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Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Asp and Gly28Glu disrupt
the disulfide-bonding arrangement of the protein and cause at least a
5-fold increase in the half-time of secretion of MBP compared with
wild-type rat serum MBP. A similar phenotype, including a 3-fold
increase in the half-time of secretion, disruption of the disulfide
bonding arrangement, and inefficient complement fixation, is observed
when nearby glucosylgalactosyl hydroxylysine residues at positions 27
and 30 are replaced with arginine residues. The results suggest that
defective secretion resulting from structural changes in the
collagen-like domain is likely to be a contributory factor for MBP
immunodeficiency. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MBP deficiency, a common genetic disorder in humans, is associated with increased susceptibility to infections (1). Individuals are particularly vulnerable before the adaptive immune system is fully developed in early childhood or when immunity is compromised, for example, in AIDS (3). This immunodeficiency is associated with three different point mutations within the collagen-like domain. These mutations cause distinct structural defects in MBP, which in turn result in a reduced ability to activate the complement cascade (4). Defective complement fixation by the mutant MBPs is further compounded by low levels of serum MBP, which arise through an unknown mechanism. The structural defects in the MBP variants could lead to aberrant secretion or increased turnover from the serum. It has also been suggested that mutations within upstream promoter regions of the MBP gene contribute to decreased synthesis of the protein (5).
Serum MBP consists of a heterogeneous mixture of covalent,
noninteracting homo-oligomers assembled from trimeric subunits
(6, 7). Structural and functional consequences of
mutations to the collagenous domain have been most extensively
characterized in a rat model system, in which the natural human
mutations have been introduced into rat serum MBP (MBP-A) expressed in
Chinese hamster ovary cells. The mutations associated with MBP
deficiency cause several distinct changes. The mutation
Arg23Cys generates adventitious disulfide
bonds and dramatically reduces formation of tetramers and trimers of
subunits. The reduced ability of this mutant to fix complement reflects
the lower efficiency of complement fixation by the smaller oligomeric
forms that predominate. The mutations Gly28Glu
and Gly25Asp cause a less severe reduction in
higher oligomer formation but have a disrupted arrangement of disulfide
bonds in the N-terminal domain adjacent to the collagenous region.
These mutations also result in reduced levels of hydroxylation and
glycosylation of lysine residues in the collagenous domains. These
disruptions to the structure of the collagenous and adjacent domains
result in inefficient complement fixation, probably because they
interfere with MASP binding.
By expressing wild-type and mutant MBP from a common promotor in Chinese hamster ovary cells, it is possible specifically to examine the effects of changes in the protein sequence on secretion rates. The results indicate that secretion is significantly impaired in the two glycine mutants associated with low protein serum levels in MBP deficiency. This defect arises as a direct consequence of the common structural alterations in these proteins that lead to aberrant assembly within the cell and can be replicated by elimination of glucosylgalactosyl hydroxylysine residues in the N-terminal portion of the collagenous domain.
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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). Tissue culture medium was from Life Technologies (Gaithersburg, MD). Yeast mannan, protein m.w. markers for gel electrophoresis, brefeldin A, methotrexate, and protein A-Sepharose were purchased from Sigma (St. Louis, MO). Guinea pig serum and sheep erythrocytes were obtained from Serotec (Oxford, U.K.). Pro-mix cell labeling mix (7:3 mixture of [35S]methionine and [35S]cysteine), [3H]galactose, and [14C]galactose were obtained from Amersham (Little Chalfont, U.K.).
Production and analysis of mutant rat MBPs
Mutant MBPs were generated by substitution of synthetic double-stranded oligonucleotides for restriction fragments into the cDNA of MBP-A. Standard molecular biology techniques were conducted as described (8). The resulting constructs were expressed in Chinese hamster ovary cells, and the proteins were purified by affinity chromatography on mannose-Sepharose as described previously (7). SDS-PAGE was performed by the method of Laemmli (9). Gel filtration chromatography was conducted on a BioSep-S3000 column (300 x 7.8 mm) as described previously for MBP-A (7). The composition of oligomers in each MBP mutant was determined by fitting the gel filtration data to a multiple gaussian curve 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 (7). Results are presented as means ± SE from three independent assays.
