A Novel Type II Complement C2 Deficiency Allele in an
African-American Family1
Zeng-Bian Zhu,* T. Prescott Atkinson,† and John E. Volanakis2*
A 9-yr-old African-American male presenting with severe recurrent pyogenic infections was found to have C2 deficiency (C2D).
Analysis of his genomic DNA demonstrated that he carried one type I C2D allele associated with the HLA-A25, B18, DR15
haplotype. Screening all 18 exons of the C2 gene by exon-specific PCR/single-strand conformation polymorphism indicated abnormal bands in exons 3, 7, and 6, the latter apparently caused by the 28-bp deletion of the typical type I C2D allele. Nucleotide
(nt) sequencing of the PCR-amplified exons 3 and 7 revealed a heterozygous G to A transition at nt 392, causing a C111Y mutation,
and a heterozygous G to C transversion at nt 954, causing a E298D mutation and a polymorphic MaeII site. Cys111 is the invariable
third half-cystine of the second complement control protein module of C2. Pulse-chase biosynthetic labeling experiments indicated
that the C111Y mutant C2 was retained by transfected COS cells and secreted only in minimal amounts. Therefore, this mutation
causes a type II C2D. In contrast, the E298D mutation affected neither the secretion of C2 from transfected cells nor its specific
hemolytic activity. Analysis of genomic DNA from members of the patient’s family indicated that 1) the proband as well as one
of his sisters inherited the type I C2D allele from their father and the novel type II C2D allele from their mother; 2) the
polymorphic MaeII site caused by the G954C transversion is associated with the type I C2D allele; and 3) the novel C111Y
mutation is associated in this family with the haplotype HLA-A28, B58, DR12. The Journal of Immunology, 1998, 161: 578 –584.
D
eficiency of the second component of complement
(C2D)3 has been studied extensively. It is inherited as an
autosomal recessive trait and is the most common genetic deficiency of a complement component among individuals of
European descent, with an estimated frequency of the null allele
(C2Q0) of approximately 1% (1, 2). More than half of C2D individuals present with systemic lupus erythematosus, systemic lupus
erythematosus-like syndromes, glomerulonephritis, or various
forms of vasculitis (1, 3). About one-third of C2D individuals have
recurrent pyogenic infections (reviewed in Ref. 4).
Two distinct molecular mechanisms were recently shown to
cause C2D (5). In type I C2D, a 28-bp deletion removes 9 bp of the
39 end of exon 6 and 19 bp of the 59 end of the adjoining intron of
the C2 gene. This deletion causes skipping of exon 6 during RNA
splicing, resulting in a shift of the reading frame and a premature
termination codon (6). The putative primary translation product of
the mutant message would contain 222 amino acid residues, but
cannot be detected by biosynthetic labeling of fibroblasts from
C2D individuals (5). Type I C2D is in strong linkage disequilibrium with the MHC haplotype HLA-A25, B18, C2Q0, BfS, C4A4,
C4B2, Drw2 (7). This extended haplotype occurs in .90% of C2D
*Division of Clinical Immunology and Rheumatology, Department of Medicine, and
†
Division of Developmental and Clinical Immunology, Departments of Medicine and
Pediatrics, University of Alabama, Birmingham, AL 35294
individuals. In contrast, individuals with type II C2D are rare, representing about 7% of all cases of C2D, and are characterized by
a selective block of C2 secretion, leading to the retention of a
full-length C2 polypeptide in the intracellular compartment (5).
Two published examples of type II C2D result from two distinct
missense mutations of amino acid residues apparently critical for
proper folding of the C2 polypeptide (8). One missense mutation
(C566T) located in exon 5 results in a Ser1893 Phe substitution
and is associated with the HLA-A11, B35, DRw1 haplotype. The
other (G1930A) is located in exon 11, results in a Gly4443 Arg
substitution, and is found in association with the HLA-A2, B5,
DRw4 haplotype.
Here we report a 9-yr-old African-American male with severe
recurrent pyogenic infections and undetectable serum C2 protein
and total hemolytic complement activity (CH50). Analysis of his
genomic DNA demonstrated that he carried two distinct C2D gene
alleles. One was a type I C2D allele and was inherited from his
father; the other was inherited from his mother and harbored a
novel G392A transition in exon 3, causing a Cys1113 Tyr substitution. As shown by biosynthetic labeling experiments using COS
cells transfected with mutant C2 cDNA, the C111Y mutation results in an almost complete block of C2 protein secretion. Thus,
the novel C111Y mutation, which in this family is associated with
the haplotype HLA-A28, B58, DR12, leads to type II C2D.
