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Department of Biochemistry, Glycobiology Institute, University of Oxford, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum MBP forms complexes with three different MASPs (MASP-1, MASP-2, and MASP-3) (5, 6, 7). MASPs have a common domain organization consisting of two N-terminal CUB domains (domains found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein 1) separated by an epidermal growth factor-like domain and followed by two complement control protein modules and a serine protease domain. MASPs circulate as zymogens attached to MBP that become activated when MBP binds to a target cell. Only MASP-2 has a clearly defined role in complement fixation (8). Upon autoactivation, it cleaves complement components C4 and C2 to generate fragments that form the C3 convertase. MASP-1 and MASP-3 are synthesized through alternative splicing of the MASP-1/3 gene (5). Their biological roles are not known. MASPs are homodimers stabilized through interactions involving the N-terminal CUB domain (9). The N-terminal CUB and epidermal growth factor-like domains are necessary and sufficient to reproduce the MBP-binding properties of the full-size proteins. MBP dimers bind single MASP dimers, whereas MBP trimers and tetramers bind up to two MASP dimers.
MBP-associated immunodeficiency is a common disorder that is linked to increased susceptibility to a wide range of infections (2). Individuals are particularly vulnerable in the first few years of life, before an effective adaptive immune response is established. The deficiency also manifests itself when adaptive immunity is compromised, for example, during HIV infection or following chemotherapy (10, 11). MBP-associated immunodeficiency can be caused by any one of three separate point mutations in the MBP gene. Each mutation leads to a single amino acid substitution within the first part of the collagenous domain. In both homozygous and heterozygous individuals, the disorder is characterized by reduced levels of serum MBP and structural changes to the N-terminal domains.
A model system in which the naturally occurring human variants have been recreated in rat serum MBP has proved to be very useful for characterizing the structural defects associated with homozygous genotypes (9, 12). The mutations decrease significantly the complement-fixing activities of rat MBP as a result of structural changes that disrupt interactions with MASPs. In homozygous individuals, immunodeficiency is caused by inefficient complement activation that compounds the effects of reduced serum MBP levels. However, the molecular basis of immunodeficiency in heterozygous individuals is poorly understood. Serum MBP levels are considerably higher than in homozygous subjects, implying that diminished ability to fix complement rather than decreased serum levels may be the major factor leading to immunodeficiency.
To determine whether MBPs of the type that would be produced by heterozygotes are functionally defective, wild-type and mutant rat MBPs have been coexpressed. The resulting heterooligomeric MBPs have significantly reduced complement-fixing activities as a result of structural changes that resemble the defects in homooligomeric mutant MBPs. Defective complement fixation resulting from the dominant effects of the mutations is likely to be a major cause of immunodeficiency in individuals with mutant MBP alleles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes were purchased from New England Biolabs
(Beverly, MA). All tissue culture media were obtained from Life
Technologies (Gaithersburg, MD). Promix cell-labeling mix (
70%
[35S]methionineand 30%
35S-labeled cysteine) was purchased from Amersham
Pharmacia Biotech (Little Chalfont, U.K.). Nitrilotriacetic
acid-agarose, Sepharose 6B, and protein molecular mass markers were
obtained from Sigma-Aldrich (St. Louis, MO). Immulon 4 microtiter
wells were purchased from Dynex Technologies (Chantilly, VA).
Nitrocellulose membranes were purchased from Schleicher & Schuell
(Keene, NH). Anti-hemagglutinin (HA) and anti-c-myc mAbs
were purchased from Roche Molecular Biochemicals (Indianapolis,
IN).
Production of fragments of MASP-1/3 and MASP-2
The N-terminal three domains of MASP-1 and MASP-2, which reproduce the MBP-binding properties of full-size MASPs, were produced in Chinese hamster ovary cells and were purified on nitrilotriacetic acid-agarose as described previously (13). The N-terminal three domains of MASP-1 and MASP-3 are identical (5).