Pulse-chase labeling of MBP
MBP-producing Chinese hamster ovary cells were grown to
confluence in 35 x 10-mm tissue culture dishes containing 2 ml of
MEM-
lacking nucleosides and supplemented with 10% dialysed FCS and
0.5 µM methotrexate. Culture medium was changed daily for an
additional 4 days. Before labeling, all culture media were prewarmed to
37°C and pre-equilibrated in 5% CO2. Cells
were incubated with 2 ml of methionine-free medium for 5 min. Labelling
was initiated with 1 ml of fresh methionine-free medium supplemented
with [35S]methionine (56 µCi/ml). After 10
min, the pulse was terminated by removing the
[35S]methionine-containing medium and
incubating the cells with medium containing a 20-fold excess of
unlabeled methionine. After various time intervals, cells were released
from the culture dish by scraping, pelleted by centrifugation at 2000
rpm in an Eppendorf centrifuge, and disrupted by resuspension in cell
lysis buffer (50 mM Tris (pH 7.4) containing 500 mM NaCl and 1% Triton
X-100). Both cells and medium were stored on ice before
immunoprecipitation.
Immunoprecipitation and quantification of labeling
Before immunoprecipitation, cell extracts and medium were incubated with 5 µl of protein A-Sepharose for 30 min at 4°C with mixing to preclear any material binding directly to protein-A. The protein A-Sepharose was removed by centrifugation, and the supernatant was incubated at 4°C with 10 µl of rabbit polyclonal antiserum raised against rat MBP-A. After 16 h, 10 µl of protein A-Sepharose was added, and the samples were incubated for an additional 3 h at 4°C with mixing. The immunoprecipitates were pelleted by centrifugation in an Eppendorf centrifuge, washed five times in cell lysis buffer, dried, and resuspended in loading buffer used for SDS-PAGE. Proteins were released from the protein A-Sepharose by boiling. Radiolabeled MBP was separated by SDS-PAGE and detected using a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).
In preliminary immunoprecipitation experiments, it was found that amount of labeled MBP isolated from the culture medium decreased with increasing chase time. This effect was caused by the large excess of unlabeled MBP produced during the chase period, which can overload the Ab. For this reason, purified 125I-labeled carbohydrate-recognition domain from MBP-A, which is also bound by the Ab, was added to each extract before the immunoprecipitation procedure. After SDS-PAGE and immunodetection, the amount of labeled MBP was normalized against this internal control in all pulse-chase experiments. The half-time of secretion was determined as the time at which the amount of labeled MBP in the medium is equal to the amount within the cells. Results shown are the mean ± SE from two separate experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that MBP secretion is slow relative to other
serum proteins because it is retained for long periods within the Golgi
apparatus of cultured rat hepatoma cell lines (10, 11).
Half-times for secretion in excess of 4 h have been reported. Rat
liver produces both MBP-A and rat liver MBP (MBP-C), and it is unclear
which protein was monitored in these experiments. For these reasons,
the rate of MBP secretion was remeasured in transfected cells
expressing only one form of the protein. After a brief (10 min) pulse
with [35S]methionine, progress of labeled MBP
through the cell and secretion into the medium were monitored by
immunoprecipitation and SDS-PAGE under reducing conditions (Fig. 1
).
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). The
half-times of secretion, 80 ± 3 min for MBP-A and 96 ± 32
min for MBP-C, are comparable to those of other secreted proteins
(12). The similarity in secretion rates between MBP-A and
MBP-C indicates that assembly of the large MBP-A oligomers does not
significantly delay secretion. The long retention times observed in
previous studies may reflect the abnormal physiology of hepatoma cells.
Previous work indicates that MBP-A most closely resembles human serum
MBP in its properties (13), so further analysis of the
biosynthesis was focussed on this protein.
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Examination of the gels shown in Fig. 1
reveals that, although
only a single form of the MBP-A polypeptide is secreted from cells, two
forms exist inside the cells. Conversion from the faster migrating
(unmodified) form to the slower migrating (modified) form is largely
complete within 20 min after pulse labeling. These results suggest that
MBP-A is posttranslationally modified early in biosynthesis, after
synthesis but before secretion.
