Title: Disease-associated Mutations in Human Mannose-binding Lectin Compromise Oligomerization and Activity of the Final Protein
Abstract:Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the bio...Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement. Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement. Mannose-binding lectin (MBL) 1The abbreviations used are: MBL, mannose-binding lectin(s); CHO, Chinese hamster ovary; MASP-1, -2, and -3, MBL-associated serine protease-1, -2, and -3, respectively; MTX, methotrexate; SDG, sucrose density gradient; sMAP, small MBL-associated protein (identical to MAp19); SELDI, surface-enhanced laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; WT, wild type; PBS, phosphate-buffered saline; HSA, human serum albumin; ELISA, enzyme-linked immunosorbent assay. has been shown to be an important component of innate immunity and is a central recognition molecule of the lectin pathway of complement (for a recent review, see Ref. 1Turner M.W. Hamvas R.M.J. Rev. Immunogenet. 2001; 2: 305-322Google Scholar). MBL binds to an array of carbohydrate structures on surfaces of bacteria (2Kawasaki N. Kawasaki T. Yamashina I. J. Biochem. (Tokyo). 1989; 106: 483-489Crossref PubMed Scopus (150) Google Scholar, 3Neth O. Jack D.L. Dodds A.W. Holzel H. 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Ezekowitz R.A. J. Exp. Med. 1989; 169: 1733-1745Crossref PubMed Scopus (391) Google Scholar), and the biological effect is mediated by direct killing via complement (10Lu J.H. Thiel S. Wiedemann H. Timpl R. Reid K.B. J. Immunol. 1990; 144: 2287-2294PubMed Google Scholar) through the lytic membrane attack complex or by promoting phagocytosis either by the MBL lectin pathway of complement or by direct binding to one or more cell surface receptors (11Tenner A.J. Robinson S.L. Ezekowitz R.A. Immunity. 1995; 3: 485-493Abstract Full Text PDF PubMed Scopus (152) Google Scholar). The lectin pathway comprises at least three MBL-associated serine proteases (MASPs), namely MASP-1 (12Sato T. Endo Y. Matsushita M. Fujita T. Int. Immunol. 1994; 6: 665-669Crossref PubMed Scopus (154) Google Scholar), MASP-2 (13Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (757) Google Scholar), and MASP-3 (14Dahl M.R. Thiel S. Matsushita M. Fujita T. Willis A.C. Christensen T. Vorup-Jensen T. Jensenius J.C. Immunity. 2001; 15: 127-135Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Furthermore, the functional MBL-MASP complex contains a small MBL-associated protein (sMAP), also named MAp19, with no serine protease activity (15Stover C.M. Thiel S. Thelen M. Lynch N.J. Vorup-Jensen T. Jensenius J.C. Schwaeble W.J. J. Immunol. 1999; 162: 3481-3490PubMed Google Scholar, 16Takahashi M. Endo Y. Fujita T. Matsushita M. Int. Immunol. 1999; 11: 859-863Crossref PubMed Scopus (171) Google Scholar). MASP-2 is a homologue of C1s of the classical complement pathway because it activates C4 and C2 (13Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (757) Google Scholar). When MBL associated with MASP-2 binds to sugar groups on the surface of microbes, the MBL-MASP2 proenzyme is activated and cleaves sequentially C4 and C2, thereby creating the C4b2a complex, a potent C3 convertase. The MBL-MASP-1 complex is suggested to activate C3 directly (12Sato T. Endo Y. Matsushita M. Fujita T. Int. Immunol. 1994; 6: 665-669Crossref PubMed Scopus (154) Google Scholar). Whether both MASP-1 and MASP-2 are bound on the same MBL molecule is still unclear. Moreover, the biological role of sMAP, as well as the substrate for the recently discovered MASP-3, remains unclear at this moment (for a recent review on MASPs, see Ref. 17Gadjeva M. Thiel S. Jensenius J.C. Curr. Opin. Immunol. 2001; 13: 74-78Crossref PubMed Scopus (100) Google Scholar). MBL is a complex of six sets of homotrimers of a single polypeptide chain containing 228 amino acids (18Sastry K. Herman G.A. Day L. Deignan E. Bruns G. Morton C.C. Ezekowitz R.A. J. Exp. Med. 1989; 170: 1175-1189Crossref PubMed Scopus (253) Google Scholar, 19Ezekowitz R.A. Day L.E. Herman G.A. J. Exp. Med. 1988; 167: 1034-1046Crossref PubMed Scopus (246) Google Scholar, 20Taylor M.E. Brickell P.M. Craig R.K. Summerfield J.A. Biochem. J. 1989; 262: 763-771Crossref PubMed Scopus (190) Google Scholar, 21Kurata H. Sannoh T. Kozutsumi Y. Yokota Y. Kawasaki T. J. Biochem. (Tokyo). 1994; 115: 1148-1154Crossref PubMed Scopus (38) Google Scholar). This polypeptide consists of four domains (Fig. 1): 1) a 20-amino acid N-terminal cysteine-rich domain involved in formation of intra- and intersubunit disulfide bonds, 2) a collagen-like domain consisting of 18-20 tandem repeats of Gly-Xaa-Yaa, 3) an α-helical coiled-coil neck region, and 4) A carbohydrate recognition domain capable of binding to a wide variety of carbohydrate arrays on the surface of microorganisms (3Neth O. Jack D.L. Dodds A.W. Holzel H. Klein N.J. Turner M.W. Infect. Immun. 2000; 68: 688-693Crossref PubMed Scopus (473) Google Scholar). Three polypeptides form a structural unit or subunit containing a triple helix at their collagen-like domain. Six of these units combine by interunit disulfide bonds to form the biologically active bouquet-like MBL protein (for a recent review on MBL structure, see Ref. 22Kawasaki T. Biochim. Biophys. Acta. 1999; 1473: 186-195Crossref PubMed Scopus (49) Google Scholar). Three different genetic polymorphisms in exon 1 of the human MBL2 gene (MBL1 is a pseudogene (23Guo N. Mogues T. Weremowicz S. Morton C.C. Sastry K.N. Mamm. Genome. 1998; 9: 246-249Crossref PubMed Scopus (68) Google Scholar)) independently lead to reduced serum concentrations of MBL. Two interrupt the tandem repeat Gly-Xaa-Yaa in the first (Gly) position, and the third introduces a cysteine residue in the second position. The designation of the MBL variant alleles is B, C, and D, whereas the normal allele is termed A. MBL-B has a G → A mutation in codon 54 (24Heise C.T. Nicholls J.R. Leamy C.E. Wallis R. J. Immunol. 2000; 165: 1403-1409Crossref PubMed Scopus (31) Google Scholar), which results in a Gly → Asp substitution in the fifth Gly-Xaa-Yaa repeat. MBL-C has a G → A mutation in codon 57 (25Lipscombe R.J. Sumiya M. Hill A.V. Lau Y.L. Levinsky R.J. Summerfield J.A. Turner M.W. Hum. Mol. Genet. 1992; 1: 709-715Crossref PubMed Scopus (382) Google Scholar), which translates into a Gly → Asp substitution of the sixth Gly-Xaa-Yaa repeat. The third mutation is MBL-D, a C → T mutation in codon 52 (25Lipscombe R.J. Sumiya M. Hill A.V. Lau Y.L. Levinsky R.J. Summerfield J.A. Turner M.W. Hum. Mol. Genet. 