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*
Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark;
Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom; and
Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 µg/ml, with wild mice tending to
show higher levels than laboratory strains. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MBL is synthesized by hepatocytes and has been isolated from the liver or serum of several vertebrate species. Only one form of human MBL has been characterized, whereas two forms are found in rabbits, rats, mice, and rhesus monkeys (9). So far MBL-A has been considered to be the serum form in rodents, whereas MBL-C has been called the liver form (10). The N-terminal segment of MBL-A comprises 21 amino acid residues which, as in human MBL, include 3 cysteine residues. MBL-C has only two cysteine residues in the equivalent segment, which has led to the assumption that MBL-A forms higher oligomers than MBL-C. This has been confirmed for the rat where MBL-C forms dimers or trimers and MBL-A forms hexamers of subunits consisting of three identical polypeptide chains. Moreover, rat MBL-C dimers or trimers, unlike rat MBL-A, are reported to be incapable of activating complement (10), which has led to the assumption that this may be a general property of MBL-Cs. In mice, the differentiation between murine MBL-A (mMBL-A) and mMBL-C is complicated by their identical mobilities on SDS-PAGE in the reduced state, corresponding to polypeptide chains of 28 kDa (11). In this study, we present the purification and characterization of both forms of MBL from mouse serum.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mannose was coupled to TSK HW/75(F) beads (Tosoh, Tokyo, Japan) activated by divinyl sulfone (12). The beads were suspended in 9.1% (v/v) divinyl sulfone in 0.25 M Na2CO3, incubated for 90 min, washed with water, incubated in 10% (w/v) mannose in 0.5 M Na2CO3 (pH 11) for 24 h at room temperature, washed, and incubated for 2 h in 0.1 M ethanolamine (pH 9.0), washed, and kept in TBS.
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% divinyl sulfone (v/v). The coupling buffer was 15 mM NaHCO3 containing 135 mM NaCl and 5% (w/v) polyethylene glycol (PEG) 20,000 (pH 8.6).
Purification of mMBL-A and mMBL-C by carbohydrate affinity chromatography
Pooled mouse serum (45 ml), obtained from inbred BALB/c mice or outbred NMRI mice, was mixed with an equal volume of precipitation buffer consisting of 10 mM barbital-HCl, 300 mM NaCl, 10 mM CaCl2, 15 mM NaN3, and 0.01% (v/v) Tween 20 (Tw) (pH 7.4; barbital-buffered saline (BBS)-1/Ca2+) containing 13% (w/v) PEG 6000. The mixture was centrifuged for 20 min at 2000 x g and the pellet was washed with BBS-1/Ca2+ containing 8.0% (w/v) PEG 6000. The pellet was redissolved in 25 ml of BBS-1/Ca2+ and applied to a 25-ml TSK 75/HW(F) precolumn connected to a 25-ml mannose-TSK 75/HW(F) column pre-equilibrated with BBS-1/Ca2+. The columns were then washed with the same buffer. The TSK precolumn was removed and the mannose-TSK column was eluted with 50 ml of BBS-1/Ca2+ containing 12 mM glucose ("glucose eluate"). It was then washed with 50 ml of BBS-1/Ca2+ and eluted with 50 ml of BBS-1/Ca2+ containing 25 mM mannose ("mannose eluate"). The absorbance of fractions at 280 nm was measured and relevant fractions were analyzed by rocket immunoelectrophoresis, acid/urea-PAGE, and N-terminal amino acid sequencing.
