Distinct molecular mechanisms underlying immunodeficiency caused by three different naturally occurring point mutations within the collagen-like domain of human mannose-binding protein (MBP; also known as mannose-binding lectin) have been revealed by introduction of analogous mutations into rat serum MBP. The change Arg23→Cys results in a lower proportion of the large oligomers most efficient at activating the complement cascade. The presence of cysteine at position 23, which forms aberrant interchain disulfide bonds, causes disruption of the normal oligomeric state. The deficiency in MBPs containing Gly25→Asp and Gly28→Glu substitutions also results in part from reduced formation of higher oligomers. However, decreased ability to interact with downstream components of the complement cascade due to changes in both the N-terminal disulfide-bonding arrangement and the local structure of the collagenous domain make more important contributions to the loss of activity in these mutants.

Serum mannose-binding protein (MBP3; also known as mannose-binding lectin), a part of the mammalian innate immune system, provides one of the first lines of defense against invasion by pathogenic microorganisms (1, 2). MBPs bind to sugar structures on the surface of bacterial, fungal, and parasitic cells and trigger complement fixation by a modification of the classical pathway, in which MBP-associated serine proteases (MASPs) activate C4 and C2 to generate fragments that have C3-convertase activity (3, 4, 5, 6, 7, 8). Microorganisms are neutralized either by formation of a host-mediated lytic complex or by clearance after stimulation of host phagocytic cells (9).

MBP deficiency is a relatively common genetic disorder in humans (1). Increased susceptibility to bacterial, fungal, and viral infections is particularly evident during the first few years of life before the adaptive immune system is fully established (10, 11). Moreover, recent studies indicate that MBP deficiency may also contribute to the progression of infectious diseases in adults. For example, acquired immunodeficiency syndrome patients both homozygous and heterozygous for MBP-deficient alleles had significantly shorter survival times than patients with a wild-type genotype (12).

The molecular basis for immunodeficiency linked to the MBP locus is not well understood. The disorder is characterized by low levels of serum MBP (10, 13). Protein isolated from patients consists of low m.w. covalent forms that do not resemble the large oligomeric structures in wild-type protein (14). The defect has been associated with three different point mutations within the MBP gene that result in amino acid substitutions in the first part of the N-terminal collagen-like domain of the protein (11, 13, 15). These findings indicate that the immunodeficiency may result from reduced activity of the protein. However, it has also been suggested that the deficiency can be explained by rapid turnover from serum or by reduced secretion of protein resulting from mutations in the promoter region upstream of the gene (1, 16).

To demonstrate that changes in MBP directly affect the activity of the protein, mutations corresponding to the known human variants have been recreated in rat serum MBP. All three mutations significantly decrease the efficiency of complement fixation by rat serum MBP (MBP-A). These changes are caused by local structural alterations that occur as a direct consequence of the amino acid substitutions. Thus, decreased ability of MBP to activate complement is likely to make a major contribution to the immunodeficient phenotype of patients with this disorder.

Restriction enzymes were purchased from New England BioLabs (Beverly, MA). Tissue culture medium was from Life Technologies (Gaithersburg, MD). Yeast mannan, Sepharose, and protein m.w. markers for gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) were purchased from Sigma (St. Louis, MO). Guinea pig serum and sheep erythrocytes were obtained from Serotec (Oxford, U.K.). Endoproteinase Arg-C was from Boehringer-Mannheim (Mannheim, Germany). Reagents for amino acid sequencing were from Beckman Instruments (Fullerton, CA). Iodo[2-3H]acetic acid was obtained from Amersham (Little Chalfont, U.K.). Unlabeled iodoacetic acid was purchased from Aldrich (Milwaukee, WI).

Amino acid sequencing was conducted on a Beckman LF3000 protein sequencer. MALDI-MS was conducted on a Lasermat mass spectrometer (Finnigan-MAT, San Jose, CA) as described previously (17). SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (18). Standard molecular biology techniques were conducted as described (19). Gel filtration chromatography was conducted on a BioSep-S3000 column (300 × 7.8 mm) (Phenomenex, Belmont, CA) as described previously for MBP-A (20). To determine the composition of oligomers in each mutant, gel filtration data were fitted to a multiple gaussian curve with the use of Microcal Origin. Results reported are the mean ± SE from two separate experiments. Scanning laser densitometery of SDS-polyacrylamide gels was performed on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). Analysis was conducted with the software supplied with the instrument. Results are means ± SE from scans performed on at least two independent protein preparations. The presence of free thiol groups was determined by incubating the Arg23→Cys mutant with iodo[2-3H]acetic acid as described previously (17). Radioactivity was detected using a PhosphorImager SI (Molecular Dynamics) and calibrated using tritium-labeled BSA after exposure of dried SDS-polyacrylamide gels for up to 30 days. Coomassie blue-stained gels were scanned by densitometry to determine the composition of oligomers.