1992; 1: 709-715Crossref PubMed Scopus (382) Google Scholar) that results in the introduction of a cysteine instead of an arginine in the protein (26Madsen H.O. Garred P. Kurtzhals J.A. Lamm L.U. Ryder L.P. Thiel S. Svejgaard A. Immunogenetics. 1994; 40: 37-44Crossref PubMed Scopus (464) Google Scholar). The presence of these mutations leads to markedly reduced MBL protein levels in the blood (27Garred P. Thiel S. Madsen H.O. Ryder L.P. Jensenius J.C. Svejgaard A. Clin. Exp. Immunol. 1992; 90: 517-521Crossref PubMed Scopus (114) Google Scholar, 28Garred P. Madsen H.O. Kurtzhals J.A. Lamm L.U. Thiel S. Hey A.S. Svejgaard A. Eur. J. Immunogenet. 1992; 19: 403-412Crossref PubMed Scopus (133) Google Scholar). The B and D alleles are seen in Eurasian and indigenous American populations with frequencies ranging from 0.1 to 0.5 and from 0.0 to 0.1, respectively (29Madsen H.O. Garred P. Thiel S. Kurtzhals J.A. Lamm L.U. Ryder L.P. Svejgaard A. J. Immunol. 1995; 155: 3013-3020PubMed Google Scholar, 30Lipscombe R.J. Beatty D.W. Ganczakowski M. Goddard E.A. Jenkins T. Lau Y.L. Spurdle A.B. Sumiya M. Summerfield J.A. Turner M.W. Eur. J. Hum. Genet. 1996; 4: 13-19Crossref PubMed Scopus (88) Google Scholar, 31Madsen H.O. Satz M.L. Hogh B. Svejgaard A. Garred P. J. Immunol. 1998; 161: 3169-3175PubMed Google Scholar). The C allele is found most frequently in sub-Saharan African populations with a frequency ranging from 0.07 to 0.3 (29Madsen H.O. Garred P. Thiel S. Kurtzhals J.A. Lamm L.U. Ryder L.P. Svejgaard A. J. Immunol. 1995; 155: 3013-3020PubMed Google Scholar, 30Lipscombe R.J. Beatty D.W. Ganczakowski M. Goddard E.A. Jenkins T. Lau Y.L. Spurdle A.B. Sumiya M. Summerfield J.A. Turner M.W. Eur. J. Hum. Genet. 1996; 4: 13-19Crossref PubMed Scopus (88) Google Scholar, 31Madsen H.O. Satz M.L. Hogh B. Svejgaard A. Garred P. J. Immunol. 1998; 161: 3169-3175PubMed Google Scholar). The presence of these alleles is associated with increased risk of infections during childhood, particularly during the vulnerable period of infancy ranging from 6 to 18 months of age (32Garred P. Madsen H.O. Hofmann B. Svejgaard A. Lancet. 1995; 346: 941-943Abstract PubMed Scopus (0) Google Scholar, 33Summerfield J.A. Sumiya M. Levin M. Turner M.W. Br. Med. J. 1997; 314: 1229-1232Crossref PubMed Scopus (361) Google Scholar, 34Koch A. Melbye M. Sorensen P. Homoe P. Madsen H.O. Molbak K. Hansen C.H. Andersen L.H. Hahn G.W. Garred P. JAMA (J. Am. Med. Assoc.). 2001; 285: 1316-1321Crossref PubMed Scopus (397) Google Scholar), in immunocompromised patients (35Garred P. Madsen H.O. Balslev U. Hofmann B. Pedersen C. Gerstoft J. Svejgaard A. Lancet. 1997; 349: 236-240Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 36Garred P. Madsen H.O. Halberg P. Petersen J. Kronborg G. Svejgaard A. Andersen V. Jacobsen S. Arthritis Rheum. 1999; 42: 2145-2152Crossref PubMed Scopus (199) Google Scholar) and is a risk factor for critically ill patients to develop sepsis (37Garred P. Strom J. Quist L. Taaning E. Madsen H.O. J. Infect. Dis. 2003; 188: 1394-1403Crossref PubMed Scopus (182) Google Scholar). Moreover, MBL variant alleles are associated with disease progression in concomitant diseases such as chronic granulomatous disease and cystic fibrosis (38Foster C.B. Lehrnbecher T. Mol F. Steinberg S.M. Venzon D.J. Walsh T.J. Noack D. Rae J. Winkelstein J.A. Curnutte J.T. Chanock S.J. J. Clin. Invest. 1998; 102: 2146-2155Crossref PubMed Scopus (226) Google Scholar, 39Garred P. Pressler T. Madsen H.O. Frederiksen B. Svejgaard A. Hoiby N. Schwartz M. Koch C. J. Clin. Invest. 1999; 104: 431-437Crossref PubMed Scopus (406) Google Scholar), Additionally, the importance of MBL deficiency in autoimmunity has been emphasized in diseases like systemic lupus erythematosus and rheumatoid arthritis (40Davies E.J. Snowden N. Hillarby M.C. Carthy D. Grennan D.M. Thomson W. Ollier W.E. Arthritis Rheum. 1995; 38: 110-114Crossref PubMed Scopus (153) Google Scholar, 41Sullivan K.E. Wooten C. Goldman D. Petri M. Arthritis Rheum. 1996; 39: 2046-2051Crossref PubMed Scopus (152) Google Scholar, 42Graudal N.A. Madsen H.O. Tarp U. Svejgaard A. Jurik G. Graudal H.K. Garred P. Arthritis Rheum. 2000; 43: 515-521Crossref PubMed Scopus (114) Google Scholar, 43Ip W.K. Lau Y.L. Chan S.Y. Mok C.C. Chan D. Tong K.K. Lau C.S. Arthritis Rheum. 2000; 43: 1679-1687Crossref PubMed Scopus (85) Google Scholar). In order to define the molecular mechanisms underlying the disease associations accompanying MBL variant alleles in more detail, we constructed and expressed recombinant wild type as well as variant MBL forms and investigated their structural and functional characteristics. Restriction enzymes and reverse transcriptase were from Amersham Biosciences. Taq polymerase was from Applied Biosystems. Cell culture utensils were from TPP (Trasadingen, Switzerland), except for triple bottom flasks that were from Nalge Nunc. Mannan, mannose, N-acetylglucosamine, trypsin (1:250), Geneticin (G418), hypoxanthine/thymidine media supplement (HT-supplement), l-glutamine solution, penicillin-streptomycin solution, methotrexate (MTX), dialyzed fetal bovine serum, RPMI 1640, and Iscove's modified Dulbecco's medium were all from Sigma. Large scale plasmid DNA isolation was performed using the Qiagen EndoFree Plasmid Maxi Kit. Small scale DNA preparations were performed using the Quantum Prep Plasmid Miniprep Kit (Bio-Rad). General methods of molecular biology were applied as described in Ref. 44Ausubel F. Brent R. Kingston K. Moore D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998Google Scholar. Cloning of the MBL cDNA—mRNA was isolated from the human hepatocellular carcinoma cell line HepG2 (obtained from ATCC (Manassas, VA) and having the MBL genotype: HYPA/LYPB) using the Dynabeads mRNA DIRECT Kit (Dynal). cDNA synthesis was done using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA). A PCR product was generated using Pfu DNA polymerase (Stratagene, La Jolla, CA) and 5′-primer (GAGATTAACCTTCCCTGAGT) and 3′-primer (GAGGGCCTGAGTGATATGAC) and cloned using the PCR-Script Amp Cloning Kit, Stratagene (La Jolla, CA). Verification of correct DNA sequence was performed on Applied Biosystems sequencing equipment. The expected DNA sequence was obtained except for the silent mutation in codon 136: AACAsn → AATAsn. This construct was used for site-directed mutagenesis. Site-directed Mutagenesis—Mutagenesis was performed according to the manufacturer's instructions using the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, WI) and the following 5′-phosphorylated primers: MBL-B, CCCTTGGTGTCATCACGCCC; MBL-C, CCCCTTTTCTTCCTTGGTGC; MBL-D, GTGCCATCACACCCATCTTTG; MBL-E, GCCATCACGCTCATCTTTGG; MBL-F, GAGAAAAGGAGGAACCAGGC (substituted nucleotides shown underlined). Transfection and MBL Expression—The MBL gene was PCR-amplified out of the pPCR-Script Amp vector using the following primers: 5′-primer, GTCTCTTCC↓ATGTCCCTGTTTCCATCAC; 3′-primer, TGCTCTTCC↓AAGTCAGATAGGGAACTCACAGA (Eam1104I recognition sequence shown underlined, and cut site indicated with an arrow). The PCR product was digested with Eam1104I and cloned into the pDual vector (Stratagene). Attempts were made to achieve expression in Escherichia coli BL21 as well as transient expression in the human HUH-7 hepatoma cells (JCRB Cell Bank) and COS-7 (ATCC) but failed or gave low expression levels. The MBL gene was moved from the pCR-Script Amp vector to pBK-CMV (Stratagene) using NotI and XhoI and from pBK-CMV to the dicistronic vector pED using SmaI and PstI. The pEDdC vector was a kind gift from the Genetics Institute (Cambridge, MA) (45Kaufman R.J. Davies M.V. Wasley L.C. Michnick D. Nucleic Acids Res. 1991; 19: 4485-4490Crossref PubMed Scopus (231) Google Scholar, 46Davies M.V. Kaufman R.J. Curr. Opin. Biotechnol. 