Mannan-Binding Lectins from Mouse Serum Purification And

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Purification and Characterization of Two Mannan-Binding Lectins from Mouse Serum Søren Hansen, Steffen Thiel, Anthony Willis, Uffe Holmskov and Jens Christian Jensenius This information is current as of October 2, 2021. J Immunol 2000; 164:2610-2618; ; doi: 10.4049/jimmunol.164.5.2610 http://www.jimmunol.org/content/164/5/2610 Downloaded from References This article cites 38 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/164/5/2610.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Puriﬁcation and Characterization of Two Mannan-Binding Lectins from Mouse Serum

Søren Hansen,1* Steffen Thiel,2* Anthony Willis,† Uffe Holmskov,‡ and Jens Christian Jensenius*

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in Downloaded from acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan- coated microtiter wells. MBL-A showed a higher affinity for D-glucose and ␣-methyl-D-glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 ␮g/ml, with wild mice tending to show higher levels than laboratory strains. The Journal of Immunology, 2000, 164: 2610–2618.

annan-binding lectin (MBL)3 is an animal C-type lec- MBL is synthesized by hepatocytes and has been isolated from tin (i.e., showing calcium-dependent carbohydrate the liver or serum of several vertebrate species. Only one form of http://www.jimmunol.org/ M binding) of the collectin family in which the carbohy- human MBL has been characterized, whereas two forms are found drate recognition domains (CRDs) are attached to collagen regions in rabbits, rats, mice, and rhesus monkeys (9). So far MBL-A has (1). The mature protein is an oligomer of subunits each composed been considered to be the serum form in rodents, whereas MBL-C of three identical polypeptide chains of about 30 kDa united by has been called the liver form (10). The N-terminal segment of disulﬁde bridges and a collagen triple helix. After binding to car- MBL-A comprises 21 amino acid residues which, as in human bohydrates located on the surface of microorganisms, MBL acti- MBL, include 3 cysteine residues. MBL-C has only two cysteine vates the complement system (2). Activation occurs via C4 and C2 residues in the equivalent segment, which has led to the assump-

and is mediated by the MBL-associated serine proteases MBL- tion that MBL-A forms higher oligomers than MBL-C. This has by guest on October 2, 2021 associated serine protease (MASP)-1 and MASP-2 (3, 4). MBL been conﬁrmed for the rat where MBL-C forms dimers or trimers may also directly opsonize microorganisms for phagocytosis (5), and MBL-A forms hexamers of subunits consisting of three iden- probably by interacting with C1q/collectin receptors found on sev- tical polypeptide chains. Moreover, rat MBL-C dimers or trimers, eral types of cells, including macrophages. In humans, low serum unlike rat MBL-A, are reported to be incapable of activating com- levels of MBL or the presence of variant alleles have been corre- plement (10), which has led to the assumption that this may be a lated with a common opsonic defect that predisposes to recurrent general property of MBL-Cs. In mice, the differentiation between infections and may be involved in recurrent abortion (6–8). murine MBL-A (mMBL-A) and mMBL-C is complicated by their identical mobilities on SDS-PAGE in the reduced state, corre-

*Department of Medical Microbiology and Immunology, University of Aarhus, Aar- sponding to polypeptide chains of 28 kDa (11). In this study, we hus, Denmark; †Medical Research Council Immunochemistry Unit, Department of present the purification and characterization of both forms of MBL Biochemistry, University of Oxford, Oxford, United Kingdom; and ‡Immunology and from mouse serum. Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark Materials and Methods Received for publication June 28, 1999. Accepted for publication December 10, 1999. Affinity beads 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 Mannose was coupled to TSK HW/75(F) beads (Tosoh, Tokyo, Japan) with 18 U.S.C. Section 1734 solely to indicate this fact. activated by divinyl sulfone (12). The beads were suspended in 9.1% (v/v) 1 Current address: Immunology and Microbiology, Institute of Medical Biology, Uni- divinyl sulfone in 0.25 M Na2CO3, incubated for 90 min, washed with versity of Southern Denmark, Odense, DK-5000 Odense C, Denmark. water, incubated in 10% (w/v) mannose in 0.5 M Na2CO3 (pH 11) for 24 h 2 Address correspondence and reprint requests to Dr. Steffen Thiel, Department of at room temperature, washed, and incubated for 2 h in 0.1 M ethanolamine Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C, (pH 9.0), washed, and kept in TBS. Denmark. E-mail address: [email protected] Affinity-purified rabbit anti-mouse-IgG Ab (50 mg; see “Preparation of Abs”) was coupled to 5 ml of TSK HW/75(F) beads activated with 3% 3 Abbreviations used in this paper: MBL, mannan-binding lectin; ␣MeGal, ␣-methyl- divinyl sulfone (v/v). The coupling buffer was 15 mM NaHCO containing D-galactose; ␣MeGlc, ␣-methyl-D-glucose; ␣MeGlcNAc, N-acetyl-␣-methyl-D-glu- 3 cosamine; ␣MeL-Fuc, ␣-methyl-L-fucose: ␣MeMan, ␣-methyl-D-mannose; BBS, bar- 135 mM NaCl and 5% (w/v) polyethylene glycol (PEG) 20,000 (pH 8.6). ␤ ␤ bital-buffered saline; MeL-Fuc, -methyl-L-fucose; CRD, carbohydrate recognition Purification of mMBL-A and mMBL-C by carbohydrate affinity domain; EIA, enzyme immunoassay; Fuc, D-fucose; Gal, D-galactose; GalN, D-galactosamine; GalNAc, N-acetyl-D-galactosamine: Glc, D-glucose; GlcN, D-glucosamine; chromatography GlcNAc, N-acetyl-D-glucosamine; HSA, human serum albumin; L-fuc, L-fucose; Man, D-mannose; ManN, mannosamine; ManNAc, N-acetyl-D-mannosamine; MASP, Pooled mouse serum (45 ml), obtained from inbred BALB/c mice or out- MBL-associated serine protease; mMBL, murine mannan-binding lectins; PEG, poly- bred NMRI mice, was mixed with an equal volume of precipitation buffer ethylene glycol; Tw, Tween 20 (polyoxyethylenesorbitan monolaureate). consisting of 10 mM barbital-HCl, 300 mM NaCl, 10 mM CaCl2,15mM