Mutant constructs of MBP-A cDNA were generated by substitution of synthetic double-stranded oligonucleotides for restriction fragments. The resulting constructs were expressed in Chinese hamster ovary cells, and the proteins were purified by affinity chromatography as described for MBP-A (20). This system generates wild-type MBP that faithfully resembles natural protein in its oligomeric composition, posttranslational modifications, and complement-fixation activity (20). Typical yields of each mutant range from 1 to 2 mg from 500 ml medium. Oligomers of the Arg23→Cys mutant were resolved by ion exchange chromatography as described previously (20).

Before proteolysis, preparations of the Arg23→Cys mutant were incubated with 1000-fold excess of unlabeled iodoacetic acid in 50 mM Tris, pH 7.5, containing 150 mM NaCl and 25 mM CaCl2 to alkylate any free cysteine residues. After incubation at 37°C for 1 h, excess iodoacetic acid was removed by dialysis. Purified single subunits of the Arg23→Cys mutant (0.5 mg) were incubated with 10% (w/w) of endoproteinase Arg-C in 50 mM MOPS, pH 6.5, containing 25 mM CaCl2 at 37°C for 18 h. The reaction mixture was passed through a mannose-Sepharose column equilibrated in reaction buffer to remove the carbohydrate recognition domains, because they are resistant to digestion under these conditions. The peptide mixture was lyophilized to dryness, resuspended in 20% acetic acid (100 μl), and applied to a C3 reverse phase column. Proteolysis of the Gly25→Asp and Gly28→Glu mutants was conducted in 50 mM Tris, pH 7.5, containing 150 mM NaCl and 25 mM CaCl2, with the used of 5% (w/w) endoproteinase Arg-C. The reaction mixture was incubated at 37°C for 18 h. Peptides were isolated as described above for the Arg23→Cys mutant.

Complement-fixation activities of MBPs were determined by a modified version of the assay described by Ikeda et al. (3) performed as in the report of Wallis and Drickamer (20). Values reported are the means ± SE from three independent assays.

Serum MBPs are members of the collectin family of animal lectins and are homooligomers of polypeptides comprised of N-terminal cysteine-rich domains, collagen-like domains, α-helical coiled coils, and C-terminal carbohydrate recognition domains (5). As shown in Fig. 1, mutations identified in patients with MBP deficiency lie in the N-terminal portion of the collagen-like domain and occur in a region of high sequence identity between the rat and human proteins. Previous studies have shown that rat MBP-A produced in Chinese hamster ovary cells faithfully resembles the native lectin with respect to its oligomeric composition, posttranslational modifications, and complement-fixation activity (20). Because MBP-A is well characterized and recombinant protein can be produced in large quantities, precisely analogous mutations were introduced into the rat protein to investigate the molecular basis of immunodeficiency in humans. In addition to revealing the molecular effects of these mutations, these studies indicate that the rat system is a viable model for examining the behavior of the mutant proteins in vivo.

FIGURE 1.

Aligned amino acid sequences of the cysteine-rich and collagen-like domains of human MBP and rat MBP-A. Amino acid residues that are mutated in MBP deficiency are boxed. Padding characters have been introduced into the sequence of MBP-A to optimize the alignment (2122 ).

FIGURE 1.

Aligned amino acid sequences of the cysteine-rich and collagen-like domains of human MBP and rat MBP-A. Amino acid residues that are mutated in MBP deficiency are boxed. Padding characters have been introduced into the sequence of MBP-A to optimize the alignment (2122 ).