Purification of mMBL-A and -C by affinity and ion-exchange chromatography
Mouse serum (135 ml) was mixed with an equal volume of buffer consisting of 20 mM barbital-HCl, 40 mM CaCl2, 1.0 M NaCl, 0.08% (v/v) emulphogene, 100 µM benzamidine, 100 µM iodoacetamide, and 100 µM cyclocaprone (pH 7.4). The diluted serum was applied to a 70-ml TSK precolumn connected to a 70-ml mannose-TSK column pre-equilibrated with 10 mM barbital-HCl, 20 mM CaCl2, 0.5 M NaCl, 0.04% (v/v) emulphogene, 50 µM benzamidine, 50 µM iodoacetamide, and 50 µM cyclocaprone (pH 7.4; BBS-2/NaCl/Ca2+). After loading, the columns were washed with BBS-2/NaCl/Ca2+ followed by the same buffer with the NaCl concentration reduced to 0.15 M NaCl (BBS-2/Ca2+). The precolumn was removed and a 10-ml rabbit anti-mouse IgG Ab column was attached to the outflow of the mannose-TSK column. The columns were washed with BBS-2/Ca2+ and eluted with BBS-2 containing 20 mM EDTA instead of calcium. Fractions with a high content of mMBL, as determined by SDS-PAGE in the reduced state, were pooled and diluted with three volumes of Mono-Q loading buffer (20 mM piperazine-HCl, 5 mM EDTA, 0.03% (v/v) Tw, 100 µM PMSF, 100 µM benzamidine, and 100 µM cyclocaprone (pH 6.2)). The pH was adjusted to 6.2 and the pool was applied to a 1-ml Mono-Q column (Amersham Pharmacia Biotech, Uppsala, Sweden). The effluent, containing mMBL-A, was collected and stored at 4°C. The column was washed with 10 ml of Mono-Q loading buffer and eluted in a stepwise manner with the same buffer containing NaCl in concentrations of 127 mM, 750 mM, and 1.0 M. The bulk of mMBL-C was found in the 750 mM NaCl eluate, which was frozen and kept at -70°C. Aliquots (120 µl) from the 750 mM NaCl eluate were analyzed by gel-permeation chromatography on a TSK 3000 SW column (0.75 x 60 cm) (Pharmacia LKB, Uppsala, Sweden) with a TSK SW precolumn (0.75 x 7.5 cm) (Pharmacia LKB) in a buffer consisting of 50 mM ammonium acetate, 450 mM NaCl, 10% (v/v) acetonitrile, 5 mM EDTA, 50 µM PMSF, 50 µM benzamidine, and 50 µM 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; Statens Serum Institut, Copenhagen, Denmark), and aldolase (158 kDa), catalase (232 kDa), and thyroglobulin (669 kDa) from Amersham Pharmacia Biotech. Fractions were stored at -70°C and analyzed by SDS-PAGE. The effluent from the Mono-Q column was concentrated on a 1-ml Resource-Q column (Amersham Pharmacia Biotech) equilibrated in Resource-Q loading buffer (20 mM Tris-HCl, 0.03% (v/v) Tw, 100 µM PMSF, 100 µM benzamidine, and 100 µM cyclocaprone (pH 8.6)). Tris was added to the Mono-Q effluent to a final concentration of 20 mM and pH was adjusted to 8.6. After loading, the column was eluted with Resource-Q loading buffer containing 750 mM NaCl. Fractions were stored at 4°C and analyzed by SDS-PAGE with silver staining. Protein concentrations of the purified preparations were estimated by their absorbance at 280 nm using extinction coefficients (E1cm, 1 mg/ml) of 0.63 for mMBL-A and 0.90 for mMBL-C calculated from the deduced amino acid sequences (13) according to Ref. 14 .
Preparation of Abs
Rabbit anti-mouse IgG (H + L chain) Abs were affinity purified from locally produced antiserum using mouse IgG coupled to TSK beads.