NaN3, and 0.01% (v/v) Tween 20 (Tw) (pH 7.4; barbital-buffered saline bifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysulfos- (BBS)-1/Ca2ϩ) containing 13% (w/v) PEG 6000. The mixture was centri- uccinimide ester (Pierce, Rockford, IL) according to Ref. 16. Rabbits were fuged for 20 min at 2000 ϫ g and the pellet was washed with BBS-1/Ca2ϩ primed by s.c. injection of 0.2 ml of bacillus Calmette-Gue`rin vaccine containing 8.0% (w/v) PEG 6000. The pellet was redissolved in 25 ml of (Statens Serum Institut). Three weeks later the peptide-purified protein ϩ BBS-1/Ca2 and applied to a 25-ml TSK 75/HW(F) precolumn connected derivative of tuberculin conjugate (ϳ35 ␮g adsorbed to 0.2 mg of alumi- to a 25-ml mannose-TSK 75/HW(F) column pre-equilibrated with BBS- num hydroxide gel (Superfos Kemi, Vedbaek, Denmark) in 0.5 ml of 145 ϩ 1/Ca2 . The columns were then washed with the same buffer. The TSK mM NaCl) was emulsified in an equal volume of Freund’s complete ad- precolumn was removed and the mannose-TSK column was eluted with 50 juvant (Difco, Detroit, MI) and half of it was injected s.c. in each of two ϩ ml of BBS-1/Ca2 containing 12 mM glucose (“glucose eluate”). It was rabbits. Booster injections were given 2 wk later with the same dose of Ag ϩ then washed with 50 ml of BBS-1/Ca2 and eluted with 50 ml of BBS- emulsified in Freund’s incomplete adjuvant (Difco) and repeated at 4-wk ϩ 1/Ca2 containing 25 mM mannose (“mannose eluate”). The absorbance of intervals. Antiserum was collected 2 wk after each boost. The antisera fractions at 280 nm was measured and relevant fractions were analyzed by raised against the synthetic peptide are referred to as rabbit anti-mMBL-CЈ. rocket immunoelectrophoresis, acid/urea-PAGE, and N-terminal amino acid sequencing. Purification of mMBL-A and -C by affinity and ion-exchange Complement activation assay chromatography Microtiter wells (FluoroNunc from Nalge-Nunc, Roskilde, Denmark) were coated overnight with 70 ng of mannan in 100 ␮l of coating buffer (15 mM Mouse serum (135 ml) was mixed with an equal volume of buffer consist- Na2CO3, 35 mM NaHCO3,30mMNaN3). Wells were emptied and incu- ing of 20 mM barbital-HCl, 40 mM CaCl2, 1.0 M NaCl, 0.08% (v/v) emul- phogene, 100 ␮M benzamidine, 100 ␮M iodoacetamide, and 100 ␮M cy- bated for 2 h with TBS/Tw/HSA (10 mM Tris-HCl, 140 mM NaCl, 15 mM NaN3, 0.05% (v/v) Tw, 0.1% (w/v) HSA (pH 7.4)). After washing with clocaprone (pH 7.4). The diluted serum was applied to a 70-ml TSK 2ϩ precolumn connected to a 70-ml mannose-TSK column pre-equilibrated TBS/Tw/Ca (TBS/Tw/HSA without HSA but including 5 mM CaCl2), duplicate wells were incubated overnight at 4°C with dilutions of purified with 10 mM barbital-HCl, 20 mM CaCl2, 0.5 M NaCl, 0.04% (v/v) emul- Downloaded from phogene, 50 ␮M benzamidine, 50 ␮M iodoacetamide, and 50 ␮M cyclo- human MBL (Statens Serum Institut), mMBL-A, or mMBL-C in BBS/Tw/ ϩ caprone (pH 7.4; BBS-2/NaCl/Ca2 ). After loading, the columns were HSA (10 mM barbital sodium, 145 mM NaCl, 15 mM NaN3, 0.05% (v/v) ϩ ␮ washed with BBS-2/NaCl/Ca2 followed by the same buffer with the NaCl Tw, 0.01% (w/v) HSA (pH 7.4)) containing enzyme inhibitors (100 M ϩ ␮ ␮ ␮ concentration reduced to 0.15 M NaCl (BBS-2/Ca2 ). The precolumn was PMSF, 100 M iodoacetamide, 100 M benzamidine, 100 M cycloca- ␮ removed and a 10-ml rabbit anti-mouse IgG Ab column was attached to the prone, 100 M phenanthroline) and either 10 mM CaCl2 or 10 mM EDTA. 2ϩ outflow of the mannose-TSK column. The columns were washed with The wells were washed with TBS/Tw/Ca or TBS/Tw/EDTA (TBS/Tw 2ϩ with 5 mM EDTA instead of CaCl2), respectively, and then with TBS/Tw/ BBS-2/Ca and eluted with BBS-2 containing 20 mM EDTA instead of ϩ