Two types of posttranslational modifications that are characteristic of
collagens have been identified in MBP: hydroxylation of proline
residues within the consensus sequence Pro-Gly-X and hydroxylation and
glycosylation of lysine residues within the consensus sequence
Lys-Gly-X (14, 15). In vertebrate collagens, both of these
modifications occur before assembly of the collagen triple helix
(16). When MBP was labeled with
[3H]galactose or
[14C]galactose, immunoprecipitated, and
analyzed by SDS-PAGE, radioactivity is associated only with the more
slowly migrating (modified) band (Fig. 1). These data suggest that the
decrease in mobility of MBP is due to glycosylation of lysine residues,
which must occur early in the biosynthetic pathway as in the case of
the true collagens.
Assembly of MBP oligomers
Analysis by SDS-PAGE under nonreducing conditions indicates that
the higher order oligomers of MBP assemble slowly relative to the time
scale of secretion (Fig. 3A).
Immediately after the pulse, most of the labeled protein consists of
covalent monomers and dimers of subunits together with small covalent
structures comprising single polypeptides and two chain species. Over
the next 4 h, the amounts of the larger oligomeric forms increase,
whereas the proportion of monomeric subunits decreases (Fig. 3B). The total amount of labeled MBP is constant after
synthesis, indicating that there is not significant degradation taking
place. These findings suggest that the observed redistribution of
oligomeric forms is due to assembly of trimers and tetramers of
oligomers from the smaller trimeric subunits.
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Posttranslational modification and assembly of MBP in the endoplasmic reticulum
To investigate the intracellular compartment in which MBPs are
posttranslationally modified and assembled into oligomers, cells
producing MBP were treated with brefeldin A to block passage from the
endoplasmic reticulum to the Golgi apparatus (17). The
decrease in the mobility of MBP that is associated with lysine
glycosylation is observed even in the presence of brefeldin A (Fig. 4A). Thus, modification of
lysine residues within the collagen-like domain must occur within the
endoplasmic reticulum, a conclusion that is consistent with the timing
of the observed mobility shift described above. This finding is
consistent with previous studies indicating that lysyl hydroxylase and
the glycosyl transferases responsible for modification of the
hydroxylysine residues are found in the endoplasmic reticulum
(18, 19).
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, B and C). No secretion of MBP was detected
over the time course examined in treated cells, confirming that
transport from the endoplasmic reticulum to the Golgi apparatus is
disrupted, and maturation of the oligomers to achieve the levels of
trimers and tetramers secreted from untreated cells is not seen in the
presence of brefeldin A.
Nearly normal secretion of the Arg23Cys mutant
Previous studies have revealed that the molecular phenotype of the
Arg23 mutant of MBP is distinct from the
phenotypes of the Gly25 and
Gly28 mutants (4). Introduction of
the cysteine residue does not appear to disrupt formation of the
collagen triple helix but prevents higher order oligomer assembly as a
result of formation of adventitious disulfide bonds. The protein is
functionally defective largely as a consequence of its oligomeric
composition, which consists of monomers of subunits together with some
dimers and only trace amounts of the larger oligomers most efficient at
activating the complement cascade. Pulse chase analysis of cells
expressing protein with the Arg23Cys mutation
reveals that the half-time for secretion is 112 ± 4 min,
indicating that this protein is secreted 
1.4-fold more slowly than
wild-type protein. Because protein containing the
Arg23 mutant consists predominantly of single
subunits, the slightly reduced secretion rate may be because of the
slower secretion of these smaller oligomers. However, the fact that the
secretion rate is only slightly reduced indicates that the rates of
secretion of monomers and higher oligomers do not differ greatly.
Defective secretion in Gly28Glu and
Gly25Asp mutants
Similar pulse chase assays reveal that rate of secretion of the
Gly25Asp and Gly28Glu
mutants are substantially reduced compared with wild-type protein and
the Arg23Cys mutant (Fig. 5). The half-time of secretion is in
excess of 6 h for both of these mutants, indicating that the rate
of secretion of these proteins is reduced by more than 5-fold. In all
cases, the total amount of labeled protein remains approximately
constant during the chase period, indicating that there is no
significant degradation of MBPs over the time course of the
experiments. Thus, mutant proteins must be retained for longer within
the cell.
