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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 concen- trations 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-galac- tosamine; 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 Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 2611 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”).