(Received for publication, January 16, 1996)
From the
The bullous pemphigoid antigen BPAG1 is required for keratin
filament linkage to the hemidesmosome, an adhesion complex in
epithelial basal cells. BPAG1 structural organization is similar to the
intermediate filament-associated proteins desmoplakin I (DPI) and
plectin. All three proteins have predicted dumbbell-like structure with
central -helical coiled-coil rod and regions of N- and C-terminal
homology. To characterize the size of the N-terminal globular domain in
BPAG1, two polypeptides spanning possible boundaries with the
coiled-coil rod domain of BPAG1 were expressed in Escherichia coli. BP-1 (M
= 111,000), containing amino
acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto,
J.(1991) J. Biol. Chem. 266, 17784-17790), and BP-1A,
with a 186 amino acid N-terminal deletion, were purified. BP-1 and
BP-1A behave as highly asymmetric dimers in aqueous solution according
to velocity sedimentation and gel filtration. Both have globular heads
with rod-like tails of roughly equal length, 55-60 nm, upon
rotary shadowing. BP-1A content of
-helix, determined by circular
dichroism, is
90%, consistent with
-helical coiled-coil
formation in the rod-like tails. The estimated rod length, 383 ±
57 amino acids (0.15 nm/amino acid), implies that globular folding in
the BPAG1 N-terminal extends to the end of N-terminal homology with DPI
and plectin. These findings support the existence of a common domain
structure in the N-terminal regions of the BPAG1/DPI/plectin family.
The intracellular bullous pemphigoid antigen (BPAG1) ()is a part of the cytoplasmic plaque of the hemidesmosome,
a supramolecular structure that links keratin intermediate filaments to
extracellular matrix in a number of epithelial cell types, including
epidermal keratinocytes(1, 2) . BPAG1 (or the 230-kDa
bullous pemphigoid antigen) was identified by autoimmune antibodies of
patients with the dermal-epidermal blistering disease, bullous
pemphigoid(3, 4) . BPAG1 is rapidly assembled into a
stable anchoring contact, or prehemidesmosome, at the ventral surface
of freshly plated keratinocytes in
culture(5, 6, 7) . Disruption of the BPAG1
gene in mice by homologous recombination prevents keratin filament
attachment to the hemidesmosome and consequently weakens
dermal-epidermal adhesion but does not affect formation of the
membrane-associated dense plaque of the hemidesmosome(8) . The
hemidesmosome also contains the high molecular weight cytoplasmic
component HD-1 (9) and two transmembrane proteins, BPAG2, which
contains an extracellular collagenous
domain(10, 11, 12, 13) , and the
integrin
(5, 14) .
BPAG1 is homologous to two proteins which associate with
intermediate filaments (IF): desmoplakin I (DPI), which is part of the
desmosome, a cell-cell junction; and plectin, a ubiquitous cytoskeletal
protein that binds IF subunits in
vitro(15, 16, 17, 18, 19, 20) .
The predicted molecular masses of the three proteins are >300 kDa,
and all three have homologous N- and C-terminal domains. Cell
transfection and molecular studies have demonstrated that the
C-terminal domains for plectin and DPI specifically interact with
several IF types(21, 22, 23) . The central
regions of all three proteins, which lack homology to one another,
nonetheless contain highly significant heptad repeats characteristic of
-helical coiled-coil rods, similar to the rod domain of myosin or
to the core of IF heterodimers(20, 24) . Purified DPI
dimerizes and has an extended dumbbell-like shape in which terminal
globular regions are separated by a central rod(25) . Plectin
and BPAG1 appear to take similar but less well-characterized
conformations(26, 27) .
