From the Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
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ABSTRACT |
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Rat serum mannose-binding protein (MBP-A)
functions as part of the innate immune system by targetting complement
toward potentially pathogenic microorganisms. In order to examine the
molecular basis for complement activation, rat MBP-A has been
overproduced in Chinese hamster ovary cells. Recombinant protein is
post-translationally modified in the same way as the native lectin.
Hydrodynamic studies indicate that MBP-A consists predominantly of
covalent oligomers containing one to four copies of a subunit that
comprises a trimer of polypeptides. These oligomers are
non-interconverting and do not assemble into higher order structures at
concentrations in excess of those normally found in serum. Disulfide
bonds formed between cysteine residues at the N-terminal end of the
collagen-like domain link polypeptides to form covalent oligomers.
Analysis of wild-type MBP-A and MBP-A containing the substitution
Cys6 Serum mannose-binding protein
(MBP)1 is an important
component in the mammalian innate immune system that binds
carbohydrates on the surfaces of pathogenic microorganisms and
activates complement in an antibody-independent manner (1, 2). It also
functions directly as an opsonin by binding to specific receptors on
the surfaces of phagocytic cells (3). Serum MBP is a member of the
collectin family of animal lectins. Each collectin polypeptide contains
an N-terminal cysteine-rich domain, a collagen-like domain, an
Complement fixation by serum MBP occurs by a mechanism similar to the
classical pathway of complement activation. MBP interacts with two
MBP-associated serine proteases (MASP-1 and MASP-2) (5-7) that are
homologs of complement components C1r and C1s. On activation, MASP-2
cleaves both C4 and C2 to form a C4b2a complex with C3 convertase
activity (8). Human MBP deficiency is a relatively common disorder
caused by point mutations in the MBP gene (9, 10). This condition is
associated with a high susceptibility to bacterial and viral infections
particularly during the first few years of life.
Two distinct rat mannose-binding proteins, MBP-A and MBP-C, have been
cloned and sequenced (11). These proteins are found predominantly in
the serum and liver, respectively. The two forms adopt distinct higher
order structures (4). MBP-C consists of a single trimer of polypeptides
whereas MBP-A assembles into higher oligomers. Rotary shadowing
electron microscopy and sucrose gradient ultracentrifugation of human
MBP suggest the presence of oligomers ranging from dimers to hexamers
of trimeric subunits (12, 13). The trimeric building blocks from which
MBPs are assembled will subsequently be referred to as subunits. The
MBP oligomers adopt a bouquet-like structure, similar to complement component C1q (12). Assembly of each subunit in MBPs occurs in a C- to
N-terminal direction (14), so that association of the CRDs and
trimerization of the In the crystal structure of a fragment of the trimeric MBP-A subunit
containing the CRD and neck region, the CRDs of each trimer are
clustered such that the binding sites are orientated in a configuration
to enable binding to bacterial and fungal cell surfaces (16). In this
study, we have used the relatively simple structure of MBP-C to examine
the organization of its more complex serum homolog. Biophysical
analysis of chimeras of MBP-A and MBP-C indicate that oligomers of
MBP-A assemble as a result of the interactions of residues in the
cysteine-rich domain and N-terminal part of the collagen-like domain.
The presence of these regions of MBP-A also correlates with the level
of complement fixing activity, indicating that correct assembly of
MBP-A is essential for its biological roles.
Materials--
Restriction enzymes were purchased from New
England BioLabs. All tissue culture medium was from Life Technologies.
Sheep erythrocytes and guinea pig serum were purchased from Serotech.
Yeast mannan, phenylthiohydantoin-derivatives, and protein molecular
weight markers for gel-filtration, SDS-polyacrylamide gel
electrophoresis, and MALDI-MS were purchased from Sigma. Proteinase
Arg-C, thermolysin, and subtilisin were obtained from
Boehringer-Mannheim. Tosylphenylalanyl chloromethyl ketone-treated
trypsin and chymotrypsin were purchased from Worthington Biochemicals.
Reagents for amino acid sequencing were from Beckman Instruments.
Iodo[2-3H]acetic acid and Amplify fluorography reagent
were obtained from Amersham.
Analytical Methods--
Amino acid sequencing was carried out on
a Beckman LF3000 protein sequencer. Amino acid analysis was performed
by the method of Heinrikson and Meredith (17). SDS-polyacrylamide gel
electrophoresis was performed by the method of Laemmli (18). Standard
molecular biology techniques were carried out as described (19). The
presence of free thiol groups was determined as described previously
(14).
Production of MBP-A--
MBP-A was produced in Chinese hamster
ovary cells using a strategy similar to that previously described for
MBP-C (14). The cDNA encoding MBP-A (11) was modified to introduce
an XhoI restriction site adjacent to the EcoRI
site at the 5' end and a SalI site near the BstYI
site (base 835) in the 3'-untranslated region. This fragment was
ligated into the unique SalI restriction site upstream of
the dihydrofolate reductase gene in the vector pED (20). Chimeric
constructs were made using restriction sites that are common to the
MBP-A and MBP-C cDNAs or by inserting synthetic oligonucleotide
linkers between appropriate restriction sites. Chinese hamster ovary
cell transfectants were generated and selected as described previously
(14). Cells were grown to confluence in 225-cm2 tissue
culture flasks containing 70 ml of medium supplemented with 0.5 µM methotrexate and 10% dialyzed fetal calf serum in which endogenous bovine lectins had been removed by passage through a
1-ml mannose-Sepharose column. Once the cells were confluent, the
medium was replaced daily. Medium was harvested every day beginning 4 days after confluence was reached. Cell debris was removed by
centrifugation (4000 × g for 4 min) and the medium was
stored at
MBP-A was purified from the culture medium of confluent cells, since
this procedure yielded protein which resembles preparations of the
native lectin (21). The larger oligomers of MBP-A are deficient in
protein preparations from the medium of subconfluent cells. Protein
isolated from the medium of subconfluent cells was also found to have a
reduced apparent molecular mass, which may reflect changes in
post-translational modification.