2 http://www.jimmunol.org/ calcium. Fractions with a high content of mMBL, as determined by SDS- Ca . The MBL-reacted wells were then incubated for3hat37°C with 2 PAGE in the reduced state, were pooled and diluted with three volumes of human serum devoid of MBL and C1q, diluted 1:90 in TBS/Tw/Ca . The Mono-Q loading buffer (20 mM piperazine-HCl, 5 mM EDTA, 0.03% serum was obtained from an MBL-deficient subject (serum MBL Ͻ10 (v/v) Tw, 100 ␮M PMSF, 100 ␮M benzamidine, and 100 ␮M cyclocap- ng/ml) and C1q was removed by chromatography on Biorex 70 (Bio-Rad, rone (pH 6.2)). The pH was adjusted to 6.2 and the pool was applied to a Bedford, MA) (17). Any remaining MBL was removed from the serum by 1-ml Mono-Q column (Amersham Pharmacia Biotech, Uppsala, Sweden). passing it through mannan-coupled Sepharose (15) in the presence of 2ϩ The effluent, containing mMBL-A, was collected and stored at 4°C. The CaCl2. Wells were then washed in TBS/Tw (TBS/Tw/Ca without CaCl2) column was washed with 10 ml of Mono-Q loading buffer and eluted in a and incubated for 3 h with 100 ␮l TBS/Tw containing 10 ␮g HSA and 100 Ј stepwise manner with the same buffer containing NaCl in concentrations of ng biotinylated F(ab )2 fragment of rabbit anti-human C4 Ab (Dako, 127 mM, 750 mM, and 1.0 M. The bulk of mMBL-C was found in the 750 Glostrup, Denmark). Wells were washed in TBS/Tw and incubated for 2 h Ϫ ␮ mM NaCl eluate, which was frozen and kept at 70°C. Aliquots (120 l) with 10 ng europium-labeled streptavidin (Wallac, Turku, Finland) in 100 by guest on October 2, 2021 from the 750 mM NaCl eluate were analyzed by gel-permeation chroma- ␮l TBS/Tw containing 25 ␮M EDTA and 0.01% (w/v) HSA. After wash- tography on a TSK 3000 SW column (0.75 ϫ 60 cm) (Pharmacia LKB, ing with TBS/Tw, bound europium was released into the fluid phase by Uppsala, Sweden) with a TSK SW precolumn (0.75 ϫ 7.5 cm) (Pharmacia incubation for 10 min with enhancement buffer (Wallac). The amount of LKB) in a buffer consisting of 50 mM ammonium acetate, 450 mM NaCl, europium in each well was assessed by time-resolved fluorometry on a 10% (v/v) acetonitrile, 5 mM EDTA, 50 ␮M PMSF, 50 ␮M benzamidine, Delphia fluorometer (Wallac). and 50 ␮M cyclocaprone (pH 6.8). Acetonitrile was included to minimize hydrophobic interactions. The column was calibrated with cytochrome c (13 kDa; Sigma, St. Louis MO), human serum albumin (HSA) (69 kDa; Carbohydrate selectivity Statens Serum Institut, Copenhagen, Denmark), and aldolase (158 kDa), catalase (232 kDa), and thyroglobulin (669 kDa) from Amersham Phar- Purified MBL was biotinylated with 40 ␮g N-hydroxysuccinimidobi- macia Biotech. Fractions were stored at Ϫ70°C and analyzed by SDS- otin/mg of MBL. Microtiter wells (FluoroNunc) were coated overnight at PAGE. The effluent from the Mono-Q column was concentrated on a 1-ml room temperature with 13 ng mannan in 100 ␮l coating buffer. Plates were Resource-Q column (Amersham Pharmacia Biotech) equilibrated in Re- blocked and washed as described above. Dilutions of monosaccharides in source-Q loading buffer (20 mM Tris-HCl, 0.03% (v/v) Tw, 100 ␮M 50 ␮l TBS/Tw/Ca2ϩ were added in duplicate to the wells by means of a PMSF, 100 ␮M benzamidine, and 100 ␮M cyclocaprone (pH 8.6)). Tris robotic pipetting system (Packard, Meriden, CT). Negative and positive was added to the Mono-Q effluent to a final concentration of 20 mM and controls consisting of TBS/Tw/EDTA or TBS/Tw/Ca2ϩ without monosac- pH was adjusted to 8.6. After loading, the column was eluted with Re- charides were included. Biotinylated MBLs at 0.15 ␮g/ml (mMBL-A), source-Q loading buffer containing 750 mM NaCl. Fractions were stored at 0.06 ␮g/ml (mMBL-C), or 0.04 ␮g/ml (human MBL) in TBS/Tw/Ca2ϩ 4°C and analyzed by SDS-PAGE with silver staining. Protein concentra- containing 0.01% (w/v) HSA were then added in duplicate 50-␮l volumes. tions of the purified preparations were estimated by their absorbance at 280 The solutions were mixed on a shaking platform and incubated overnight nm using extinction coefficients (E1cm, 1 mg/ml) of 0.63 for mMBL-A and at 4°C. The monosaccharides tested comprised D-mannose (Man), ␣-meth- 0.90 for mMBL-C calculated from the deduced amino acid sequences (13) yl-D-mannose (␣MeMan), D-mannosamine (ManN), N-acetyl-D-man- according to Ref. 14. nosamine (ManNAc), D-glucose (Glc), ␣-methyl-D-glucose (␣MeGlc), D- Preparation of Abs glucosamine (GlcN), N-acetyl-D-glucosamine (GlcNAc), D-galactose (Gal), ␣-methyl-D-galactose (␣MeGal), D-galactosamine (GalN), N-acetyl- Rabbit anti-mouse IgG (H ϩ L chain) Abs were affinity purified from D-galactosamine (GalNAc), D-fucose (Fuc), and L-fucose (L-fuc); all were locally produced antiserum using mouse IgG coupled to TSK beads. purchased from Sigma. All were tested at concentrations ranging from Rabbit antiserum raised against mMBL was produced as described pre- 0.184 to 100 mM. The wells were washed and developed with europium- viously (15). This antiserum recognizes mMBL-C as well as mMBL-A and labeled streptavidin as described above. The background was defined as the is referred to as anti-mMBL antiserum. A specific mMBL-C Ab was raised average signal of three wells incubated with biotinylated MBL in the pres- against a synthetic peptide (Immune Systems, Bristol, U.K.) representing ence of EDTA; the maximum signal was that obtained in buffer without hydrophilic loops 1 and 2 of the mMBL-C CRD. The peptide comprised monosaccharide. Fluorescence intensities from 103 to 106 counts/s were amino acid residues 184–202 (DVRVEGSFEDLTGNRVRYT) from the obtained with Ͻ3% variation between duplicates. Each monosaccharide deduced sequence (13) with an additional cysteine residue at its C termi- was tested in at least five different experiments, and various combinations nus. The peptide (0.24 mg) was conjugated to 1 mg of purified protein of five different monosaccharides were tested on the same microtiter plate derivative of tuberculin (Statens Serum Institut) by means of the hetero- to determine their individual ranking. 2612 MANNAN-BINDING LECTIN A AND C IN MOUSE SERUM

