From the Department of Medicine, Division of Dermatology, University of Southern California, Los Angeles, California 90033 and the Shriners Hospital for Children, Portland, Oregon 97201
Received for publication, January 9, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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Type VII collagen, the major
component of anchoring fibrils, consists of a central collagenous
triple-helical domain flanked by two noncollagenous domains, NC1 and
NC2. The NC2 domain has been implicated in catalyzing the antiparallel
dimer formation of type VII procollagen. In this study, we produced the
entire 161 amino acids of the NC2 domain plus 186 amino acids of
adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of
collagen VII. Removal of the collagenous helical domain from NC2/COL by
collagenase digestion abolished the antiparallel dimer formation. Using
site-directed mutagenesis, we found that mutation of either cysteine
2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel
dimer formation. In contrast, a single cysteine mutation, 2634, within
the collagenous helical domain had no effect. A generated methionine to
lysine substitution, M2798K, that is associated with recessive
dystrophic epidermolysis bullosa, was unable to form antiparallel
dimers. Furthermore, autoantibodies from epidermolysis bullosa
acquisita patients also reacted with NC2/COL. We conclude that NC2 and
its adjacent collagenous segment mediate antiparallel dimer formation
of collagen VII. Epidermolysis bullosa acquisita autoantibodies
bound to this domain may destabilize anchoring fibrils by interfering
with antiparallel dimer assembly leading to epidermal-dermal disadherence.
Type VII collagen, a genetically distinct member of the collagen
family, is found within the basement membrane zone beneath stratified
squamous epithelium (1, 2). Type VII collagen is a major component of
anchoring fibrils, attachment structures within the basement membrane
between the epidermis and dermis of human skin (3, 4). In inherited
forms of dystrophic epidermolysis bullosa
(DEB),1 anchoring fibrils are
diminutive and/or reduced in number (5-7). In addition to inherited
DEB, epidermolysis bullosa acquisita (EBA) is an acquired autoimmune
form of epidermolysis bullosa. EBA is characterized by circulating and
tissue-bound IgG autoantibodies to type VII collagen (8, 9). Like DEB,
ultrastructural studies have demonstrated a dramatic paucity of
anchoring fibrils in EBA skin (10). These observations suggest that
type VII collagen plays an important role in maintaining
epidermal-dermal adherence. Type VII collagen has been cloned, and a
genetic linkage has been established between inherited DEB and
mutations in the gene that encodes for type VII collagen, COL7A1
(11-14). There have been over 100 distinct COL7A1 gene mutations
identified in patients with DEB, and these mutations have occurred
within NC1, NC2, and the helical domain (15, 16).
Type VII collagen is composed of three identical NC2 consists of 161 amino acids. The NC2 domain has a segment that is
homologous with the Kunitz-type protease inhibitor (12). It also
contains four potential sites for phosphorylation by casein kinase I
and II (12). The overall conservation between mouse and human NC2
domain sequences is 90% at the amino acid level. The relatively high
degree of conservation of this segment is highlighted by the presence
of 67-amino acid residues spanning the junction of the collagenous and
NC2 domains which is 100% conserved between hamster, mouse, and human
sequences (20). It has been proposed that this region contains the
cleavage site for proteolytic removal of the NC2 domain. This region
also contains a pair of cysteines in amino acid positions 2802 and 2804 which are likely to be involved in the disulfide-bond linkages that stabilize antiparallel dimers. In addition, there is a conserved single
cysteine residue in amino acid position 2634 within the adjacent
collagenous domain which may be involved in interchain disulfide
bonding (20).
The NC2 domain of type VII collagen has been implicated in initiating
the triple-helical assembly of type VII collagen and catalyzing the
antiparallel dimer formation of procollagen (2). However, the exact
function of the NC2 domain is not known. This is at least partly due to
the extremely low quantities of procollagen VII in skin and cell
culture. Furthermore, there has been a very limited amount of NC2
available because of its susceptibility to nonspecific proteolytic
degradation during biochemical purification. Therefore, there has been
great difficulty obtaining sufficient quantities of pure NC2.
The major antigenic epitopes for autoantibodies in patients with EBA
and bullous systemic lupus erythematosus (BSLE) reside within the NC1
domain of type VII collagen (21-23). However, recently a novel EBA
subgroup with a milder clinical presentation was defined in children
with tissue bound and circulating autoantibodies targeting only the
triple-helical central domain of type VII collagen (24). These data
suggest that EBA may exhibit a wider, more heterogeneous spectrum of
autoantibody reactivities than previously assumed.