Analytical methods
SDS-PAGE was performed using the method of Laemmli (14). Gel filtration chromatography was conducted on a BioSep-S3000 column (300 x 7.8 mm; Phenomenex, Belmont, CA ) as described previously for rat serum MBP (4). The composition of oligomers in each MBP was determined by fitting the gel filtration data to multiple Gaussian curves using Microcal Origin. Data are presented as means ± SE from two independent experiments. Complement-fixation activities of MBPs were determined using mannan-coated erythrocytes as targets, following the protocol described previously (4). Results are presented as means ± SE from two independent assays. Solid-phase competition assays were performed by incubating increasing concentrations of MBPs with immobilized fragments comprising the N-terminal three domains of either MASP-1/3 or MASP-2 using 35S-labeled MBP as the reporter ligand (13). Results are presented as means ± SE from two independent assays performed in duplicate.
Production and purification of MBPs
Mutations were introduced into the cDNA encoding rat serum MBP
by substitution of synthetic double-stranded oligonucleotides for
restriction fragments. Standard molecular biology techniques were
conducted as described elsewhere (15). High selectivity
for galactose was introduced into the CRD of MBP by incorporation of
the following changes: Glu185
Gln,
Asn187Asp, His189Trp,
Gly190Tyr, Ser191Gly,
and insertion of the sequence Gly-Leu-Gly-Gly between residues
Ser191 and Gly192. The
resulting CRDs have previously been designated QPDWG (16).
In some cases, epitope tags were attached to the C termini of MBP
polypeptides. Synthetic double-stranded oligonucleotides encoding the
sequences Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, derived from influenza
HA and Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, from the human
p62c-myc protein (c-myc), were
inserted immediately before the stop codon at the 3' end of the
cDNA.
To coexpress wild-type and mutant MBPs, wild-type and modifiedcDNAs
were inserted in tandem into the polylinker region of expression
plasmid pEE14.1 using convenient restriction sites (17). A
separate promoter derived from CMV was introduced upstream of each cDNA
to ensure equivalent, high-level expression (Fig. 1
). The resulting
plasmids were used to transfect Chinese hamster ovary cell line K-1,
using the calcium phosphate method. Cells containing the constructs
were selected by growth in Glasgow MEM supplemented with the glutamine
synthetase inhibitor methionine sulfoximine (25 µM)
(17). For large-scale protein preparations, cells were
grown to confluence in 225-cm2 tissue culture
flasks containing 50 ml of culture medium. Medium was harvested and
stored as described previously for wild-type MBP (4).
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supplemented with 10% dialyzed FCS, 0.5 µM
methotrexate, and 0.1 mCi/ml [35S]methionine
for 16 h. Radiolabeled protein was purified as for unlabeled
wild-type MBP. Quantification of the amounts of wild-type and mutant polypeptides in MBPs
Relative amounts of wild-type and mutant polypeptides in MBP preparations were determined by separating known amounts of purified HA and c-myc epitope-tagged proteins on 17.5% polyacrylamide gels under reducing conditions. WT* MBP, containing a HA tag and the homozygous R23C/R23C protein with a c-myc tag, were also loaded on the gels as standards. Duplicate immunoblots were probed with Abs specific for the HA and c-myc epitopes. Radioactivity was quantified using a PhosphorImager SI from Molecular Dynamics (Sunnyvale, CA) after exposure for at least 18 h. Relative amounts of polypeptides were calculated from the levels of radioactivity normalized to the amounts of radioactivity detected in the corresponding standard. The proportion of WT* polypeptides in preparations of MBP purified from two separate cell lines were 0.40 ± 0.09 for the WT*/R23C protein, 0.52 ± 0.07 for the WT*/G25D protein, and 0.51 ± 0.07 for the WT*/G28E protein. Thus, there is no intracellular selection against MBPs containing mutant polypeptides.