Estimation of mMBL by rocket immunoelectrophoresis Rocket immunoelectrophoresis was performed as described (15) in agarose gels containing 6.7% (v/v) rabbit anti-mMBL antiserum. Samples of pu- riﬁed mMBL-A and two mouse sera were used as standards and controls. The mMBL-A in serum could be quantiﬁed since this precipitate was stron- ger and of a shape distinct from that of mMBL-C. The presence of two independent precipitates shows that separate Abs recognize mMBL-A and mMBL-C.

Gel-permeation chromatography of mouse serum Serum from BALB/C mice (100 ␮l) was subjected to gel-permeation chromatography on a Superose 6 column (30 ϫ 1.0 cm, HR 10/30; Amersham Pharmacia Biotech) equilibrated with running buffer (TBS/Tw containing 2.5 mM EDTA). Chromatography was performed at a ﬂow rate of 0.5 ␮ ml/min, and 0.5-ml fractions were collected. Each fraction was precipitated FIGURE 1. A, Rocket immunoelectrophoresis of 10 l mouse serum with 0.6 ml acetone for 2–3 h at Ϫ20°C and centrifuged at 10,000 ϫ g for (I), 10 ␮l glucose eluate (II), and 10 ␮l mannose eluate (III) from a man- 20 min at 4°C. The pellets were dried in an evaporating centrifuge and nose-TSK column loaded with mouse serum. The agarose gel contained redissolved in 25 ␮l sample buffer for SDS-PAGE and Western blotting. rabbit antiserum raised against puriﬁed mMBL. B, Acid/urea-PAGE anal- The column was calibrated with the following proteins: thyroglobulin (669 ysis of EDTA eluate (I) (2 ␮g protein), glucose eluate (II) (2 ␮g protein), kDa), ferritin (330 kDa; Amersham Pharmacia Biotech), catalase (232 and mannose eluate (III) (2 ␮g protein) from the mannose-TSK column. kDa), and HSA (70 kDa). The elution volume of human C1q and IgM was The marker lane contains HSA (69 kDa), catalase (65 kDa), aldolase (40 assessed by gel permeation of human serum in the presence of 10 mM Downloaded from kDa), and ferritin (23 kDa). Samples were reduced before electrophoresis EDTA. The content of C1q and IgM in the fractions was measured by enzyme immunoassay (EIA). and the gel was silver stained.

PAGE SDS-PAGE was performed in a discontinuous buffer system (18) on 6.5– 20% gradient gels. Proteins bands were silver stained as described (19) Mice http://www.jimmunol.org/ with the following modifications: Formalin fixation, rinsing with H2O, and Sera from laboratory strains of mice were obtained from Bommice (Bom- dehydration in acetone were prolonged to 15 min each, and silver impreg- holtgaard Breeding and Research Center, Ry, Denmark), whereas those nation was conducted with a solution of 0.2% (w/v) AgNO3 and 0.25% from wild mice were obtained from the Laboratoire Ge`nome et Populations (v/v) Formalin. Molecular weights were estimated by comparison with (Universite` de Montepellier II, Montpellier, France). Sera were kept at prestained marker proteins (Amersham Pharmacia Biotech). Ϫ20°C. Acid/urea-PAGE was performed as described previously (20, 21) on uniform vertical slab gels (15% (w/v) acrylamide and 0.1% (w/v) bis- acrylamide) in 6.0 M urea and 5.4% (v/v) acetic acid (pH 2.5). The elec- Results trophoresis buffer was 5.4% (v/v) acetic acid (pH 2.5). Gels were prerun Two MBL in mouse serum overnight in electrophoresis buffer at a fixed current of 1 mA. Samples Two serum forms of mMBL were found during work to improve containing 1–2 ␮g protein were dried in an evaporating centrifuge and by guest on October 2, 2021 redissolved in 15 ␮l 20 mM sodium borate buffer (pH 9.0) containing 8 M the purification of mMBL-A. The eluates obtained from differen- urea and 30 mM DTT. Sample solutions were then boiled for 3 min and tial monosaccharide elution of a mannose affinity column loaded acidified with acetic acid to a final concentration of 10% (v/v). Electro- with the 6.5% PEG 6000 precipitate of mouse serum were ana- ϳ phoresis was performed at a fixed current of 9 mA for 5h. lyzed by rocket immunoelectrophoresis (Fig. 1). The MBL in the 10 mM glucose eluate formed rockets of a shape and size consis- Western blotting tent with the mMBL-A content found in previous mMBL-A puri- Protein bands from PAGE were electroblotted onto polyvinylidene diflu- fications (15). Murine MBL-A eluted over a relatively large num- oride membranes (Immobilon P; Millipore, Bedford, MA) (22). Mem- ber of fractions and the recovery was ϳ30% of the amount applied branes were blocked with TBS containing 0.1% (v/v) Tw for 30 min, cut to the column as determined by rocket immunoelectrophoresis. into strips, incubated with Ab dilution, and developed by the alkaline phosphatase method (23). Strips were stained with colloidal gold to visualize When the mannose eluate was analyzed, precipitates of weak in- the proteins on the blot (24). For estimating the apparent molecular size of tensity and with a pointed shape unlike that of mMBL-A rockets mMBL by gel-permeation chromatography of mouse serum, the Western were observed. These pointed rockets varied in character and in- blots of the fractions were incubated with biotinylated second Ab and de- tensity between different runs and were also occasionally observed veloped by enhanced chemiluminescence using HRP-labeled streptavidin when whole mouse serum was analyzed. SDS-PAGE of aliquots (Dako) at 0.2 ␮g/ml and luminescence reagent (Pierce). Stripping of blots developed by enhanced chemiluminescence was performed by incubation from the glucose and mannose eluates showed that the major pro- in denaturing and reducing buffer (62.5 mM Tris-HCl, 2.0% (w/v) SDS, tein in each eluate had a mobility corresponding to 28 kDa in the 0.078% (v/v) 2-ME (pH 6.9)) at 70°C for 45 min. Blots were then washed reduced state (data not shown). To explain the different appear- overnight in TBS and developed with another Ab. ances on rocket immunoelectrophoresis, the 28-kDa protein bands were blotted onto Problot membranes and subjected to N-terminal N-terminal sequencing sequencing. Analysis of the protein in the glucose eluate yielded The proteins obtained by differential glucose and mannose elution of a the first 25 amino acid residues of mMBL-A, whereas the sequence mannose-TSK column were subjected to N-terminal amino acid sequenc- of the protein in the mannose eluate corresponded to the first 20 ϳ ␮ ing. Eluate (0.5 ml) containing 30 g reduced mMBL was applied to amino acid residues of mMBL-C. In both cases, the identity to the SDS-PAGE. After electrophoresis gels were equilibrated for 10 min in transfer buffer (10 mM 3-cyclohexylamino-1-propanesulfonic acid and published deduced amino acid sequence was 100% and no cross- 10% (v/v) methanol (pH 11)) and the protein bands were transferred to contamination was observed. Acid/urea-PAGE of the glucose and Problot membranes (PE Applied Biosystems, Foster City, CA) at 7.5 mannose eluates confirmed the presence of two different forms of volt/cm for 10 h. Protein bands were visualized with Ponceau S, and bands mMBL (Fig. 1). The electrophoretic mobilities of the two proteins appearing at 28 kDa were cut out and sequenced on an Applied Biosystems Ͼ 470/120A sequencer. Similarly, the N-terminal sequence of the 21-kDa in this system (mMBL-A mMBL-C) were consistent with the band appearing in the mMBL-C preparations after the ion-exchange chro- pIs calculated from their deduced amino acid compositions (7.6 for matography (see above) was obtained. mMBL-A and 4.8 for mMBL-C). The Journal of Immunology 2613