1992; 3: 512-517Crossref PubMed Scopus (21) Google Scholar). This vector carries a cloning sequence for insertion of the target gene followed by the selectable and amplifiable marker gene dehydroxyfolate reductase (dhfr). The cell line used for recombinant expression of human MBL was Chinese hamster ovary (CHO) DG 44. This CHO clone is a double deletion mutant that contains no copies of the hamster dhfr gene and was a kind gift from professor Lawrence Chasin (Columbia University, New York) (47Urlaub G. Mitchell P.J. Kas E. Chasin L.A. Funanage V.L. Myoda T.T. Hamlin J. Somatic Cell Mol. Genet. 1986; 12: 555-566Crossref PubMed Scopus (188) Google Scholar). Untransfected cells were cultivated in Iscove's modified Dulbecco's medium supplemented by 10% dialyzed fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 2 mm l-glutamine, 10 mm hypoxanthine, and 1.6 mm thymidine (designated hereafter as complete Iscove's modified Dulbecco's medium) in a 37 °C humidified atmosphere containing 5% CO2. Cells were passaged using 0.05% trypsin in PBS. Stable transfections were performed using the LipofectAMINE PLUS reagent kit from Invitrogen. Transfection was done by seeding 8 × 105 cells in 6-cm Petri dishes on day 0. On day 1, cell medium was replaced and transfected according to the manufacturer's protocol, except that 24 μl of LipofectAMINE, 0.2 μg of pSV2NEO (Clontech, Palo Alto, CA), and 20 μg of the pED-MBL vector were used. On day 3, cells were moved to a T25 flask, and on day 5, cells were moved to complete Iscove's modified Eagle's medium containing 0.5 mg/ml G418 and omitting hypoxanthine and thymidine. Cells were moved to larger bottles when dense. When a G418-resistant pool of clones was obtained (after 10-14 days), selection and gene amplification with MTX was initiated by cultivating cells in complete Iscove's modified Eagle's medium containing 50 nm MTX and omitting hypoxanthine and thymidine. After cells had regained normal growth rate and morphology, the concentration of MTX was increased to 200 nm, 4 μm, 20 μm, and finally 80 μm. No significant amplification of MBL secretion into medium was seen at levels of MTX above 200 nm. Western Immunoblotting—SDS-PAGE was performed using 3-8% NuPAGE Tris acetate gels and Tris acetate running buffer both from Novex (San Diego, CA). Western blotting was done using the XCell II Mini-Cell blot apparatus and NuPAGE transfer buffer, both from Novex, and Hybond ECL nitrocellulose membranes from Amersham Biosciences (Hørsholm, Denmark). SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) was used for development of immunoblots utilizing horseradish peroxidase-conjugated antibodies or horse-radish peroxidase-conjugated streptavidin. As an Mr standard, the Precision prestained protein standard (Bio-Rad) was used. Quantization of MBL by ELISA—To determine expression levels of MBL in cell growth medium, in elutes from gel filtration experiments, and in fractions from SDG centrifugation, ELISA was performed as previously described (28Garred P. Madsen H.O. Kurtzhals J.A. Lamm L.U. Thiel S. Hey A.S. Svejgaard A. Eur. J. Immunogenet. 1992; 19: 403-412Crossref PubMed Scopus (133) Google Scholar). In brief, microtiter wells were coated overnight with a capture antibody at 4 °C (1 mg/liter anti-human MBL mouse monoclonal antibody HYB 131-01 or HYB 131-11; State Serum Institute, Copenhagen, Denmark) in PBS and washed five times with a buffer of 10 mm Tris-HCl, 150 mm NaCl, 0.05% Tween 20, pH 7.4. All subsequent washes were performed with this buffer. Cell growth supernatant, eluates from gel filtration, or fractions from SDG centrifugation and, as a reference, different dilutions of a serum pool diluted in the above buffer containing 10 mm EDTA were incubated at room temperature for 3 h. Wells were washed and incubated overnight at 4 °C with a mouse monoclonal biotin-labeled anti human MBL antibody (biotinylated HYB 131-01, 0.1 ng/ml, State Serum Institute). Wells were rinsed and incubated for 2 h at room temperature with a 1:2,000 dilution of a streptavidin-horseradish peroxidase conjugate (RPN 1231; Amersham Biosciences) in the above buffer, developed using OPD tablets from Dako (Glostrup, Denmark), according to the manufacturer's instructions, and the absorbance was read at 490 nm. MBL-mediated Complement Activation—Assay for complement activation was performed by coating microtiter plates (MaxiSorp; Nalge Nunc) overnight at 4 °C with mannan (0.1 g/liter) in a buffer of 15 mm Na2CO3, 35 mm NaHCO3, pH 9.6. Wells were washed five times in a buffer of 0.4 mm sodium barbital, 0.15 m NaCl, 2.6 mm CaCl2, 2.12 mm MgCl2, 0.05% Tween 20, pH 7.4. All subsequent incubations and washings were done with this buffer. Wells were incubated overnight at 37 °C with cell growth medium containing recombinant MBL. In control experiments, MBL binding to mannan was inhibited by the addition of 10 mm mannose, 10 mm N-acetylglucosamine, or 10 mm EDTA. Wells were incubated 1 h at 37 °C with serum from an MBL-deficient person diluted 1:400 (as a source of complement components, MBL genotype HYPD/HYPD (i.e. functionally defective MBL-D variant) (48Garred P. Larsen F. Madsen H.O. Koch C. Mol. Immunol. 