Binding of MBPs on mannose-Sepharose and galactose-Sepharose affinity columns
MBPs (0.1 mg) were loaded onto mannose-Sepharose and galactose-Sepharose affinity columns (1 ml) in 1 ml of loading buffer (50 mM Tris (pH 7.8) containing 1.25 M NaCl and 25 mM CaCl2). Columns were washed with loading buffer (3 x 1-ml fractions), and protein was eluted in 50 mM Tris (pH 7.8) containing 1.25 M NaCl and 2.5 mM EDTA (3 x 1-ml fractions). Galactose (6 mM) was included in the loading buffer for the mannose-Sepharose affinity columns to prevent weak interactions with the CRDs of WT* MBP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first step toward understanding why MBPs produced by
heterozygous individuals are functionally defective was to determine
whether wild-type and mutant polypeptide chains associate with each
other during biosynthesis. To address this question, the CRDs of
wild-type chains, but not mutant chains, were modified to confer
selectivity to galactose (Fig. 1
). This
strategy enabled selective purification of homooligomeric wild-type and
mutant MBPs by affinity chromatography on galactose-Sepharose or
mannose-Sepharose columns while heterooligomeric MBPs bound to both
columns. Because CRDs are not involved in oligomerization of subunits,
the changes were not expected to disrupt assembly of the MBPs.
As expected, wild-type MBP binds to a mannose-Sepharose column and can
be eluted by chelating the Ca2+ required for
binding (Fig. 2). It elutes in the
unbound wash fractions from a galactose-Sepharose column, indicating
that it does not interact significantly with galactose. In contrast,
MBP with galactose-binding specificity (WT*) is completely
retained on a galactose-Sepharose column, whereas it elutes in the wash
fractions on a mannose-Sepharose column, confirming that it binds
selectivity to galactose. As shown in Fig. 2, 
95% of the WT*/R23C,
WT*/G25D, and WT*/G28E proteins bind to both mannose-Sepharose and
galactose-Sepharose affinity columns, with only trace amounts of
protein eluting in the wash fractions. Because WT* CRDs are selective
for galactose whereas mutant CRDs are specific for mannose, nearly all
of the MBP oligomers must contain both mutant and wild-type
polypeptides. Thus, the proteins are secreted predominantly as
heterooligomers.
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To assess the ability of heteroligomeric MBPs to fix complement on
mannose-containing surfaces, it was necessary to produce additional
heterooligomeric MBPs containing mannose-binding CRDs in both types of
polypeptides and without C-terminal tags. Complement-activation assays
were conducted using mannan-coated sheep erythrocytes as targets for
the MBPs. Compared with wild-type MBP, higher concentrations of
heterooligomeric MBPs were required to cause equivalent lysis of the
target erythrocytes, demonstrating that the heterooligomers have
reduced complement-fixing activities (Fig. 3). The complement-fixing activity of the
WT/R23C protein is reduced by 1.5- to 4-fold while the activities of
the WT/G25D and WT/G28E proteins are reduced by 5-fold and over
30-fold, respectively. By comparison, the complement-fixing
activities of the homooligomeric R23C/R23C, G25D/G25D, and G28E/G28E
proteins are reduced by 10-, 7-, and 100-fold, respectively. Thus,
mutations to just some polypeptide chains lead to dominant phenotypes.
Because individuals heterozygous for the human disorder are likely to
produce heterooligomeric MBPs, defective complement fixation is
expected to be a major cause of immunodeficiency in such patients.
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Previous studies have revealed that the overall complement-fixing
activity of MBP is a function of the distribution of oligomers and the
specific activities of each oligomer (4). Wild-type rat
MBP comprises covalent oligomers ranging from monomers to tetramers of
subunits, of which trimers and tetramers have the highest
complement-fixing activities. By contrast, the R23C/R23C protein
consists of a lower proportion of dimers, trimers, and tetramers of
subunits and higher amounts of single subunits, which leads to a
reduction in the overall complement-fixing activity (18).
Gel filtration analysis shows that the proportion of MBP trimers and
tetramers is also reduced in the WT/R23C protein, although not to the
same extent as in the R23C/R23C protein (Fig. 4). The major species consist of covalent
oligomers that resemble those of wild-type MBP on SDS-polyacrylamide
gels, indicating that the disulfide bonds that link individual
polypeptide chains together at the N terminus are not disrupted
significantly (Fig. 5). Based on the
relative complement-fixing activities of oligomers in wild-type MBP
(4), the WT/R23C protein would be expected to have 50%
of the activity of wild-type protein if the defect were due to changes
in the relative proportion of oligomers alone. This value is consistent
with the 1.5- to 4-fold reduction in complement-fixing activity
observed, suggesting that the primary defect is due to aberrant
assembly during biosynthesis, resulting in lower amounts of trimers and
tetramers of subunits.