Rabbit antiserum raised against mMBL was produced as described
previously (15). This antiserum recognizes mMBL-C as well
as mMBL-A and is referred to as anti-mMBL antiserum. A specific
mMBL-C Ab was raised against a synthetic peptide (Immune Systems,
Bristol, U.K.) representing hydrophilic loops 1 and 2 of the mMBL-C
CRD. The peptide comprised amino acid residues 184–202
(DVRVEGSFEDLTGNRVRYT) from the deduced sequence (13) with
an additional cysteine residue at its C terminus. The peptide (0.24 mg)
was conjugated to 1 mg of purified protein derivative of tuberculin
(Statens Serum Institut) by means of the heterobifunctional
cross-linking reagent
m-maleimidobenzoyl-N-hydroxysulfosuccinimide
ester (Pierce, Rockford, IL) according to Ref. 16 . Rabbits
were primed by s.c. injection of 0.2 ml of bacillus
Calmette-Guèrin vaccine (Statens Serum Institut). Three weeks
later the peptide-purified protein derivative of tuberculin conjugate
(
35 µg adsorbed to 0.2 mg of aluminum hydroxide gel (Superfos
Kemi, Vedbaek, Denmark) in 0.5 ml of 145 mM NaCl) was emulsified in an
equal volume of Freund’s complete adjuvant (Difco, Detroit, MI) and
half of it was injected s.c. in each of two rabbits. Booster injections
were given 2 wk later with the same dose of Ag emulsified in Freund’s
incomplete adjuvant (Difco) and repeated at 4-wk intervals. Antiserum
was collected 2 wk after each boost. The antisera raised against the
synthetic peptide are referred to as rabbit anti-mMBL-C'.
Complement activation assay
Microtiter wells (FluoroNunc from Nalge-Nunc, Roskilde, Denmark) were coated overnight with 70 ng of mannan in 100 µl of coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 30 mM NaN3). Wells were emptied and incubated 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 TBS/Tw/Ca2+ (TBS/Tw/HSA without HSA but including 5 mM CaCl2), duplicate wells were incubated overnight at 4°C with dilutions of purified human MBL (Statens Serum Institut), mMBL-A, or mMBL-C in BBS/Tw/HSA (10 mM barbital sodium, 145 mM NaCl, 15 mM NaN3, 0.05% (v/v) Tw, 0.01% (w/v) HSA (pH 7.4)) containing enzyme inhibitors (100 µM PMSF, 100 µM iodoacetamide, 100 µM benzamidine, 100 µM cyclocaprone, 100 µM phenanthroline) and either 10 mM CaCl2 or 10 mM EDTA. The wells were washed with TBS/Tw/Ca2+ or TBS/Tw/EDTA (TBS/Tw with 5 mM EDTA instead of CaCl2), respectively, and then with TBS/Tw/Ca2+. The MBL-reacted wells were then incubated for 3 h at 37°C with human serum devoid of MBL and C1q, diluted 1:90 in TBS/Tw/Ca2. The serum was obtained from an MBL-deficient subject (serum MBL <10 ng/ml) and C1q was removed by chromatography on Biorex 70 (Bio-Rad, Bedford, MA) (17). Any remaining MBL was removed from the serum by passing it through mannan-coupled Sepharose (15) in the presence of CaCl2. Wells were then washed in TBS/Tw (TBS/Tw/Ca2+ without CaCl2) and incubated for 3 h with 100 µl TBS/Tw containing 10 µg HSA and 100 ng biotinylated F(ab')2 fragment of rabbit anti-human C4 Ab (Dako, Glostrup, Denmark). Wells were washed in TBS/Tw and incubated for 2 h with 10 ng europium-labeled streptavidin (Wallac, Turku, Finland) in 100 µl TBS/Tw containing 25 µM EDTA and 0.01% (w/v) HSA. After washing with TBS/Tw, bound europium was released into the fluid phase by incubation for 10 min with enhancement buffer (Wallac). The amount of europium in each well was assessed by time-resolved fluorometry on a Delphia fluorometer (Wallac).