2003; 40: 73-84Crossref PubMed Scopus (361) Google Scholar)), rinsed, and incubated for 45 min at 37 °C with a rabbit anti-human C4 antibody (0.3 ng/ml) (Dako). Wells were rinsed three times and incubated for 45 min at 37 °C with a 1:2,000 dilution of a donkey anti-rabbit Ig horseradish peroxidase-linked F(ab′)2 fragment, rinsed, and developed as described above. Quantization of MBL-mediated Complement Deposition Versus MBL Binding—Microtiter plates (MaxiSorp; Nalge Nunc) were coated with an MBL binding antibody (NImoAb001, 1.2 μg/ml, a kind gift from NatImmune, in PBS) and incubated overnight at 4 °C. Plates were washed three times in TBS-T plus calcium (10 mm Tris-HCl, 150 mm NaCl, 0.05% Tween 20, and 10 mm CaCl2) and blocked for 2 h in TBS-T plus calcium containing 1 mg/ml human serum albumin (TBS-T plus calcium and HSA; HSA was from the State Serum Institute). Culture supernatants were diluted 1 + 3, 1 + 7, 1 + 15, 1 + 31, 1 + 63, and 1 + 119 in the above buffer and added to microtiter wells in triplicates. Plates were incubated overnight at 4 °C. and washed three times in TBS-T plus calcium. Human purified recombinant MASP-2 (activated during purification) was added to a final concentration of 0.5 μg/ml (a kind gift from NatImmune) in TBS-T plus calcium and HSA and incubated overnight at 4 °C. Plates were washed and divided into two. One set of plates (A) was developed by measuring MBL binding, and the other set of plates (B) was developed by measuring deposition of C4. In plate A, MBL binding was measured by incubating wells with a mouse monoclonal biotin-labeled anti-human MBL antibody (diluted 1:3000 in TBS-T plus calcium, biotinylated HYB 131-01; State Serum Institute) for 2 h at room temperature. Wells were washed, and europium-labeled streptavidin (Wallac, Turku, Finland) was added at a concentration of 0.1 mg/liter in the above washing buffer except that calcium was omitted, and 50 μm EDTA was included. Wells were incubated 1 h at room temperature, washed, and developed by adding 100 μl of Delfia Enhancement Solution (PerkinElmer Life Sciences) and incubated on an orbital shaker for 5 min. at room temperature. Then wells were counted in a Wallac Victor 2d multicounter 1420 (Wallac, Turku, Finland). In plate B, deposition of C4 was measured by incubation 1.5 h at 37 °C with purified human complement component C4 (∼1.5-2 ng/ml) in a buffer of sodium barbital (5 mm), NaCl (181 mm), CaCl2 (2.5 mm), MgCl2 (1.25 mm), pH 7.4, 1 mg/ml human albumin (State Serum Institute). Wells were washed three times, and 1 mg/liter biotinylated rabbit anti-human complement component C4c was added (Dako), biotinylated according to standard procedures. Wells were incubated for 1 h at room temperature and washed. Europium-labeled streptavidin was added, and development continued as described above. Purification of rMBL from Tissue Culture Supernatant—MBL was purified from tissue culture medium using both affinity and ion exchange chromatography. 200 ml of tissue culture medium was centrifuged (15,000 × g, 20 min) and filtered (0.2 μm) before incubation end-over-end at 4 °C with anti-MBL-Sepharose (HYB 131-11; State Serum Institute). The beads were packed into a chromatography column (Econo-Column (25 mm); Bio-Rad) and washed with 10-30 ml of PBS (1 ml/min on a BioLogic LP chromatography system; Bio-Rad) until the A280 base line was reached. Bound rMBL was eluted with 1 ml/min of 4.5 m MgCl2, 50 mm Tris-HCl, pH 7.5, and ELISA-positive fractions were pooled and dialyzed against 20 mm Tris-HCl, pH 8.0. Samples were concentrated using Centricon Plus-20 (regenerated cellulose, Mr cut-off 10,000; Millipore Corp., Bedford, MA). The concentrated samples were loaded on an ion exchange column (Resource Q (1 ml), flow rate 1 ml/min; Amersham Biosciences) mounted in a BioLogic LP chromatography system (Bio-Rad). The column was washed with 5 ml of 20 mm Tris-HCl, pH 8.0, and eluted using a 10-ml linear gradient of 0-100% 1 m NaCl in 20 mm Tris-HCl, pH 8.0. ELISA-positive fractions were pooled and dialyzed against PBS, and purified recombinant MBL was stored at -20 °C. Gel Filtration—Purified MBL or tissue culture medium containing MBL was centrifuged (16,000 × g for 5 min) before being fractionated on a Superose 6 column (300 × 10 mm) equilibrated in 20 mm HEPES, 130 mm NaCl, 0.5 mm EDTA, using the fast protein liquid chromatography system (Amersham Biosciences) operated at a flow rate of 0.7 ml/min. The void volume of the column was 8 ml. The column was equilibrated using a range of standards with known Stokes radii: human C1q (10.7 nm), human factor H (7.2 nm), human IgG (5.4 nm), chicken ovalbumin (2.9 nm). Standard protein Stokes radii were calculated as described in Ref. 49Ackers G.K. Biochemistry. 1964; 3: 723-730Crossref PubMed Scopus (428) Google Scholar from diffusion coefficients taken from Ref. 50Smith M.H. Handbook of Biochemistry and Selected Data for Molecular Biology. CRC Press, Inc., Cleveland, OH2001: C3-C28Google Scholar, a=KT/6πZD(Eq. 1) where a represents the Stokes radius, K is the Bolzman constant, T is the temperature in kelvin, Z is the viscosity of water at 20 °C, and D is the diffusion coefficient of the protein in water at 20 °C. The Stokes radii of the different MBL mutations were determined by their elution from the gel filtration column relative to the protein standards (49Ackers G.K. Biochemistry. 1964; 3: 723-730Crossref PubMed Scopus (428) Google Scholar). Sucrose Density Gradient Centrifugation—Two aliquots of sucrose solution (20 and 10% sucrose in 20 mm HEPES, 130 mm NaCl, 0.5 mm EDTA) were layered into centrifuge tubes, and the linear gradient was generated by centrifugation at 35,000 rpm for 17 h at 4 °C (Beckman SW40Ti rotor) to form a linear sucrose density gradient. Protein samples (500 μl) were loaded onto the gradient and centrifuged at 35,000 rpm for 16 h at 4 °C. Gradients were fractionated into ∼20 fractions by peristaltic pumping from the base of the gradient. Individual fractions were analyzed by SDS-PAGE and by measuring A280. MBL was detected in fractions by ELISA as described above. Sedimentation coefficients (s20,w) were estimated by comparison of their mobilities with those of the following standard proteins: thyroglobulin (19.2 S), bovine liver catalase (11.2 S), bovine serum albumin (4.2 S), and equine skeletal muscle myoglobin (2.0 S). All proteins were from Sigma, and sedimentation coefficients were taken from Ref. 50Smith M.H. Handbook of Biochemistry and Selected Data for Molecular Biology. CRC Press, Inc., Cleveland, OH2001: C3-C28Google Scholar. Estimation of Molecular Weight under Nondenaturing Conditions (51Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar)—The Stokes radius and sedimentation coefficient values obtained were used to caRead More
Title: $Disease-associated Mutations in Human Mannose-binding Lectin Compromise Oligomerization and Activity of the Final Protein