Ion-exchange Chromatography--
Oligomers of MBP-A and MBP-A
Cys6 Hydrodynamic Studies--
Gel-filtration chromatography was
carried out on a Biosep Sec-S3000 column (300 × 7.8 mm,
Phenomenex) equilibrated in 50 mM potassium phosphate
buffer, pH 7.0, at a flow rate of 0.5 ml/min. Analytical
ultracentrifugation was carried out as described previously for MBP-C
(14). Values reported are mean ± S.D. for at least two
independent experiments. The partial specific volume of MBP-A (0.7208 ml/g) was calculated from the amino acid and carbohydrate composition
using the method of Cohn and Edsall (22).
Proteolysis--
For thermolysin and chymotrypsin digestion,
MBP-A was incubated with 10-20% (w/w) of enzyme in 50 mM
Tris-HCl, pH 7.5, containing 150 mM NaCl and 25 mM CaCl2 at 37 °C for 1 h. Samples were
separated on a 17.5% polyacrylamide gel under reducing conditions and
blotted onto polyvinylidene difluoride membranes for sequence analysis (23). Trypsin digestion of MBP-A was carried out in 50 mM
Tris, pH 7.5, containing 150 mM NaCl, 25 mM
CaCl2, and 2.5 mM dithiothreitol. MBP-A (1 mg)
was incubated with trypsin (5% w/w) at 37 °C with mixing for
18 h. The reaction mixture was passed through a mannose-Sepharose column equilibrated in reaction buffer without dithiothreitol to remove
the CRDs. The peptide mixture was lyophilized to dryness, resuspended
in 20% acetic acid (200 µl), and applied to a C3 reverse-phase column. Subtilisin digestion of N-terminal tryptic fragments, reverse
phase high performance liquid chromatography, and MALDI-MS were
performed as described previously (14).
Complement Fixation Activity--
Complement fixation activity
was determined by measuring MBP-dependent hemolysis of
mannan-coated erythrocytes, using a slightly modified version of the
assay described by Ikeda et al. (1). For coating of sheep
erythrocytes with mannan by the chromium chloride method (24), 4 × 1010 cells were washed three times by centrifugation
(2000 × g for 2 min) and resuspended in 10 ml of
Veronal-buffered saline, pH 7.3, (4.4 mM barbituric acid,
1.8 mM sodium barbitone, 145 mM NaCl). Aliquots
of mannan (5 ml at 100 µg/ml) were mixed with 5 ml of erythrocytes
(1 × 1010 cells) and 5 ml of
CrCl3·6H20 (0.5 mg/ml) in Veronal-buffered saline and incubated with mixing for 5 min at 25 °C. The reaction was stopped by the addition of 20 ml of ice-cold gelatin-Veronal buffer
(Veronal buffer containing 0.1% gelatin, 5 mM
CaCl2, and 5 mM MgCl2). The
mannan-coated erythrocytes were washed three times in the same buffer
and resuspended at 1 × 109 cells/ml. MBP-depleted
serum was prepared by passing guinea pig serum (15 ml) through two
mannose-Sepharose columns (1 ml), equilibrated in Veronal-buffered
saline containing 10 mM CaCl2 at 4 °C. In addition, prior to setting up each complement assay, serum (1 ml) was
incubated with mannan-coated erythrocytes (2 × 108
cells) for 1 h at 37 °C to remove any remaining
endogenous lectins. Complement fixation assays were performed in
duplicate. Aliquots (0.4 ml) of MBP were incubated with 0.1 ml of cells
(1 × 108 cells) for 1 h in gelatin-Veronal
buffer at 25 °C. The cells were washed with 0.5 ml of buffer and
incubated with MBP-depleted guinea pig serum in a total volume of 0.5 ml for 1 h with mixing at 37 °C. Sufficient guinea pig serum
was used to give complete lysis when incubated with 1 µg of MBP-A.
Gelatin-Veronal buffer (700 µl) was added to each sample and the
cells were pelleted by centrifugation. The absorbance was measured at
541 nm and expressed as a percentage of the absorbance of an equivalent
volume of cells totally lysed in water, correcting for lysis observed
in the absence of MBP. The data were fitted to a sigmoidal curve using
Microcal Origin. Relative complement fixing activities were calculated from the concentrations of MBP required for 50% hemolysis compared with MBP-A in assays performed using the same batch of mannan-coated erythrocytes. Values reported are the mean ± S.E. for at least three independent assays.
Production of MBP-A--
Mannose-binding protein has generally
been purified from mammalian sera by affinity chromatography on
immobilized mannan or invertase, or on columns of mannose linked to
agarose (25-27). The purified protein consists of multiple covalent
species detected by SDS-polyacrylamide gel electrophoresis under
nonreducing conditions. Rat MBP-A comprises three major covalent forms
which have been interpreted as dimers, trimers, and tetramers of the
trimeric polypeptide (21). Quantities of MBP-A sufficient to enable a detailed study of its structural organization were produced in Chinese
hamster ovary cells. Typical yields of 1.5-2.5 mg from 500 ml of
serum-supplemented medium and 0.5-1 mg from 500 ml of serum-free
medium were obtained. In contrast, yields reported from serum
preparations range from 0.02 to 0.5 mg for human MBP (12, 26) and 0.2 mg for rat MBP-A (27) from an equivalent volume of serum. MBP-A
purified from the expression system is not contaminated with other
serum lectins as has been reported for serum preparations (27).
Post-translational Modifications--
Amino acid sequencing of
MBP-A revealed that the signal sequence is processed correctly (11).
The MALDI-MS profile of reduced MBP-A is consistent with a single
species of mass 24,730 Da. This value is considerably greater than the
calculated mass based on the amino acid sequence (23,526.4 Da),
indicating that the polypeptide has undergone extensive
post-translational modification (11). Amino acid analysis revealed
4-hydroxyproline and 5-hydroxylysine contents of 1.6 and 1.8 mol %,
respectively. These data are consistent with 3.5 residues of
4-hydroxyproline and 4.0 residues of 5-hydroxylysine for each
polypeptide chain. Examination of the amino acid sequence of MBP-A
indicates that most of the potential sites for hydroxylation must be
fully derivatized since there are four sites for both proline and
lysine hydroxylation, within the consensus sequences Gly-X-Pro and Gly-X-Lys.
These results are consistent with previous studies in which both
4-hydroxyproline and 5-hydroxylysine have been detected in preparations
of the native lectin (11).