In this study, we prepared a cDNA construct for the NC2
domain plus its immediately adjacent collagenous domain (COL). This construct was used in an eukaryotic expression system to produce large
quantities of purified NC2/COL, a 50-kDa carboxyl terminus of the type
VII collagen Expression Vector Construction and Transfection--
The
eukaryotic expression vector pRC/CMV (Invitrogen, San Diego, CA) which
contains the cytomegalovirus (CMV) promoter and enhancer was used to
express a chimeric cDNA (Fig. 1). The cDNA contains a 16-amino
acid signal peptide sequence derived from human type VII collagen, a
186-amino acid COL domain, and the entire 161-amino acid NC2 domain.
To generate the expression vector, a 1.8-kilobase pair of COL
and NC2 cDNA fragment was released from pGEX-PCR10 by
BamHI/EcoRI digestion (21) and made blunt-ended
by DNA polymerase I (Klenow fragment) following a standard protocol.
This fragment was then ligated to the KasI-digested and
blunt-ended pRC/CMV to generate a construct designated CMV/NC2*. Then,
a cDNA fragment containing signal peptide, COL domain, and NC2
domain was released from CMV/NC2* by NruI/MscI
digestion and ligated into the NruI/XbaI
(blunt-ended) pRC/CMV vector, resulting in the final expression
construct as shown in Fig. 1. We designated the protein product of this
construct NC2/COL. All the constructs were verified by DNA
sequencing. The expression vector was used to transfect a human
embryonic kidney cell line 293 (ATCC, Rockville, MD) using Lipofectin
(Life Technologies, Inc., Gaithersburg, MD), and stable clones were
selected using 500 µg of G418/ml as previously described (25).
For Northern blot analysis, total RNA was extracted using guanidine
isothiocyanate-CsCl2 density gradient centrifugation as previously described (26). The total RNA was size fractionated and
transferred to a nylon membrane (GeneScreen Plus, DuPont, Boston, MA)
and hybridized to a 32P-labeled (random-primer labeling
kit, BMB, Indianapolis, IN) 1.4-kilobase pair human type VII collagen
cDNA probe containing NC2/COL domain.
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed on the NC2/COL chimeric cDNA in the pRC/CMV vector using
a commercial kit (QuikChangeTM site-directed mutagenesis
kit, Stratagene Inc., La Jolla, CA) according to the manufacturer's
instructions. Briefly, a pair of complementary primers with 39 bases
was designed, and mutation to change cysteine to serine or methione to
lysine was placed in the middle of the primers. A double cysteine
mutant was created by changing cysteine at a given site to serine and
using this mutant DNA as a template in the next round of mutagenesis.
Parental cDNA inserted in pRC/CMV was amplified using
Pyrococcus furiosus DNA polymerase with these primers for 12 cycles in a DNA thermal cycler (PerkinElmer Life Sciences). After
digestion of the parental DNA with DpnI, the amplified DNA
with nucleotide substitution incorporated was transformed into
Escherichia coli (XL1-Blue). The mutations were confirmed by
automated DNA sequencing.
Protein Purification and Analysis--
For Western blot
analysis, clonal cell lines resistant to G418 were grown to confluence,
the medium was changed to serum-free medium, and the cultures were
maintained for an additional 24 h. The media were collected,
concentrated 10-fold by Centricon-30 (Amicon, Beverly, MA), and
subjected to 10% SDS-PAGE. Proteins were then electrotransferred onto
a nitrocellulose membrane. The presence of recombinant NC2/COL was
detected with a rabbit polyclonal antibody prepared from our laboratory
against the NC2/COL domain of type VII collagen followed by a
horseradish peroxidase-conjugated goat anti-rabbit IgG and enhanced
chemiluminescence detection reagent (Amersham Corp.) (27).
For large-scale purification of recombinant NC2/COL, serum-free media
were equilibrated to 5 mM EDTA, 50 µM
phenylmethylsulfonyl fluoride, and 50 µM
N-ethylmaleimide, and precipitated with 400 mg/ml ammonium
sulfate at 4 °C overnight with stirring. Precipitated proteins were
collected by centrifuging at 13,000 rpm for 1 h, resuspended and
dialyzed in buffer A containing 500 mM NaCl, 25 mM Tris-HCl, pH 7.8, and 1 mM EDTA and passed
over a Q-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ)
equilibrated in the same buffer. Elution was then carried out with a
linear gradient from 0.5 to 1.0 M NaCl of appropriate
volume size. The proteins that eluted between 0.8 and 1 M
NaCl were pooled and further purified on a Superdex 200 (HR 10/30,
Pharmacia) equilibrate in 25 mM Tris-HCl, pH 7.8, 1 mM EDTA, and 200 mM NaCl.