Abstract: Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement. Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement. Mannose-binding lectin (MBL) 1The abbreviations used are: MBL, mannose-binding lectin(s); CHO, Chinese hamster ovary; MASP-1, -2, and -3, MBL-associated serine protease-1, -2, and -3, respectively; MTX, methotrexate; SDG, sucrose density gradient; sMAP, small MBL-associated protein (identical to MAp19); SELDI, surface-enhanced laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; WT, wild type; PBS, phosphate-buffered saline; HSA, human serum albumin; ELISA, enzyme-linked immunosorbent assay. has been shown to be an important component of innate immunity and is a central recognition molecule of the lectin pathway of complement (for a recent review, see Ref. 1Turner M.W. Hamvas R.M.J. Rev. Immunogenet. 2001; 2: 305-322Google Scholar). MBL binds to an array of carbohydrate structures on surfaces of bacteria (2Kawasaki N. Kawasaki T. Yamashina I. J. Biochem. (Tokyo). 1989; 106: 483-489Crossref PubMed Scopus (150) Google Scholar, 3Neth O. Jack D.L. Dodds A.W. Holzel H. Klein N.J. Turner M.W. Infect. Immun. 2000; 68: 688-693Crossref PubMed Scopus (473) Google Scholar, 4Townsend R. Read R.C. Turner M.W. Klein N.J. Jack D.L. Clin. Exp. Immunol. 2001; 124: 223-228Crossref PubMed Scopus (42) Google Scholar), yeast, viruses (5Haurum J.S. Thiel S. Jones I.M. Fischer P.B. Laursen S.B. Jensenius J.C. AIDS. 1993; 7: 1307-1313Crossref PubMed Scopus (130) Google Scholar, 6Hartshorn K.L. Sastry K. White M.R. Anders E.M. Super M. Ezekowitz R.A. Tauber A.I. J. Clin. Invest. 1993; 91: 1414-1420Crossref PubMed Scopus (183) Google Scholar), and parasitic protozoa (7Green P.J. Feizi T. Stoll M.S. Thiel S. Prescott A. McConville M.J. Mol. Biochem. Parasitol. 1994; 66: 319-328Crossref PubMed Scopus (82) Google Scholar, 8Kelly P. Jack D.L. Naeem A. Mandanda B. Pollok R.C. Klein N.J. Turner M.W. Farthing M.J. Gastroenterology. 2000; 119: 1236-1242Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). MBL functions as an opsonin (9Kuhlman M. Joiner K. Ezekowitz R.A. J. Exp. Med. 1989; 169: 1733-1745Crossref PubMed Scopus (391) Google Scholar), and the biological effect is mediated by direct killing via complement (10Lu J.H. Thiel S. Wiedemann H. Timpl R. Reid K.B. J. Immunol. 1990; 144: 2287-2294PubMed Google Scholar) through the lytic membrane attack complex or by promoting phagocytosis either by the MBL lectin pathway of complement or by direct binding to one or more cell surface receptors (11Tenner A.J. Robinson S.L. Ezekowitz R.A. Immunity. 1995; 3: 485-493Abstract Full Text PDF PubMed Scopus (152) Google Scholar). The lectin pathway comprises at least three MBL-associated serine proteases (MASPs), namely MASP-1 (12Sato T. Endo Y. Matsushita M. Fujita T. Int. Immunol. 1994; 6: 665-669Crossref PubMed Scopus (154) Google Scholar), MASP-2 (13Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (757) Google Scholar), and MASP-3 (14Dahl M.R. Thiel S. Matsushita M. Fujita T. Willis A.C. Christensen T. Vorup-Jensen T. Jensenius J.C. Immunity. 2001; 15: 127-135Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Furthermore, the functional MBL-MASP complex contains a small MBL-associated protein (sMAP), also named MAp19, with no serine protease activity (15Stover C.M. Thiel S. Thelen M. Lynch N.J. Vorup-Jensen T. Jensenius J.C. Schwaeble W.J. J. Immunol. 1999; 162: 3481-3490PubMed Google Scholar, 16Takahashi M. Endo Y. Fujita T. Matsushita M. Int. Immunol. 1999; 11: 859-863Crossref PubMed Scopus (171) Google Scholar). MASP-2 is a homologue of C1s of the classical complement pathway because it activates C4 and C2 (13Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (757) Google Scholar). When MBL associated with MASP-2 binds to sugar groups on the surface of microbes, the MBL-MASP2 proenzyme is activated and cleaves sequentially C4 and C2, thereby creating the C4b2a complex, a potent C3 convertase. The MBL-MASP-1 complex is suggested to activate C3 directly (12Sato T. Endo Y. Matsushita M. Fujita T. Int. Immunol. 1994; 6: 665-669Crossref PubMed Scopus (154) Google Scholar). Whether both MASP-1 and MASP-2 are bound on the same MBL molecule is still unclear. Moreover, the biological role of sMAP, as well as the substrate for the recently discovered MASP-3, remains unclear at this moment (for a recent review on MASPs, see Ref. 17Gadjeva M. Thiel S. Jensenius J.C. Curr. Opin. Immunol. 2001; 13: 74-78Crossref PubMed Scopus (100) Google Scholar). MBL is a complex of six sets of homotrimers of a single polypeptide chain containing 228 amino acids (18Sastry K. Herman G.A. Day L. Deignan E. Bruns G. Morton C.C. Ezekowitz R.A. J. Exp. Med. 1989; 170: 1175-1189Crossref PubMed Scopus (253) Google Scholar, 19Ezekowitz R.A. Day L.E. Herman G.A. J. Exp. Med. 1988; 167: 1034-1046Crossref PubMed Scopus (246) Google Scholar, 20Taylor M.E. Brickell P.M. Craig R.K. Summerfield J.A. Biochem. J. 1989; 262: 763-771Crossref PubMed Scopus (190) Google Scholar, 21Kurata H. Sannoh T. Kozutsumi Y. Yokota Y. Kawasaki T. J. Biochem. (Tokyo). 1994; 115: 1148-1154Crossref PubMed Scopus (38) Google Scholar). This polypeptide consists of four domains (Fig. 1): 1) a 20-amino acid N-terminal cysteine-rich domain involved in formation of intra- and intersubunit disulfide bonds, 2) a collagen-like domain consisting of 18-20 tandem repeats of Gly-Xaa-Yaa, 3) an α-helical coiled-coil neck region, and 4) A carbohydrate recognition domain capable of binding to a wide variety of carbohydrate arrays on the surface of microorganisms (3Neth O. Jack D.L. Dodds A.W. Holzel H. Klein N.J. Turner M.W. Infect. Immun. 2000; 68: 688-693Crossref PubMed Scopus (473) Google Scholar). Three polypeptides form a structural unit or subunit containing a triple helix at their collagen-like domain. Six of these units combine by interunit disulfide bonds to form the biologically active bouquet-like MBL protein (for a recent review on MBL structure, see Ref. 22Kawasaki T. Biochim. Biophys. Acta. 1999; 1473: 186-195Crossref PubMed Scopus (49) Google Scholar). Three different genetic polymorphisms in exon 1 of the human MBL2 gene (MBL1 is a pseudogene (23Guo N. Mogues T. Weremowicz S. Morton C.C. Sastry K.N. Mamm. Genome. 1998; 9: 246-249Crossref PubMed Scopus (68) Google Scholar)) independently lead to reduced serum concentrations of MBL. Two interrupt the tandem repeat Gly-Xaa-Yaa in the first (Gly) position, and the third introduces a cysteine residue in the second position. The designation of the MBL variant alleles is B, C, and D, whereas the normal allele is termed A. MBL-B has a G → A mutation in codon 54 (24Heise C.T. Nicholls J.R. Leamy C.E. Wallis R. J. Immunol. 2000; 165: 1403-1409Crossref PubMed Scopus (31) Google Scholar), which results in a Gly → Asp substitution in the fifth Gly-Xaa-Yaa repeat. MBL-C has a G → A mutation in codon 57 (25Lipscombe R.J. Sumiya M. Hill A.V. Lau Y.L. Levinsky R.J. Summerfield J.A. Turner M.W. Hum. Mol. Genet. 