of the expression system employed. Nevertheless, the formation of higher order oligomers observed for recombinant MBL variants in the current analysis are in agreement with the findings of other groups that have shown large oligomers to be present in plasma from donors homozygous for MBL alleles [42,43]. Studies employing pMBL/A or rMBL/A have shown that higher order oligomers of MBL are required for high affinity binding to mannan and efficient activation of complement [62–64]. In the present study, although purification of rMBL structural variants was achieved by affinity chromatography on mannan–Sepharose columns (Fig. 3) and the purified preparations exhibited formation of higher order oligomers by Western blot analysis (Fig. 2A and B) and gel filtration chromatography (Fig. 8), the rMBL structural variants did not bind mannan as determined by ELISA (Fig. 4B). It is likely that the weaker avidity (18- to 52-fold) of the primary detect-

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ing antibody for rMBL structural variants than rMBL/A may have contributed to the poor interaction observed with mannan. Other studies that have reported poor mannan binding for MBL present in the serum of individuals with variant MBL genotypes [40,42,43] or for rMBL variants generated in other expression systems [45] used ELISA. The MBL variant B/B has been reported to bind mannan but only under conditions that employed prolonged incubation of serum with mannan [28]. The study attributed the weak avidity of MBL/B variant for mannan to its reduced capacity to form stable higher order oligomers although it may be pointed out that mannan binding was measured by ELISA. Yet another study that has shown rMBL/B to bind mannan employed radiolabeled MBL [65]. Our observations when considered together with the published reports indicate the drawback of using antibodies to analyze physiological functions of MBL structural variants. In the present study, although rMBL structural variants exhibited formation of higher order oligomers (Figs. 2 and 8), they activated complement to different extents. This was evident from the data in Fig. 6, which showed rMBL/C to be ineffective while rMBL/B activated complement weakly. In contrast, rMBL/D activated complement almost to the same extent as rMBL/A. And because rMBL/B and rMBL/C showed formation of higher order oligomers (Figs. 2 and 8) their inability to activate complement (Fig. 6) may be attributed to their weak interaction with mannan (Fig. 4B) while the ability of rMBL/D to activate complement suggested that rMBL/D should bind to mannan. But mannan binding determined by ELISA was found to be weak (Fig. 4B). The weak binding to mannan is attributed to the weak avidity of mAb 13101 used in the ELISA method. Moreover, low expression levels by insect cells together with low yields obtained up on affinity chromatography limited the amount of pure rMBL structural variants generated. This precluded the generation of an array of binding curves required to determine KD values for the binding of rMBL structural variants to mannan by surface plasmon resonance. And although not determined in this study, the results (Fig. 6) also imply that the binding of MASP-2 to MBL/B and MBL/C but not rMBL/D may be ineffective or result in a less active conformation leading to poor complement activation. Support for this notion comes from the study of Matsushita et al., which showed that the mutation of Gly54Asp in pMBL/A did not affect the ability of rMBL/B to form higher order oligomers or bind bacteria, but its ability to activate complement was compromised due to its failure to bind MASP-2 [65]. Other studies that have reported poor complement activation for rMBL/B expressed in COS-1 cells [19] or rMBL/B, C, and D expressed in CHO cells [45] also showed poor formation of higher order oligomers by the rMBL variant. Nevertheless, since the normal median concentration of MBL/A in blood is 995 ng/ml (Fig. 6, vertical dashed line) [17] and that for variant MBL alleles is 500 ng/ml [43], the results shown in Fig. 6 suggest that at physiological concentrations rMBL/A and rMBL/D will be fully active, rMBL/B weakly active, and rMBL/C inactive in activating complement. The prevalence of lower order oligomers over higher order oligomers may result in MBL interacting weakly with mannan and consequently activating complement poorly. In the present study, this was observed to be the case when the structural/functional properties of rMBL/A were compared to that of pMBL/A. As seen in Figs. 7 and 8, native PAGE electrophoresis and gel filtration chromatography show lower order oligomers to be prevalent in rMBL/A while pMBL/A essentially exhibits higher order oligomers (Table 1). These structural differences between rMBL/A and pMBL/A may be responsible for the 8- and 24-fold weaker mannan-binding capacity of rMBL/A than pMBL/A determined by BIAcore analysis (KD = 13.1 nM versus 1.56 nM) (Fig. 5) and ELISA (Fig. 4A) and also for the 5-fold weaker activity of rMBL/A than pMBL/A in activating complement (Fig. 6). The implications of these findings are important because lower order oligomers are also found to be prevalent