Protease-inhibitor Assay--
The inhibitory activity of
recombinant NC2/COL for various proteases was examined. We tested
whether NC2/COL could inhibit selected proteases from degrading
recombinant NC1 domain of type VII collagen. The recombinant NC1 (200 µg/ml) as substrate was dissolved in 50 mM Tris-HCl, pH
7.8, 100 mM NaCl, and 2 mM CaCl2 as
described previously (28). The proteases (2 µg/ml) were incubated with inhibitors in 10- or 50-fold excess (by molar) for 1 h at 37 °C prior to the addition of recombinant NC1. Digestions were allowed to incubate for 4 h at 37 °C and then analyzed by
SDS-PAGE.
Digestion with Collagenase--
Purified recombinant NC2/COL (5 µg) was dialyzed against 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM CaCl2, and 2 mM N-ethylmaleimide and then subjected to 10 units of bacterial collagenase (clostridiopeptidase A, type III, Sigma)
digestion at 37 °C for 1 h.
Enzyme-linked Immunosorbent Assay (ELISA) Using Recombinant
NC2/COL--
For testing IgG reactivity with NC2/COL, EBA and BSLE
sera were used. The diagnosis of EBA was based on clinical findings, characteristic histology, demonstration of linear IgG deposits at the
dermal-epidermal junction by direct immunofluorescence, and
demonstration of IgG deposits at the dermal floor of the patients skin
when dermal-epidermal junction was fractured though the lamina lucida
by treatment with 1 M NaCl (29, 30). We showed previously that these sera contained autoantibodies against the NC1 domain of type VII collagen (31). ELISA was performed as described previously
except that purified recombinant NC2/COL (0.15 µg/per well) was used
as antigen to coat 96-well microtiter plates (Immulon-4, Dynatch
Laboratory Inc., Alexandria, VA) (31).
Immunoblotting of Recombinant NC2/COL--
Purified recombinant
NC2/COL protein (100 ng/well) was subjected to a 10% SDS-PAGE, then
electrotransferred to a nitrocellulose membrane. Cut strips of the
nitrocellulose were blocked for 1 h at room temperature with 5%
nonfat dry milk in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20 (TTBS). After washing with TTBS
buffer, the strips were incubated for 1 h at room temperature with
individual patient antisera or control antisera diluted in TTBS with
5% nonfat dry milk (1:200). The strips were then washed as before with
TTBS three times. The immunoreactivity was detected with a horseradish
peroxidase-conjugated goat anti-human IgG (Organon Teknika-Cappel,
Durham, NC) diluted in TTBS with 5% nonfat dry milk (1:5,000) for 30 min at room temperature and enhanced chemiluminescence detection
reagent (ECL, Amersham Pharmacia Biotech).
Rotary Shadowing--
To determine directly if the purified
recombinant NC2/COL could form hexameric structures (antiparallel
dimers), the purified protein was subjected to rotary shadowing
electron microscopy by an established protocol (18). Briefly, 10 µg/ml of the purified NC2/COL protein was dialyzed against 0.2 M ammonium bicarbonate, pH 7.8, and then sprayed onto
freshly cleaved mica. The mica was then transferred to a Balzers BAE
250 evaporator and coated while rotating with a mixture of Pt-C at an
angle of 6°. The surface replica was floated free from the mica, then
transferred to grids in preparation for examination using a Philips 410 transmission electron microscope.
Recombinant Production of NC2/COL Domain of Type VII
Collagen--
A potent eukaryotic expression vector pRC/CMV was used
to express the entire 161 amino acids of the NC2 domain plus 186 amino acids of the adjacent collagenous domain of type VII collagen attached
to the type VII collagen signal peptide (Fig.
1A). The signal peptide
sequence from human type VII collagen was used because efficient
expression and secretion was obtained for the recombinant NC1 domain
using the same expression vector (25).
The expression vector NC2/COL was initially examined by transient
transfection of human 293 embryonic kidney cells. Western blot analysis
with a polyclonal antibody against the NC2/COL domain of type VII
collagen detected the expression of a 50-kDa doublet in both serum-free
medium and cellular extracts of transfected 293 cells (Fig.
1B, lanes 2 and 4). These bands were
absent in the control, vector alone transfected 293 cells (Fig. 1B,
lanes 1 and 3). The parental 293 cells did not
produce endogenous type VII collagen. Stable cell clones were then
established by G418 selection.
Northern hybridization of the NC2/COL stably transfected 293 cell
clones demonstrated abundant amounts of a 1.5-kilobase pair exogenous
mRNA which corresponds in size to that of the cDNA insert (Fig.
1C, lane 2). No hybridization, even after longer exposure, was observed with parental 293 cell RNA, indicating that there is very
little or no production of endogenous type VII collagen in 293 cells
(lane 1).