The actual size of the
N-terminal globular domains in BPAG1, DPI, or plectin have not been
measured directly. In the case of BPAG1, this is of particular interest
since the presence of heptad repeats in its N-terminal homology domain
has suggested that coiled-coil rod formation may extend as far as 450
amino acids into this domain from the central, nonhomologous rod
region(16, 18, 20) . In plectin and DPI, the
N-terminal homology region lacks certain characteristics generally
associated with coiled-coil structures, such as a high (>1.0) ratio
of charged/apolar amino acids, and, thus, rod formation in these
domains is considered unlikely(19, 20) . In N-terminal
conserved regions of BPAG1, however, the presence of heptad repeats
along with a high level of predicted interchain charge interactions has
led to predictions that -helical coiled-coil formation initiates
at residue 708 or 875 (16, 18) according to the
numbering of Sawamura et al.(18) rather than near the
boundary of the N-terminal homology domain (residue 1145) as is the
case with plectin and DPI(20) . In order to directly establish
the boundary of the N-terminal globular and central rod domains in
BPAG1, two BPAG1-derived polypeptides containing the predicted
N-terminal rod domain transitions were expressed in Escherichia
coli and purified. The renatured polypeptides dimerize and, by
rotary shadowing, are shown to have highly asymmetric structures in
which a globular N-terminal conincides with the N-terminal homology
region defined by comparison with plectin and DPI.
Figure 1:
Purification of BP-1 and BP-1A. A, BP-1 extracted from bacterial inclusion bodies in 8 M urea (lane a) was purified on DEAE-chromatography (lane b), and renatured by dialysis from urea. The 200,000
g supernatant following dialysis is shown in lane
c. B, BP-1A in the high speed supernatant of crude bacterial cell
lysate (lane a) was purified on DEAE-chromatography (lane
b), followed by Sephacryl HR 500 gel filtration (lane c).
The molecular mass standards used were (from top to bottom): myosin heavy chain, 200 kDa; phosphorylase B, 97.4
kDa; bovine serum albumin, 68 kDa; and ovalbumin, 43
kDa.
Analytical gel filtration
chromatography of BP-1 and BP-1A was performed on a 1.0 30 cm
Superose 6 column (Pharmacia FPLC) in 20 mM Hepes-Na, pH 7.0,
0.1 M NaCl, and 1 mM DTT buffer at a flow rate of 0.1
ml/min, and the column was cooled and equilibrated in an ice bath. The
marker proteins (and Stokes radii, R
) used in the
gel filtration study include catalase (52 Å),
-galactosidase
(69 Å), thyroglobulin (86 Å)(33) , fibrinogen (107
Å)(34) , and erythrocyte spectrin dimer (123
Å)(35) . Spectrin dimer was obtained from human red blood
cell ghost spectrin tetramer as described(36) : red blood cell
ghosts were dialyzed overnight at 4 °C in 0.1 mM EDTA,
0.01 mM DTT at a pH of 8-9.5 adjusted with diluted
NH
OH. After dialysis, the ghosts were centrifuged at
200,000
g for 1 h at 4 °C. The supernatant was
incubated at 37 °C for 15 min, spun again on an Eppendorf
centrifuge at top speed, and stored at -20 °C prior to gel
filtration. The elution positions for the marker proteins were
determined on Coomassie Blue-stained SDS-PAGE gels according to
distinctive subunit molecular masses: thyroglobulin (670 kDa), which
gives rise to a family of 10- to 330-kDa polypeptides upon reduction
for SDS-PAGE, fibrinogen (340 kDa) with 68 kDa, 55-kDa and 46-kDa
subunit polypeptides, and spectrin dimer, comprised of a 220- and a
240-kDa polypeptide. BP-1 and BP-1A were identified by a rabbit
polyclonal antibody raised to BP-1 in this laboratory.