Received for publication February 12, 1998. Accepted for publication March
10, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by U.S. Public Health Service Grants AI21067 and
AR44505.
2
Address correspondence and reprint requests to Dr. John E. Volanakis, Division of
Clinical Immunology and Rheumatology, THT 437 University of Alabama, Birmingham, AL 35294-0006.
3
Abbreviations used in this paper: C2D, deficiency of the second component of
complement; SSCP, single-strand conformation polymorphism; nt, nucleotide; wt,
wild-type; CCP, complement control protein; ER, endoplasmic reticulum.
Copyright © 1998 by The American Association of Immunologists
Materials and Methods
Case report
A 9-yr-old African-American male was referred for evaluation of his complement system because of his third serious bacterial infection. He had
developed meningitis at 4 and 6 yr of age caused by Hemophilus influenzae
and Streptococcus pneumoniae, respectively. His most recent hospitalization was for septic arthritis of the right shoulder, again due to S. pneumoniae. All three infections had responded well to antibiotics. Laboratory
evaluation had demonstrated undetectable total serum hemolytic complement activity (CH50).
0022-1767/98/$02.00
The Journal of Immunology
ELISA for C2 and factor B
The concentration of C2 was measured by a solid phase ELISA using the
IgG fraction of a rabbit anti-C2a serum as first Ab and the anti-C2b mAb
3A3.3 (9) as second Ab. The assay was developed with affinity-purified
alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL), followed by Sigma 104 phosphatase
substrate (St. Louis, MO). Color development was measured at 405 nm by
using a Vmax kinetic microplate reader (Molecular Devices, Menlo Park,
CA). The concentration of factor B was measured by a similar ELISA,
using the IgG fraction of rabbit anti-Bb serum and the anti-Ba mAb
HA4 –1A (10) as the first and second Abs, respectively. A standardized
serum was used to construct standard curves for both assays.
Analysis of genomic DNA for type I C2D
Genomic DNA was isolated from the buffy coat fraction of blood samples
collected in EDTA (11). The genomic DNA from each member of the
proband’s family (J. C. kindred) was subjected to PCR using the oligonucleotide pair, 59-GCCTGGGCCGTAAAATCCAAAT-39 and 59-GCA
CAGGAAGGCCTCTGCTGCA-39, which was designed to amplify exon 6
of the C2 gene and its downstream boundary. Agarose gel electrophoresis
of PCR products amplified from normal and type I C2D alleles yielded
fragments of 174 and 146 bp, respectively. All oligonucleotides were synthesized in a model 394 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA).
PCR/single-strand conformation polymorphism (SSCP)
PCR/SSCP was used as a first step in the search for mutations in the C2
gene. Primers for exon-specific PCR for all 18 exons of the C2 gene were
synthesized on the basis of the flanking intronic sequences (12). PCR was
conducted using 0.2 mg of genomic DNA, 1 mM of each oligonucleotide
primer, 200 mM of each dNTP, 2 mCi of [a-32P]dCTP (Amersham, Arlington Heights, IL), and 0.5 U of Taq polymerase (AmpliTaq, PerkinElmer/Cetus, Norwalk, CT) in a final reaction volume of 25 ml. Samples
were overlaid with 25 ml of mineral oil to avoid evaporation and were
subjected to 30 amplification cycles using a Tempcycler (Coy Laboratory
Products, Ann Arbor, MI). Each cycle consisted of 1-min denaturation at
95°C, 1-min annealing at different temperatures depending on the melting
temperature (Tm) of each primer, and 1- to 1.5-min extension at 72°C.
Electrophoresis of the PCR products was conducted at 20 watts and 25°C
for 4 to 4.5 h in 6% nondenaturing acrylamide gels containing 5% glycerol
or at 4°C without glycerol, using 45 mM Tris-borate/1 mM EDTA buffer,
pH 8.3. The single-stranded DNA fragments were visualized by
autoradiography.