N-terminal sequencing of the intact protein and of chymotrypsin and
thermolysin fragments indicate that Pro44,
Pro50, and Pro56 are fully derivatized, since
only phenylthiohydantoin-4-hydroxyproline was detected at these
positions (Fig. 1). However,
Pro33 was found to be unmodified, since only
phenylthiohydantoin-proline is detected in the 33rd sequence cycle of
Edman degradation of the intact polypeptide. These results were
confirmed by MALDI-MS and N-terminal sequencing of tryptic peptides of
MBP-A separated by reverse-phase chromatography. Peptide
Gly39-Lys46 elutes in the unbound fraction from
the reverse-phase column and peptide
Leu47-Arg68 elutes as a single peak from the
column. The masses of these peptides are consistent with complete
hydroxylation of Pro44, Pro50, and
Pro56. The peptide Asp24-Arg38 also
elutes as a single peak from the reverse-phase column. However, the
mass of this peptide indicates that Pro33 is unmodified,
confirming the sequencing data. This residue occurs immediately before
the break in the collagen consensus sequence. Previous studies have
shown that the proline residue at the corresponding position in MBP-C
is only partially hydroxylated (14).
MALDI-MS analysis of peptides purified from trypsin digests of MBP-A
indicates that the 4 lysine residues within the consensus sequence for
hydroxylation are at least partially modified. In each case, the mass
of the peptide is consistent with further derivitization of the
hydroxylysine residue by the addition of 2 hexose units. In addition,
no phenylthiohydantoin-derivative could be detected at these positions
by N-terminal sequencing suggesting glycosylation (28).
Glucosylgalactosyl 5-hydroxylysine has previously been identified
in preparations of MBP (15). Additional minor peaks were observed in
the spectra of glycosylated peptides observed by MALDI-MS analysis. The
masses were consistent with the loss of single hexose units (
Altogether, the post-translational modifications of MBP-A produced in
Chinese hamster ovary cells account for the increased mass of the MBP-A
polypeptide determined by MALDI-MS. These modifications, which are
characteristic of those found in vertebrate collagens, are consistent
with those previously reported for protein purified from rat serum.
Oligomeric Structure of MBP-A--
Recombinant MBP-A, when
analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing
conditions, migrates as four major bands with apparent molecular masses
of 85, 185, 280, and 355 kDa (Fig.
2B). Similar bands were
detected in preparations of protein purified by affinity chromatography
and in radiolabeled material immunoprecipitated using an antibody
specific for the CRD of
MBP-A.2 These findings
indicate that multiple covalent forms of MBP-A are secreted from the
cells and are not created by rearrangement of disulfide bonds during
the purification procedure.
Under reducing conditions, MBP-A migrates as a single band with an
apparent molecular mass of 29 kDa. Thus, the bands observed under
nonreducing conditions are compatible with the presence of
disulfide-linked monomers, dimers, trimers, and tetramers of subunits,
with dimers and trimers being the most abundant covalent forms. The
composition and ratio of multimers is consistent with previous studies
of MBP-A purified from rat serum (21). Although the apparent molecular
masses of the trimer and tetramer are beyond the range of the molecular
weight markers, the identities of the these species were confirmed by
analytical ultracentrifugation (see below). Minor peaks could be
observed on SDS-polyacrylamide gels overloaded with MBP-A under
nonreducing conditions. The apparent molecular masses of 26, 45, 122, 155, 226, 255, 310, and 340 kDa are consistent with the presence of low
levels of intermediate covalent forms comprising 1, 2, 4, 5, 7, 8, 10, and 11 polypeptide chains, respectively.
MBP-A analyzed by gel-filtration chromatography elutes at several
positions: a well resolved peak with an apparent molecular mass of 250 kDa and three overlapping peaks with apparent molecular masses in the
range 450-800 kDa (Fig. 2A). SDS-polyacrylamide gel
electrophoresis of fractions collected across the gel-filtration profile indicates that the larger covalent species elute as multiple overlapping peaks from the column (Fig. 2B). The order of
elution corresponds to the molecular mass of each covalent oligomer.
The elution profile of MBP-A is similar over the entire range of
concentrations examined (up to 1 mg/ml). There was no evidence of
assembly of the four major oligomers to form more complex, higher order structures.
The elution of intermediate covalent forms of MBP-A on gel-filtration
chromatography indicates that in some cases the three polypeptides in
the trimeric subunits are not covalently linked to each other. For
example, small amounts of monomeric and dimeric MBP-A polypeptide elute
at the same position as the covalent trimeric subunit, suggesting the
presence of some noncovalent monomeric subunits (Fig. 2B).
Similar heterogeneity has been observed in preparations of MBP-C, which
consists of both covalent and noncovalent trimers of polypeptide chains
(14).
Previous studies have revealed that collectins show anomalous behavior
on gel-filtration because of the highly extended conformation of the
molecules, which in turn is due to the presence of a triple-helical collagen-like domain (13, 14). For this reason, analytical ultracentrifugation was used to determine the accurate mass of oligomers of MBP-A, independent of shape. The equilibrium distribution of purified MBP-A at loading concentrations of up to 1 mg/ml is consistent with a non-interacting heterogeneous mixture (data not
shown). The apparent molecular mass as a function of concentration increases throughout the sample cell. However, the data for different loading concentrations, rather than overlapping as would be expected for a self-associating system, show similar apparent molecular mass
distributions. Furthermore, the apparent weight-averaged molecular mass
is dependent on the rotor speed, since a systematic decrease in
apparent molecular mass with increasing rotor speed was observed. This
result is also consistent with the presence of heterogeneity.