1992; 1: 709-715Crossref PubMed Scopus (382) Google Scholar), which translates into a Gly → Asp substitution of the sixth Gly-Xaa-Yaa repeat. The third mutation is MBL-D, a C → T mutation in codon 52 (25Lipscombe R.J. Sumiya M. Hill A.V. Lau Y.L. Levinsky R.J. Summerfield J.A. Turner M.W. Hum. Mol. Genet. 1992; 1: 709-715Crossref PubMed Scopus (382) Google Scholar) that results in the introduction of a cysteine instead of an arginine in the protein (26Madsen H.O. Garred P. Kurtzhals J.A. Lamm L.U. Ryder L.P. Thiel S. Svejgaard A. Immunogenetics. 1994; 40: 37-44Crossref PubMed Scopus (464) Google Scholar). The presence of these mutations leads to markedly reduced MBL protein levels in the blood (27Garred P. Thiel S. Madsen H.O. Ryder L.P. Jensenius J.C. Svejgaard A. Clin. Exp. Immunol. 1992; 90: 517-521Crossref PubMed Scopus (114) Google Scholar, 28Garred P. Madsen H.O. Kurtzhals J.A. Lamm L.U. Thiel S. Hey A.S. Svejgaard A. Eur. J. Immunogenet. 1992; 19: 403-412Crossref PubMed Scopus (133) Google Scholar). The B and D alleles are seen in Eurasian and indigenous American populations with frequencies ranging from 0.1 to 0.5 and from 0.0 to 0.1, respectively (29Madsen H.O. Garred P. Thiel S. Kurtzhals J.A. Lamm L.U. Ryder L.P. Svejgaard A. J. Immunol. 1995; 155: 3013-3020PubMed Google Scholar, 30Lipscombe R.J. Beatty D.W. Ganczakowski M. Goddard E.A. Jenkins T. Lau Y.L. Spurdle A.B. Sumiya M. Summerfield J.A. Turner M.W. Eur. J. Hum. Genet. 1996; 4: 13-19Crossref PubMed Scopus (88) Google Scholar, 31Madsen H.O. Satz M.L. Hogh B. Svejgaard A. Garred P. J. Immunol. 1998; 161: 3169-3175PubMed Google Scholar). The C allele is found most frequently in sub-Saharan African populations with a frequency ranging from 0.07 to 0.3 (29Madsen H.O. Garred P. Thiel S. Kurtzhals J.A. Lamm L.U. Ryder L.P. Svejgaard A. J. Immunol. 1995; 155: 3013-3020PubMed Google Scholar, 30Lipscombe R.J. Beatty D.W. Ganczakowski M. Goddard E.A. Jenkins T. Lau Y.L. Spurdle A.B. Sumiya M. Summerfield J.A. Turner M.W. Eur. J. Hum. Genet. 1996; 4: 13-19Crossref PubMed Scopus (88) Google Scholar, 31Madsen H.O. Satz M.L. Hogh B. Svejgaard A. Garred P. J. Immunol. 1998; 161: 3169-3175PubMed Google Scholar). The presence of these alleles is associated with increased risk of infections during childhood, particularly during the vulnerable period of infancy ranging from 6 to 18 months of age (32Garred P. Madsen H.O. Hofmann B. Svejgaard A. Lancet. 1995; 346: 941-943Abstract PubMed Scopus (0) Google Scholar, 33Summerfield J.A. Sumiya M. Levin M. Turner M.W. Br. Med. J. 1997; 314: 1229-1232Crossref PubMed Scopus (361) Google Scholar, 34Koch A. Melbye M. Sorensen P. Homoe P. Madsen H.O. Molbak K. Hansen C.H. Andersen L.H. Hahn G.W. Garred P. JAMA (J. Am. Med. Assoc.). 2001; 285: 1316-1321Crossref PubMed Scopus (397) Google Scholar), in immunocompromised patients (35Garred P. Madsen H.O. Balslev U. Hofmann B. Pedersen C. Gerstoft J. Svejgaard A. Lancet. 1997; 349: 236-240Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 36Garred P. Madsen H.O. Halberg P. Petersen J. Kronborg G. Svejgaard A. Andersen V. Jacobsen S. Arthritis Rheum. 1999; 42: 2145-2152Crossref PubMed Scopus (199) Google Scholar) and is a risk factor for critically ill patients to develop sepsis (37Garred P. Strom J. Quist L. Taaning E. Madsen H.O. J. Infect. Dis. 2003; 188: 1394-1403Crossref PubMed Scopus (182) Google Scholar). Moreover, MBL variant alleles are associated with disease progression in concomitant diseases such as chronic granulomatous disease and cystic fibrosis (38Foster C.B. Lehrnbecher T. Mol F. Steinberg S.M. Venzon D.J. Walsh T.J. Noack D. Rae J. Winkelstein J.A. Curnutte J.T. Chanock S.J. J. Clin. Invest. 1998; 102: 2146-2155Crossref PubMed Scopus (226) Google Scholar, 39Garred P. Pressler T. Madsen H.O. Frederiksen B. Svejgaard A. Hoiby N. Schwartz M. Koch C. J. Clin. Invest. 1999; 104: 431-437Crossref PubMed Scopus (406) Google Scholar), Additionally, the importance of MBL deficiency in autoimmunity has been emphasized in diseases like systemic lupus erythematosus and rheumatoid arthritis (40Davies E.J. Snowden N. Hillarby M.C. Carthy D. Grennan D.M. Thomson W. Ollier W.E. Arthritis Rheum. 1995; 38: 110-114Crossref PubMed Scopus (153) Google Scholar, 41Sullivan K.E. Wooten C. Goldman D. Petri M. Arthritis Rheum. 1996; 39: 2046-2051Crossref PubMed Scopus (152) Google Scholar, 42Graudal N.A. Madsen H.O. Tarp U. Svejgaard A. Jurik G. Graudal H.K. Garred P. Arthritis Rheum. 2000; 43: 515-521Crossref PubMed Scopus (114) Google Scholar, 43Ip W.K. Lau Y.L. Chan S.Y. Mok C.C. Chan D. Tong K.K. Lau C.S. Arthritis Rheum. 2000; 43: 1679-1687Crossref PubMed Scopus (85) Google Scholar). In order to define the molecular mechanisms underlying the disease associations accompanying MBL variant alleles in more detail, we constructed and expressed recombinant wild type as well as variant MBL forms and investigated their structural and functional characteristics. Restriction enzymes and reverse transcriptase were from Amersham Biosciences. Taq polymerase was from Applied Biosystems. Cell culture utensils were from TPP (Trasadingen, Switzerland), except for triple bottom flasks that were from Nalge Nunc. Mannan, mannose, N-acetylglucosamine, trypsin (1:250), Geneticin (G418), hypoxanthine/thymidine media supplement (HT-supplement), l-glutamine solution, penicillin-streptomycin solution, methotrexate (MTX), dialyzed fetal bovine serum, RPMI 1640, and Iscove's modified Dulbecco's medium were all from Sigma. Large scale plasmid DNA isolation was performed using the Qiagen EndoFree Plasmid Maxi Kit. Small scale DNA preparations were performed using the Quantum Prep Plasmid Miniprep Kit (Bio-Rad). General methods of molecular biology were applied as described in Ref. 44Ausubel F. Brent R. Kingston K. Moore D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998Google Scholar. Cloning of the MBL cDNA—mRNA was isolated from the human hepatocellular carcinoma cell line HepG2 (obtained from ATCC (Manassas, VA) and having the MBL genotype: HYPA/LYPB) using the Dynabeads mRNA DIRECT Kit (Dynal). cDNA synthesis was done using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA). A PCR product was generated using Pfu DNA polymerase (Stratagene, La Jolla, CA) and 5′-primer (GAGATTAACCTTCCCTGAGT) and 3′-primer (GAGGGCCTGAGTGATATGAC) and cloned using the PCR-Script Amp Cloning Kit, Stratagene (La Jolla, CA). Verification of correct DNA sequence was performed on Applied Biosystems sequencing equipment. The expected DNA sequence was obtained except for the silent mutation in codon 136: AACAsn → AATAsn. This construct was used for site-directed mutagenesis. Site-directed