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in rMBL structural variants (Table 1). And when considered with the potential therapeutic application of recombinant MBL in MBLdeficient patients, our studies emphasize the need to thoroughly understand structural/functional differences between MBL and its variant forms and their functional role in immunity. The present findings on rMBL structural variants generated in insect cells, when considered together with our published report showing lectin pathway activation to be four times more efficient than the classical pathway in generating C3/C5 convertases [13], imply that impaired activation of the lectin pathway and hence production of pro-inflammatory products C3a, C4a, and C5a that are required to mount an effective innate immune response may put individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D at increased risk to infections but may also provide them with a biological advantage against certain infections such as parasitic that require complement activation (i.e., C4b/C3b opsonization) for infectivity. Disclosures Dr. N. Rawal is an officer of and has a financial interest in CompTech, a supplier of complement reagents. Acknowledgement We express our appreciation to Padmaja Paidipally for her excellent technical work. References [1] Hoppe H-J, Reid KBM. Collectins-soluble proteins containing collagenous regions and lectin domains-and their roles in innate immunity. Protein Science 1994;3:1143–58. [2] Turner MW. The role of mannose-binding lectin in health and disease. Molecular Immunology 2003;40:423–9. [3] Takahashi K, Ezekowitz RAB. The role of the mannose-binding lectin in innate immunity. Clinical Infectious Diseases 2005;41:S440–4. [4] Worthley DL, Bardy PG, Gordon DL, Mullighan CG. Mannose-binding lectin and maladies of the bowel and liver. World Journal of Gastroenterology 2006;12:6420–8. [5] Heise CT, Nicholls JR, Leamy CE, Wallis R. Impaired secretion of rat mannosebinding protein resulting from mutations in the collagen-like domain. Journal of Immunology 2000;165:1403–9. [6] Petersen SV, Thiel S, Jensenius JC. The mannan-binding lectin pathway of complement activation: biology and disease association. Molecular Immunology 2001;38:133–49. [7] Kilpatrick DC. Mannan-binding lectin: clinical significance and applications. Biochimica et Biophysica Acta 2002;1572:401–13. [8] Roos A, Bouwman LH, Munoz J, Zuiverloon T, Faber-Krol MC, Fallaux-van den Houten FC, et al. Functional characterization of the lectin pathway of complement in human serum. Molecular Immunology 2003;39:655–68. [9] Fujita T, Matsushita M, Endo Y. The lectin-complement pathway-its role in innate immunity and evolution. Immunological Reviews 2004;198:185–202. [10] Worthley DL, Brady PG, Mullighan CG. Mannose-binding lectin: biology and clinical implications. Internal Medicine Journal 2005;35:548–55. [11] Dommett RM, Klein N, Turner MW. Mannose-binding lectin in innate immunity:past, present and future. Tissue Antigens 2006;68:193–209. [12] Rawal N. Complement. In: Laurent GJ, Shapiro SD, editors. Encyclopedia of Respiratory medicine. Elsevier Ltd.; 2006. p. 546–52. [13] Rawal N, Rajagopalan R, Salvi VP. Activation of complement component C5: comparison of C5 convertases of the lectin pathway and the classical pathway of complement. Journal of Biological Chemistry 2008;283:7853–63. [14] Kawasaki T. Structure and biology of mannan-binding protein, MBP, an important component of innate immunity. Biochimica et Biophysica Acta 1999;1473:186–95. [15] Teillet F, Dublet B, Andrieu J-P, Gaboriaud C, Arlaud GJ, Thielens NM. The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases. Journal of Immunology 2005;174:2870–7. [16] Garred P, Larsen F, Seyfarth J, Fujita R, Madsen HO. Mannose-binding lectin and its genetic variants. Genes and Immunity 2006;7:85–94. [17] Super M, Gillies SD, Foley S, Sastry K, Schweinle JE, Silverman VJ, et al. Distinct and overlapping functions of allelic forms of human mannose binding protein. Nature Genetics 1992;2:50–5. [18] Lu J, Thiel S, Wiedemann H, Timpl R, Reid KBM. Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the

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