Purification of NC2/COL Domain of Type VII Collagen--
The
recombinant NC2/COL was purified from serum-free culture medium from a
high expressing 293 cell clone by a two-step procedure. An ammonium
sulfate precipitate of the conditioned medium proteins (Fig.
2, lane 1) was passed over a
Q-Sepharose column. Most of the NC2/COL proteins eluted from the column
in high salt fractions (0.8-1 M NaCl) (lane 2).
Further purification was achieved using molecular sieve chromatography
on a Superdex-200 (lane 3). The final purified yields were
2-5 mg/liter of culture medium.
Formation of Hexamers or Antiparallel Dimers by
NC2/COL--
Previous studies suggested that dimerization is catalyzed
by binding of the NC2 domain to a specific region of the triple helix
(2). We assessed the ability of recombinant NC2/COL to assemble into
disulfide-bonded oligomers. Fig. 3 shows
that prior to reduction, the recombinant NC2/COL migrated as a
disulfide-bonded 300-kDa hexamer, equivalent to one antiparallel dimer
(that is, a molecular dimer) (lane 3). Reduction effectively
dissociated the aggregate into a 50-kDa monomer of NC2/COL (lane
1). These results indicate that the recombinant NC2/COL is capable
of forming a hexameric complex such as those that comprise antiparallel
dimers consisting of six
To examine whether the adjacent COL domain is required for the hexamer
formation, we subjected purified recombinant NC2/COL to collagenase
digestion. As also shown in Fig. 3, digestion of NC2/COL by bacterial
collagenase converted the 50-kDa NC2/COL into a 32-kDa fragment under
reducing conditions (lane 2). This apparent molecule mass
corresponds to that expected for the NC2 domain of type VII collagen.
Under nonreducing conditions, the 32-kDa NC2 runs as a 64-kDa dimer (D)
and a 100-kDa trimer (T), but not as a hexameric complex of 192 kDa
(lane 4). The NC2 domain is capable of forming trimers, but
not hexamers. Therefore, the 186-amino acid collagenous sequences
adjacent to NC2 are essential for the formation of hexamers and the
initiation of antiparallel dimers.
To determine more directly if NC2/COL formed hexameric structures akin
to antiparallel dimers, we subjected purified recombinant protein to
rotary shadowing. Rotary-shadowed images of the recombinant NC2/COL
showed an extended overlapping rod COL domain with two distinct small
globular folding NC2 domains at both ends (Fig. 4A). Within the field of
rotary-shadowed structures, some of the molecules were separated along
their length and appeared to be held together at their globular ends
(Fig. 4B). The rotary-shadowed molecules measured ~60 nm
within the overlapping COL domains, similar to the 60-nm lengths
estimated from intact native type VII collagen dimers (18).
Identification of Cysteine Residues Involved in Antiparallel Dimer
Formation--
To further identify the cysteine residues involved in
the formation of the disulfide linkages to stabilize the antiparallel type VII collagen dimers, we generated four site-directed Cys-Ser mutant constructs and transfected them into 293 cells. As shown in the
immunoblot analysis in Fig.
5A, transfection with C2634S, C2802S, C2804S, and C2802S/C2804S constructs resulted in the secretion of the 50-kDa NC2/COL at a similar level as wild type (compare lanes 2-5 to 1). Under nonreducing conditions,
the protein product of two single cysteine mutations within the NC2
domain (C2802S and C2804S) runs as a 50-kDa monomer, a 100-kDa dimer,
and a 150-kDa trimer (lanes 8 and 9). A double
cysteine mutation within the NC2 domain (C2802S/C2804S) runs
predominately as a 50-kDa monomer, but some 100-kDa dimers are also
observed (lane 10). In contrast, a single cysteine mutation
within the collagenous domain (C2634S) runs as a 300-kDa hexamer
similar to the wild type (lanes 6 and 7). Thus,
both cysteine residues at amino acid 2802 and 2804 within the NC2
domain are vital for hexamer (antiparallel dimer) formation.
Formation of Antiparallel Dimer Is Impaired by a Methionine to
Lysine Mutation--
Previous studies have identified a homozygous
methionine to lysine missense mutation at the amino acid residue 2798 in a patient with recessive DEB (32). To investigate the molecular
defect underlying this mutation, site-directed mutagenesis was used to generate the same mutation in the NC2/COL construct. As shown in the
immunoblot analysis in Fig. 5B, transfection of the M2798K construct into 293 cells resulted in the secretion of a 50-kDa NC2/COL
at a similar level as the wild type (compare lanes 1 and 2). Under nonreducing conditions, the M2789K mutation
protein product runs mostly as a 50-kDa monomer and a 150-kDa trimer
(lane 4). However, the mutation protein product was
incapable of forming hexameric complexes. These data indicate that a
methione to lysine mutation within the NC2 domain impairs the ability
of NC2/COL to form antiparallel dimers.