Mutagenesis—Mutagenesis was performed according to the manufacturer's instructions using the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, WI) and the following 5′-phosphorylated primers: MBL-B, CCCTTGGTGTCATCACGCCC; MBL-C, CCCCTTTTCTTCCTTGGTGC; MBL-D, GTGCCATCACACCCATCTTTG; MBL-E, GCCATCACGCTCATCTTTGG; MBL-F, GAGAAAAGGAGGAACCAGGC (substituted nucleotides shown underlined). Transfection and MBL Expression—The MBL gene was PCR-amplified out of the pPCR-Script Amp vector using the following primers: 5′-primer, GTCTCTTCC↓ATGTCCCTGTTTCCATCAC; 3′-primer, TGCTCTTCC↓AAGTCAGATAGGGAACTCACAGA (Eam1104I recognition sequence shown underlined, and cut site indicated with an arrow). The PCR product was digested with Eam1104I and cloned into the pDual vector (Stratagene). Attempts were made to achieve expression in Escherichia coli BL21 as well as transient expression in the human HUH-7 hepatoma cells (JCRB Cell Bank) and COS-7 (ATCC) but failed or gave low expression levels. The MBL gene was moved from the pCR-Script Amp vector to pBK-CMV (Stratagene) using NotI and XhoI and from pBK-CMV to the dicistronic vector pED using SmaI and PstI. The pEDdC vector was a kind gift from the Genetics Institute (Cambridge, MA) (45Kaufman R.J. Davies M.V. Wasley L.C. Michnick D. Nucleic Acids Res. 1991; 19: 4485-4490Crossref PubMed Scopus (231) Google Scholar, 46Davies M.V. Kaufman R.J. Curr. Opin. Biotechnol. 1992; 3: 512-517Crossref PubMed Scopus (21) Google Scholar). This vector carries a cloning sequence for insertion of the target gene followed by the selectable and amplifiable marker gene dehydroxyfolate reductase (dhfr). The cell line used for recombinant expression of human MBL was Chinese hamster ovary (CHO) DG 44. This CHO clone is a double deletion mutant that contains no copies of the hamster dhfr gene and was a kind gift from professor Lawrence Chasin (Columbia University, New York) (47Urlaub G. Mitchell P.J. Kas E. Chasin L.A. Funanage V.L. Myoda T.T. Hamlin J. Somatic Cell Mol. Genet. 1986; 12: 555-566Crossref PubMed Scopus (188) Google Scholar). Untransfected cells were cultivated in Iscove's modified Dulbecco's medium supplemented by 10% dialyzed fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 2 mm l-glutamine, 10 mm hypoxanthine, and 1.6 mm thymidine (designated hereafter as complete Iscove's modified Dulbecco's medium) in a 37 °C humidified atmosphere containing 5% CO2. Cells were passaged using 0.05% trypsin in PBS. Stable transfections were performed using the LipofectAMINE PLUS reagent kit from Invitrogen. Transfection was done by seeding 8 × 105 cells in 6-cm Petri dishes on day 0. On day 1, cell medium was replaced and transfected according to the manufacturer's protocol, except that 24 μl of LipofectAMINE, 0.2 μg of pSV2NEO (Clontech, Palo Alto, CA), and 20 μg of the pED-MBL vector were used. On day 3, cells were moved to a T25 flask, and on day 5, cells were moved to complete Iscove's modified Eagle's medium containing 0.5 mg/ml G418 and omitting hypoxanthine and thymidine. Cells were moved to larger bottles when dense. When a G418-resistant pool of clones was obtained (after 10-14 days), selection and gene amplification with MTX was initiated by cultivating cells in complete Iscove's modified Eagle's medium containing 50 nm MTX and omitting hypoxanthine and thymidine. After cells had regained normal growth rate and morphology, the concentration of MTX was increased to 200 nm, 4 μm, 20 μm, and finally 80 μm. No significant amplification of MBL secretion into medium was seen at levels of MTX above 200 nm. Western Immunoblotting—SDS-PAGE was performed using 3-8% NuPAGE Tris acetate gels and Tris acetate running buffer both from Novex (San Diego, CA). Western blotting was done using the XCell II Mini-Cell blot apparatus and NuPAGE transfer buffer, both from Novex, and Hybond ECL nitrocellulose membranes from Amersham Biosciences (Hørsholm, Denmark). SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) was used for development of immunoblots utilizing horseradish peroxidase-conjugated antibodies or horse-radish peroxidase-conjugated streptavidin. As an Mr standard, the Precision prestained protein standard (Bio-Rad) was used. Quantization of MBL by ELISA—To determine expression levels of MBL in cell growth medium, in elutes from gel filtration experiments, and in fractions from SDG centrifugation, ELISA was performed as previously described (28Garred P. Madsen H.O. Kurtzhals J.A. Lamm L.U. Thiel S. Hey A.S. Svejgaard A. Eur. J. Immunogenet. 1992; 19: 403-412Crossref PubMed Scopus (133) Google Scholar). In brief, microtiter wells were coated overnight with a capture antibody at 4 °C (1 mg/liter anti-human MBL mouse monoclonal antibody HYB 131-01 or HYB 131-11; State Serum Institute, Copenhagen, Denmark) in PBS and washed five times with a buffer of 10 mm Tris-HCl, 150 mm NaCl, 0.05% Tween 20, pH 7.4. All subsequent washes were performed with this buffer. Cell growth supernatant, eluates from gel filtration, or fractions from SDG centrifugation and, as a reference, different dilutions of a serum pool diluted in the above buffer containing 10 mm EDTA were incubated at room temperature for 3 h. Wells were washed and incubated overnight at 4 °C with a mouse monoclonal biotin-labeled anti human MBL antibody (biotinylated HYB 131-01, 0.1 ng/ml, State Serum Institute). Wells were rinsed and incubated for 2 h at room temperature with a 1:2,000 dilution of a streptavidin-horseradish peroxidase conjugate (RPN 1231; Amersham Biosciences) in the above buffer, developed using OPD tablets from Dako (Glostrup, Denmark), according to the manufacturer's instructions, and the absorbance was read at 490 nm. MBL-mediated Complement Activation—Assay for complement activation was performed by coating microtiter plates (MaxiSorp; Nalge Nunc) overnight at 4 °C with mannan (0.1 g/liter) in a buffer of 15 mm Na2CO3, 35 mm NaHCO3, pH 9.6. Wells were washed five times in a buffer of 0.4 mm sodium barbital, 0.15 m NaCl, 2.6 mm CaCl2, 2.12 mm MgCl2, 0.05% Tween 20, pH 7.4. All subsequent incubations and washings were done with this buffer. Wells were incubated overnight at 37 °C with cell growth medium containing recombinant MBL. In control experiments, MBL binding to mannan was inhibited by the addition of 10 mm mannose, 10 mm N-acetylglucosamine, or 10 mm EDTA. Wells were incubated 1 h at 37 °C with serum from an MBL-deficient person diluted 1:400 (as a source of complement components, MBL genotype HYPD/HYPD (i.e. functionally defective MBL-D variant) (48Garred P. Larsen F. Madsen H.O. Koch C. Mol. Immunol. 