Close modal

Complement-fixation assays were conducted using mannan-coated erythrocytes as targets for the mutant proteins expressed in Chinese hamster ovary cells. The complement-fixation assays reveal that activities of the mutant MBPs are substantially reduced compared with wild-type, although the effects of each mutation are distinct (Fig. 2). The activities of the Gly25→Asp and Arg23→Cys mutants are 7- and 10-fold lower than wild-type MBP-A, whereas the activity of the Gly28→Glu mutant is reduced by >100-fold. Because these results indicate that inefficient complement fixation in patients carrying these mutations could contribute directly to the observed immunodeficiency compounding the effects of low serum MBP levels, the molecular basis for the reduced complement-fixation activity was investigated.

FIGURE 2.

Complement activation by mutant MBP-A proteins. Numbers indicate the relative complement-fixation activities, calculated from the amount of each mutant MBP required for 50% hemolysis compared with the amount of wild-type MBP-A required for 50% hemolysis (20 ).

FIGURE 2.

Complement activation by mutant MBP-A proteins. Numbers indicate the relative complement-fixation activities, calculated from the amount of each mutant MBP required for 50% hemolysis compared with the amount of wild-type MBP-A required for 50% hemolysis (20 ).

Close modal

The MBP polypeptides assemble to form trimeric subunits, which in turn form higher oligomers. MBP-A consists of mixtures of oligomers ranging from monomers to tetramers of trimeric subunits, of which trimers and tetramers are most efficient at activating the complement cascade (20). Oligomer formation is controlled by residues within the cysteine-rich domain and the first part of the collagen-like domain (20), so it could be imagined that the mutations might reduce the population of the larger oligomers, which would in turn reduce complement-fixation activity. Gel filtration analysis indicates that the Cys23 mutant consists mainly of single trimeric subunits with a small amount of dimer (Fig. 3). As expected from this finding, the major species observed by SDS-PAGE under nonreducing conditions are monomers and dimers of subunits that migrate as broad bands (Fig. 4). Minor bands reflect the presence of low levels of covalent trimers and tetramers seen on the gel filtration column. Analysis of fractions collected across the gel filtration peaks confirms that most of the oligomers are covalent (data not shown).

FIGURE 3.

Gel filtration analysis of mutant MBP-A proteins. Protein was loaded onto the column in 100 μl at a concentration of 0.5 mg/ml. The elution positions of monomers (M), dimers (D), trimers (T), and tetramers (TET) of subunits of wild-type MBP-A are indicated in each case. Rechromatography of fractions of the Asp25 and Glu28 mutants even after storage for several weeks at 4°C confirms that, as for wild-type protein, the oligomers are stable and noninterconverting.

FIGURE 3.

Gel filtration analysis of mutant MBP-A proteins. Protein was loaded onto the column in 100 μl at a concentration of 0.5 mg/ml. The elution positions of monomers (M), dimers (D), trimers (T), and tetramers (TET) of subunits of wild-type MBP-A are indicated in each case. Rechromatography of fractions of the Asp25 and Glu28 mutants even after storage for several weeks at 4°C confirms that, as for wild-type protein, the oligomers are stable and noninterconverting.

Close modal
FIGURE 4.

SDS-PAGE of mutant MBP-A proteins. Proteins were separated on a 10% polyacrylamide gel under nonreducing conditions. Protein was detected by staining with Coomassie blue. Migration positions of molecular mass markers and covalent oligomeric forms of MBP-A are shown on the left and right, respectively.

FIGURE 4.

SDS-PAGE of mutant MBP-A proteins. Proteins were separated on a 10% polyacrylamide gel under nonreducing conditions. Protein was detected by staining with Coomassie blue. Migration positions of molecular mass markers and covalent oligomeric forms of MBP-A are shown on the left and right, respectively.

Close modal

Trimers and tetramers account for ∼90% of the complement-fixing activity in wild-type MBP-A. Quantification of the results in Figs. 3 and 4 reveals that ∼5% of the Cys23 mutant exists as the trimer and tetramer species (Table I). Calculations based on the known relative activities of different oligomeric forms of wild-type MBP (20) indicate that this mutant would be expected to have ∼15% of the activity of the wild-type protein if the loss in complement-fixation activity is solely a result of the altered oligomer composition. This prediction is consistent with the observed 8- to 14-fold loss in activity, suggesting that the deficiency in complement fixation caused by this mutation results largely from its failure to assemble into trimers and tetramers.

Table I.

