(Received for publication, September 1, 1995; and in revised form, November 6, 1995)
From the
Type II complement protein C2 deficiency is characterized by a
selective block in C2 secretion. The Type II C2 null allele (C2Q0) is
linked to two major histocompatibility haplotypes (MHC) that differ
from the MHC of the more common Type I C2 deficiency. To determine the
molecular basis of Type II deficiency the two Type II C2Q0 genes were
isolated and transfected separately into L-cells. Subsequent molecular
biology, biosynthetic, and immunofluorescence studies demonstrated that
C2 secretion is impaired in Type II C2 deficiency because of different
missense mutations at highly conserved residues in each of the C2Q0
alleles. One is in exon 5 (nucleotide C
T;
Ser
Phe) of the C2Q0 gene linked to the MHC
haplotype A11,B35,DRw1,BFS, C4A0B1. The other is in exon 11 (G
A; Gly
Arg) of the C2Q0 gene linked
to the MHC haplotype A2,B5, DRw4,BFS,C4A3B1. Each mutant C2 gene
product is retained early in the secretory pathway. These mutants
provide models for elucidating the C2 secretory pathway.
The complement system consists of about 30 soluble and membrane
proteins that constitute one of several important mediators of host
defenses against microbial infection. The complement protein C2 ()is a M
100,000 serine proteinase that
functions in the classical activation pathway of the complement system.
It is encoded by a 20-kb gene of 18 exons that is tightly linked to the
homologous 6-kb gene encoding the complement protein factor
B(1, 2, 3) . Both genes comprise part of the
class III gene cluster (4) located on the short arm of
chromosome 6 between the HLA-D and HLA-B loci of the major
histocompatibility complex (MHC)(5, 6) .
Deficiency of the second component (C2D) is the most common genetic deficiency of the complement system. In populations of western European origin, the C2 null gene (C2Q0) frequency is about 1%(7, 8) . Molecular heterogeneity in C2 deficiency was recently recognized based on expression of the protein in cell culture of fibroblasts from affected individuals(9) . In Type I C2D, there is no detectable translation of C2-specific mRNA. Multiple C2D families from different geographic regions have been examined, and to date the Type I phenotype in each case results from a 28-bp deletion in the C2Q0 gene that removes 9 bp of the 3`-end of exon 6 and 19 bp of the 5`-end of the adjoining intron(10, 11) . This deletion generates a mature C2 transcript from which exon 6 is deleted, creating a downstream premature stop codon and a failure to synthesize detectable C2 protein (10) . Additionally, all C2Q0 genes examined containing the 28-bp deletion are linked to at least part of the same MHC haplotype/complotype (extended haplotype) A25,B18,C2Q0,BFS,C4A4B2,DRw2 (12, 13) .
In
contrast, Type II C2D is characterized by a selective block in C2
secretion (9) and is found in the context of two different MHC
extended haplotypes that differ from that associated with Type I C2D,
suggesting the possibility of more than one molecular mechanism leading
to the secretory block. Accordingly, to examine the molecular genetic
basis of Type II C2D, the two C2Q0 genes associated with the Type II
extended haplotypes were isolated, transfected separately into L-cells,
and the corresponding C2 cDNA sequenced. The data reported here
establish that Type II C2D within the HLA haplotype A2,B5,DRw4
complotype C2Q0,BFS,C4A3B1 is due to a single missense mutation
(nucleotide G
A) leading to a Gly to Arg change
at amino acid residue 444. Type II C2D in the context of the HLA
haplotype A11,B35,DRw1 complotype C2Q0,BFS,C4A0B1 is due to a different
missense mutation (C
T) leading to an amino acid
change from serine to phenylalanine at residue 189. These single amino
acid substitutions result in a marked inhibition of secretion of the
respective C2 proteins, although the secretory block is more profound
for the Arg
mutant.
Figure 1: Core pedigree of the C2D type II family. This family has been described previously(9) . Circles denote female family members, and squares male family members. Open symbols represent the normal C2 gene. Black symbols represent the Type I C2Q0 gene. Shaded and hatched symbols represent the Type II C2Q0 genes. The HLA haplotypes and complotypes linked to each C2 gene are indicated at the bottom of the figure.