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). However, MASP binding by WT/R23C is
less perturbed than MASP binding by the R23C/R23C protein (Table I), an effect that parallels the degree
of disruption to oligomerization in homooligomeric and heterooligomeric
MBPs (Fig. 4). Thus, defective binding to MASP-2 by the WT/R23C protein
can also be accounted for by changes in the distribution of oligomers
and probably leads directly to aberrant complement fixation.
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Structural defects in the WT/G25D and WT/G28E proteins
Previous studies have shown that reductions in the proportion of
trimers and tetramers of MBP subunits account for an 3-fold decrease
in the complement-fixing activities of the G25D/G25D and G28E/G28E
proteins (18). Gel filtration analysis reveals that the
distribution of oligomers in the WT/G25D and WT/G28E proteins is also
perturbed compared with wild-type MBP (Fig. 4), although percentages of
trimers and tetramers of subunits are somewhat higher than for the
G25D/G25D and G28E/G28E proteins. Changes in the distribution of
oligomers alone could only account for a reduction of 25% in the
complement-fixing activities, which is much less than the 5-fold and
>30-fold reductions observed. Therefore, individual heterooligomers of
the WT/G25D and WT/G28E proteins must also have lower specific
complement-fixing activities than corresponding homooligomers of their
wild-type counterparts.
Reduced specific complement-fixing activities of individual oligomers
of the G25D/G25D and G28E/G28E proteins reflect perturbation of the
MASP-2 binding site caused by local disruption to the collagen-like
domain and the adjacent cysteine-rich domain (18). These
changes lead to a 4-fold decrease in complement-fixing activity.
Comparable structural defects in the cysteine-rich domains are detected
in the WT/G25D and WT/G28E proteins, as evidenced by the pattern of
bands observed on SDS-polyacrylamide gels, in which the predominant
covalent species comprise two and four polypeptide chains (Fig. 5). The
covalent structures are also comparable to proteins isolated from the
serum of immunodeficient individuals that consist of low molecular mass
covalent forms.
A 4-fold decrease in complement-fixing activity due to structural perturbations in the N-terminal domains that disrupt the interaction with MASP-2 combined with the small reduction in complement-fixing activity due to the changes in the distribution of oligomers would account for the overall 5-fold decrease in complement-fixing activity of the WT/G25D protein. However, the WT/G28E protein is a further 8- to 10-fold less active at fixing complement than the WT/G25D protein. This reduction mirrors the 10-fold difference in complement-fixing activities between the G25D/G25D and G28E/G28E proteins. Thus, the additional defect caused by the G28E mutation is also a dominant effect.
As expected, the structural changes in the WT/G25D and WT/G28E proteins lead to defective interactions with MASPs. Indeed, solid-phase competition assays reveal that the affinities of the complexes are broadly comparable to those of complexes between the corresponding homooligomeric mutant MBPs and MASPs. Thus, the dominant nature of the phenotypes implies that replacing a glycine residue at the 25 or 28 position by an acidic residue in just one or two polypeptides of the collagen triple helix is almost as disruptive as when all three polypeptide chains contain the mutation. The mutation at the 28 position affects binding to either MASP-1 or MASP-2 more than the mutation at the 25 position. The simplest explanation to account for differences in affinities and complement-fixing activities between the two glycine mutants is that the MASPs bind closer to the 28 position, near the point at which individual subunits diverge from the central core in the bouquet-like structures.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that immunodeficiency in heterozygous individuals arises because only a small fraction of the MBP oligomers will be normal (19). Thus, only 1 in 8 (1 in 2 x 2 x 2) single subunits, 1 in 64 dimers, 1 in 512 trimers, and 1 in 4096 tetramers will contain exclusively wild-type polypeptides, assuming random association of chains. This hypothesis is entirely consistent with data for the WT/G25D and WT/G28E proteins, because the specific complement-fixing activities of these heteroligomeric MBPs are equivalent to the activities of their homooligomeric mutant counterparts. The complement-fixing activity of the WT/R23C protein is less impaired than would be predicted from the model, implying that the defect results from the presence of more than one mutant polypeptide in an MBP oligomer.