Composition of oligomersa in wild-type and mutant MBPs determined by gel filtration and scanning densitometry of SDS-polyacrylamide gels (in parentheses)

MutationMonomer (%)Dimer (%)Trimer (%)Tetramer (%)
Wild type 15 ± 1 28 ± 5 24 ± 3 33 ± 2 
 (6 ± 3) (19 ± 7) (34 ± 2) (29 ± 7) 
Arg23 → Cys 78 ± 4 17 ± 2 4 ± 2 1 ± 0.5 
 (63 ± 6) (14 ± 3) (4 ± 1) (1 ± 0.5) 
Arg23 → Ala 23 ± 2 34 ± 14 20 ± 5 23 ± 8 
 (14 ± 6) (28 ± 1) (28 ± 4) (19 ± 5) 
Gly25 → Aspb 39 ± 2 43 ± 2 10 ± 3 8 ± 0.5 
Gly28 → Glub 31 ± 4 39 ± 3 13 ± 4 17 ± 3 
MutationMonomer (%)Dimer (%)Trimer (%)Tetramer (%)
Wild type 15 ± 1 28 ± 5 24 ± 3 33 ± 2 
 (6 ± 3) (19 ± 7) (34 ± 2) (29 ± 7) 
Arg23 → Cys 78 ± 4 17 ± 2 4 ± 2 1 ± 0.5 
 (63 ± 6) (14 ± 3) (4 ± 1) (1 ± 0.5) 
Arg23 → Ala 23 ± 2 34 ± 14 20 ± 5 23 ± 8 
 (14 ± 6) (28 ± 1) (28 ± 4) (19 ± 5) 
Gly25 → Aspb 39 ± 2 43 ± 2 10 ± 3 8 ± 0.5 
Gly28 → Glub 31 ± 4 39 ± 3 13 ± 4 17 ± 3 
a

Data are expressed as a percentage of the total protein in each case.

b

Oligomeric compositions of the Gly25 → Asp and Gly28 → Glu mutants are calculated from the gel filtration data only, because oligomers of these mutants are assembled primarily from smaller covalent forms as discussed in the text. Based on scanning densitometry of gels, the four polypeptide species comprise 35–45% of the total protein in each of these mutants, whereas the two polypeptide species comprise 30–35%, single trimeric subunits constitute 10–20%, and single polypeptide chains constitute 5–15%. Because all of the highly oligomeric forms are composed of the same covalent species, it is not possible to calculate the distribution of oligomers from the densitometry data.

To assess whether the altered oligomer composition of the Arg23→Cys mutant is a consequence of loss of the arginine side chain or a result of incorporation of the cysteine residue, Arg23 was also changed to an alanine residue. Complement-fixation activity of the Arg23→Ala mutant is similar to the activity of wild-type protein (Fig. 2), and gel filtration and gel electrophoresis confirm that the distribution of oligomers in preparations of the Arg23→Ala mutant is similar to that in MBP-A (Table I). Thus, the reduced activity of the Arg23→Cys mutant is largely a result of the introduction of the thiol group into the collagen-like domain.

Because the presence of the cysteine residue is disruptive in the Cys23 mutant, the chemical state of Cys23 was investigated. The possibility that it contains a free sulfhydryl group was tested by incubating the mutant with iodo[2-3H]acetic acid in the presence and absence of guanidine hydrochloride. Based on the degree of alkylation detected after nonreducing SDS-PAGE, an upper limit could be calculated for the number of free thiol groups within each oligomeric form. The results indicate that <2% of polypeptides within trimers and dimers of subunits and that only trace amounts (<1%) of polypeptides within monomeric subunits contain free thiols. No labeling of tetramers could be detected. Similar levels of labeling was observed under denaturing and nondenaturing conditions. Thus, within each oligomeric form, most Cys23 residues must form disulfide bonds with other cysteines in mutant polypeptides or with small sulfhydryl compounds such as cysteine or glutathione.