Carbohydrate selectivity
Purified MBL was biotinylated with 40 µg
N-hydroxysuccinimidobiotin/mg of MBL. Microtiter wells
(FluoroNunc) were coated overnight at room temperature with 13 ng
mannan in 100 µl coating buffer. Plates were blocked and washed as
described above. Dilutions of monosaccharides in 50 µl
TBS/Tw/Ca2+ were added in duplicate to the wells
by means of a robotic pipetting system (Packard, Meriden, CT). Negative
and positive controls consisting of TBS/Tw/EDTA or
TBS/Tw/Ca2+ without monosaccharides were
included. Biotinylated MBLs at 0.15 µg/ml (mMBL-A), 0.06 µg/ml
(mMBL-C), or 0.04 µg/ml (human MBL) in
TBS/Tw/Ca2+ containing 0.01% (w/v) HSA were then
added in duplicate 50-µl volumes. The solutions were mixed on a
shaking platform and incubated overnight at 4°C. The monosaccharides
tested comprised D-mannose (Man),
-methyl-D-mannose (MeMan),
D-mannosamine (ManN),
N-acetyl-D-mannosamine (ManNAc),
D-glucose (Glc),
-methyl-D-glucose (MeGlc),
D-glucosamine (GlcN),
N-acetyl-D-glucosamine (GlcNAc),
D-galactose (Gal),
-methyl-D-galactose (MeGal),
D-galactosamine (GalN),
N-acetyl-D-galactosamine (GalNAc),
D-fucose (Fuc), and
L-fucose (L-fuc); all were
purchased from Sigma. All were tested at concentrations ranging from
0.184 to 100 mM. The wells were washed and developed with
europium-labeled streptavidin as described above. The background was
defined as the average signal of three wells incubated with
biotinylated MBL in the presence of EDTA; the maximum signal was that
obtained in buffer without monosaccharide. Fluorescence intensities
from 103 to 106 counts/s
were obtained with <3% variation between duplicates. Each
monosaccharide was tested in at least five different experiments, and
various combinations of five different monosaccharides were tested on
the same microtiter plate to determine their individual ranking.
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 purified mMBL-A and two mouse sera were used as standards and controls. The mMBL-A in serum could be quantified since this precipitate was stronger 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 µl) was subjected to gel-permeation chromatography on a Superose 6 column (30 x 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 with 0.6 ml acetone for 2–3 h at -20°C and centrifuged at 10,000 x g for 20 min at 4°C. The pellets were dried in an evaporating centrifuge and redissolved in 25 µl sample buffer for SDS-PAGE and Western blotting. The column was calibrated with the following proteins: thyroglobulin (669 kDa), ferritin (330 kDa; Amersham Pharmacia Biotech), catalase (232 kDa), and HSA (70 kDa). The elution volume of human C1q and IgM was assessed by gel permeation of human serum in the presence of 10 mM EDTA. The content of C1q and IgM in the fractions was measured by enzyme immunoassay (EIA).
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) with the following modifications: Formalin fixation, rinsing with H2O, and dehydration in acetone were prolonged to 15 min each, and silver impregnation was conducted with a solution of 0.2% (w/v) AgNO3 and 0.25% (v/v) Formalin. Molecular weights were estimated by comparison with prestained marker proteins (Amersham Pharmacia Biotech).
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 electrophoresis buffer was 5.4% (v/v) acetic acid (pH
2.5). Gels were prerun overnight in electrophoresis buffer at a fixed
current of 1 mA. Samples containing 1–2 µg protein were dried in an
evaporating centrifuge and redissolved in 15 µl 20 mM sodium borate
buffer (pH 9.0) containing 8 M urea and 30 mM DTT. Sample solutions
were then boiled for 3 min and acidified with acetic acid to a final
concentration of 10% (v/v). Electrophoresis was performed at a fixed
current of 9 mA for 5 h.