FIGURE 2. Specificity of the anti-mMBL antisera by Western blotting. ␮ A, Reduced mMBL-A, 2 g. B, Reduced EDTA eluate from mannose-TSK Downloaded from (containing both mMBL-A and mMBL-C). Strips: stained with gold (I), FIGURE 4. Identification of mMBL-C split product by SDS-PAGE and incubated with normal rabbit serum (II), incubated with rabbit anti-mMBL Western blotting. Strips show reduced 50-␮l samples of the 750 mM NaCl antiserum (III), and incubated with anti-mMBL-CЈ antiserum (IV and V) eluate from Mono-Q stained with gold (I), incubated with biotinylated from each of the two rabbits. Bound Ab was detected with alkaline phos- rabbit anti-mMBL-CЈ Ab (II), and incubated with biotinylated normal rab- phatase-conjugated anti-rabbit IgG. bit IgG (III). Strips II and III were developed with alkaline phosphatase- conjugated avidin. http://www.jimmunol.org/ Anti-mMBL-CЈ Ab The specificity of the anti-mMBL-CЈ antisera was tested by EIA EDTA, removal of Ig by passage through an anti-mouse IgG col- and Western blotting. EIA showed high Ab binding in wells coated umn, ion-exchange chromatography at pH 6.2 on a Mono-Q col- with the synthetic peptide Ag and no binding to wells coated with umn, gel-permeation chromatography on TSK 3000 column, and irrelevant peptides. No specific binding was observed in wells ion-exchange chromatography on a Resource-Q column. Fractions coated with preparations of native mMBL. Western blotting of from the different eluates were analyzed by SDS-PAGE and silver previously purified mMBL-A (15) and a preparation containing staining (Fig. 3). The two forms of mMBL were well separated by anion-exchange chromatography at pH 6.2. The effluent con- both mMBL-A and mMBL-C (the EDTA eluate of a mannose- by guest on October 2, 2021 TSK column loaded with mouse serum) showed that antisera from tained mMBL-A but no mMBL-C, as judged by Western blotting Ј the two rabbits recognized mMBL-C but not mMBL-A (Fig. 2). using the biotinylated anti-mMBL-C Ab (Fig. 2A), while the bulk of the mMBL-C was eluted with 750 mM NaCl. This eluate con- Purification of mMBL-A and mMBL-C tained no mMBL-A when analyzed by rocket immunoelectrophoresis, even when concentrated 10-fold by acetone precipitation. The purification of mMBL-A and mMBL-C was monitored by Controls showed nearly 100% recovery of MBL-A after acetone rocket immunoelectrophoresis against rabbit anti-mMBL anti- precipitation. serum and by Western blotting against biotinylated rabbit anti- After concentrating mMBL-A on the Resource-Q column, a mMBL-CЈ Ab, respectively. The two proteins were purified by faint band at 65 kDa was observed in the reduced state (Fig. 3C). affinity chromatography on a mannose-TSK column eluted with This probably represents incompletely reduced mMBL-A, as it bound to the rabbit anti-mMBL-A Ab (results not shown).