In wild-type MBP-A, most subunits are linked by disulfide bonds between Cys13 and Cys18 arranged in an asymmetrical configuration, whereas disulfide bonds involving Cys6 residues link subunits to form higher order covalent oligomers (20). Because the Cys23 mutant also consists predominantly of covalent oligomers, this disulfide bonding arrangement may be conserved. If, as these results suggest, the N-terminal cysteine residues are arranged in a wild-type configuration, they would not be available to become linked to Cys23. The possibility that Cys23 residues are linked to each other was examined by isolating disulfide-linked peptides from purified monomeric subunits of the mutant. A fragment corresponding to peptide Asp21–Arg38 was isolated as a disulfide-linked homodimer (Fig. 5,A), and the identity of this species was confirmed by MALDI-MS analysis under reducing conditions (Fig. 5 B). Thus, some Cys23 residues form interchain disulfide bonds with corresponding residues within trimeric subunits. However, the recovery of this species is low, suggesting that a significant proportion of Cys23 residues may form other disulfide bonds. Although no species corresponding to peptides in which Cys23 is linked to free cysteine or glutathione were detected, certain peptide configurations are resistant to cleavage; therefore, the possibility of other disulfide arrangements cannot be ruled out.

FIGURE 5.

MALDI-MS of disulfide-linked peptide Asp21-Arg38 from the Arg23→Cys mutant. A, The [M + H]+ observed under nonreducing conditions corresponds to the mass of a dimer. The calculated value (in parentheses) assumes hydroxylation and O-linked glycosylation of Lys27 and Lys30. Pro33 is underivatized as in wild-type MBP-A. The other marked peaks correspond to loss of between one and four hexose moieties (−162.1 Da). This effect may reflect heterogeneity of glycosylation or may be a consequence of the desorption/ionization process. B, Peptide Asp21–Arg38 under reducing conditions. Minor peaks correspond to loss of one and two hexose moieties. A minor peak was also detected with [M + H]+ of 2141.8 Da, indicating that trace amounts of polypeptides contain only one modified lysine residue.

FIGURE 5.

MALDI-MS of disulfide-linked peptide Asp21-Arg38 from the Arg23→Cys mutant. A, The [M + H]+ observed under nonreducing conditions corresponds to the mass of a dimer. The calculated value (in parentheses) assumes hydroxylation and O-linked glycosylation of Lys27 and Lys30. Pro33 is underivatized as in wild-type MBP-A. The other marked peaks correspond to loss of between one and four hexose moieties (−162.1 Da). This effect may reflect heterogeneity of glycosylation or may be a consequence of the desorption/ionization process. B, Peptide Asp21–Arg38 under reducing conditions. Minor peaks correspond to loss of one and two hexose moieties. A minor peak was also detected with [M + H]+ of 2141.8 Da, indicating that trace amounts of polypeptides contain only one modified lysine residue.

Close modal

Heterogeneity caused by variations in the degree of posttranslational modifications of proline and lysine residues within the collagen-like domain might account for the broad bands observed for monomers of subunits on nonreducing SDS-polyacrylamide gels. However, MALDI-MS of peptides isolated from the collagen-like region of the Cys23 mutant indicate that the modifications are similar to wild-type MBP-A (20), because lysine residues close to the mutation at positions 27 and 30 are largely modified while Pro33 is underivatized (Fig. 5). Thus, it seems unlikely that variations in collagen-specific posttranslational modifications alone can account for the migration pattern observed on gels. It is more likely that the altered mobility compared with wild-type protein is due to heterogeneity associated with the presence of extra material attached to Cys23 or the presence of additional interchain disulfide bonds involving Cys23, which might hinder unfolding of the polypeptides in SDS and thus affect their migration.

If the sole defect in the Gly25→Asp and Gly28→Glu mutants were a result of altered oligomer composition as in the case of the Cys23 mutant, the observed complement-fixation activity should be predicted from the distribution of oligomers (Table I). Gel filtration reveals that all the oligomers are present in these mutants, and the amounts of trimers and tetramers are only slightly lower than for wild type (Fig. 3). These results predict only a 50% decrease in activity, much less than the observed 7- and 100-fold decreases; thus, the observed shift toward smaller oligomers cannot account for the reduced activity of either mutant. Moreover, the activity of the Glu28 mutant is >10-fold lower than that of the Asp25 mutant, despite similar oligomeric compositions. Thus, oligomers of both the Asp25 and Glu28 mutants must be less effective than their wild-type counterparts in activating downstream components of the complement cascade.