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Asp
and Gly28Glu mutants disrupts collagen
triple helix formation, leading to structural alterations in the
N-terminal cysteine-rich domain and aberrant disulfide bond formation.
The distributions of oligomeric forms of these two mutant proteins
recovered from the medium are very similar to each other. Compared with
wild type, the proportion of trimers and tetramers is reduced by 2- to
3-fold. The relatively rapid secretion of monomeric forms of MBP-C and
the Arg23Cys mutant of MBP-A indicates that
this shift in oligomeric structure would have little effect on the
half-time of secretion. Therefore, it is likely that the disrupted
structure near the N terminus of the Gly25 and
Gly28 mutants directly affects the secretion
rates by causing accumulation inside the cells. Effect of lysine modifications on secretion rates
In addition to altering the oligomeric state of MBP, mutations to
Gly25 or Gly28 prevent
hydroxylation and glycosylation of residues Lys27
and Lys30 because of their close proximity to
these residues (Fig. 6). Because the
effects of mutations at the glycine positions on oligomer assembly and
secretion rates could be mediated by changes in the posttranslational
modifications of the lysine residues, it was of interest to examine the
effects of eliminating these posttranslational modifications by
changing the lysine residues to arginine. The arginine side chains
preserve the positive charge of the natural amino acids but cannot be
hydroxylated or glycosylated. Analysis of protein containing either the
Gly25 or Gly28 mutation
indicates that hydroxylation of both lysine residues is inhibited by
either mutation, so arginine substitutions were made at both positions
simultaneously.
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). The half-time for secretion is
266 ± 40 min, more than 3-fold greater than for wild-type MBP.
Analysis of mutant proteins in which Lys27 and
Lys30 were individually changed to arginine
reveals that these proteins are secreted rapidly from producing cells
(Fig. 7), with half-times of secretion (94 ± 10 and 116 ± 4
min) similar to wild-type protein. The results indicate that
modification of neither of these residues per se is required for
efficient secretion. Instead, these findings suggest that simultaneous
elimination of both glycosylated hydroxylysine residues results in a
physical change to the protein that reduces the rate of secretion.
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Gel filtration shows that formation of tetramers and trimers of
subunits by MBP containing single lysine to arginine mutations is at
least as efficient as for wild-type protein (Fig. 8A). However, the formation of
higher oligomers is reduced when both mutations are present
simultaneously. The double mutant comprises a greater proportion of
monomers and dimers of trimeric subunits and is thus very similar to
the naturally occurring Gly25 and
Gly28 mutants. As discussed above, this shift in
oligomer composition is unlikely to have a large effect on the
secretion rate.
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B). In contrast to wild-type MBP, which comprises a mixture
of covalent oligomers, oligomers in the double mutant are assembled
predominantly from smaller covalent structures. Although some monomeric
subunits are covalent as in wild-type MBP, other oligomers are formed
from two- and four-chain species together with some single polypeptide
chains. Because correct disulfide bond formation is dependent on prior
formation of the collagen triple helix (6), these findings
indicate that the presence of both mutations together significantly
disrupts assembly of the collagenous domain. Analysis of the single
lysine mutants shows that these proteins have essentially wild-type
covalent composition, again indicating that significant structural
disruption only occurs when both modified lysine residues are
absent. The structural defects in the double lysine mutant correspond precisely to those in the Gly25 and Gly28 mutants associated with immunodeficiency. Therefore, it seems likely that defective secretion in all of these mutants is a direct result of the common structural defects that arise as a consequence of defective assembly of the collagen triple helix.
Effect of lysine mutations on complement fixation
Because simultaneous changes to Lys27 and
Lys30 affect the structure and secretion of MBP
in much the same way as changes to Gly25 or
Gly28, it was of interest to determine the effect
of these changes on the activity of MBP in complement-fixation assays.
The results of such assays indicate that MBP containing both lysine
mutations is significantly impaired in its ability to activate the
complement cascade (Fig. 9). The activity
of this mutant is 10-fold lower than wild-type MBP, whereas the
single-lysine mutants fix complement as efficiently as wild-type MBP.
The decrease in activity in the double mutant is comparable to the loss
in activity observed for the Gly25Asp
mutant.