Nucleotide (nt) sequencing
PCR products of 280 and 241 bp were amplified from exons 3 and 7,
respectively, using the oligonucleotide pairs, 59-ATCCAGTCCTATAT
TCCCCAC-39 and 59-AGGTTCCCCAGGAGACCCCAGC-39 for exon 3
and 59-TCCCCTTTGGCTTCAGGGCCC-39 and 59-AGAGGGTCCATC
TTCTCCTCTC-39 for exon 7. PCR products were purified by electrophoresis in 2.5% low melting point agarose gel (Sea Plaque Agarose, FMC,
Rockland, ME) and subcloned into the pCR II vector using the TA cloning
kit (Invitrogen, San Diego, CA). Recombinant plasmids were purified and
alkali denatured to produce templates. The nt sequencing was performed by
the dideoxy chain termination method, using modified bacteriophage T7
DNA polymerase (13). The complete nt sequences, including boundaries,
of exons 3 and 7 of the C2 gene were determined in both orientations.
Sequence data were analyzed using the MacVector Sequence Analysis
Software (International Biotechnologies, New Haven, CT). The nt sequencing of exon 3 amplified from genomic DNA of family members was
performed using an ABI PRISM 377 DNA Sequencer and the ABI PRISM
Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer,
Foster City, CA), according to the manufacturer’s instructions.
Construction and expression of mutant C2 cDNA
The C2 cDNA C2SacR (14) cloned into the expression vector pcDNA3
(Invitrogen) was used to construct mutant C2 cDNA. C2SacR was derived
from the full-length C2 cDNA C2HL5–3 (15) by deletion of the first 352
bp from the 59 untranslated region and by elimination of the internal EcoRI
site by site-directed mutagenesis to facilitate transfer between vectors.
Since the mutation is silent, C2SacR is considered wild-type (wt) C2
cDNA. Two mutant C2 cDNAs, termed C2-G392A and C2-G954C, were
constructed by site-specific mutagenesis (16), using the mutagenic oligonucleotides 59-GTTGGGGCGATACTGACGCAC-39 and 59-TGCTGAT
CACGTCAGTCATAT-39, respectively. Both mutations were verified by
nt sequencing.
579
Ten micrograms of pcDNA3 containing wt or mutant C2 cDNA was
transfected into 5 3 105 COS cells using lipofectin (Life Technologies,
Gaithersburg, MD). Transfected COS cells were grown in serum-free
DMEM (Mediatech, Washington DC) at 37°C for 5 h. The medium was
then replaced with fresh DMEM supplemented with 10% heat-inactivated
FCS (Irvine Scientific, Santa Ana, CA), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, and cell culture was continued at 37°C
in 6% CO2. After 48 h, supernatants were collected and stored at 290°C
until assayed. The C2 concentration was measured by ELISA, and the
hemolytic activity was determined using EAC14 cells prepared as previously described (17).
Pulse-chase biosynthetic labeling and immunoprecipitation
COS cells (3.0 3 107) were transfected by electroporation with 20 mg of
the indicated recombinant plasmid. Transfected cells were divided equally
into seven 60-mm petri dishes and cultured at 37°C for 72 h. The cells were
then washed and incubated in methionine-free DMEM for 10 min. Biosynthetic labeling was performed by incubating the cells in 1 ml of methionine-free DMEM containing 125 mCi/ml [35S]methionine (DuPontNew England Nuclear, Boston, MA) for 15 min at 37°C in 6% CO2. Cells
were washed and chased in 1 ml of complete medium (DMEM with 10%
heat-inactivated FCS) for 0, 15, 30, 60, 120, 240, and 480 min at 37°C. At
each time point, culture supernatant was collected from one culture dish,
and the cells were washed with Dulbecco’s PBS (Mediatech) twice and
lysed in 1 ml of Dulbecco’s PBS, pH 7.2, containing 0.5% sodium deoxycholate (Boehringer Mannheim, Indianapolis, IN), 1% SDS, 1% Triton
X-100 (Sigma), 10 mM EDTA, 2 mM PMSF (Sigma), and 1 mg/ml each
of leupeptin, aprotinin (Boehringer Mannheim), and pepstatin A (Sigma;
lysis buffer). Lysates and supernatants were cleared of cellular debris by
centrifugation and were stored at 290°C until used. Immunoprecipitation,
SDS-PAGE, and autoradiography were conducted exactly as described previously (18). Autoradiographs were scanned by using a Scanjet 3C/T
(Hewlett Packard, Wilmington, DE), and the data were analyzed with the
National Institutes of Health Image program (version 1.58, Bethesda, MD).