The C2 cDNA derived from the other Type II allele was
generated by RT-PCR amplification using RNA isolated from L-cells
transfected with the genomic cosmid clone, C. Single-stranded cDNA was
synthesized from 1 mg of total RNA using the ``cDNA Cycle
Kit'' (Invitrogen) and antisense C2 oligonucleotide primers 922
and 034C (see below for sequences). The cDNA was subsequently amplified
in four overlapping fragments by the polymerase chain
reaction(24) , using the first strand cDNA as template and the
following pairs of oligonucleotide primers, which were designed
according to the published human C2 cDNA sequence(25) :
fragment 1, 395B and 310B; fragment 2, 311D and 034C; fragment 3, 923
and 277; and fragment 4, 922B and 282B. The PCR oligonucleotide
sequences are shown below and were constructed with either BamHI and HindIII restriction sites near the 5`- and
3`-ends to facilitate subcloning. The first strand cDNA was initially
denatured at 95 °C for 1 min with 50 pmol of each oligonucleotide
in a 50-ml solution containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl, 0.1% gelatin, 300
µM dNTPs, and 0.5 units of KlenTaq 1 DNA
polymerase(26) . Following initial denaturation, the cDNA was
amplified by melting at 94 °C for 1 min, annealing at 55-64
°C for 2 min, and polymerizing at 72 °C for 2 min using a
programmable Hybaid OmniGene thermal cycler (Labnet Corp., Woodbridge,
NJ). The amplified cDNA was digested with BamHI and HindIII, purified by low melted agarose extraction using
NuSieve GTG-agarose (FMC Bioproducts, Rockland, ME), and subcloned into
pBluescript II (Stratagene). Competent Sure cells (Stratagene) were
transformed with the ligations, and plasmid DNA was isolated from the
recombinants using the alkaline lysis procedure(27) .
Figure 2: Synthesis of C2 and factor B by fibroblasts and transfected L-cell. Shown are autoradiograms of SDS-polyacrylamide gels (7.5% in reducing conditions) of methionine-labeled C2 and factor B immunoprecipitated from intracellular lysates (I) and culture media (X) as described under ``Experimental Procedures.'' The normal lanes are from skin fibroblasts of a homozygous C2-sufficient female (A-III-2 in (9) ). The Type II lanes are from skin fibroblasts of the homozygous C2D Type II father (II.8 in Fig. 1). The clone B lanes are from L-cells transfected with a cosmid genomic clone containing complete C2 and factor B genes corresponding to one of the Type II C2D alleles. L-cells transfected with clone C gave the same results as shown here for clone B (see text for details). Arrows indicate the major C2 and factor B intracellular and extracellular polypeptides identified in previous studies(29) .
To examine C2
biosynthesis from each of the C2D Type II genes, murine L cells were
separately transfected with cosmid clones bearing the entire factor B
and C2 genes corresponding to normal and each of the Type II C2Q0 genes
(described in detail under ``Experimental Procedures''). The
latter were separable by the presence of an EcoRI RFLP in
intron 1 of the C2 gene. The M 84,000
intracellular C2 protein was abundant in lysates of L cells transfected
with cosmid clones derived from each of the Type II C2Q0 genes, but as
in the primary fibroblasts only a trace amount of C2 protein was
present extracellularly (Fig. 2, right two lanes, top panel). The apparent defect in secretion of C2 in Type II
C2D fibroblasts and the transfectants is selective because factor B is
synthesized and secreted normally in the deficient fibroblasts and the
transfectants (Fig. 2, bottom panel).
Figure 3: Rate of C2 secretion from normal and C2D Type II skin fibroblasts. Fibroblasts were metabolically labeled, washed, and incubated in medium containing excess unlabeled methionine for the times indicated as described under ``Experimental Procedures.'' At the indicated time points, C2 was immunoprecipitated from intracellular lysates and extracellular medium and subjected to SDS-polyacrylamide gel electrophoresis (7.5% in reducing conditions). Skin fibroblasts were from the normal and homozygous C2D Type II individuals studied in Fig. 2. Shown are autoradiograms from 72-h exposures at -70 °C with enhancing screens. Arrows indicate the major C2 intracellular and extracelullar polypeptides.