Lack of Protease Inhibitor Activity--
Sequence analysis of the
NC2 domain reveals the presence of a segment with homology to
Kunitz-type protease inhibitors (12). However, it is unclear whether
NC2 functions as a protease inhibitor. To examine this issue, trypsin,
chymotrypsin, and several other proteases with a trypsin-like
specificity or other specificities were incubated with the NC1 domain
of type VII collagen with and without the presence of purified
recombinant NC2/COL added at 10- or 50-fold excess. Recombinant NC1
domain of type VII collagen was selected as the protein substrate
simply because of its large size (145 kDa) and the presence of many
protease cleavage sites. As shown in Fig.
6, degradation of NC1 by either trypsin
or chymotrypsin was completely protected by a 50-fold excess of
aprotinin (lanes 2 and 6). However, no protective
effect could be observed with recombinant NC2/COL added at 10-fold
excess (lanes 3 and 7) or 50-fold excess
(lanes 4 and 8). The same lack of inhibitory
activity of NC2/COL was also found for thrombin and plasmin (data not
shown).
NC2/COL Is Targeted by Autoantibodies in Bullous
Diseases--
Previously, we and others have identified the 4 immunodominant epitopes within the NC1 domain of type VII collagen for
EBA and BSLE autoantibodies (21-23). To investigate whether NC2/COL contained any antigenic epitopes for EBA and BSLE autoantibodies, recombinant purified NC2/COL was used as an antigen in an ELISA and
immunoblot analysis. We then tested sera from 24 EBA patients and 5 BSLE patients. All of these sera were shown previously to react with
the NC1 domain of type VII collagen (31). The results of the NC2/COL
ELISA are shown in Fig. 7 as a scatter
plot. Twenty-two negative control sera (12 NHS, 10 BP) were analyzed to
set an appropriate threshold for this assay. All the control sera
showed very low reactivity in the ELISA with values less than 0.2 optical density. We set 0.25 optical density as the threshold for the ELISA (horizontal line in Fig. 7). In contrast to the
controls, 4 of the 24 EBA sera and 4 of the 5 BSLE sera exhibited
reactivity with the recombinant NC2/COL in the ELISA with optical
density values ranging from 0.5 to 1.6.
To confirm the ELISA, the EBA and BSLE sera that were positive in the
ELISA were further analyzed by immunoblotting with NC2/COL. As shown in
Fig. 8, sera from 3 EBA patients
(lanes 3-5) and 3 BSLE patients (lanes 8-10)
strongly recognized the 50-kDa recombinant NC2/COL while serum from 1 EBA patient (lane 6) and 1 BSLE patient (lane 7)
demonstrated very weak reactivity. These two sera also had low optical
density values in the ELISA. Control normal human sera (lanes
1 and 2) did not react with NC2/COL. Taken together, these data show strong concordance between the results of the ELISA and
the immunoblot analysis.
In the present study, we developed a versatile eukaryotic
expression system that allowed us to join the 161-amino acid of NC2
domain plus 186 amino acids of the adjacent collagenous domain of type
VII collagen to a potent signal peptide sequence derived from the NC1
domain of type VII collagen. This approach directs the recombinant
protein products to the secretory pathway, facilitating their
purification. The efficient production and secretion of recombinant
NC2/COL was achieved in a human cell line 293, which showed no
endogenous production of type VII collagen. The production of NC2/COL
has allowed us to obtain sufficient quantities of the NC2/COL segment
for structural and functional studies.
The cDNA sequence analysis of the NC2 domain reveals the
presence of a region homologous with the large family of Kunitz-type serine protease inhibitors. There is no evidence to date, however, that
the NC2 actually can serve as a protease inhibitor. We tested the
recombinant NC2/COL and NC2 for inhibitory activity against trypsin and
trypsin-like proteases. We were unable to demonstrate any protease
inhibitory activity with either NC2/COL or NC2 obtained biochemically
from collagenase digestion of NC2/COL (data not shown). All protease
inhibitors with the Kunitz motif contain an important region that is
responsible for the antiprotease function. This region has a critical
lysine or arginine at the P1 site in all protease inhibitors with
antitrypsin activity (33, 34). In what would be the homologous NC2
region (residue 2882-2289), there is a threonine (residue 2886) rather
than either lysine or arginine. This single amino acid difference may
account for the lack of inhibition of trypsin by NC2. Some proteins
have a homologous region without a basic residue at the P1 site and yet still have antiprotease activity against chymotrypsin. However, we were
unable to demonstrate any anti-chymotrypsin activity in our recombinant
NC2. Although we were unable to demonstrate any antiprotease activity
in NC2, our studies do not exclude the possibility that the Kunitz
motif in NC2 could have an inhibitory activity for a protease that is
yet to be identified. Or, perhaps, in vivo NC2 is processed
to an active form.