Trimerization of MBP polypeptides is initiated by interactions between
the C-terminal CRDs and the helices that form the neck regions and
proceeds in a C- to N-terminal direction (20, 21). Because
WT/mutant MBPs form trimeric subunits and contain both wild-type and
mutant polypeptides in comparable proportions, the mutations do not
prevent subunit formation. All three mutations are near the N terminus
of MBP and so it is likely that only the N-terminal part of the
collagenous domain and the cysteine-rich domain are misfolded.
Reduced complement-fixing activities of the WT/R23C and R23C/R23C proteins can be accounted for by changes in the population of oligomers, resulting in a reduction in the amounts of the larger species, although oligomerization of the WT/R23C protein is less impaired. Previous studies have shown that at least some cysteine 23 residues are disulfide bonded to corresponding residues within individual subunits of the homooligomeric mutant MBPs (18). The disulfide bonds probably disrupt interactions between subunits by distorting the collagen triple helix, thus preventing oligomerization of subunits. In heteroligomeric MBPs, in which just half of the polypeptides contain cysteine 23 residues, only a fraction of subunits are likely to contain aberrant interchain disulfide bonds. This distinction probably accounts for the differences in the distribution of oligomers between the WT/R23C and R23C/R23C proteins.
Each protomer of a MASP dimer must bind to a separate subunit of a MBP oligomer to form a stable complex capable of fixing complement (9). Therefore, disruption of even a single MBP subunit due to the presence of a polypeptide containing a mutation is likely to destabilize a MBP-MASP complex. The dominant phenotype resulting from the G25D and G28E mutations in heterozygous individuals probably arises because almost all of MBPs will be heteroligomers that bind defectively to MASP-2.
Reduction in the affinities of MBP/MASP-2 complexes alone cannot
account for the lower complement-fixing activities of the
homooligomeric and heterooligomeric MBPs (Table I). Binding of MASP-1/3
is also affected by the mutations and the possibility that MASP-1,
MASP-2, and MASP-3 function cooperatively cannot be completely ruled
out. Because MASP-2 in complex with MBP autoactivates and can trigger
complement activation, it is more probable that MASP-2 activation
itself is defective in complexes with mutant MBPs. Defective
interactions between MBP and MASP-2 probably prevent efficient zymogen
activation, thus precluding complement fixation.
There is a complex relationship between disruption of the structure of
the collagenous domain of MBP, formation of larger oligomers, MASP-2
binding, and complement activation. For example, the WT/G25D and
WT/R23C proteins bind to MASP-2 with broadly comparable affinities but
the complement-fixing activity of the WT/G25D protein is 2.5-fold
lower than the activity of the WT/R23C protein (Fig. 3). The
discrepancy between affinities and complement-fixing activities
probably arises because the principal molecular defect is different in
these two MBPs. In the WT/R23C protein, reduced complement-fixing
activity is due largely to defective oligomerization of subunits,
whereas in the WT/G25D protein it is caused by structural changes in
the collagenous domain. Although both changes affect MASP-2 binding
affinity to comparable extents, the effects on MASP-2 activation must
be different.
Serum MBP concentrations are considerably higher in heterozygous individuals than in homozygotes. For example, British Caucasoids heterozygous for the mutation equivalent to G25D in rat MBP have only 4- to 5-fold lower serum MBP levels than wild-type individuals, while trace amounts of MBP could be detected in the serum of homozygotes (22). Data reported here indicate that complement-fixation activities of WT/mutant MBPs are all significantly reduced. Thus, defective complement activation may make a more important contribution to the immunodeficient phenotype of heterozygotes than of homozygotes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Russell Wallis, Department of Biochemistry, Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. E-mail address: rwallis{at}glycob.ox.ac.uk
3 Abbreviations used in this paper: MBP, serum mannose-binding protein; WT, wild type; WT* MBP, MBP with galactose-binding selectivity; CRD, carbohydrate-recognition domain; MASP, MBP-associated serine protease; HA, influenza hemagglutinin.
Received for publication January 8, 2002. Accepted for publication March 6, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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