Western blotting
Protein bands from PAGE were electroblotted onto polyvinylidene difluoride membranes (Immobilon P; Millipore, Bedford, MA) (22). Membranes were blocked with TBS containing 0.1% (v/v) Tw for 30 min, cut into strips, incubated with Ab dilution, and developed by the alkaline phosphatase method (23). Strips were stained with colloidal gold to visualize the proteins on the blot (24). For estimating the apparent molecular size of mMBL by gel-permeation chromatography of mouse serum, the Western blots of the fractions were incubated with biotinylated second Ab and developed by enhanced chemiluminescence using HRP-labeled streptavidin (Dako) at 0.2 µg/ml and luminescence reagent (Pierce). Stripping of blots developed by enhanced chemiluminescence was performed by incubation in denaturing and reducing buffer (62.5 mM Tris-HCl, 2.0% (w/v) SDS, 0.078% (v/v) 2-ME (pH 6.9)) at 70°C for 45 min. Blots were then washed overnight in TBS and developed with another Ab.
N-terminal sequencing
The proteins obtained by differential glucose and mannose
elution of a mannose-TSK column were subjected to N-terminal amino acid
sequencing. Eluate (0.5 ml) containing 30 µg reduced mMBL was
applied to SDS-PAGE. After electrophoresis gels were equilibrated for
10 min in transfer buffer (10 mM 3-cyclohexylamino-1-propanesulfonic
acid and 10% (v/v) methanol (pH 11)) and the protein bands were
transferred to Problot membranes (PE Applied Biosystems, Foster City,
CA) at 7.5 volt/cm for 10 h. Protein bands were visualized with
Ponceau S, and bands appearing at 28 kDa were cut out and sequenced on
an Applied Biosystems 470/120A sequencer. Similarly, the N-terminal
sequence of the 21-kDa band appearing in the mMBL-C preparations after
the ion-exchange chromatography (see above) was obtained.
Mice
Sera from laboratory strains of mice were obtained from Bommice (Bomholtgaard Breeding and Research Center, Ry, Denmark), whereas those from wild mice were obtained from the Laboratoire Gènome et Populations (Universitè de Montepellier II, Montpellier, France). Sera were kept at -20°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two serum forms of mMBL were found during work to improve the
purification of mMBL-A. The eluates obtained from differential
monosaccharide elution of a mannose affinity column loaded with the
6.5% PEG 6000 precipitate of mouse serum were analyzed by rocket
immunoelectrophoresis (Fig. 1
). The MBL
in the 10 mM glucose eluate formed rockets of a shape and size
consistent with the mMBL-A content found in previous mMBL-A
purifications (15). Murine MBL-A eluted over a relatively
large number of fractions and the recovery was 30% of the amount
applied to the column as determined by rocket immunoelectrophoresis.
When the mannose eluate was analyzed, precipitates of weak intensity
and with a pointed shape unlike that of mMBL-A rockets were observed.
These pointed rockets varied in character and intensity between
different runs and were also occasionally observed when whole mouse
serum was analyzed. SDS-PAGE of aliquots from the glucose and mannose
eluates showed that the major protein in each eluate had a mobility
corresponding to 28 kDa in the reduced state (data not shown). To
explain the different appearances on rocket immunoelectrophoresis, the
28-kDa protein bands were blotted onto Problot membranes and subjected
to N-terminal sequencing. Analysis of the protein in the glucose eluate
yielded the first 25 amino acid residues of mMBL-A, whereas the
sequence of the protein in the mannose eluate corresponded to the first
20 amino acid residues of mMBL-C. In both cases, the identity to the
published deduced amino acid sequence was 100% and no
cross-contamination was observed. Acid/urea-PAGE of the glucose and
mannose eluates confirmed the presence of two different forms of mMBL
(Fig. 1). The electrophoretic mobilities of the two proteins in this
system (mMBL-A > mMBL-C) were consistent with the pIs calculated
from their deduced amino acid compositions (7.6 for mMBL-A and 4.8 for
mMBL-C).
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The specificity of the anti-mMBL-C' antisera was tested by EIA
and Western blotting. EIA showed high Ab binding in wells coated with
the synthetic peptide Ag and no binding to wells coated with irrelevant
peptides. No specific binding was observed in wells coated with
preparations of native mMBL. Western blotting of previously purified
mMBL-A (15) and a preparation containing both mMBL-A and
mMBL-C (the EDTA eluate of a mannose-TSK column loaded with mouse
serum) showed that antisera from the two rabbits recognized mMBL-C but
not mMBL-A (Fig. 2).