Analysis of the novel MaeII polymorphic restriction site in exon
7
A 241-bp DNA fragment containing exon 7 of the C2 gene was amplified
from genomic DNA and digested with MaeII. Resulting fragments were
separated on 4% agarose gels and visualized with ethidium bromide. In the
absence of the novel polymorphic MaeII site, fragments of 205 and 36 bp
were detected, while in its presence the 205-bp fragment was split into 155and 50-bp fragments.
Results
Pedigree analysis
Figure 1 shows the HLA haplotypes and serum concentrations of
C2 and factor B of all living members of the proband’s family.
Laboratory normal ranges (mean 6 2 SD) for serum C2 and factor
B are 11 to 35 and 74 to 286 mg/ml, respectively. As the propositus’ father was deceased, his HLA haplotypes were deduced from
those of his offspring. The propositus (II.2) and an asymptomatic
female sibling (II.6) inherited haplotype a from their father (I.1)
and haplotype c from their mother (I.2); both had undetectable
serum C2, while their factor B levels were at the low range of
normal. Haplotype a, HLA-A25, B18, DR15, is identical with that
associated with type I C2D (7). The propositus’ mother (I.2) and
another female sibling (II.1) who had inherited haplotype c from
the mother had low serum C2. All other siblings had normal C2
and factor B levels. These results indicated that haplotypes a and
c carry C2D alleles, that heterozygosity for haplotype c, but not a,
is associated with low serum C2, and that compound heterozygosity for C2D is associated with low normal factor B levels. In contrast to haplotype a, which is frequently associated with C2D, no
such association has been reported for haplotype c. It thus seemed
likely that haplotype c harbored a novel C2D allele.
Screening for type I C2D alleles
To confirm that haplotype a carried the type I C2D allele, genomic
DNA from each family member was subjected to PCR using the
580
NOVEL C2 DEFICIENCY ALLELE
of the type I C2D allele. PCR-amplified exon 3 from the proband
and the control individual were subcloned into the pCR vector.
Four independent clones derived from the proband and three from
the control were subjected to nt sequencing in both orientations. A
G to A transition at nt position 392 (numbering from the translational start site) was present in two of the proband’s clones, while
the other two had the same sequence as the control clone and the
normal gene (12) (Fig. 3). The G392A mutation results in the
substitution of Tyr for Cys at amino acid residue 111 (C111Y).
Thus, the propositus is heterozygous for a novel C2 mutation,
C111Y. To verify that the C111Y mutation is associated with haplotype c, PCR-amplified exon 3 from all family members carrying
haplotype c (I.2, II.1, II.2, and II.6) and that from a control sibling
(II.5) without haplotype c were subjected to automatic nt sequencing. All four individuals carrying haplotype c had double A and G
nt peaks at position 392, indicating heterozygosity for the G392A
mutation. The control sequence had a single G nt peak at the same
position. These data confirm that the novel C111Y mutation is
associated with haplotype c.
Translation and secretion of C111Y C2
FIGURE 1. Family tree. a to d are HLA haplotypes described in the box
at the bottom of the figure. The serum concentrations of C2 and factor B
were measured by ELISA and are given in micrograms per milliliter. Normal laboratory ranges for C2 and factor B are 11 to 35 and 74 to 286 mg/ml
(means 6 2 SD), respectively. The propositus (II.2) and his youngest sister
(II.6) are C2 deficient. Therefore, the paternal haplotype a and the maternal
haplotype c carry C2Q0 alleles.
oligonucleotide pair designed to amplify exon 6, and its downstream boundary and the size of the resulting PCR products were
assessed by agarose gel electrophoresis. As shown in Figure 2, all
family members who had inherited haplotype a from the father
(II.2, II.4, II.5, and II.6) had, in addition to the 174-bp fragment
amplified from normal C2 alleles, a 146-bp fragment, which is
amplified from alleles carrying the 28-bp deletion that causes type
I C2D.
A novel C111Y mutation located in exon 3 of the C2 gene
Exon-specific PCR/SSCP was used to screen all 18 exons of the
proband’s C2 gene for possible mutations. DNA from a C2-sufficient individual was run in parallel as a control. Mobility shifts
suggesting nt sequence alterations were present in exons 3, 7, and
6 of the proband, the latter apparently caused by the 28-bp deletion
To assess the functional consequences of the C111Y mutation, we
performed pulse-chase biosynthetic labeling experiments using
COS cells transfected with C2-G392A cDNA. Cells were pulsed
with [35S]methionine for 15 min 72 h after transfection, then
chased over an 8-h period. The results of a representative experiment are shown in Figure 4. In cells transfected with wt cDNA, C2
protein was detected as a single intracellular band of about 97 kDa.