In order to simplify analysis of MBP-A, the oligomers were separated by
ion-exchange chromatography on a Mono-Q column. Four major overlapping
peaks were detected. On SDS-polyacrylamide gel electrophoresis these
peaks were found to consist predominantly of covalent monomers, dimers,
trimers, and tetramers of subunits. Low levels of intermediate
oligomers are also detected, suggesting the presence of noncovalent
oligomers of subunits. The elution positions of these latter species
correspond to their covalent counterparts. Individual oligomers were
further purified where necessary by reapplying fractions to the column
and eluting using a shallow salt gradient. A sample of each of the
purified oligomers is shown in Fig. 2C. Each sample was
analyzed by analytical ultracentrifugation to determine the native
molecular mass. The equilibrium distributions of apparent molecular
mass as a function of concentration of purified dimer and trimer of
subunits are shown in the upper and lower panels
of Fig. 3. Data from three initial
loading concentrations are shown in each case. No self-association can
be detected. The weight-averaged molecular mass of each oligomer
corresponds closely to the value calculated from the mass of the
modified polypeptide (Table I). The
sedimentation coefficient of each oligomer is also shown in Table I,
together with the Stokes radius and the frictional ratio, calculated
from the sedimentation coefficient, and the weight-averaged molecular
mass. These values indicate that the oligomers of MBP-A are highly
asymmetrical, consistent with their domain organization, and provide an
explanation for the anomalous behavior observed on gel-filtration
columns. The gel-filtration and analytical ultracentrifugation analysis
indicate that MBP-A consists of abundant covalent oligomers with trace amounts of noncovalent oligomers of subunits. These oligomers show no
self-association at concentrations in excess of those normally present
in serum.
Disulfide-bonding Pattern--
Comparison of the sequences of
MBP-A and MBP-C reveals that each protein contains 2 cysteine residues
at the N-terminal junction of the collagen-like domain (11). Previous
studies indicate that protomers of MBP-C are linked by interchain
disulfide bonds between these residues arranged in an asymmetrical
configuration (14). MBP-A contains an additional cysteine residue at
the N terminus of the protein. One possibility is that additional
disulfide bonds formed through this extra cysteine residue link
polypeptides in separate subunits to form the large covalent structures
observed on nonreducing polyacrylamide gels. No labeling of protein
with iodo[2-3H]acetic acid could be detected on
SDS-polyacrylamide gels under nonreducing conditions. Thus, the 3 cysteine residues at the N terminus of MBP-A must either form internal
disulfide bonds or are modified, for example, by addition of free
cysteine or glutathione.
To examine the disulfide bonding pattern in MBP-A, the cysteine residue
(Cys6) proposed to link polypeptides in separate subunits
was mutated to a serine residue. Comparison of the gel-filtration
profile of MBP-A Cys6
The single subunit of MBP-A Cys6
The interchain disulfide bonding pattern in the MBP-A Cys6
The disulfide-bonding pattern of the single subunit of MBP-A
Cys6
While the data are consistent with these conclusions for most of the
oligomers, higher order oligomers are detected in preparations of MBP-A
Cys6 Contribution of Multiple Domains in MBP-A to Higher Oligomer
Formation--
Measurements of bouquet-like forms of human MBP
obtained by electron microscopy are consistent with the central core
region consisting of the cysteine-rich domain and the N-terminal part of the collagen-like domain of each protomer, up to the Gly-Gln-Gly interruption (12). Thus, this region is likely to contain residues responsible for assembly of trimeric subunits of serum MBPs into large
oligomers. Since MBP-C is a single subunit, while MBP-A forms higher
oligomers, analysis of chimeras of the two proteins enables
identification of residues that determine the oligomeric state of the molecules.
Three sets of reciprocal chimeras of MBP-A and MBP-C were created (Fig.
5). In each case the site of
recombination was chosen to be within a region of high sequence
identity, in order to minimize disruption of the protein structure.
Chimeras were constructed at the junctions of the collagen-like domain
and at the Gly-Gln-Gly interruption of the collagen-consensus sequence.
MBP-C contains an insertion for which there are no corresponding
residues present in MBP-A, at the start of the collagen-like domain.
For this reason, two sets of reciprocal chimeras were created at this
site, in which this sequence is either included or omitted. Since MBP-A contains an additional cysteine residue (Cys6) within the
cysteine-rich domain, one further construct was made consisting of
MBP-C with the N-terminal 4 amino acid residues replaced by the first 6 residues of MBP-A. The resulting chimeric proteins were overproduced
and purified as described for MBP-A.
The native structures of the purified chimeric proteins were examined
by a combination of gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. Analysis by gel-filtration chromatography indicates that exchanging the C-terminal half of the
collagen-like domain, the neck and CRDs of the MBPs has little effect
on assembly of the chimeras (Fig. 6).
However, both the cysteine-rich domain and the N-terminal part of the
collagen-like domain of MBP-A are required for full assembly into
higher order oligomers. The presence of only one of these regions from
MBP-A (constructs 2AC, 2CA, 3AC, and 3CA) gives an intermediate
phenotype, suggesting that residues in both domains contribute to
assembly of MBP-A.
Gel-filtration peaks corresponding to low molecular weight species
(elution volume
The conclusion that residues in the cysteine-rich domain and in the
N-terminal part of the collagen-like domain of MBPs determine their
oligomeric structure is supported by analysis of the chimeras by
SDS-polyacrylamide gel electrophoresis under nondenaturing conditions
(Fig. 7). The apparent molecular masses
of the covalent oligomers observed for each chimera is dependent on the
origin of both the cysteine-rich domain and the N-terminal part of the collagen-like domain. Thus, the source of these regions within each
chimera determines the overall covalent structure. Again, chimeras
containing one of these regions from each protein have an intermediate
phenotype, indicating that residues from both domains contribute to the
assembly of MBPs.
The N-terminal part of the collagen-like domain of MBP-C contains an
insertion of 9 amino acid residues for which there are no corresponding
residues present in MBP-A (Fig. 5). The presence of this insertion does
not exclude the formation of higher order oligomers, since large
covalent structures are detected in chimeras containing this region.
However, construct 2CA in which the insertion is adjacent to the
collagen-like domain of MBP-A comprises a higher proportion of
noncovalent oligomers than constructs in which it is not present (Fig.
7). Thus, this region appears to modulate oligomer formation, while not
preventing the assembly of higher order structures.
The collagen-like domain of MBP-A up to the Gly-Gln-Gly interruption
appears to promote the formation of higher order oligomers, since
chimeras containing this region (2CA and 3CA) comprise a higher
proportion of the larger covalent structures than those containing the
corresponding region of MBP-C (Figs. 6 and 7). This part of the
collagen-like domain contains only three differences between the MBPs,
in which Lys27, His30, and Ala33 in
MBP-C correspond to Arg20, Arg23, and
Pro26 in MBP-A, respectively (Fig. 5). Thus, one or more of
these residues in MBP-A must contribute to the differences in the
phenotypes of the MBPs conferred by the collagen-like domains and are
determinants of the oligomeric state of the molecules.