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The purification of mMBL-A and mMBL-C was monitored by rocket
immunoelectrophoresis against rabbit anti-mMBL antiserum and by
Western blotting against biotinylated rabbit anti-mMBL-C' Ab,
respectively. The two proteins were purified by affinity chromatography
on a mannose-TSK column eluted with EDTA, removal of Ig by passage
through an anti-mouse IgG column, ion-exchange chromatography at pH
6.2 on a Mono-Q column, gel-permeation chromatography on TSK 3000
column, and ion-exchange chromatography on a Resource-Q column.
Fractions from the different eluates were analyzed by SDS-PAGE and
silver staining (Fig. 3). The two forms
of mMBL were well separated by anion-exchange chromatography at pH
6.2. The effluent contained mMBL-A but no mMBL-C, as judged by Western
blotting using the biotinylated anti-mMBL-C' Ab (Fig. 2
A), while the bulk of the mMBL-C was eluted with 750 mM
NaCl. This eluate contained no mMBL-A when analyzed by rocket
immunoelectrophoresis, even when concentrated 10-fold by acetone
precipitation. Controls showed nearly 100% recovery of MBL-A after
acetone precipitation.
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C). This
probably represents incompletely reduced mMBL-A, as it bound to the
rabbit anti-mMBL-A Ab (results not shown).
SDS-PAGE of the 750 mM NaCl eluate containing mMBL-C (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
sequence of the 21-kDa band was identical to that of mMBL-C and the
band was stained on Western blots with rabbit anti-mMBL-C' Ab (Fig. 4). An aliquot from the 750 mM NaCl
eluate was subjected to gel-permeation chromatography on a TSK 3000 SW
column. Two major absorbance peaks at 280 nm, of approximately equal
size, were observed (data not shown). The first peak emerged with the
void volume (>600 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 presence of 28-kDa and 21-kDa
bands in fractions corresponding to molecular masses from >600 to 230
kDa. In these fractions, the intensity of the 21-kDa band paralleled
that of the 28-kDa mMBL-C band. Two fractions in the void volume
contained >95% pure mMBL-C, as judged by SDS-PAGE and silver
staining, with only trace amounts of other bands at 70 and 100 kDa in
the reduced state (Fig. 3D). Fractions from the other major
peak at 350 kDa contained bands corresponding to mMBL-C as well as
other bands at 40, 70, and 100 kDa under reducing conditions. The
>600-kDa peak was used in subsequent analyses for complement
activation and carbohydrate-binding specificity.
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23 µg/ml. The amount of mMBL-C
recovered in the void volume fractions was estimated as 20 µg by
absorbance measurements. If all of the 750 mM NaCl eluate had been
applied to gel-permeation chromatography, 0.25 mg mMBL-C would have
been recovered in these fractions. Complement activation
The capacity of mMBL-A and mMBL-C to activate complement was
determined by measuring C4 deposition in mannan-coated microtiter
wells, with human serum depleted of MBL and C1q as the complement
source. As shown in Fig. 5, both proteins
as well as human MBL mediated C4 deposition, but mMBL-C was less active
than mMBL-A and human MBL. Although mMBL-A at 3 ng/ml produced a
fluorescence intensity of 105 counts/s, 16 ng
mMBL-C/ml was required to produce the same signal. In the presence of
EDTA, none of the MBLs produced C4 deposition.
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The relative potencies of monosaccharides in inhibiting the
binding of biotinylated mMBL-A, mMBL-C, and human MBL are given in
Table I and compared with previous
published observations in Table II. Note
the different sensitivity toward inhibition with Glc, MeGlc, and
L-fuc.