Figure 4: Rate of C2 secretion from L-cells transfected with cosmid clones containing normal C2 or Type II C2Q0 genes. L-cells that were transfected with cosmid clones containing the normal and Type II C2Q0 genes were metabolically labeled, washed, and incubated in medium containing excess unlabeled methionine for the times indicated as described under ``Experimental Procedures.'' At the indicated time points, C2 was immunoprecipitated from intracellular lysates (lanes on the left side of each panel) and extracellular medium (lanes on the right side of each panel) and subjected to SDS-polyacrylamide gel electrophoresis (7.5% in reducing conditions). Clones were isolated from cosmid libraries constructed using DNA from the normal and homozygous C2D Type II individuals studied in Fig. 2and Fig. 3(see text for details). Shown are autoradiograms from 48-h exposures at -70 °C with enhancing screens. Arrows indicate the C2 intracellular and extracellular polypeptides.
Figure 5:
Indirect immunofluorescence microscopy of
L-cells transfected with normal C2 and Type II C2Q0 genes. Shown are
photomicrographs (magnification, 1000 of L-cells transfected
with cosmid clones containing the normal (panel a) and Type II
C2Q0 genes. L-cells transfected with Type II clones B and C are shown
in panels c and d, respectively. Untransfected L-cell
controls are shown in panel b. The photomicrographs in panels a and b were obtained from a 10-s exposure and
those in panels c and d were obtained from a 1-s
exposure. C2-specific immunofluorescence was performed as outlined
under ``Experimental
Procedures.''
Figure 6:
Detection of C2 RNA in C2D Type II
fibroblasts and transfected L-cells by Northern blot analysis.
Twenty-five micrograms of total RNA isolated from fibroblasts and
transfected L-cells were subjected to Northern blot analysis as
described under ``Experimental Procedures.'' A full-length P-radiolabeled human C2 cDNA was used as a hybridization
probe to detect C2-specific RNA. Shown is an autoradiogram that was
developed after a 24-h exposure at -70 °C with an enhancing
screen. The lanes correspond to RNA isolated from the following
cultures: lane 1, skin fibroblasts isolated from the C2D
homozygous Type II individual II.8 in Fig. 1; lane 2,
L-cells transfected with a cosmid clone containing a normal C2 gene; lane 3, L-cells transfected with cosmid clone B that contains
one of the Type II C2Q0 genes; lane 4, L-cells transfected
with cosmid clone C that contains the other Type II C2Q0 gene; lane
5, untransfected L-cells. The quantities of RNA loaded in each
lane were comparable as judged by ethidium staining (data not shown).
The normal size (approximately 2.7 kb) C2 mRNA is indicated by the arrow on the left. The mobilities of the 28 and 18 S
ribosomal RNA are indicated by the arrows on the right.
Figure 7:
Location of missense mutations in the Type
II C2Q0 genes. Shown at the top of this figure is the exon/intron
organization of the human C2 gene(3) . Exons are depicted by
the numbered boxes with untranslated sequences indicated by shorter boxes. Exons encoding short consensus repeats (SCRs) are indicated by stippled boxes, those
encoding the von Willebrand factor type A-like domain by striped
boxes, and those encoding the serine esterase domain by solid
boxes. The SINE-R.C2 retroposon is represented by the horizontal open box in intron 3. The position of the C1 s
cleavage site is shown by an arrow. The exon 5 missense
mutation (C
T; Ser
Phe) and
proximal nucleotide and amino acid sequences are shown on the left. The exon 11 missense mutation (G
A; Gly
Arg) and proximal nucleotide and amino acid
sequences are shown on the right. The B and C
boxes depict the C2D Type II cosmid clone that was used to
determine the corresponding missense mutation. No mutations, other than
the missense mutations, are present in the cDNA derived from the B and
C clones.