The construct 4AC, containing the first 6 amino acid residues of MBP-A
in place of the N-terminal 4 residues of MBP-C, consists mainly of a
single trimeric subunit. Despite the additional cysteine residue
(Cys6), only low levels of higher order structures are
detected (Fig. 6). This finding suggests that the contribution of the
N-terminal cysteine-rich domain of MBP-A toward the formation of large
covalent oligomers is not due simply to the presence of an extra
cysteine residue but that additional residues within the C-terminal
half of this domain are involved.
Complement Activation--
In order to assess the complement
fixing activity of the recombinant MBPs, complement activation was
measured by hemolysis of erythrocytes coated with yeast mannan (Fig.
8). The complement fixing activity of
recombinant MBP-A is consistent with that described previously for
preparations of the native lectin, using a similar assay system (1).
Interestingly, complement-dependent hemolysis was also
detected in the presence of MBP-C, but only at concentrations 20 times
higher than for MBP-A. The amount of MBP-C required to cause detectable
hemolysis is greater than that tested in previous studies, since more
protein was available due to production in the expression system. Thus,
these results are consistent with previous studies of MBP-A and suggest
that MBP-C can activate complement but with lower activity that its
serum homolog.
Chimeras of MBP-A and MBP-C were tested for their ability to activate
complement in order to identify regions of MBP-A responsible for its
enhanced activity. Specific hemolysis as a function of concentration
for samples of recombinant MBP-A, MBP-C, and a selection of chimeras is
shown in Fig. 8. As with oligomerization, the complement fixing
activities of the chimeras correlate with the presence of the
cysteine-rich domain and the N-terminal part of the collagen-like domain of MBP-A. These data suggest that the higher oligomeric forms of
MBP-A are more efficient at complement activation in this assay. These
findings are consistent with previous studies of partially purified
oligomers of native MBP isolated from mammalian sera (12, 21).
Since the contribution to the overall complement-dependent hemolytic
activity of MBP-A is determined both by the relative abundance and the
specific activity of each of the component oligomers, the hemolytic
activities of these oligomers were determined individually. Based on
SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, the
most abundant forms of MBP-A are dimers and trimers of subunits.
Specific hemolysis is shown as a function of concentration for purified
oligomers of MBP-A in Fig. 9. The
complement fixing activities of the tetramer, trimer, and dimer of
subunits relative to total MBP-A are 0.95 ± 0.05, 1.18 ± 0.23, and 0.24 ± 0.06, respectively. These data suggest that most
of the hemolytic activity of MBP-A is due to the trimer of subunits.
The tetramer and dimer of subunits also contribute to the complement
fixing activity of MBP-A, although to a lesser extent due to lower
abundance and lower activity of these oligomers, respectively. As noted
above, MBP-C, which consists of a single subunit, also activates
complement in this assay. Therefore, these findings suggest that each
oligomeric form of MBP can interact with and activate downstream
components of the complement cascade.
No complement-dependent hemolytic activity could be detected for the
single subunit of MBP-A under the conditions tested. It is possible
that the binding affinity of this oligomer for mannan-coated
erythrocytes is insufficient to enable complement activation in the
hemolytic assay. Binding studies show that the affinities of MBP-A and
MBP-C for monosaccharide ligands are very similar. However, isolated
CRDs of MBP-A have a much lower affinity than CRDs of MBP-C for
multivalent ligands containing clusters of mannose moieties (29). The
difference in the affinities of MBP-A and MBP-C for multivalent ligands
is likely to be most apparent for smaller oligomers of MBP-A that
contain fewer binding sites. This finding emphasizes that the hemolytic
assay used to detect complement activation reflects a complex series of
molecular interactions, including both ligand binding and binding to
and activation of the downstream components of the complement cascade.
Heterogeneity in MBPs--
The biochemical and biophysical data
reported here indicate that MBP-A produced in Chinese hamster ovary
cells faithfully resembles protein isolated from rat serum. MBP-A
comprises a heterogenous mixture of oligomers consisting predominantly
of dimers and trimers of trimeric subunits, along with lower levels of
tetramers and monomers. Equilibrium ultracentrifugation indicates that
these oligomers do not interact even at high protein concentrations, suggesting that multiple forms of MBP-A exist in the serum. Detailed hydrodynamic analysis of serum MBPs has not been possible previously due to the relatively low amounts of protein isolated from mammalian sera. However, biophysical analysis suggests that human MBP can form
larger structures than its rat counterpart, with oligomers ranging from
dimers to octamers of subunits (13), and electron microscopy
reveals mixtures of oligomers ranging from trimers to hexamers of
subunits (12).
Many purification procedures described for MBPs have incorporated steps
such as gel-filtration chromatography in order to separate serum MBP
from liver MBP and from other contaminating proteins. A consequence of
the heterogeneity of serum MBPs is that these procedures are likely to
introduce a bias in the oligomeric composition, by selectively
purifying certain oligomeric species. This problem may explain
discrepancies in the reported compositions of MBPs in preparations
isolated by different purification strategies.
Assembly of MBP-A--
Analysis of chimeras between MBP-A and
MBP-C indicates that assembly of MBP-A subunits to form larger
oligomers is mediated by amino acid residues within the cysteine-rich
domain and the N-terminal part of the collagen-like domain. Images of
human MBP observed by rotary shadowing electron microscopy are
consistent with this region forming a core which links the trimeric
stems of separate subunits (12). The mechanisms by which collagen triple helices assemble into larger oligomers is unclear, although electrostatic interactions involving charged residues have been implicated, both in the association of collagen triple helices to form
fibrils and in the binding of various molecules to collagen (30-32).
The N-terminal region of the collagen-like domain of MBP-A is rich in
both acidic and basic residues. This region also contains glucosygalactosyl-5-hydroxylysine residues, which may mediate or
modulate oligomer formation through interactions involving the
relatively large, derivatized side chains.