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Asp mutant is partly a consequence of the
change in oligomeric composition, resulting in a lower proportion of
the larger oligomers that are most efficient at activating the
complement cascade (4). However, based on the composition
of oligomers together with the known relative activities of each
wild-type oligomeric form, only a 3-fold decrease in activity would be
predicted for the Gly25Asp and double-lysine
mutants, which is much less than the 10-fold decrease detected. Thus,
for both of these mutants, the oligomers that are present must be less
efficient at fixing complement than their wild-type counterparts.
Inefficient activation of downstream components of the complement
cascade is presumably a result of disruption of the MASP binding site
within the collagenous domain. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present studies indicate that naturally occurring mutations in
the collagen-like domain of MBP can affect rates of secretion as well
as complement-fixing activity. However, there is a complex relationship
among disruption of the structure of the collagen-like domain,
formation of larger oligomers, and the secretion and activity of the
protein (Table I and Fig. 10).
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Cys mutant, it has
been shown previously that the presence of this extra cysteine residue
causes formation of adventitious disulfide bonds and dramatically
reduces formation of trimers and tetramers of subunits
(4). Because these higher oligomers account for most of
the complement-fixing activity in MBP-A, the reduction in the
proportion of these forms accounts for a 10-fold decrease in
complement-fixing activity in preparations of the mutant protein. The
present results indicate that secretion of the mutant protein is only
slightly impaired compared with the wild-type protein, indicating that
changes in oligomeric state do not have a major effect on secretion
rates.
Changes to glycine residues in the collagen-like domain have more
substantial effects on secretion, suggesting that defective secretion
of such mutants would make a larger contribution to immunodeficiency.
The effect of these mutations arises from alterations in the collagen
triple helix that are propagated toward the N terminus of the protein,
causing disruption in the intrachain and interchain disulfide bonds in
the N-terminal domain (4). In the case of the
Gly25Asp mutant, approximately half of the
10-fold decrease in complement-fixing activity is caused by decreased
formation of higher oligomers, whereas the remainder is a more direct
result of the structural alterations in the N-terminal portion of the
protein.
As shown here, similar disruption of the N-terminal domain can be
induced by elimination of glycosylated hydroxylysine residues at
positions 27 and 30, resulting in a similar loss in complement-fixing
activity. Although insertion of arginine residues at these positions
could have unexpected disruptive effects, arginine side chains in the Y
positions of Gly-X-Y repeats would normally be expected to stabilize
the triple helix (21). Thus, it is reasonable to propose
that the effect of the lysine-to-arginine changes reflects removal of
the cluster of 12 saccharide residues that would normally surround the
helix in this region. These results suggest that reduced hydroxylation
and glycosylation of lysine residues caused by the
Gly25Asp (and
Gly28Glu) mutations might be the proximal
cause of the structural disruptions at the N-terminal end of the
protein.
The fact that native MBP-C, which is a monomer, as well as the largely
monomeric Arg23Cys mutant of MBP-A are
secreted efficiently indicates that reduced formation of the trimer and
tetramer probably does not account for slow secretion of the
Gly25Asp mutant. Therefore, it seems likely
that the slower secretion results directly from the disruption to the
structure of the N-terminal domain and may be related to the
misformation of disulfide bonds. For example, a misfolded segment of
the protein might cause prolonged retention by chaperones, such as
Hsp47, Ig heavy chain-binding protein, protein disulfide isomerase, and
prolyl 4-hydroxylase, all of which are known to interact with
collagenous proteins during their assembly (22, 23, 24, 25).
Impairment of secretion of the Gly28Glu mutant
appears to be quite similar to the Gly25Glu
mutant, which probably reflects the similar nature of the structural
alterations in the N-terminal domains evident from the similarly
aberrant disulfide bond patterns and oligomer distributions. This
finding contrasts with the extreme defect in complement-fixing activity
for the Gly28Glu mutant, which has been
hypothesized to reflect a more specific disruption of the MASP
binding site.
The observation that MBP-C is secreted at a rate similar to that of MBP-A indicates that there are no special retention signals within the protein sequence. Thus, its location within the liver must be mediated through specific molecular interactions. Although the function of MBP-C is not known, it is able to activate complement in an in vitro assay system (7). These findings suggest that it may play a role similar to that of its serum counterpart in host defense, but within the liver.