Analysis of the covalent structure of the Asp25 and Glu28 mutants indicates that the arrangement of disulfide bonds within the cysteine-rich domains of oligomers is severely disrupted (Fig. 4.). Under nonreducing conditions, MBP-A containing either mutation appears as two major bands at 44 and 104 kDa (Fig. 4). Based on the molecular masses, these bands must represent covalent structures comprising two and four polypeptide chains, a distribution that is quite distinct from the wild-type profile. Additional bands reflect the presence of monomers of subunits and single polypeptide chains. SDS-PAGE of fractions collected across gel filtration peaks reveals that whereas single subunits are largely covalent, as in wild-type MBP, dimers, trimers, and tetramers of subunits are each assembled predominantly from the two- and four-chain covalent species as well as single polypeptide chains. As shown in Fig. 6, formation of the novel two- and four-chain species must result from a substantially altered conformation of the N-terminal domain as evidenced by the different arrangement of disulfide bonds in both of these mutants. The structural perturbations may prevent efficient complement fixation by disrupting the interaction with MASPs, because these proteases are thought to bind within the cysteine-rich domain and the first part of the collagen-like domain (20).

FIGURE 6.

Disruption of the disulfide bond pattern in the Gly25→Asp and Gly28→Glu mutants. Top, Proposed wild-type configuration in a trimer of subunits results from a specific set of intra- and interchain disulfide bonds involving Cys6, Cys13, and Cys18 (20 ). The arrangement of bonds between Cys13 and Cys18 reflects the asymmetrical assembly of this portion of the molecule. Only some of the potential disulfide bonds between Cys6 residues are shown. The remaining Cys6 residues form additional Cys6-Cys6 disulfide bonds within the oligomers or are linked to small sulfhydryl compounds such as cysteine or glutathione. Bottom, Two of the many possible configurations for a trimer of subunits in the Asp25 or Glu28 mutants. The oligomers depicted are assembled from four-chain covalent species together with a single polypeptide chain (left) and four-chain and two-chain covalent species together with a single polypeptide chain (right). The disulfide bonding arrangement at Cys13 and Cys18 must be disrupted in at least one trimeric subunit to account for the covalent structure of the mutant. Similar arguments can be made for mutant dimers and tetramers of subunits.

FIGURE 6.

Disruption of the disulfide bond pattern in the Gly25→Asp and Gly28→Glu mutants. Top, Proposed wild-type configuration in a trimer of subunits results from a specific set of intra- and interchain disulfide bonds involving Cys6, Cys13, and Cys18 (20 ). The arrangement of bonds between Cys13 and Cys18 reflects the asymmetrical assembly of this portion of the molecule. Only some of the potential disulfide bonds between Cys6 residues are shown. The remaining Cys6 residues form additional Cys6-Cys6 disulfide bonds within the oligomers or are linked to small sulfhydryl compounds such as cysteine or glutathione. Bottom, Two of the many possible configurations for a trimer of subunits in the Asp25 or Glu28 mutants. The oligomers depicted are assembled from four-chain covalent species together with a single polypeptide chain (left) and four-chain and two-chain covalent species together with a single polypeptide chain (right). The disulfide bonding arrangement at Cys13 and Cys18 must be disrupted in at least one trimeric subunit to account for the covalent structure of the mutant. Similar arguments can be made for mutant dimers and tetramers of subunits.

Close modal

The data described above indicate that the Glu28 mutation causes a loss in complement-fixation activity far beyond what would be expected based on reduced oligomer formation. Further, because changes in the covalent structure of the N-terminal disulfide-bonded region are similar in the Glu28 and Asp25 mutants, there must be additional defects in the Glu28 mutant not found in the Asp25 mutant. These results suggest that local disruption of the collagen-like domain may contribute significantly to the phenotype of the Glu28 mutant, because the binding site for the MASPs is believed to include the N-terminal portion of the collagen-like domain (20). Within this region, Lys27 and Lys30 are at least partially hydroxylated and glycosylated to form glucosylgalactosyl-5-hydroxylysine. Because Gly28 forms part of the Lys-Gly-X consensus sequence for hydroxylation of Lys27, lack of glycosylation of this residue might contribute to loss of MASP binding.