FIGURE 3. SDS-PAGE of fractions from the puriﬁcation of the mMBLs. A, The EDTA eluate from linked mannose-TSK and antimouse FIGURE 5. Complement activation. C4 deposition in mannan-coated IgG columns. B, The 750 mM NaCl eluate from the Mono-Q column. C, microtiter wells mediated by mMBL-A, mMBL-C, and human MBL in the The Resource-Q eluate. D, Void-volume fraction from gel-permeation presence of Ca2ϩ or EDTA. Immobilized C4b was measured by time- chromatography. Samples were reduced and gels were silver stained. resolved ﬂuorometry. 2614 MANNAN-BINDING LECTIN A AND C IN MOUSE SERUM

Table I. Monosaccharide inhibition of MBL-binding to mannana

Monosaccharide mMBL-A I50 mMBL-C I50 Human MBL I50

L-fuc 1.1 (0.79) 7.8 (3.9) 18 (1.3) ␣MeMan 1.4 (1.0) 2.0 (1.0) 14 (1.0) GlcNAc 1.5 (1.1) 11 (5.5) 16 (1.1) Man 1.5 (1.1) 4.0 (2.0) 14 (1.0) ␣MeGlc 1.8 (1.3) 34 (17) 49 (3.5) Glc 2.0 (1.4) 29 (15) 63 (4.5) ManNAc 3.8 (2.7) 9.8 (4.9) 26 (1.9) Fuc 38 (27) 85 (43) Ͼ100 (Ͼ71) Gal 52 (37) Ͼ100 (Ͼ71) Ͼ100 (Ͼ71) GlcN 56 (40) Ͼ100 (Ͼ71) Ͼ100 (Ͼ71) ManN 78 (56) 76 (41) Ͼ100 (Ͼ71) ␣MeGal Ͼ100 (Ͼ71) Ͼ100 (Ͼ71) Ͼ100 (Ͼ71) GalN Ͼ100 (Ͼ71) Ͼ100 (Ͼ71) Ͼ100 (Ͼ71) GalNAc Ͼ100 (Ͼ71) Ͼ100 (Ͼ71) Ͼ100 (Ͼ71)

a Concentration (mM) of monosaccharide required for 50% inhibition (I50). I50 ␣ values in parentheses are calculated relative to the I50 value of -methyl-mannose.

SDS-PAGE of the 750 mM NaCl eluate containing mMBL-C Downloaded from (Fig. 3B) revealed the presence of new protein bands between 30 and 40 kDa and a strong band at 21 kDa not present in the EDTA eluate from the mannose-TSK column (Fig. 3A). The N-terminal FIGURE 6. Oligomerization of mMBLs analyzed by Western blotting. sequence of the 21-kDa band was identical to that of mMBL-C and Lane I, 0.5 ␮g mMBL-A, reduced (glucose eluate from mannose-TSK); the band was stained on Western blots with rabbit anti-mMBL-CЈ lane II, the same, unreduced; lane III, mMBL-A and mMBL-C, reduced

Ab (Fig. 4). An aliquot from the 750 mM NaCl eluate was sub- (10% PEG 6000 precipitate of mouse serum); and lane IV, the same, un- http://www.jimmunol.org/ jected to gel-permeation chromatography on a TSK 3000 SW col- reduced. Left panel, incubated with biotinylated anti-mMBL Ab. Right umn. Two major absorbance peaks at 280 nm, of approximately panel, incubated with biotinylated anti-mMBL-CЈ Ab. Blots were devel- equal size, were observed (data not shown). The first peak emerged oped with alkaline phosphatase-conjugated avidin. with the void volume (Ͼ600 kDa), while the second peak emerged at a volume corresponding to 350 kDa. Minor peaks were observed in later fractions. SDS-PAGE of reduced fractions showed the by rocket immunoelectrophoresis, whereas the final recovery was presence of 28-kDa and 21-kDa bands in fractions corresponding 52%. The protein content in 135 ml serum was measured to 8060 to molecular masses from Ͼ600 to 230 kDa. In these fractions, the mg (according to the method of Lowry), and the mMBL-A content intensity of the 21-kDa band paralleled that of the 28-kDa was measured by rocket immunoelectrophoresis to 3.01 mg. The by guest on October 2, 2021 mMBL-C band. Two fractions in the void volume contained specific MBL-A content is thus 0.37 ␮g MBL-A/mg protein. Sim- Ͼ95% pure mMBL-C, as judged by SDS-PAGE and silver stain- ilarly, the specific MBL-A content of the purified mMBL-A can be ing, with only trace amounts of other bands at 70 and 100 kDa in calculated to 1.6 mg mMBL-A/mg protein, estimated according to the reduced state (Fig. 3D). Fractions from the other major peak at the Lowry method with HSA as standard. The purification factor 350 kDa contained bands corresponding to mMBL-C as well as can thus be calculated at 4251. Based on these recoveries, the other bands at 40, 70, and 100 kDa under reducing conditions. The concentration of mMBL-A in serum was calculated at ϳ23 ␮g/ml. Ͼ600-kDa peak was used in subsequent analyses for complement The amount of mMBL-C recovered in the void volume fractions activation and carbohydrate-binding specificity. was estimated as 20 ␮g by absorbance measurements. If all of the Approximately 1.6 mg mMBL-A was purified from 135 ml 750 mM NaCl eluate had been applied to gel-permeation chroma- mouse serum as estimated by absorbance at 280 nm. Recovery of tography, ϳ0.25 mg mMBL-C would have been recovered in these mMBL-A in the eluate from the mannose-TSK column was 59% fractions.