Mutations within the N-terminal region of the collagen-like domain are
associated with MBP deficiency in humans (9, 10). This common genetic
defect results in an increased susceptibility to infections
particularly during the first few years of life. Protein isolated from
patients with this disorder has an altered oligomeric structure
consisting predominantly of lower molecular weight forms (13). The
mutations known to cause MBP deficiency are all localized within the
region of the protein identified as being critical for assembly of
MBP-A. Thus, it seems likely that the altered oligomeric composition of
MBP from patients with this disorder arises due to defective assembly
of the protein.
The covalent structure of MBP-A, consisting of trimeric subunits linked
by disulfide bonds, indicates that these subunits must associate at
some stage during assembly of the protein. However, analytical
ultracentrifugation experiments indicate that no self-association of
the secreted oligomeric forms occurs at high protein concentrations. It
is possible that the conformation of MBP-A oligomers changes during
assembly, so that the secreted forms can no longer self-associate. MBPs
are thought to assemble in a C- to N-terminal direction in which one of
the last steps involves formation of disulfide bonds that link the
polypeptide chains (14). This step may prevent further self-assembly of
subunits. Biochemical analysis indicates that there are no free
cysteine residues within any of the oligomeric forms of MBP-A. This
finding implies that at least some of the cysteine residues within the
cysteine-rich domain must be derivatized, perhaps by linkage to
cysteine or glutathione as has been observed for certain secreted
proteins (33). For example, in the form of MBP-A consisting of a single
subunit of three polypeptide chains, each polypeptide contains three
N-terminal cysteine residues. At least one of these residues cannot
form an intrachain or interchain disulfide bond and must be
derivatized. Analysis of MBP-A Cys6
Vertebrate collagens are known to interact with many different proteins
within the endoplasmic reticulum during their assembly. For example,
collagens have been shown to bind to protein disulfide isomerase, which
is thought to mediate efficient folding by suppressing aggregation
during collagen biosynthesis (34). Thus, it is conceivable that the
interaction of MBP polypeptides with certain molecules during folding
may be necessary for the correct assembly of MBP oligomers to form the
larger structures secreted from the cell. Once the MBP structures have
left the endoplasmic reticulum, these molecules will no longer be
available to assist in assembly, therefore preventing further MBP
oligomer formation.
Complement Fixation by MBPs--
The role of serum MBPs in the
innate immune system is well established (1-3). In contrast, the
function of liver MBP is unknown. MBP-C is able to bind to certain
mammalian glycoproteins, indicating that it may interact with
endogenous glycoproteins within the liver (35, 29). However, the high
degree of sequence identity with MBP-A implies that MBP-C may have a
role in innate immunity. The results reported here indicate that MBP-C
is able to activate complement in an in vitro assay,
although with lower activity than its serum homolog. While the
physiological relevance of this observation is unclear, its seems
possible that within the liver MBP-C may have a role similar to that of
MBP-A in serum. Since MBP-C is smaller than MBP-A, the enhanced
affinity for complex sugar ligands observed for isolated CRDs of MBP-C
may reflect an alternative mechanism for achieving high affinity
binding to carbohydrate structures on the surfaces of microorganisms.
The formation of large oligomeric structures is critical for efficient
complement fixation by MBP-A. However, each oligomeric form appears to
be able to interact with downstream components of the complement
cascade leading to some degree of complement fixation. Since oligomers
do not interact to form higher order structures, the total complement
fixation activity is a function of the individual molecular activities
of oligomers and their relative abundance within the serum. The mutant
MBPs associated with MBP deficiency in humans have an altered
composition, consisting predominantly of lower molecular weight forms
(13). The decreased activity associated with these structures may
provide an explanation for the low complement fixing activity of serum
isolated from patients with this disorder. Alternatively, mutations
within the collagen-like domain of serum MBP may disrupt the binding
site for MASP-1 or MASP-2, thus directly preventing activation of the complement cascade.
Ser suggests that polypeptides within each
trimeric structural unit are mostly linked by disulfide bonds between
cysteine residues at positions 13 and 18 arranged in an asymmetrical
configuration. Disulfide bonds involving Cys6 connect
polypeptides within separate trimers. Analysis of chimeras between
MBP-A and rat liver MBP (MBP-C) indicates that residues within the
N-terminal region of the collagenous domain and the cysteine-rich
domain of MBP-A enable assembly of trimers into higher order oligomers.
The activity of MBP-A in a hemolytic complement fixation assay using
mannan-coated sheep erythrocytes was approximately 20-fold greater than
the activity of MBP-C. Analysis of the MBP chimeras and isolated
oligomers of MBP-A reveals that the larger oligomers are more efficient
at complement activation. These data indicate that the overall
complement fixing activity of MBP-A is a function of the individual
molecular activities of oligomers and their relative abundance within
the serum.
INTRODUCTION
Top
Abstract
Introduction
References
-helical coiled-coil neck region and a C-terminal
carbohydrate-recognition domain (CRD) (4).
-helical coiled-coil region enables formation
of the collagen triple helix. The collagen-like domain contains
4-hydroxyproline and 5-hydroxylysine residues, which are further
derivatized to form glucosylgalactosyl-5-hydroxylysine (11, 15).
MBP-A and MBP-C each contain a single break in the Gly-Xaa-Yaa repeat
pattern within the collagenous domain (11). This interruption is
thought to introduce a kink or region of flexibility into the protein,
which in MBP-A enables the trimeric stems to angle away from the
central core of the bouquet-like structure. The polypeptides are linked
by cysteine residues within the N-terminal domain which form disulfide
bonds within and between subunits in the larger MBP-A structures
(11).
EXPERIMENTAL PROCEDURES
80 °C. MBP was isolated following the method previously used for MBP-C (14). For complement assays, protein was isolated from
serum-free medium. Two days after cell confluence was reached, the
medium from each tissue culture flask was replaced with 70 ml of
CHO-S-SFM II without nucleosides supplemented with 50 mM HEPES, pH 7.55, and 0.5 µM methotrexate. This medium was
harvested daily from 4 days after confluence and MBP-A was purified as
described above.