The extent of lysine modification in the mutant MBPs was examined by analyzing peptides from the N-terminal part of the collagenous domain by MALDI-MS. Peptide Asp21–Arg38 from each protein elutes from the reverse phase column as two overlapping peaks. The first peaks correspond to peptides in which only one of the two lysine residues is modified (Fig. 7, A and C). Edman degradation of these peptides indicates that Lys27 is largely unmodified in each protein, because phenylthiohydantoin-lysine is detected in the 7th cycle. Because glucosylhydroxylysine derivatives are not usually detected by Edman degradation, the absence of a peak in the 10th cycle suggests that Lys30 is derivatized. The masses of the second peaks to elute from the column correspond to the masses of underivatized peptides (Fig. 7 B and D). These results indicate that both Gly25→Asp and Gly28→Glu substitutions prevent hydroxylation and subsequent glycosylation of Lys27 and may reduce the degree of derivatization of Lys30 in MBP-A. Thus, defective posttranslational modifications may contribute to the altered structural composition and reduced complement-fixation activities of both mutants. However, because the two mutants appear to be underhydroxylated to the same extent, this change does not account for the more extreme phenotype of the Glu28 mutant.

FIGURE 7.

Posttranslational modifications of peptide Asp21-Arg38 from the Gly25→Asp and Gly28→Glu mutants, analyzed by MALDI-MS. Detected [M + H]+ values correspond closely to calculated values (in parentheses). No peaks in the reverse phase separation of peptide corresponding to peptides Asp24–Arg38 were detected, indicating that the peptide bond between Arg23 and Asp24 is relatively resistant to cleavage. The sequence corresponding to the major peak is shown. K* denotes glucosylgalactosyl-5-hydroxylysine. A and B, Peptides from the Gly25→Asp mutant. The minor peak in A corresponds to underivatized peptide, and the minor peak in B corresponds to the partially derivatized peptide shown in A. C and D, Peptides from the Gly28→Glu mutant. The minor peak in C corresponds to loss of two hexose moieties. The minor peak in D corresponds to the partially derivatized peptide shown in C.

FIGURE 7.

Posttranslational modifications of peptide Asp21-Arg38 from the Gly25→Asp and Gly28→Glu mutants, analyzed by MALDI-MS. Detected [M + H]+ values correspond closely to calculated values (in parentheses). No peaks in the reverse phase separation of peptide corresponding to peptides Asp24–Arg38 were detected, indicating that the peptide bond between Arg23 and Asp24 is relatively resistant to cleavage. The sequence corresponding to the major peak is shown. K* denotes glucosylgalactosyl-5-hydroxylysine. A and B, Peptides from the Gly25→Asp mutant. The minor peak in A corresponds to underivatized peptide, and the minor peak in B corresponds to the partially derivatized peptide shown in A. C and D, Peptides from the Gly28→Glu mutant. The minor peak in C corresponds to loss of two hexose moieties. The minor peak in D corresponds to the partially derivatized peptide shown in C.

Close modal

All three mutations that lead to human MBP deficiency result in aberrant complement fixation when reproduced in rat MBP-A. The reduced activity of the Cys23 mutant can be accounted for by altered composition resulting in lower levels of the oligomers most efficient at activating the complement cascade. These changes, in turn, are principally consequences of the presence of a cysteine residue within the collagen-like domain. Although some of these cysteines are disulfide bonded to corresponding residues within individual subunits, others appear to be derivatized in some other way. Because the N-terminal part of the collagen-like domain is essential for correct oligomer formation, the presence of extra material attached to MBP-A polypeptides within this region would disrupt higher order oligomer formation in the mutant.

A number of mutations within the genes of vertebrate fibrillar collagens have been identified that result in replacement of the naturally occurring amino acid residues by cysteine residues (23). In some mutations, glycine within the Gly-X-Y repeat is replaced. Some of the introduced cysteine residues form disulfide bonds that may contribute to the disease phenotype (24, 25). Although Cys23 is in the X position rather than the Gly position of a Gly-X-Y triplet, the data reported here indicate that it can form interchain disulfide bonds with corresponding residues in other polypeptides within a collagen triple helix. Thus, the presence of Cys at the X position would not necessarily in itself disrupt triple helix formation, but the conformational constraints imposed by the extra disulfide bonds might prevent correct assembly of higher order oligomers.