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The oligomer state of mMBLs was examined by SDS-PAGE under
nonreducing conditions and Western blotting (Fig. 6). The lack of a specific mMBL-A Ab made
it necessary to use a preparation of mMBL-A which contained no mMBL-C.
The glucose eluate from mannose-TSK was chosen since this preparation
had been subjected to the least handling. In parallel, a 10% PEG 6000
precipitate of mouse serum was blotted against rabbit anti-mMBL-C'
Ab. Both mMBL-A and mMBL-C were observed as a complex mixture of
oligomers, varying from dimers of polypeptide chains at 60 kDa to
complexes of more than 200 kDa. The pattern was similar for the two
types of mMBL. Normal mouse serum was subjected to gel-permeation
chromatography and fractions were analyzed by SDS-PAGE and Western
blotting. The blots were developed with rabbit anti-mMBL-C' Abs,
stripped, and then developed with rabbit anti-mMBL Abs (Fig. 7). mMBL-C was found in two peaks
corresponding to a molecular size of 600 kDa and 150 kDa (for
globular protein markers, Fig. 7a), the first being the main
(Fig. 7b). mMBL-A also emerged at a position corresponding
to a molecular mass of about 600 kDa, but without the second peak
observed for mMBL-C (Fig. 7c). Purified mMBL-A was analyzed
on the same column. The UV chromatogram and SDS-PAGE of fractions
showed that it emerged as a single peak corresponding to an average
molecular mass of 600 kDa.
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Concentrations of mMBL-A in sera from laboratory strains
of mice and wild mouse species and strains were estimated by rocket
immunoelectrophoresis in gels containing the anti-mMBL antiserum
and with purified mMBL-A as standards (Fig. 8). Serum mMBL concentrations determined
by this method varied between 5 and 50 µg/ml in laboratory strains of
mice. The lowest concentration was found in the DW/DW homozygous dwarf
strain. Greater variation was found among individuals in wild strains.
The least concentration observed in wild mice was 5 µg/ml (Mus
musculus domesticus and Mus musculus musculus). The
highest concentration of >120 µg/ml was found in a single mouse of
the species M. caroli. In general, wild mice showed higher
serum mMBL-A concentrations than laboratory strains.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two murine serum forms of MBL have been observed before (11). The initial identification of mMBL-C in serum was based on affinity isolation using Ra chemotypes of Salmonella and analysis by acid/urea-PAGE. Two forms of MBL have also been identified in rat serum, where the MBL-C form is described as a minor component compared with MBL-A (10, 31). There has been a tendency in the literature to regard MBL-C as a hepatic protein involved in the intracellular transport of high mannose-type oligosaccharides (32). In contrast to this notion, we find that mMBL-C is a circulating protein present at serum levels comparable to those of mMBL-A. Judged from the recovery and the total yield of mMBL-A purified, an approximate serum concentration of 23 µg/ml was calculated. mMBL-C could not be quantified, but the experiences gathered suggest a concentration similar to that of mMBL-A.
It is possible that MBL-C may differ in abundance and function among different rodent species. Another possibility is that MBL-C often escapes detection because of its sensitivity to proteolytic degradation. Purification of mMBL-C was only possible in the presence of protease inhibitors and preliminary attempts to purify rat MBL-C showed this to be even more susceptible to degradation.
After ion-exchange chromatography at pH 6.2, a 21-kDa fragment of mMBL-C was observed. The relative amount of the 21-kDa fragment increased if purification of mMBL-C was conducted at room temperature or in the absence of protease inhibitors, or when purified mMBL-C was kept at 4°C. The 21-kDa fragment has a N-terminal sequence identical to the terminus of mMBL-C.