The band’s intensity increased during the first 30 min of chase and
subsequently decreased, with ,20% of peak levels detected intracellularly after 4 h of chase. Substantial secretion started at 60 min
with the appearance of an approximately 99-kDa band in the extracellular fluid and was essentially complete at the end of the 8-h
chase. In contrast, C111Y C2 was not secreted in detectable
amounts in the extracellular medium, although very faint bands
were visible after exposure of the film for 3 days, indicating minimal secretion. For the first 30 min of chase, mutant C2 was detected intracellularly as a single band of a size equal to that of wt
C2. The band’s intensity reached peak levels at 30 min of chase
and subsequently decreased slowly, so that after 8 h about 65% of
the mutant C2 was still present intracellularly. An additional band
of about 93 kDa appeared within the intracellular fraction after 60
min of chase, possibly representing a proteolytic degradation fragment of the mutant C2.
A new polymorphic site G954C in exon 7 of the C2 gene
Exon 7, which also displayed mobility shifts on PCR/SSCP, was
amplified from the proband’s genomic DNA and subjected to nt
FIGURE 2. PCR analysis for the presence of the 28-bp deletion associated with type I C2D. Genomic DNA from each member of the family was
subjected to PCR using oligonucleotides designed to amplify exon 6 of the C2 gene. The PCR products were separated on a 2.5% agarose gel and visualized
by ethidium bromide. M, markers of the indicated size in base pairs. The 174- and 146-bp PCR products indicated by arrows are amplified from the normal
C2 and the type I C2D allele, respectively. The results demonstrate that in this family the type I C2D allele is associated with HLA haplotype a.
The Journal of Immunology
581
FIGURE 3. A novel C111Y mutation in exon 3
of one of the proband’s C2 gene alleles. Partial nt
sequences of an exon 3 clone of the C2 gene of the
propositus (C2D, II.2) and an unrelated C2-sufficient individual (C2S) are shown. The sequences
depicted are those of the sense strands and show a
G to A transition at nt 392 in the C2D individual.
The mutated nt is boxed, and the substituted Tyr
for Cys at position 111 is in italics.
sequencing. Two of five independent clones isolated from the proband’s PCR product carried a G954C transversion (Fig. 5). The
remaining three clones had the same sequence as clones from a
C2-sufficient individual used as a control. The G954C mutation
generates a MaeII restriction site and causes a Glu for Asp substitution at amino acid residue 298 (E298D). Apparently, the propositus is heterozygous for the mutation. To investigate whether
the G954C mutation is associated with haplotype a or c, exon 7 of
the C2 gene was amplified from genomic DNA from each member
of the proband’s family. The PCR products were digested with
MaeII, and the digests were analyzed by agarose gel electrophoresis. As shown in Figure 6, all living family members carrying HLA
haplotype a (II.2, II.4, II.5, and II.6) also had the MaeII RFLP,
while none of the other family members did. Therefore, in this
kindred the G954C mutation and the resulting polymorphic MaeII
site of exon 7 are associated with haplotype a and the type I C2D
allele. Subsequent studies have demonstrated that type I C2D alleles from additional unrelated individuals also carried the MaeII
polymorphic site. The possible functional effects of this mutation
were investigated by constructing the G954C mutant C2 cDNA
FIGURE 4. Pulse-chase analysis of the rate of secretion of C111Y C2.
COS cells transfected with 20 mg of wt or C111Y C2 cDNA, were pulselabeled with [35S]methionine for 15 min and chased for various time intervals for up to 8 h. Media and lysates were analyzed by immunoprecipitation, 7.5% SDS-PAGE, and autoradiography. IC, intracellular, EC,
extracellular. The length of chase in hours is indicated at the top of each
lane. C111Y C2 is not secreted in detectable amounts, indicating that this
mutation causes type II C2D.
and testing it in transfection experiments in comparison with wt
C2. Transiently transfected COS cells secreted about equal
amounts of mutant and wt C2. Moreover the specific hemolytic
activity of E298D C2 (4.43 U/ng) was almost identical with that of
wt C2 (4.39 U/ng). Therefore, the E298D mutation is functionally
silent.