Both Gly28→Glu and Gly25→Asp substitutions cause significant local disruption to the collagen-like domain in MBP-A. These substitutions result in altered arrangements of disulfide bonds between cysteine residues within the adjacent N-terminal domains. In each case, the structural perturbations probably result from incorporation of the relatively bulky acidic side chains into the collagen triple helix. This suggestion is consistent with studies of synthetic collagen peptides in which substitution of other residues for glycine destabilizes the collagen structure (26).

Previous studies using human recombinant MBP indicate that the mutation corresponding to Gly25→Asp in rat MBP-A has reduced complement-fixation activity compared with wild-type protein (27, 28). Furthermore, binding studies suggest that recombinant protein is unable to bind to purified MASP (29). However, it is unclear from these studies whether the properties of this mutant result from altered oligomeric composition (14) or reflect an inability of oligomers to fix complement (27). The biochemical data described here indicate that MBP-A containing either the Gly28→Glu or Gly25→Asp substitution is able to activate complement and hence interact with downstream components of the complement cascade, but both mutants are inefficient compared with wild-type protein. Changes in the oligomer composition can account for a small part of the reduction in activity, but disruption of the binding site for downstream serine proteases is probably more important. Residues within the N-terminal part of the collagen-like domain together with the cysteine-rich domain directly contribute to MASP binding (20). Both the Gly28→Glu and Gly25→Asp substitutions cause significant disruption to the cysteine-rich domain as evidenced by the altered arrangement of disulfide bonds. These changes are consistent with the contention that the MASPs interact near this domain in MBP-A. However, the simplest explanation to account for the large difference in complement-fixing activities of these two mutants is that the binding site is located toward the C-terminal end of the first part of the collagenous domain, near the Gly-Gln-Gly interruption of the collagen consensus sequence, because MBP containing the Glu28 mutation within this region is more functionally defective.

The covalent structures observed in preparations of the Asp25 and Glu28 mutants are comparable with human protein isolated from the serum of patients homozygous for point mutations in codons 54 and 57 of the human gene (14). SDS-polyacrylamide gels of serum preparations from such individuals reveal mostly material with an apparent molecular mass of 120–130 kDa. The results with the MBP-A mutants suggest that these covalent species may be assembled into larger oligomers within the serum. Thus it is likely that higher oligomers are present in immunodeficient patients in spite of the absence of covalent oligomers detected by gel electrophoresis.

MBP deficiency in humans is characterized by low serum levels of MBP (10). For example, the median serum MBP concentration of British Caucasoids homozygous for the codon 54 mutation (Gly→Asp) is ∼100-fold less than typical concentrations in individuals with the wild-type genotype (15). The reason for these low levels of serum MBP is not known, although it is thought that the mutations may disrupt MBP secretion or lead to increased turnover from the serum. One possibility is that failure to interact with downstream serine proteases leads to increased turnover. Because MBP is believed to form a complex with MASPs, unbound MBP may be rapidly targeted for removal from the serum. The studies described here suggest that the problems associated with decreased serum levels in MBP deficiency are compounded by a reduction in the ability of the protein to activate the complement cascade. Thus, both factors are likely to contribute to the immunodeficient phenotype of homozygous patients with MBP deficiency.

Previous studies suggest that individuals heterozygous for the mutant MBP alleles are also more susceptible to infections (1). Increased susceptibility is in part the result of reduced serum MBP levels, although the serum concentrations are generally higher than in homozygous individuals (13, 15). Analysis of the composition of MBP isolated from the serum of heterozygotes indicates that a significant proportion of MBP polypeptides are derived from the mutant allele (14). These findings suggest that some oligomers may comprise polypeptides of both wild-type and mutant origin. Because mutant MBPs are inefficient at fixing complement, it seems likely that hybrid MBPs may also have a decreased ability to activate the complement cascade. Thus, inefficient complement fixation by MBP oligomers may significantly contribute to the risk of infection in individuals both homozygous and heterozygous for the mutant alleles.

We thank Kurt Drickamer, in whose laboratory these experiments were conducted, for critical reading of the manuscript and for many helpful discussions regarding all aspects of this work; Maureen Taylor for critical reading of the manuscript; and David Harvey for use of the mass spectrometer.

1

This work was supported by Grant 041845 from the Wellcome Trust.

3

Abbreviations used in this paper: MBP, mannose-binding protein; MBP-A, rat serum MBP; MASP, MBP-associated serine protease; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry.

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