The
full-length C2 cDNA sequence corresponding to the other C2D Type II
allele was delineated from overlapping subcloned cDNA fragments
generated by RT-PCR using RNA isolated from the L-cells transfected
with cosmid clone C (see ``Experimental Procedures''). The
nucleotide sequence of this cDNA was also identical to published human
C2 sequences except for a single base change. In this case, a C
T substitution occurs at nucleotide position 566, resulting in a
predicted serine to phenylalanine amino acid change at residue position
189, which is located in exon 5 of the C2Q0 gene (Fig. 7). The
presence of this nucleotide substitution was confirmed by sequence
analysis of PCR fragments generated from genomic DNA isolated from the
father of the propositus. These results together with the biosynthetic
data demonstrate that the T
and A
nucleotide substitutions in exons 5 and 11 of the Type II C2Q0
genes are missense mutations that ultimately result in the synthesis of
mutant full-length C2 precursor proteins. Because of each amino acid
substitution (either Phe
or Gly
), the C2
mutant precursor is retarded in transit through the normal C2 secretory
pathway.
Figure 8:
Determination of HLA haplotype linkage by
RFLP analysis. Shown is an ethidium-stained 2% agarose gel in which PCR
generated genomic DNA was subjected to PstI RFLP analysis and
electrophoresis as described under ``Experimental
Procedures.'' The DNA samples used to amplify the PCR products are
indicated by the square boxes above the corresponding lanes.
The cosmid DNA was purified from the Type II C2D genomic clones B and
C. Genomic DNA was purified from peripheral blood leukocytes of the
indicated Type II C2D family members (see Fig. 1for family
pedigree). DNA samples not digested(-) or digested (+) with PstI are indicated at the bottom of the agarose gel. The arrows indicate the four DNA bands of interest (see text for
details). The DNA size markers used are X174 cut with HaeIII. The strategy employed in the RFLP analysis is shown at
the bottom of this figure. Oligonucleotides used in the PCR
amplifications are indicated by arrows above exons 10 and 12. Shown are
the natural PstI site in exon 10 and the PstI
polymorphism resulting from the exon 11 missense mutation (indicated by
an asterisk). DNA fragments predicted from the RFLP strategy
are drawn as horizontal lines. The numbers indicate
the predicted size (bp) of each fragment.
Type II C2 deficiency is characterized by a selective block
in C2 secretion and has been found in the context of two different MHC
haplotypes(9) . Using L-cells transfected with the two separate
Type II-associated C2Q0 genes, it is demonstrated here that C2
secretion is impaired in Type II cells because of two distinct C2Q0
allele-specific missense mutations that result in critical amino acid
substitutions in the C2 protein structure. One missense mutation is in
exon 11 (G
A) in the Type II C2Q0 gene linked to
the HLA haplotype A2,B5,DRw4, complotype BFS,C4A3B1. This mutation
results in a Gly
Arg substitution. The other
missense mutation is in exon 5 (C
T) in the type
II C2Q0 gene linked to the HLA haplotype A11,B35,DRw1, complotype
BFS,C4A0B1 and results in a Ser
Phe substitution.
During the past decade, the molecular genetic basis of numerous
protein deficiencies has been determined. The mutations that cause
these deficiencies are of several different types and include various
nonsense mutations, splice site mutations, transcriptional promoter
sequence mutations, and missense mutations. As in the case of Type II
C2 deficiency, recent studies have demonstrated that several protein
deficiencies result from missense mutations that cause critical amino
acid changes, which directly impair secretion of the affected protein.
For example, secretory defects due to single amino acid substitutions
have been reported to cause protein deficiencies of Type IIA von
Willebrand factor(31) , high molecular weight
kininogen(32) ,
-antichymotrypsin(33) , human hepatic
lipase(34) , protein C(35) , murine
I light
chain(36) ,
-antitrypsin(37) ,
lysosomal
-glucosidase(38) , and complement component
C3(39) . In some of these cases, the molecular/cellular basis
of the secretory defect has been examined. Some missense mutations
appear to impair secretion by disrupting critical structural domains
that cause misfolding of the protein. In other cases, missense
mutations do not cause large structural changes but instead alter
important recognition determinants in the protein required for
efficient processing, transport, and secretion. An example of the
former case occurs in PiZZ
-antitrypsin deficiency in
which a single amino acid substitution, lysine for glutamate 342,
results in the synthesis of an improperly folded protein that cannot
readily be transported through the secretory pathway. The mutant PiZZ
-antitrypsin molecule instead remains bound in the
lumen of the endoplasmic reticulum, where it ultimately undergoes
degradation. In contrast, a single serine for phenylalanine 62
substitution in a conserved region of the variable domain of
I
light chain does not induce obvious structural changes. The mutant
I light chain still assembles with the heavy chain forming a
functional antigen-binding antibody and is still recognized by several
polyclonal and monoclonal anti-
antibodies(36) . However,
the mutant
I light chain is not secreted but is arrested in the
endoplasmic reticulum in association with two lumenal endoplasmic
reticulum stress/chaperon proteins, BiP/GRP78 and
GRP94(40, 41) . These chaperon proteins are involved
in the normal folding of
light chains by transient interactions;
however,
I mutants appear to bind BiP/GRP78 and GRP94 more avidly,
thereby inhibiting the normal processing and secretion of the mutant
light chain.