It is of interest that the Kunitz motif in the NC2 domain of type VII
collagen is most similar to the motif in the carboxyl-terminal noncollagenous domain of the The cDNA sequence analysis of the NC2 domain reveals an 88%
conservation at the amino acid level between the human and mouse. Furthermore, there is 100% conservation in the 67-amino acid segment spanning the junction of the triple helical and the NC2 domain which
contains a pair of cysteine residues. It has been postulated that NC2
within one It has been suggested that a pair of cysteine residues at positions
2802 and 2804 within the NC2 domain may be involved in the interchain
and intermolecular disulfide bonds which stabilize type VII collagen
antiparallel dimers. We directly tested the importance of these
cysteine residues by site-directed mutagenesis studies. We showed that
a single cysteine mutant protein in either C2802S or C2804S within the
NC2 domain failed to form hexamers. Furthermore, mutation of both
cysteines 2802 and 2804, but not mutation of either alone, abolished
the trimer formation. Interestingly, a single cysteine mutation C2634S
within the collagenous domain had no effect on hexamer formation, even
though this cysteine has been proposed to be involved in interchain
disulfide bonding (20).
In a study by Christiano and colleagues (32), a family with members who
had recessive DEB was described in which a gene defect in type VII
collagen was identified as a single amino acid substitution from
methionine to lysine at residue 2789 within the NC2 domain.
Specifically, this substitution was 4 residues upstream from cysteines
2802 and 2804 within the NC2 domain (32). Because of this report and
the resultant complete lack of anchoring fibrils in the skin of
affected patients, we attempted to duplicate this gene defect in
vitro using site-directed mutagenesis. We introduced the same
mutation in the NC2/COL construct which rendered the protein product
unable to form hexameric, disulfide-bonded aggregates (antiparallel
dimers). It is possible that the replacement of a non-polar amino acid
methionine with a positively charged amino acid lysine might interfere
with the ability of the two adjacent cysteine residues, 2802 and 2804, to form a disulfide bond and stabilize antiparallel dimers. Such a
mechanism could result in deficient formation of type VII collagen
antiparallel dimers and assembly of anchoring fibrils. This would lead
to a paucity of anchoring fibrils which is characteristic of recessive DEB.
Several biochemical studies employing rotary shadowing and
immunoelectron microscopy have suggested that the NC2 domain is cleaved
off of type VII collagen The IgG autoantibodies in the sera of patients with EBA recognize
distinct antigenic epitopes in the NC1 domain (21-23). Nevertheless, at least three EBA patients have been identified with autoantibodies against the helical domain (24). These patients were Japanese children
who had the inflammatory, bullous-pemphigoid-like variety of EBA. We
examined the immunoreactivity of EBA sera with recombinant NC2/COL
using ELISA and Western blot analysis. We found four bullous SLE and
four EBA sera that recognized both the NC1 domain and NC2/COL. The
reactivity was abolished with collagenase treatment of NC2/COL which
suggested that the antigenic epitope is within the 186-amino acid
helical collagenous segment immediately juxtaposed to NC2 (data not
shown). All of the EBA patients in this study had the classical
mechanobullous form of EBA. In our previous study, the majority of EBA
patients with classic clinical features had autoantibodies reactive
with the NC1 domain of type VII collagen except one patient with
bullous SLE who showed immunoreactivity to both NC1 and the collagenous
domain (19). In this study, we demonstrate coexistence of
autoantibodies to both NC1 and the collagenous domains in a subset of
EBA patients.
It has been hypothesized that EBA autoantibodies may contribute to
defective epidermal-dermal adhesion by binding to the NC1 domain of
type VII collagen. Autoantibodies bound to type VII collagen would
interfere with the normal interactions between type VII collagen and
its ECM ligand(s) in the basement membrane zone or the papillary dermis
(25, 36). In this study, we identified EBA and BSLE sera that bound to
NC2/COL. In this regard, it is interesting to note that we have
previously mapped the 187-amino acid collagenous sequences in the
NC2/COL construct as a fibronectin-binding site (37). It is possible
that perturbation of the type VII collagen-fibronectin interaction may
contribute to the pathogenesis of EBA. Alternatively, binding of EBA
autoantibodies to the COL domain may destabilize anchoring fibrils by
interfering with the antiparallel assembly of procollagen dimers
leading to epidermal-dermal disadherence.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chains, each
consisting of a 145-kDa central collagenous triple-helical segment,
flanked by a large 145-kDa amino-terminal non-collagenous domain (NC1),
and a smaller 34-kDa carboxyl-terminal non-collagenous domain (NC2) (4,
17). In the extracellular space, individual type VII collagen molecules
form antiparallel tail-to-tail dimers stabilized by disulfide bonding
through a small carboxyl-terminal overlap (NC2), and a portion of the
NC2 domain is proteolytically removed (18, 19). The antiparallel dimers
then aggregate laterally in a nonstaggered manner to form anchoring
fibrils (4).