Discussion
Hereditary C2D is the most common complement component deficiency among individuals of European descent, but is relatively
rare among other ethnic groups. Here we report a case of C2D in
an African-American family. The proband of this family and his
asymptomatic youngest sister were compound heterozygotes for
type I and type II C2D. The type I C2D allele was inherited from
their father, and the type II C2D allele was inherited from their
mother. The former was associated with the HLA-A25, -B18,
-DR15 haplotype and carried the 28-bp deletion in exon 6, which
characterizes the common type I C2D seen among Europeans. The
type II C2D allele carried a novel G392A mutation in exon 3 of the
C2 gene, which led to a Cys1113 Tyr substitution. Cys111 is the
invariable third half-cystine of the second CCP module of C2, and
its substitution with Tyr was shown to be responsible for blocking
the secretion of the C2 polypeptide by COS cells transfected with
mutant cDNA.
The mechanism leading to type II C2D was originally defined
using L cells transfected with two different type II-associated
C2Q0 genes from unrelated individuals (5, 8). One of these was
due to a single missense mutation, C566T, located in exon 5 and
resulting in a Ser1893 Phe substitution, while the other was due to
a different missense mutation, G1930A, located in exon 11 and
leading to a Gly4443 Arg substitution. These amino acid substitutions were apparently directly responsible for a marked inhibition of secretion of the respective mutant C2 proteins, but the
molecular mechanism of inhibition was not investigated further.
However, it seems likely that these two mutations as well as the
one described in this report lead to misfolding of the polypeptide
chain, resulting in its retention in the endoplasmic reticulum (ER)
and eventual degradation. Thus, type II C2D appears to be another
example of a growing number of human diseases caused by protein
folding defects (reviewed in Ref. 19).
Classic examples of diseases caused by protein folding defects
are cystic fibrosis and a1-antitrypsin deficiency. In the most common form of cystic fibrosis, deletion of a single phenylalanine at
residue 508 of the cystic fibrosis transmembrane conductance regulator causes retention in the ER and inadequate expression of the
582
NOVEL C2 DEFICIENCY ALLELE
FIGURE 5. A new polymorphic site
G954C in exon 7 of the C2 gene. Partial nt
sequence of an exon 7 clone of the C2 gene
showing a G to C transversion at nt 954.
PCR-amplified exon 7 products derived from
genomic DNA of the propositus (C2D, II.2)
and an unrelated C2-sufficient individual
(C2S) were cloned and subjected to nt sequencing. The sequences depicted are those
of the sense strands. The changed nt is boxed,
and the substituted Asp for Glu at position
298 is in italics.
protein in the cell membrane (20). A common form of emphysema-related a1-antitrypsin deficiency is the result of a Glu3423 Lys
substitution (21), which has been shown to cause a folding defect
apparently responsible for ER retention (22). Other examples of
presumed misfolding leading to deficiency are missense mutations
causing type IIA von Willebrand factor disease (23, 24) and several different missense and nonsense mutations of the A subunit of
factor XIII causing deficiency of the enzyme and bleeding disorders (25, 26).
A generally accepted model for the folding and processing of
membrane-associated and secreted proteins has emerged from
studies on the intracellular folding of a small number of proteins
(27–29). Initial cotranslational folding involving secondary and
some tertiary structure occurs during translocation of the polypeptide chain into the cisternal space of the ER. Addition and initial
processing of N-linked glycans and formation of disulfide bonds
occurs next, and native conformation is attained. Proteins are
translocated to the Golgi apparatus, further processed, and then are
either translocated to the cell surface or secreted (19). ER chaperones, including BiP (30), calnexin (28, 31), and calreticulin (32),
play a key role in the process by facilitating protein folding, oligomerization, and translocation (reviewed in Ref. 33). In addition
to facilitating folding and maturation of polypeptides, ER molecular chaperones constitute an important component of the ER
“quality control” system, which ensures that incompletely or incorrectly folded proteins are retained in the ER until they fold
correctly or are degraded (34). Indeed, prolonged association with
chaperones constitutes a signal for degradation of aberrant proteins. A number of recent reports have indicated that degradation
of misfolded proteins takes place in proteasomes in the cytoplasm
(35–38). In some, but not all, cases, proteasome digestion is preceded by ubiquitination.