The Stokes
radii of the standards were plotted against erf(1
- K
), which gives a linear plot with
standard proteins(37) . erf
is the inverse
error function, and K
is a partition coefficient
or measure of relative elution volume. 1 - K
is defined as: (V
Estimated protein molecular weights were calculated as:
where all values are under standard conditions (20 °C in
water): (viscosity) = 1.004; N =
Avogadro's number;
= protein partial specific volume
(estimated 0.725 cm
/g);
= density of water
(0.998 g/cm
). The ratio of actual frictional coefficient (f) to the coefficient for a spherical protein of the same
molecular weight (f
) can be estimated as
follows(34) :
Further
purification of BP-1 and BP-1A by heat treatment was attempted since
many water-soluble coiled-coil proteins, such as tropomyosin and light
meromyosin, renature spontaneously when cooled following heating to 95
°C(43, 44) . Interestingly, both BP-1 and BP-1A
precipitated out of solution when heat-denatured (Fig. 2).
Horowitz and co-workers (45) have shown that a detergent, such
as Zwittergent 3-16, when near its critical micelle
concentration, can facilitate native protein folding during rapid
renaturation by binding to exposed hydrophobic surfaces that otherwise
induce aggregation. In the presence of 0.1 mg/ml Zwittergent 3-16, BP-1
and BP-1A renature primarily to soluble forms (Fig. 2) following
heat treatment. While overall protein purity is not improved by this
procedure, the detergent requirement for renaturation of BP-1 and BP-1A
following heat treatment is consistent with the presence of complex
globular folding domains in addition to the predicted -helical
coiled-coil.
Figure 2:
Heat
treatment of BP-1 and BP-1A in the presence or absence of the nonionic
detergent, Zwittergent 3-16. DEAE-purified and renatured BP-1 and
purified BP-1A, were heated to 95 °C in the absence(-) or
presence (+) of the detergent Zwittergent 3-16 at a concentration
of 0.1 mg/ml. After cooling on ice, the protein solutions were
centrifuged at 200,000 g for 30 min, and both the
supernatant (S) and pellets (P) were analyzed by
SDS-PAGE and Coomassie Blue staining.
Figure 3:
Sedimentation of BP-1A on a sucrose
density gradient. The s value of BP-1A was
determined by comparison with known marker proteins (1,
fumarase; 2, lactate dehydrogenase; 3, bovine serum
albumin; 4, human hemoglobin; 5, carbonic anhydrase).
BP-1A (higher band) comigrated with bovine serum albumin (lower band) as shown on the gel and has an estimated
s
value of 4.4.
Figure 4:
Gel filtration analysis of BP-1 and BP-1A
on Superose 6. The Stokes radii of the maker proteins are plotted
against the erf (1 - K
), which is related to the elution
volume. BP-1 and BP-1A eluted close together between fibrinogen and the
spectrin dimer. The Stokes radii for BP-1 (118 Å) and BP-1A (111
Å) were predicted by interpolation. Some experimental variation
in BP-1 was observed (arrows) and an average Stokes radius (R
) is used. The markers were: 1,
catalase; 2,
-galactosidase; 3, thyroglobulin; 4, fibrinogen; and 5, spectrin
dimer.
Figure 5: Rotary shadowed images of BP-1 and BP-1A. A representative field of BP-1A is shown in the top panel (a). Most molecules have a long rod-like tail with a globular head. BP-1 has similar features: a prominent head and a well-resolved tail. The tail in BP-1 was either straight (b) or bent in the middle (c). For BP-1A, not all molecules had an obvious head (d), but, in most cases, the head was clearly visible with a long tail attached (e). Some had a clearly visible kink in the tail domain (f). Bar = 50 nm.
Figure 6: Distribution of BP-1 and BP-1A tail length determined by rotary shadowing. Rod length is measured from the end of the tail to the junction of the head of rotary shadowed images as displayed in Fig. 5.
Figure 7:
Far UV circular dichroism spectrum of
BP-1A. The CD spectrum of BP-1A was recorded in a 1-mm path length
cuvette at room temperature. The protein concentration was 0.134 mg/ml.
The two minima at 222 nm and 208 nm and the maximal peak at 193 nm are
characteristic of -helices.