-chain. We demonstrate herein that purified NC2/COL
readily forms disulfide-bonded hexameric aggregates, each representing
one anti-parallel dimer of collagen VII. We also demonstrate that the
adjacent COL domain is essential for the formation of antiparallel
dimers. Using site-directed mutagenesis, we found that mutating either
cysteine 2802 or cysteine 2804 within the NC2 domain inhibits
antiparallel dimer formation. In contrast, a single cysteine mutation,
cysteine 2634, within the collagenous domain had no effect.
Furthermore, a methione to lysine substitution mutation, M2798K, within
the NC2 domain also caused an inability to form antiparallel dimers.
Interestingly, this mutation has been reported to be associated with
recessive DEB.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic organization and expression of the
NC2/COL cDNA construct. A, the deduced 363-amino acid
sequence of the cDNA insert consists of a 16-amino acid signal
peptide derived from type VII collagen, a 186-amino acid collagenous
region (COL), and a 161-amino acid of entire non-collagenous NC2
domain. The cDNA insert was cloned into a eukaryotic expression
vector pRC/CMV. B, immunoblot analysis of conditioned media
(lanes 1 and 2) and cellular extracts
(lanes 3 and 4) from either 293 cells transfected
with vector alone (lanes 1 and 3) or 293 cells
transfected with NC2/COL construct (lanes 2 and
4). The positions of molecular mass markers and the 50-kDa
NC2/COL are indicated. C, lanes were loaded with 20 µg of
total RNA from parental 293 cells (lane 1) and 293 clone,
stably transfected with the NC2/COL (lane 2). The blot was
hybridized with a cDNA probe specific for the NC2 domain of type
VII collagen. The positions of the exogenous NC2/COL mRNA (1.5 kilobase pairs (kb)) and the ribosomal RNA (28 S, 18 S) are
indicated.
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Fig. 2.
Purification of recombinant NC2/COL from
conditioned media. 10% SDS-PAGE and Coomassie Blue
staining of NC2/COL. Lane 1, ammonium sulfate precipitate of
the conditioned media of the NC2/COL stably transfected 293 cell clone;
lanes 2 and 3, NC2/COL purified after Q-Sepharose
and Superdex-200 gel filtration, respectively. The positions of the
molecular mass markers and the 50-kDa NC2/COL are indicated.
-chains of type VII collagen.
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Fig. 3.
Involvement of the collagenous domain in the
formation of antiparallel dimers. Purified recombinant NC2/COL was
either untreated (lanes 1 and 3) or treated with
bacterial collagenase (lanes 2 and 4) and
analyzed by 8-16% SDS-PAGE followed by immunoblot analysis with a
polyclonal antibody to NC2 domain. Proteins were either reduced with
2-mercaptoethanol (lanes 1 and 2) or nonreduced
(lanes 3 and 4). The positions of molecular
weight markers, monomer (M), dimer (D), trimer
(T), and hexamer (H) of NC2/COL and NC2 are
indicated. The migration of full-length type VII collagen served as a
300-kDa marker.
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Fig. 4.
Transmission electron microscopic images of
NC2/COL obtained following rotary shadowing. Representative fields
are shown. A, images of two trimeric NC2/COL molecules in an
antiparallel arrangement with little if any gap between the overlapping
COL domains. One molecule consists of three identical NC2/COL peptides
in a helix. In the image, two such helixes are bound together in an
antiparallel aggregation which represents six NC2/COL peptides.
B, images of the hexameric structures in A which
exhibit some dissociation in COL domains. Bar, 50 nm.
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Fig. 5.
Formation of antiparallel dimers by cysteine
mutants. A, 293 cells were transfected with either wild
type NC2/COL (WT) or with cysteine mutants, and conditioned
media were concentrated and subjected to 8% SDS-PAGE followed by
immunoblot analysis with a polyclonal antibody to NC2 domain. Proteins
were either reduced with 2-mercaptoethanol (lanes 1-5) or
nonreduced (lanes 6-10). The positions of molecular weight
markers, monomer (M), dimer (D), trimer
(T), and hexamer (H) of NC2/COL are indicated.