Ser were resolved by ion-exchange chromatography
on a Mono-Q HR 5/5 column (Pharmacia) equilibrated in 50 mM
Tris, pH 8.2, containing 10 mM EDTA at a flow rate of 1 ml/min at 25 °C. Oligomers were eluted from the column using a 500 mM NaCl gradient in the same buffer over 45 min. The
absorbance was monitored at 280 nm and 0.5-ml fractions were collected.
RESULTS
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Fig. 1.
Post-translational modifications of
MBP-A. Peptides generated by chymotrypsin and thermolysin
digestion were blotted on to polyvinylidene difluoride membrane
and the N-terminal sequences were determined by amino acid sequencing
(bold). The remaining fragments were generated by trypsin
digestion and separated by reverse-phase chromatography. These peptides
were identified by MALDI-MS and their N-terminal sequences were
confirmed by amino acid sequencing (bold). The calculated
[M + H]+ values are shown in parentheses.
POH denotes 4-hydroxyproline and
K* denotes glucosylgalactosyl-5-hydroxylysine.
The most abundant modified forms of peptides are shown as judged by
amino acid sequencing and from the absorbance of peaks resolved by
reverse-phase chromatography.
162.1
Da). However, it is not clear whether these peaks reflect heterogeneity
of the sample or occur as a result of the ionization/desorption process.
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Fig. 2.
Gel-filtration and SDS-polyacrylamide gel
electrophoresis of recombinant MBP-A. A, gel-filtration
chromatography of MBP-A (100 µl at 0.8 mg/ml). The elution positions
of bovine thyroglobulin (T, 669 kDa), horse spleen
apoferritin (F, 443 kDa), sweet potato -amylase
(A, 200 kDa), yeast alcohol dehydrogenase (D, 150 kDa), and bovine serum albumen (S, 66 kDa) are indicated.
B, fractions (0.25 ml) collected across the protein peaks
separated on a 10%-polyacrylamide gel under nonreducing conditions.
Protein was detected by staining with Coomassie Blue. Molecular mass
markers are shown at the left. Monomers, dimers, trimers,
and tetramers of the trimeric unit have apparent molecular masses of
85, 185, 280, and 355 kDa, respectively. C, oligomers of the
trimeric unit of MBP-A, purified by ion-exchange chromatography on a
Mono-Q column, were analyzed on a 10%-polyacrylamide gel under
nonreducing conditions. Protein was detected by staining with Coomassie
Blue. Molecular mass markers are shown at the left.
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Fig. 3.
Equilibrium sedimentation ultracentrifugation
of purified oligomers of MBP-A. Apparent weight-averaged molecular
mass distribution as a function of concentration for three loading
concentrations are shown. The calculated masses of dimers, trimers,
tetramers, and hexamers of subunits, based on the amino acid sequence
of MBP-A, are indicated by an arrow. In each case, oligomers
were purified by ion-exchange chromatography on a Mono-Q column.
Top, dimer of the trimeric unit (7,000 r.p.m.). The loading
concentrations were approximately 0.4 mg/ml ( ), 0.2 mg/ml (
), and
0.1 mg/ml (
). Bottom, trimer of the trimeric unit
(5,500 r.p.m.). The loading concentrations were approximately 0.2 mg/ml (
), 0.1 mg/ml (
), and 0.05 mg/ml (
).
Hydrodynamic properties of purified oligomers of MBP-A
Ser with that of wild-type MBP-A
reveals that most of the protein elutes from the column as a single
peak at the position corresponding to a single subunit (Fig.
4A). Only low levels of the
larger oligomers are detected. The major peak consists mainly of a
covalent trimer of polypeptides based on SDS-polyacrylamide gel
electrophoresis of fractions collected across the gel-filtration peaks
(data not shown). Minor bands corresponding to dimers and monomers of
polypeptides could also be detected, which suggest that a small
proportion the trimeric units are noncovalent. As shown in Fig.
4B, the major band in the MBP-A Cys6
Ser
lane consists of disulfide-linked trimers of polypeptides, although
dimers and trimers of covalent subunits can also be detected at low
abundance. Thus, mutation of Cys6 to serine results
predominantly in formation of a single subunit. These data are
consistent with the suggestion that Cys6 forms disulfide
bonds between separate subunits, thus creating the larger covalent
forms observed in preparations of wild-type MBP-A.
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Fig. 4.
Gel-filtration and SDS-polyacrylamide gel
electrophoresis of MBP-A Cys6 Ser. A, Gel-filtration chromatography of MBP-A
(top) and MBP-A Cys6
Ser
(bottom). In each case, 100 µl of protein was loaded onto
the column at a concentration of 0.8 mg/ml. B,
SDS-polyacrylamide gel electrophoresis of MBP-A and MBP-A
Cys6
Ser on a 10% gel under nonreducing conditions.
Protein was detected by staining with Coomassie Blue. The migration
positions of molecular mass markers and the oligomeric forms of MBP-A
are shown on the left and right,
respectively.
Ser was analyzed by
analytical ultracentrifugation (data not shown). The apparent molecular mass was 75,500 ± 1,500 Da in two independent experiments. This value is very similar to the calculated value (approximately 74,200 Da)
based on the mass of individual MBP-A polypeptides determined by
MALDI-MS. No self-association could be detected over the entire concentration range examined (up to 2 mg/ml). These data therefore confirm that the most abundant form of MBP-A Cys6
Ser
is a single subunit. This result further supports the conclusion that
disulfide bonds between Cys6 residues link
polypeptides in separate subunits to form larger covalent structures in
wild-type MBP-A.
Ser trimeric subunit was determined by analysis of an N-terminal fragment generated by digestion with proteinase Arg-C. Peptides were
separated by reverse-phase chromatography and identified by MALDI-MS
and amino acid sequencing. Under nonreducing conditions, the mass of
peptide Ser1-Arg20 determined by MALDI-MS
(6,168.0 Da) corresponds closely to the mass of a disulfide-linked
trimer based on the amino acid sequence of MBP-A (6,165.8 Da).
Disulfide-linked dimers and monomers of peptide
Ser1-Arg20 were also detected by MALDI-MS.