We have previously shown that C2D(17), the product of one of
several naturally occurring alternatively spliced C2 transcripts
(39), which is missing the region encoded by exon 17, is not secreted by transfected COS cells but is retained in the ER, apparently because it is misfolded (18). We have also demonstrated that
both C2D(17) and wt C2 are associated with calnexin, which
seems to be responsible for the ER retention of the deletional mutant (40). Misfolding and calnexin-mediated retention in the ER
are the likely causes of the inhibition of secretion of the C111Y
mutant described here, although other chaperones may participate
as well. As mentioned, Cys111 is the third of four invariable halfcystines of the second of three CCP modules of C2. In native C2,
Cys111 is thought to be disulfide linked to Cys69. CCP modules are
widely distributed and occur in multiple copies in many complement receptors and regulatory proteins as well as other complement and noncomplement proteins, such as b2-glycoprotein I and
clotting factor XIII (41). Solution structures of CCP modules of
factor H (42, 43) and vaccinia virus CCP (44) have shown a common structural fold that is probably shared by all CCP modules.
The framework of a CCP module consists of a b sandwich in
which b strands surround a compact hydrophobic core. The structure is stabilized by a pair of disulfide bonds between Cys1-Cys3
and Cys2-Cys4. The present data indicate that at least the first of
these two disulfide bonds is necessary for the correct folding of the
CCP module and/or for maintaining its native conformation. The
structural importance of the CCP module disulfides was also indicated by the finding that mutation of Cys430 within the seventh
CCP module of the b subunit of clotting factor XIII caused ER
FIGURE 6. The G954C transversion generates a MaeII restriction site
in exon 7 and is associated with the type I C2D allele. A 241-bp DNA
fragment containing exon 7 of the C2 gene was amplified from genomic
DNA from each member of the proband’s family. The PCR products were
digested with MaeII, and the resulting fragments were separated on a 4%
agarose gel and visualized with ethidium bromide. The novel polymorphic
MaeII site caused by the G954C transversion splits the 205-bp fragment
into 155- and 50-bp fragments. All family members carrying HLA haplotype a also have the novel polymorphic site. Therefore, the polymorphic
MaeII site of exon 7 is associated with the type I C2D allele of this kindred.
The Journal of Immunology
retention of the polypeptide and protein deficiency (45). Similarly,
mutations leading to substitution of two different cysteine residues
within CCP modules of the control protein factor H also resulted
in ER retention of the mutant polypeptides and protein
deficiency (46).
Disulfide bonds are important for stabilizing the tertiary structure of secreted proteins. They are also thought to play a significant
role in the folding process per se (19). This proposal is based on in
vitro studies of the folding of bovine pancreatic trypsin inhibitor
(47) and on the intracellular folding of the b subunit of human
chorionic gonadotropin (48, 49). In both cases, it was observed that
during folding, transient disulfides form, which are not present in
the native protein, but represent important intermediates in the
folding pathway. These findings indicate that disulfide rearrangement takes place during maturation of the protein and imply that
failure to form certain transient disulfides during folding could
result in trapping of the polypeptide at an intermediate stage of the
folding pathway. In modular proteins such as C2, individual modules are believed to fold autonomously. Thus, it would be expected
that the C111Y mutation would influence only the folding and/or
tertiary structure of the second CCP module. However, a more
widespread effect on the polypeptide cannot be excluded. Misfolding of C2 also could cause the secretory defect associated with the
two previously described mutations, S189F and G444R, that lead
to type II C2D (8). Ser189 is within a 30-residue segment that
connects the third CCP to the von Willebrand factor type A module of C2 and Gly444 within the 14-residue segment connecting the
von Willebrand factor type A module to the serine protease domain (12). It seems more likely that defective overall folding
and/or conformation of C2, rather than a conformational defect
localized in the connecting segments, is the immediate cause of the
ER retention of these two mutants.
The second missense mutation, E298D, revealed by the PCR/
SSCP analysis of the proband’s genomic DNA, was functionally
silent, as would be expected from the conservative nature of the
amino acid substitution. This mutation is associated with a MaeII
polymorphic site of the type I C2D allele of this family. Our preliminary screening data indicate that this polymorphic restriction
site is also present in type I C2D of other unrelated kindreds. It
therefore provides a simple screening test for type I C2D. Additional testing is necessary to establish the association between the
MaeII polymorphic site and other genetic polymorphisms linked to
the typical type I C2D allele (50).
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Acknowledgments
We are grateful to Dr. Robert Benak for referral of this patient, and to Mrs.
Paula Kiley for assistance in preparing the manuscript.
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