B, immunoblot analysis of either wild type NC2/COL
(WT) or methionine to lysine substitution (M2798K). Proteins
were either reduced with 2-mercaptoethanol (lanes 1 and
2) or nonreduced (lanes 3 and
4).
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Fig. 6.
Inhibition of NC1 cleavage by trypsin and
chymotrypsin with protease inhibitors and NC2/COL. The cleavage
products from either trypsin (lanes 1-4) or chymotrypsin
(lanes 5-8) were separated by 6% SDS-PAGE followed by
Coomassie Blue staining. Inhibitors were added as follows: lanes
1 and 5, none; lanes 2 and 6,
10-fold excess of aprotinin; lanes 3 and 7,
10-fold excess of NC2/COL (by molar); lanes 4 and
8, 50-fold excess of NC2/COL. The position of intact NC1 is
indicated.
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Fig. 7.
Scatter plot representation of ELISA results
using recombinant NC2/COL. Patient and control sera (as indicated
along the horizontal axis) (1:200 dilution) were incubated
with immobilized purified NC2/COL and the bound antibodies were
detected with an alkaline phosphatase-conjugated antibody against human
IgG whole molecule. Each sample was run in triplicate and the points
plotted on this graph are the average of the
A405 obtained from study sera. Similar results
were obtained in three other independent studies.
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Fig. 8.
Immunoblot of the recombinant NC2/COL
proteins by EBA and control sera. The purified recombinant NC2/COL
(100 ng/well) protein was separated on 10% SDS-PAGE and transferred to
nitrocellulose membranes before incubation with sera at a dilution of
1:200 and horseradish peroxidase-conjugated anti-human IgG (1:5000)
followed by ECL detection. The location of 50 kDa of recombinant
NC2/COL is indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-chain of type VI collagen
(20). Although there is lysine at the P1 site in type VI collagen, a recombinant Kunitz domain of the type VI
3-chain did not
demonstrate protease inhibitor activity (28). The Kunitz motif in
protease inhibitors have also been shown to play a role in binding to
active sites. For example, dendrotoxins are speculated to act by
binding to some component of the presynaptic membranes. In this regard, it is interesting to note that type VI collagen, like type VII collagen, forms anti-parallel dimers as the first step in the formation
of the macromolecular structure (35). Thus, the non-active Kunitz-type
serine protease inhibitory motif in both collagens may contribute to
the initial intermolecular binding needed for the formation of
antiparallel dimers.
-chain may bind to an appropriate site on the
triple-helical domain of a second molecule and align cysteine residues,
which subsequently becomes stabilized by intermolecular disulfide bonds
(2). This model is consistent with the observation that native
pepsin-treated type VII collagen dimers do not remain associated
following disulfide bond reduction even under physiological conditions
(18). In this study, we provide direct evidence that recombinant
NC2/COL is capable of forming disulfide-bonded hexamers in
vitro. Furthermore, rotary-shadowed images of the recombinant NC2/COL demonstrated directly that the molecules align into hexameric aggregations that represent antiparallel dimers. By subjecting the
NC2/COL segment to bacterial collagenase digestion, we showed that the
NC2 domain alone was unable to form oligomers larger than a trimer.
These results suggest that the 186-amino acid collagenous sequences
adjacent to the NC2 domain are required for antiparallel dimer formation.
-chains in the process of anchoring fibril
formation (4, 17). In addition, using NC2-specific antibody, it was
shown that this domain was absent from normal human skin (19). Because
of its protease sensitivity and the low yield of purified type VII
collagen using biochemical purification methods, detailed studies to
identify the peptide bond involved in the physiologic NC2 removal have
not been possible. It is interesting that in this study, we repeatedly
observed two protein bands of 30 and 20 kDa in our purified fractions
of recombinant NC2/COL. Whether this represents the natural cleavage
process which occurs in the physiologic removal of NC2 during anchoring
fibrils assembly is not known.
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FOOTNOTES |
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* This work was supported in part by Grant RO1 AR33625 (to D. T. W.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Dermatology Foundation Career Development Award.
§ To whom correspondence should be addressed: Dept. of Medicine, Div. of Dermatology, University of Southern California, CRL 108, 1303 Mission Rd., Los Angeles, CA 90033. Tel.: 323-224-7056; Fax: 323-224-7679.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M100180200
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ABBREVIATIONS |
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The abbreviations used are: DEB, dystrophic epidermolysis bullosa; NC1, NH2-terminal noncollagenous domain of type VII collagen; NC2, COOH-terminal noncollagenous domain of type VII collagen; EBA, epidermolysis bullosa acquisita; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; BSLE, bullous systemic lupus erythematosus; CMV, cytomegalovirus.
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