2003; 40: 73-84Crossref PubMed Scopus (361) Google Scholar)), rinsed, and incubated for 45 min at 37 °C with a rabbit anti-human C4 antibody (0.3 ng/ml) (Dako). Wells were rinsed three times and incubated for 45 min at 37 °C with a 1:2,000 dilution of a donkey anti-rabbit Ig horseradish peroxidase-linked F(ab′)2 fragment, rinsed, and developed as described above. Quantization of MBL-mediated Complement Deposition Versus MBL Binding—Microtiter plates (MaxiSorp; Nalge Nunc) were coated with an MBL binding antibody (NImoAb001, 1.2 μg/ml, a kind gift from NatImmune, in PBS) and incubated overnight at 4 °C. Plates were washed three times in TBS-T plus calcium (10 mm Tris-HCl, 150 mm NaCl, 0.05% Tween 20, and 10 mm CaCl2) and blocked for 2 h in TBS-T plus calcium containing 1 mg/ml human serum albumin (TBS-T plus calcium and HSA; HSA was from the State Serum Institute). Culture supernatants were diluted 1 + 3, 1 + 7, 1 + 15, 1 + 31, 1 + 63, and 1 + 119 in the above buffer and added to microtiter wells in triplicates. Plates were incubated overnight at 4 °C. and washed three times in TBS-T plus calcium. Human purified recombinant MASP-2 (activated during purification) was added to a final concentration of 0.5 μg/ml (a kind gift from NatImmune) in TBS-T plus calcium and HSA and incubated overnight at 4 °C. Plates were washed and divided into two. One set of plates (A) was developed by measuring MBL binding, and the other set of plates (B) was developed by measuring deposition of C4. In plate A, MBL binding was measured by incubating wells with a mouse monoclonal biotin-labeled anti-human MBL antibody (diluted 1:3000 in TBS-T plus calcium, biotinylated HYB 131-01; State Serum Institute) for 2 h at room temperature. Wells were washed, and europium-labeled streptavidin (Wallac, Turku, Finland) was added at a concentration of 0.1 mg/liter in the above washing buffer except that calcium was omitted, and 50 μm EDTA was included. Wells were incubated 1 h at room temperature, washed, and developed by adding 100 μl of Delfia Enhancement Solution (PerkinElmer Life Sciences) and incubated on an orbital shaker for 5 min. at room temperature. Then wells were counted in a Wallac Victor 2d multicounter 1420 (Wallac, Turku, Finland). In plate B, deposition of C4 was measured by incubation 1.5 h at 37 °C with purified human complement component C4 (∼1.5-2 ng/ml) in a buffer of sodium barbital (5 mm), NaCl (181 mm), CaCl2 (2.5 mm), MgCl2 (1.25 mm), pH 7.4, 1 mg/ml human albumin (State Serum Institute). Wells were washed three times, and 1 mg/liter biotinylated rabbit anti-human complement component C4c was added (Dako), biotinylated according to standard procedures. Wells were incubated for 1 h at room temperature and washed. Europium-labeled streptavidin was added, and development continued as described above. Purification of rMBL from Tissue Culture Supernatant—MBL was purified from tissue culture medium using both affinity and ion exchange chromatography. 200 ml of tissue culture medium was centrifuged (15,000 × g, 20 min) and filtered (0.2 μm) before incubation end-over-end at 4 °C with anti-MBL-Sepharose (HYB 131-11; State Serum Institute). The beads were packed into a chromatography column (Econo-Column (25 mm); Bio-Rad) and washed with 10-30 ml of PBS (1 ml/min on a BioLogic LP chromatography system; Bio-Rad) until the A280 base line was reached. Bound rMBL was eluted with 1 ml/min of 4.5 m MgCl2, 50 mm Tris-HCl, pH 7.5, and ELISA-positive fractions were pooled and dialyzed against 20 mm Tris-HCl, pH 8.0. Samples were concentrated using Centricon Plus-20 (regenerated cellulose, Mr cut-off 10,000; Millipore Corp., Bedford, MA). The concentrated samples were loaded on an ion exchange column (Resource Q (1 ml), flow rate 1 ml/min; Amersham Biosciences) mounted in a BioLogic LP chromatography system (Bio-Rad). The column was washed with 5 ml of 20 mm Tris-HCl, pH 8.0, and eluted using a 10-ml linear gradient of 0-100% 1 m NaCl in 20 mm Tris-HCl, pH 8.0. ELISA-positive fractions were pooled and dialyzed against PBS, and purified recombinant MBL was stored at -20 °C. Gel Filtration—Purified MBL or tissue culture medium containing MBL was centrifuged (16,000 × g for 5 min) before being fractionated on a Superose 6 column (300 × 10 mm) equilibrated in 20 mm HEPES, 130 mm NaCl, 0.5 mm EDTA, using the fast protein liquid chromatography system (Amersham Biosciences) operated at a flow rate of 0.7 ml/min. The void volume of the column was 8 ml. The column was equilibrated using a range of standards with known Stokes radii: human C1q (10.7 nm), human factor H (7.2 nm), human IgG (5.4 nm), chicken ovalbumin (2.9 nm). Standard protein Stokes radii were calculated as described in Ref. 49Ackers G.K. Biochemistry. 1964; 3: 723-730Crossref PubMed Scopus (428) Google Scholar from diffusion coefficients taken from Ref. 50Smith M.H. Handbook of Biochemistry and Selected Data for Molecular Biology. CRC Press, Inc., Cleveland, OH2001: C3-C28Google Scholar, a=KT/6πZD(Eq. 1) where a represents the Stokes radius, K is the Bolzman constant, T is the temperature in kelvin, Z is the viscosity of water at 20 °C, and D is the diffusion coefficient of the protein in water at 20 °C. The Stokes radii of the different MBL mutations were determined by their elution from the gel filtration column relative to the protein standards (49Ackers G.K. Biochemistry. 1964; 3: 723-730Crossref PubMed Scopus (428) Google Scholar). Sucrose Density Gradient Centrifugation—Two aliquots of sucrose solution (20 and 10% sucrose in 20 mm HEPES, 130 mm NaCl, 0.5 mm EDTA) were layered into centrifuge tubes, and the linear gradient was generated by centrifugation at 35,000 rpm for 17 h at 4 °C (Beckman SW40Ti rotor) to form a linear sucrose density gradient. Protein samples (500 μl) were loaded onto the gradient and centrifuged at 35,000 rpm for 16 h at 4 °C. Gradients were fractionated into ∼20 fractions by peristaltic pumping from the base of the gradient. Individual fractions were analyzed by SDS-PAGE and by measuring A280. MBL was detected in fractions by ELISA as described above. Sedimentation coefficients (s20,w) were estimated by comparison of their mobilities with those of the following standard proteins: thyroglobulin (19.2 S), bovine liver catalase (11.2 S), bovine serum albumin (4.2 S), and equine skeletal muscle myoglobin (2.0 S). All proteins were from Sigma, and sedimentation coefficients were taken from Ref. 50Smith M.H. Handbook of Biochemistry and Selected Data for Molecular Biology. CRC Press, Inc., Cleveland, OH2001: C3-C28Google Scholar. Estimation of Molecular Weight under Nondenaturing Conditions (51Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar)—The Stokes radius and sedimentation coefficient values obtained were used to ca