The molecular and cellular mechanisms by which
the Type II missense mutations cause impaired C2 secretion are
currently not known. The three-dimensional structure of C2 has not been
determined; it is therefore difficult to predict what these two
mutations might do to the overall structure of the C2 molecule.
However, comparison of the murine and human C2 sequences indicate that
both mutations are located in highly conserved regions of the C2
molecule, suggesting the importance of these regions in the normal
expression of a functional C2 protein. For example, there is 74%
overall amino sequence identity between murine and human C2. In
contrast, the phylogenetic identity proximal to the exon 5 and 11
missense mutations is much greater, with 100% identity observed in the
19 and 16 amino acids immediately surrounding the Phe and
Arg
mutations in exon 5 and 11,
respectively,(2, 3) . Moreover, exon 11, that encodes
part of the C2 serine protease domain, is one of the most conserved
exons in the C2 gene, with 94% sequence identity shared between the
human and murine amino acid sequences. In addition to its location in a
highly conserved region, the exon 11 Arg
mutation is only
three amino acids upstream of a possible N-linked
glycosylation site at Asn
. The charge change resulting
from the Arg
substitution could disrupt the overall
structure of this conserved region or inhibit proper glycosylation of
Asn
. Either of these possibilities could affect
interactions of the mutant C2 protein with resident endoplasmic
reticulum proteins and cause retention in this compartment. The
substitution of the small polar Ser
with a large aromatic
nonpolar Phe residue in exon 5 could also disrupt structural features
important in the secretion of C2, especially since the substitution
occurs between two aromatic Tyr residues (Fig. 7).
Hereditary
C2 deficiency is the most common complement deficiency in individuals
of western European descent, with approximately 1 person in 10,000
being homozygous C2-deficient. More than half of homozygous C2D
individuals have rheumatological disorders, including systemic lupus
erythematosus(7, 42) . In addition, many are
predisposed to recurrent pyogenic bacterial infections(43) .
Current data indicate that the majority (93%) of C2 deficiency
(C2Q0) genes contain the Type I mutation (28-bp partial gene deletion),
and almost all Type I C2Q0 genes are linked to the extended haplotype
HLA-A25,B18,BFS,C4A4B2,DRw2 (10, 11, 44) .
All remaining Type I C2Q0 genes are associated with parts of this
haplotype, suggesting that the 28-bp deletion originated 600-1300 years
ago with the complete haplotype(12) . Recent reports have
indicated that there is no apparent correlation with these different
clinical manifestations and variations in the Type I C2Q0-associated
HLA extended haplotypes(11, 45) .
In contrast to Type I C2Q0 genes, it has been assumed that Type II C2Q0 genes are rare and comprise no more than the remaining 7% of C2D Caucasian individuals who do not contain the Type I mutation(10) . However, the possibility that the abundance of Type II C2Q0 genes has been underestimated as the result of ascertainment bias should be considered. For example, the majority of C2D families have been discovered by the manifestation of one of the associated clinical problems in a homozygous Type I C2D family member. Since Type II C2D individuals contain some serum C2, it is possible that Type II homozygous C2D individuals do not develop clinical problems as readily as Type I homozygous individuals who lack detectable C2 in their serum. Now that the molecular genetic mutations causing Type II C2D have been delineated, it is possible to examine individuals who contain all or part of the two Type II C2D-associated MHC haplotypes for the Type II missense mutations. Such an investigation should yield a more definite picture regarding the abundance of Type II C2Q0 genes and clinical manifestations associated with Type II C2D. Moreover, continued study of Type II C2D cells will reveal additional insights regarding folding, processing, and secretion of C2 as well as other secretory proteins in general.