Table II. Monosaccharide speciﬁcities of different MBLsa

Monosaccharide

Human MBL (serum) (25) GlcNAc Ͼ Man, L-fuc Ͼ ManNAc ϾϾ Glc Human MBL (serum) ␣MeMan, Man Ͼ GlcNAc Ͼ L-Fuc Ͼ ManNAC ϾϾ ␣MeGlc Ͼ Glc Chicken MBL (serum) (26) ManNAc Ͼ L-fuc Ͼ Man Ͼ GalN Ͼ GlcNAc ϾϾ Glc Bovine MBL (serum) (27) L-fuc, Man, ManNAc ϾϾ GlcNAc ϾϾϾ Glc Rat MBL-A (serum) (28) L-fuc, ManNAc, ␣MeMan Ͼ Man Ͼ GlcNAc Ͼ Glc ϾϾ GlcN Ͼ Gal Rat MBL-A (rCRD) (29) L-fuc ϾϾ Man Ͼ Glc Ͼ GlcNAc Rat MBL-A (rCRD) (30) ␣MeL-Fuc ϾϾϾ ␣MeMan ϾϾ ␤MeL-Fuc, ␣MeGlcNAc ϾϾϾ Gal mMBL-A (serum) (15) L-fuc Ͼ Man, GlcNAc ϾϾ Glc Ͼ ManNAc ϾϾϾ Gal mMBL-A (serum) L-fuc Ͼ ␣MeMan, Man, GlcNAc Ͼ ␣MeGlc Ͼ Glc Ͼ ManNAc ϾϾϾ Fuc Ͼ Gal, GlcN Ͼ ManN Rat MBL-C (rCRD) (30) ␣MeMan Ͼ ␣MeL-Fuc ϾϾ ␣MeGlcNAc ϾϾ ␣MeL-Fuc ϾϾϾ Gal mMBL-C (serum) ␣MeMan Ͼ Man Ͼ L-fuc Ͼ ManNAc Ͼ GlcNAc ϾϾϾ Glc Ͼ ␣MeGlc ϾϾϾ ManN Ͼ Fuc a Ͼ Monosaccharide speciﬁcities based on 50% inhibition (I50)orKd values of different MBLs, denotes more than 10% higher I50 value than the previous monosaccharide, ϾϾ ϾϾϾ denotes more than 50% higher I50 value than the previous monosaccharide, and denotes more than 100% higher I50 value than the previous monosaccharide. Results from the present work are underlined. The Journal of Immunology 2615 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 7. Oligomerization of mMBLs analyzed by gel-permeation chromatography. Mouse serum (100 ␮l) was applied to a Superose 6 column precalibrated with IgM (1; 970 kDa), human C1q (2; 462 kDa), thyroglobulin (3; 669 kDa), ferritin (4; 330 kDa), catalase (5; 232 kDa), and HSA (6; 69 kDa). A, Absorbance at 280 nm. B, Fractions analyzed by Western blotting against biotinylated rabbit anti-mMBL-CЈ Ab. C, The same blots stripped and incubated with biotinylated rabbit anti-mMBL Ab. Blots were developed with streptavidin-conjugated HRP followed by enhanced chemiluminescence detection.

Complement activation Oligomer state of mMBL-A and mMBL-C The capacity of mMBL-A and mMBL-C to activate complement The oligomer state of mMBLs was examined by SDS-PAGE under was determined by measuring C4 deposition in mannan-coated nonreducing conditions and Western blotting (Fig. 6). The lack of microtiter wells, with human serum depleted of MBL and C1q as a specific mMBL-A Ab made it necessary to use a preparation of the complement source. As shown in Fig. 5, both proteins as well mMBL-A which contained no mMBL-C. The glucose eluate from as human MBL mediated C4 deposition, but mMBL-C was less mannose-TSK was chosen since this preparation had been sub- active than mMBL-A and human MBL. Although mMBL-A at 3 jected to the least handling. In parallel, a 10% PEG 6000 precip- ng/ml produced a fluorescence intensity of 105 counts/s, 16 ng itate of mouse serum was blotted against rabbit anti-mMBL-CЈ Ab. mMBL-C/ml was required to produce the same signal. In the pres- Both mMBL-A and mMBL-C were observed as a complex mix- ence of EDTA, none of the MBLs produced C4 deposition. ture of oligomers, varying from dimers of polypeptide chains at 60 kDa to complexes of more than 200 kDa. The pattern was similar Carbohydrate specificity for the two types of mMBL. Normal mouse serum was subjected The relative potencies of monosaccharides in inhibiting the bind- to gel-permeation chromatography and fractions were analyzed by ing of biotinylated mMBL-A, mMBL-C, and human MBL are SDS-PAGE and Western blotting. The blots were developed with given in Table I and compared with previous published observa- rabbit anti-mMBL-CЈ Abs, stripped, and then developed with rab- tions in Table II. Note the different sensitivity toward inhibition bit anti-mMBL Abs (Fig. 7). mMBL-C was found in two peaks with Glc, ␣MeGlc, and L-fuc. corresponding to a molecular size of ϳ600 kDa and ϳ150 kDa 2616 MANNAN-BINDING LECTIN A AND C IN MOUSE SERUM Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 8. Serum mMBL-A concentrations in laboratory strains of mice (A) and wild mice (B), estimated by rocket immunoelectrophoresis.

(for globular protein markers, Fig. 7a), the first being the main Concentrations of mMBL-A in mouse sera (Fig. 7b). mMBL-A also emerged at a position corresponding to a molecular mass of about 600 kDa, but without the second peak Concentrations of mMBL-A in sera from laboratory strains of observed for mMBL-C (Fig. 7c). Purified mMBL-A was analyzed mice and wild mouse species and strains were estimated by rocket on the same column. The UV chromatogram and SDS-PAGE of immunoelectrophoresis in gels containing the anti-mMBL anti- fractions showed that it emerged as a single peak corresponding to serum and with purified mMBL-A as standards (Fig. 8). Serum an average molecular mass of 600 kDa. mMBL concentrations determined by this method varied between The Journal of Immunology 2617