MBP levels in immunodeficiency
Analysis of serum MBP levels in subjects homozygous for the Gly25 and Gly28 mutations reveals that protein levels are reduced by more than 20-fold in each case (1, 26). Although defective secretion is likely to be a contributing cause of low serum levels, additional factors probably further reduce the amount of circulating MBP. Changes in the N-terminal region of MBP caused by mutations in the collagen-like domain probably affect clearance rates, either because of the changes in the oligomer distribution or because of the reduced affinity of the mutant proteins for MASPs. In addition, some promoter haplotypes linked to the mutations in the coding region are associated with altered levels of MBP expression (5). Increased turnover and reduced rates of synthesis and secretion would combine to produce the overall reduction in serum levels of MBP observed in homozygous individuals. Because MBP is assembled from multiple polypeptide chains, assembly in cells containing one wild-type and one mutant allele is probably also affected, which is consistent with the finding that serum levels are also reduced in heterozygous individuals.
Because no information is currently available regarding serum MBP
levels in individuals homozygous for the human ArgCys variant, it is
not clear whether low protein levels contribute to the immunodeficiency
in this case. Analysis of MBP levels in subjects heterozygous for this
mutant indicates that the amount of protein in the serum is only
slightly reduced (27). Combined with the results from this
study showing that the Arg23Cys mutation
causes only a small decrease in secretion rates, these results suggest
that immunodeficiency resulting from this mutation may arise largely as
a consequence of defective complement fixation by the altered
protein.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Russell Wallis, Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.
3 Abbreviations used in this paper: MBP, mannose-binding protein; MBP-A, rat serum MBP; MBP-C, rat liver MBP; MASP, MBP-associated serine protease.
Received for publication February 14, 2000. Accepted for publication May 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chains with mutations in the carboxyl-terminal propeptide synthesized by cells from patients with osteogenesis imperfecta. J. Biol. Chem. 268:18226.This article has been cited by other articles:
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  N. Rawal, R. Rajagopalan, and V. P. Salvi Activation of Complement Component C5: COMPARISON OF C5 CONVERTASES OF THE LECTIN PATHWAY AND THE CLASSICAL PATHWAY OF COMPLEMENT J. Biol. Chem., March 21, 2008; 283(12): 7853 - 7863. [Abstract] [Full Text] [PDF]
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L. Sipila, H. Ruotsalainen, R. Sormunen, N. L. Baker, S. R. Lamande, M. Vapola, C. Wang, Y. Sado, A. Aszodi, and R. Myllyla Secretion and Assembly of Type IV and VI Collagens Depend on Glycosylation of Hydroxylysines J. Biol. Chem., November 16, 2007; 282(46): 33381 - 33388. [Abstract] [Full Text] [PDF]
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F. Larsen, H. O. Madsen, R. B. Sim, C. Koch, and P. Garred Disease-associated Mutations in Human Mannose-binding Lectin Compromise Oligomerization and Activity of the Final Protein J. Biol. Chem., May 14, 2004; 279(20): 21302 - 21311. [Abstract] [Full Text] [PDF]
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R. Wallis, J. M. Shaw, J. Uitdehaag, C.-B. Chen, D. Torgersen, and K. Drickamer Localization of the Serine Protease-binding Sites in the Collagen-like Domain of Mannose-binding Protein: INDIRECT EFFECTS OF NATURALLY OCCURRING MUTATIONS ON PROTEASE BINDING AND ACTIVATION J. Biol. Chem., April 2, 2004; 279(14): 14065 - 14073. [Abstract] [Full Text] [PDF]
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G. S. Butler, D. Sim, E. Tam, D. Devine, and C. M. Overall Mannose-binding Lectin (MBL) Mutants Are Susceptible to Matrix Metalloproteinase Proteolysis. POTENTIAL ROLE IN HUMAN MBL DEFICIENCY J. Biol. Chem., May 10, 2002; 277(20): 17511 - 17519. [Abstract] [Full Text] [PDF]
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 R. Wallis Dominant Effects of Mutations in the Collagenous Domain of Mannose-Binding Protein J. Immunol., May 1, 2002; 168(9): 4553 - 4558. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and medium (
) is plotted
as a percentage of the total labeled MBP based on the average of the
last three time points. Data are averaged from two separate
experiments.