However, the abundance of these species was relatively low. Mass values
detected for the monomer and the covalent dimer and trimer were
consistent with values calculated from the sequence of MBP-A,
indicating that the 2 cysteine residues within these peptides
(Cys13 and Cys18) are not modified by linkage
to free cysteine or glutathione. Furthermore, the trimeric and dimeric
peptides could be reduced to monomers on incubation with
dithiothreitol, confirming that the polypetide chains are linked by
disulfide bonds. As in the case of MBP-C (14), two possible isomers can
be considered for the covalent trimer, each containing three interchain
disulfide bonds. These disulfide bonds can be arranged either in a
symmetrical pattern, where each polypeptide is linked by
Cys13-Cys18, or in an asymmetrical pattern.
Cleavage of the trimeric peptide between Cys13 and
Ser14 by subtilisin indicates that the cysteine residues
must be linked by disulfide bonds arranged in an asymmetrical pattern.
Peaks were detected by MALDI-MS corresponding to all three dimeric
peptides: (Ser1-Cys13)2 ([M + H]+ = 2740.2 Da),
(Ser14-Arg20)2 (1408.6 Da), and
Ser1-Cys13/Ser14-Arg20
(2074.2 Da). A similar strategy was used to determine the
disulfide-bonding pattern of the noncovalent trimer of MBP-A
Cys6
Ser. Peptides
(Ser1-Cys13)2 and
(Ser14-Arg20)2 but not peptide
Ser1-Cys13/Ser14-Arg20
could be detected by MALDI-MS following digestion of the dimeric peptide with subtilisin. This result indicates that this peptide is
linked by disulfide bonds Cys13-Cys13 and
Cys18-Cys18 and suggests that the noncovalent
trimer comprises disulfide-linked dimer associated with monomer
containing an intrachain disulfide bond.
Ser mirrors the bonding pattern described
previously for MBP-C (14). Thus, the majority of the polypeptides in
individual trimeric subunits of MBP-A are probably linked by disulfide
bonds between Cys13 and Cys18 arranged in an
asymmetrical pattern. Disulfide bonds between Cys6 residues
in separate subunits form the larger covalent structures observed by
SDS-polyacrylamide gel electrophoresis under nonreducing conditions.
Each subunit contains 3 Cys6 residues each of which can
potentially form disulfide bonds with polypeptides in separate trimers.
Ser, although with low abundance, indicating that
there must be an alternative bonding arrangement in these oligomers.
One possible explanation of this observation is that the larger
covalent oligomers are formed as a result of the close proximity of
N-terminal cysteine residues in noncovalently associated subunits
during assembly of the protein in the endoplasmic reticulum. As an
alternative proposal, the higher order structures observed in
preparations of MBP-A Cys6
Ser may indicate that a
small proportion of oligomers of MBP-A are linked by a different
disulfide bonding arrangement, reflecting further heterogeneity in
MBP-A.
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Fig. 5.
Construction of chimeras of MBP-A and
MBP-C. Top, schematic representation of mannose-binding
proteins. Each domain is represented by a separate segment. The
collagen-like domain is divided into two segments, with the junction
corresponding to the break in the collagen consensus sequence
(Gly-Gln-Gly). The aligned amino acid sequences of the N-terminal and
collagen-like domains of MBP-A and MBP-C are shown. Padding characters
have been included in MBP-A to optimize the sequence alignment.
Bottom, schematic representation of the stable chimeras of
MBP-A and MBP-C. For each hybrid, the junction between sequence derived
from MBP-A (white) and MBP-C (black) is indicated
by an arrow. An additional segment is included at the start
of the collagen-like region in MBP-C to represent an insertion in the
sequence, for which there are no corresponding residues in MBP-A.
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Fig. 6.
Gel-filtration analysis of chimeras of MBP-A
and MBP-C. Gel-filtration chromatography of chimeric proteins. In
each case, 100 µl of protein was loaded onto the column at a
concentration of 0.2 mg/ml. Chimeras are represented in the schematic
form described in Fig. 5.
8.0 ml) are detected in preparations of MBP-C and
chimera 4AC (Fig. 6). These peaks were found to consist of a
proteolytic fragment of MBP-C by N-terminal sequencing, comprising the
C terminus from residue Val54. This proteolytic fragment
has been previously identified in preparations of MBP-C (14).
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Fig. 7.
SDS-polyacrylamide gel electrophoresis of
chimeras of MBP-A and MBP-C. Chimeras were separated on a 10%
polyacrylamide gel under nonreducing conditions. Protein was detected
by staining with Coomassie Blue. The migration positions of molecular
mass markers and the oligomeric forms of MBP-A are shown on the
left and right, respectively. Chimeras are
represented in the schematic form shown in Fig. 5.
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Fig. 8.
Complement activation by chimeras of MBP-A
and MBP-C. Complement fixing activity was measured by hemolysis of
mannan-coated sheep erythrocytes. Only data from MBP-A, MBP-C, and four
chimeras are included for clarity. Complement fixing activities are
expressed relative to the activity of MBP-A. The activities of chimeras
2AC, 2CA, and 4AC (Fig. 5) are 0.91 ± .03, 0.22 ± 0.02, and
0.020 ± 0.003, respectively. Chimeras are represented in the
schematic form shown in Fig. 5.
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Fig. 9.
Complement activation by MBP-C and oligomers
of MBP-A. MBP-A oligomers were purified by ion-exchange
chromatography. Single subunits of MBP-A showed no
complement-dependent hemolytic activity at concentrations
up to 10 µg.
DISCUSSION
Ser suggests
that disulfide bonds between Cys13 and Cys18
link MBP-A polypeptides within subunits, while bonds between Cys6 of certain polypeptides link separate subunits. Thus,
based on this model, modification is likely to occur to
Cys6 residues which do not form disulfide bonds. These
modifications may prevent assembly of MBP-A subunits in the secreted protein.
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FOOTNOTES |
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* This work was supported by a grant from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Glycobiology
Institute, Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom. Tel.: 44-1865-275762; Fax: 44-1865-275339; E-mail: rwallis{at}glycob.ox.ac.uk.
§ Wellcome Principal Research Fellow.
The abbreviations used are: MBP, mannose-binding protein; CRD, carbohydrate-recognition domain; MASP, MBP-associated serine protease; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry.
2 R. Dodd, E. Leamy, R. Wallis, and K. Drickamer, unpublished observations.
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REFERENCES |
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