Figure 8: Probability of coiled-coil formation as a function of amino acid residue according to the algorithm of Lupas et al. (42). A window size of 28 amino acids was used to estimate a coiled-coil score at each residue, and a probability of coiled-coil formation was assigned based on the behavior of known proteins in this algorithm.
Previous estimates that
coiled-coil rod extends into the N-terminal homology domain of BPAG1
were based upon the high frequency (70%) of hydrophobic amino
acids in the a and d positions of heptad repeats (a, b, c, d, e, and f) found in this domain as well as higher than average
intrachain charge interactions consistent with coiled-coil
formation(17, 20) . These characteristics may instead
give rise to non-rod
-helix (such as
-helical bundles) in the
BP-1A head domain instead of coiled-coil rod(20) . This
conclusion is also consistent with the very high content of
-helix
in BP-1A as estimated by circular dichroism.
Figure 9: Domain structure of BPAG1. BP-1 and BP-1A are shown as striped or shaded bars aligned with a BPAG1 open reading frame of 2,649 amino acids. The N-terminal globular domain GN is shown adjacent to the rod region (open bar) as defined in this study. B and C are homologous to one another and to comparable C-terminal repeats in plectin and the desmoplakins. Arrows 1, 2, and 3 represent start sites of the rod region according to the predictions of Tanaka et al.(16) , Sawamura et al.(18) , and Green et al.(20) .
Although analysis of soluble BPAG1
polypeptides was a necessity in this study, the approach can be
justified on the grounds that BPAG1 is expressed in cultured
keratinocytes in a soluble or cytosolic form which may act as a
precursor for incorporation into the detergent-resistant plaques found
in culture(3, 6, 50) . DPI and DPII are also
soluble once purified(25) , and they are present largely in
soluble form in epithelial cells cultured in low (<0.05 mM)
Ca; induction of desmosome formation by raising
extracellular Ca
causes a shift from cytosolic to
desmosome-associated forms, suggesting a precursor-product relationship
for the soluble and insoluble forms of the desmoplakins as
well(51, 52) . We presume that once BPAG1 has been
incorporated into the hemidesmosome, major tertiary folding patterns
are unchanged. Ultrastructural and genetic evidence suggest that the
BPAG1 C-terminal is associated with the cytoplasmic plate of the
hemidesmosome, which is a junction for keratin filament attachment. In
BPAG1 knockout mice, not only is the plate absent but keratin
attachment to the hemidesmosome is also abrogated(8) . The
cytoplasmic plate is found at a distance of about 0.1 µm from what
is the most prominent feature of the hemidesmosome, the plasma
membrane-associated dense plaque(2, 8, 53) .
Labeling with bullous pemphigoid patient autoantibodies directed to
BPAG1 shows predominance of staining at an average of 90 nm from the
plasma membrane, and two rabbit antisera to the BPAG1 C-terminal also
bind at the approximate location of the keratin attachment
plate(6, 12, 54) . Ultrastructural
localization of the BPAG1 N-terminal has not been addressed
experimentally, although studies in cultured normal human keratinocytes (6) and SCC-12 cells (13) strongly suggest that BPAG1
is exclusively intracellular, based on its resistance to degradation by
extracellular protease. The central discontinuous rod of BPAG1 may
provide a flexible link to another cytoplasmic structure such as the
membrane-associated dense plaque, where the N-terminal domain itself
could in turn bind to transmembrane hemidesmosome components such as
BPAG2 or the integrin
.
In summary, we have carried out high resolution mapping of the N-terminal homology region of BPAG1, empirically localizing a boundary between the N-terminal globular and central rod domain. Comparison of two polypeptides differing only in an N-terminal deletion has enabled us to specify the absolute orientation of the rotary shadowing images of these molecules unambiguously. These data on BPAG1 domain structure should facilitate more rational design of polypeptides for in vitro analysis and cell transfection studies of N-terminal globular domain function in all members of the BPAG1/DPI/plectin protein family.