Both forms of mMBL mediated C4 activation. mMBL-A was as active as
human MBL, whereas mMBL-C was approximately one-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-kDa fragment during incubation
at 37°C, with impairment of complement activation. The finding that
mMBL-C activates complement contrasts with the previous observation
that MBL-C isolated from rat liver was incapable of complement
activation (2). The same has been reported to be the case
for human MBL isolated from liver, and also some recombinant human MBL
shows a low activating potential (33, 34). The reported
lack of complement activation by rat MBL-C and human MBL isolated from
liver could be due to incomplete processing with respect to
oligomerization and other posttranslational modifications
(35). The mMBL-C analyzed for complement activation in
this report had an apparent molecular size similar to that of mMBL-A. A
similar oligomeric state of mMBL-A and MBL-C was observed by SDS-PAGE
under nonreducing conditions followed by Western blotting. Murine MBL-A
has previously been observed to emerge from gel-permeation columns at a
position corresponding to penta- and hexamers with an average size of
600 kDa, in agreement with the size of rat MBL-A (10, 15).
Analysis of mouse serum by gel-permeation chromatography and Western
blotting showed that mMBL-C emerged primarily as a peak at 600 kDa,
probably representing hexamers, with a lower amount emerging at 150
kDa. Purified mMBL-C was found in fractions corresponding to 230–600
kDa 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
rat serum MBL-C was purified by a different procedure in which it was
separated from MBL-A by gel-permeation chromatography.
High-molecular-weight rat MBL-C may have been lost in these experiments
and the question of the size of MBL-C in rat serum has so far not been
directly addressed. Judged from the complex pattern of oligomers
observed by SDS-PAGE, the disulfide bonding observed in mMBL-C clearly
deviates from the pattern found in recombinant rat MBL-C
(37).
Analysis of the carbohydrate specificity showed that human MBL
resembles that of mMBL-C more than that of mMBL-A, in agreement with
the suggested evolution of MBL genes (38). Likewise, the
specificities of mMBLs resemble those of their rat analogues and that
of mMBL-A concords with previously reported data (Table II)
(15). Remarkable is the difference in the specificities of
mMBL-A and MBL-C, especially the high affinity of the former for Glc
and -MeGlc. It is likely that the differences in monosaccharide
specificity leads to preferential binding to different microorganisms,
depending on the composition of glycoconjugates in their outer
wall.
Serum concentrations of mMBL-A, estimated by rocket immunoelectrophoresis against mMBL-A standards, were found to range from 5 to 40 µg/ml in laboratory strains of mice and, with a single exception, in wild mice from 5 to 80 µg/ml. The finding of higher mMBL levels in many wild mice is in line with previous measurement (15) in wild yellow-necked mice (M. cervicolor). The variation in serum mMBL in different mice, while significant, is small in comparison with the 500-fold variation (from <10 ng/ml to 5 µg/ml) observed in humans.
The demonstration of both mMBL-A and mMBL-C as serum forms capable of activating the complement system should be remembered when considering animal models for MBL deficiency.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steffen Thiel, Department of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C, Denmark. E-mail address:
3 Abbreviations used in this paper: MBL, mannan-binding lectin; MeGal, -methyl-D-galactose; MeGlc, -methyl-D-glucose; MeGlcNAc, N-acetyl--methyl-D-glucosamine; MeL-Fuc, -methyl-L-fucose: MeMan, -methyl-D-mannose; BBS, barbital-buffered saline; ßMeL-Fuc, ß-methyl-L-fucose; CRD, carbohydrate recognition domain; EIA, enzyme immunoassay; Fuc, D-fucose; Gal, D-galactose; GalN, D-galactosamine; GalNAc, N-acetyl-D-galactosamine: Glc, D-glucose; GlcN, D-glucosamine; GlcNAc, N-acetyl-D-glucosamine; HSA, human serum albumin; L-fuc, L-fucose; Man, D-mannose; ManN, mannosamine; ManNAc, N-acetyl-D-mannosamine; MASP, MBL-associated serine protease; mMBL, murine mannan-binding lectins; PEG, polyethylene glycol; Tw, Tween 20 (polyoxyethylenesorbitan monolaureate).
Received for publication June 28, 1999. Accepted for publication December 10, 1999.
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