5 and 50 ␮g/ml in laboratory strains of mice. The lowest concen- ation columns at a position corresponding to penta- and hexamers tration was found in the DW/DW homozygous dwarf strain. with an average size of 600 kDa, in agreement with the size of rat Greater variation was found among individuals in wild strains. The MBL-A (10, 15). Analysis of mouse serum by gel-permeation least concentration observed in wild mice was 5 ␮g/ml (Mus mus- chromatography and Western blotting showed that mMBL-C culus domesticus and Mus musculus musculus). The highest con- emerged primarily as a peak at ϳ600 kDa, probably representing centration of Ͼ120 ␮g/ml was found in a single mouse of the hexamers, with a lower amount emerging at ϳ150 kDa. Purified species M. caroli. In general, wild mice showed higher serum mMBL-C was found in fractions corresponding to 230–600 kDa mMBL-A concentrations than laboratory strains. on gel-permeation chromatography. MBL-C isolated from rat serum or liver was earlier reported to have an average size of 200 kDa as estimated by gel-permeation chromatography (10, 36). The Discussion rat serum MBL-C was purified by a different procedure in which Two forms of MBL were identified in mouse serum by their N- it was separated from MBL-A by gel-permeation chromatography. terminal amino acid sequences, mobilities on acid/urea PAGE, and High-molecular-weight rat MBL-C may have been lost in these reactivities with an Ab to a synthetic peptide from the deduced experiments and the question of the size of MBL-C in rat serum amino acid sequence of mMBL-C. Both forms of murine MBL has so far not been directly addressed. Judged from the complex gave bands at 28 kDa on SDS-PAGE in the reduced state. pattern of oligomers observed by SDS-PAGE, the disulfide bond- Two murine serum forms of MBL have been observed before ing observed in mMBL-C clearly deviates from the pattern found (11). The initial identification of mMBL-C in serum was based on in recombinant rat MBL-C (37). affinity isolation using Ra chemotypes of Salmonella and analysis Analysis of the carbohydrate specificity showed that human by acid/urea-PAGE. Two forms of MBL have also been identified MBL resembles that of mMBL-C more than that of mMBL-A, in Downloaded from in rat serum, where the MBL-C form is described as a minor com- agreement with the suggested evolution of MBL genes (38). Like- ponent compared with MBL-A (10, 31). There has been a tendency wise, the specificities of mMBLs resemble those of their rat ana- in the literature to regard MBL-C as a hepatic protein involved in logues and that of mMBL-A concords with previously reported the intracellular transport of high mannose-type oligosaccharides data (Table II) (15). Remarkable is the difference in the specific- (32). In contrast to this notion, we find that mMBL-C is a circu- ities of mMBL-A and MBL-C, especially the high affinity of the lating protein present at serum levels comparable to those of former for Glc and ␣-MeGlc. It is likely that the differences in http://www.jimmunol.org/ mMBL-A. Judged from the recovery and the total yield of monosaccharide specificity leads to preferential binding to differ- mMBL-A purified, an approximate serum concentration of 23 ent microorganisms, depending on the composition of glycocon- ␮g/ml was calculated. mMBL-C could not be quantified, but the jugates in their outer wall. experiences gathered suggest a concentration similar to that of Serum concentrations of mMBL-A, estimated by rocket immu- mMBL-A. noelectrophoresis against mMBL-A standards, were found to It is possible that MBL-C may differ in abundance and function range from 5 to 40 ␮g/ml in laboratory strains of mice and, with among different rodent species. Another possibility is that MBL-C a single exception, in wild mice from 5 to 80 ␮g/ml. The finding often escapes detection because of its sensitivity to proteolytic of higher mMBL levels in many wild mice is in line with previous by guest on October 2, 2021 degradation. Purification of mMBL-C was only possible in the measurement (15) in wild yellow-necked mice (M. cervicolor). presence of protease inhibitors and preliminary attempts to purify The variation in serum mMBL in different mice, while significant, rat MBL-C showed this to be even more susceptible to is small in comparison with the 500-fold variation (from Ͻ10 degradation. ng/ml to 5 ␮g/ml) observed in humans. After ion-exchange chromatography at pH 6.2, a 21-kDa frag- The demonstration of both mMBL-A and mMBL-C as serum ment of mMBL-C was observed. The relative amount of the 21- forms capable of activating the complement system should be re- kDa fragment increased if purification of mMBL-C was conducted membered when considering animal models for MBL deficiency. at room temperature or in the absence of protease inhibitors, or when purified mMBL-C was kept at 4°C. The 21-kDa fragment Acknowledgments has a N-terminal sequence identical to the terminus of mMBL-C. Both forms of mMBL mediated C4 activation. mMBL-A was as We thank Dr. Palle Holt for providing the rabbit anti-mMBL antiserum and active as human MBL, whereas mMBL-C was approximately one- Professor Dr. Otto Uttenthal for advice and critical comments on this manuscript. fifth as active. Whether the lower activity of mMBL-C reflects an inferior activity in vivo is open to question, as mMBL-C is susceptible to proteolytic degradation and may be split into the 21- References kDa fragment during incubation at 37°C, with impairment of com- 1. Turner, M. W. 1996. Mannose-binding lectin: the pluripotent molecule of the plement activation. The finding that mMBL-C activates innate immune system. Immunol. Today 17:532. 2. Ikeda, K., T. Sannoh, N. Kawasaki, T. Kawasaki, and I. Yamashina. 1987. Serum complement contrasts with the previous observation that MBL-C lectin with known structure activates complement through the classical pathway. isolated from rat liver was incapable of complement activation (2). J. Biol. Chem. 262:7451. The same has been reported to be the case for human MBL isolated 3. Matsushita, M., and T. Fujita. 1992. Activation of the classical complement pathway by mannose- binding protein in association with a novel C1s-like serine from liver, and also some recombinant human MBL shows a low protease. J. Exp. Med. 176:1497. activating potential (33, 34). The reported lack of complement ac- 4. Thiel, S., T. Vorup Jensen, C. M. Stover, W. Schwaeble, S. B. Laursen, K. Poulsen, A. C. Willis, P. Eggleton, S. Hansen, U. 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silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem. Mbl1 and Mbl2 reveals features common to other collectin genes. Mamm. Ge- by guest on October 2, 2021 145:315. nome 6:103.