From the Medizinische Universität zu
Lübeck, Institut für Medizinische Molekularbiologie,
Ratzeburger Allee 160, D-23538 Lübeck, Germany, ** Dermatology
Service, Veterans Affairs Palo Alto Health Care System, Palo Alto,
California 94304 and Program in Epithelial Biology, Stanford University
School of Medicine, Stanford, California 94305, and
Orthopädische Klinik, Universität Rostock,
Ulmenstrasse 44, D-18057 Rostock, Germany
Received for publication, September 22, 2000, and in revised form, October 20, 2000
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ABSTRACT |
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Collagen XVII is a transmembrane component of
hemidesmosomal cells with important functions in epithelial-basement
membrane interactions. Here we report on properties of the
extracellular ectodomain of collagen XVII, which harbors multiple
collagenous stretches. We have recombinantly produced subdomains of the
collagen XVII ectodomain in a mammalian expression system. rColXVII-A
spans the entire ectodomain from Collagen XVII, also known as the 180-kDa bullous pemphigoid
antigen (BP180), is a transmembrane protein that is widely known as a
structural component of hemidesmosomes, although structures at
cell-tissue interfaces other than hemidesmosomes may also contain collagen XVII (1, 2). Mutations in the collagen XVII gene, COL17A1, lead to junctional epidermolysis bullosa, a
hereditary blistering skin disease with epidermal detachment from
the basement membrane (3).
The cDNA sequence of collagen XVII encodes a type II integral
transmembrane protein of 1497 amino acid residues (4). It consists of
an intracellular domain of 466 residues, a transmembrane domain of 23 residues, and an extracellular collagenous domain of 1008 amino acids
with multiple non-collagenous interruptions. The length of the
individual collagenous regions varies from 14 to 242 amino acid
residues (4). Collagen XVII exists in two molecular forms,
i.e. as a full-length transmembrane homotrimer of three
180-kDa Some information about the molecular shape of collagen XVII under
physiological conditions can be deduced from rotary shadowing electron
microscopy of collagen XVII from bovine cell lines or from recombinant
fragments. These studies revealed asymmetric molecules with an
elongated shape and a globular, ball-like structure at one end (6, 7).
A 90-kDa pepsin/trypsin fragment of collagen XVII in detergent extracts
of keratinocytes was resistant to further trypsin digestion at
physiological temperatures, therefore suggesting that it was of
triple-helical structure (5).
The entire collagen XVII as well as the C15 collagenous domain could be
recombinantly produced in protease-resistant conformation, apparently
not requiring propeptides (7). Triple helix formation of fiber-forming
collagens was defined as a two-step process initiated by annealing and
aggregation of the C-propeptides followed by C- to N-terminal folding
of the three Opposite to this, there is also experimental evidence that
C-propeptides can be substituted for by other peptide sequences or can
be deleted without apparent harm for helix formation (9-11). In
fibril-associated collagens with interrupted triple helices, C-propeptides are completely missing, and even if nonhelical ends at
the C terminus are deleted in recombinantly produced collagenous molecules, as shown for collagen XII, correct triple helix formation takes place (12).
The present study was designed to address the role of subdomains of
collagen XVII ectodomain in triple helix formation and stability, in
particular to delineate information on the directionality of the
assembly process. With a variety of recombinant polypeptides expressed
in a mammalian system, we obtained structural information by circular
dichroism studies, protease sensitivity assays, and electron microscope
studies that strongly suggest triple helix formation from the N to the
C terminus of collagen XVII.
Construction of Expression Plasmids for Subdomains of the
Collagen XVII Ectodomain--
Human cDNA coding for collagen XVII
(4) was used as the template for amplification by the polymerase chain
reaction with appropriate primers. The numbering of nucleotides in
cDNAs are according to Giudice et al. (Ref. 4,
GenBankTM accession number M91669), and the deduced
protein sequence was counted from the methionine start codon (position
36 in M91669). All fragments were digested by appropriate restriction
enzymes and were cloned into an episomal expression vector pCEP-Pu
containing the signal peptide sequence of BM-40 and a puromycin
resistance gene (13). Cloning of inserts into pCEP-Pu via a
NheI restriction site resulted in secreted polypeptides with
four additional N-terminal amino acid residues (APLA) preceding the
expressed sequence (13). Sequences and correct in-frame insertions of
all constructs were verified by DNA sequencing (Medigenomix).
An expression plasmid for rColXVII-A corresponding to the extracellular
domain of collagen XVII (amino acid residues 527-1497) was constructed
by amplification of the cDNA with the sense primer 5'-TTCGCTAGCTATGGCACCCGCGGCGGGAGCAGAC-3' (nucleotides 1684 to 1707) and
the antisense primer 5'-ACGCGTCGACTCACGGCTTGACAGCAATACTTCTTC-3' (nucleotides 4574 to 4596). The NheI-SalI
fragment of the product was subcloned into pCEP-Pu, resulting in
plasmid pCEP-rColXVII-A. This expression plasmid generates a 971-amino
acid residue protein with the N terminus within the NC16a domain and
the C terminus at the cognate end of the ectodomain.
The expression plasmid for rColXVII-B (amino acid residues 527-1482)
was constructed by amplification of the cDNA with sense primer
5'-TTCGCTAGCTATGGCACCCGCGGCGGGAGCAGAC-3' (nucleotides 1684 to 1707) and
antisense primer 5'-ACGCGTCGACTCATTGGTCACCTTTGTCTCCTTTTTCTC-3' (nucleotides 4526 to 4551). The
NheI-SalI-restricted fragment from the
amplification product was ligated into pCEP-Pu, resulting in plasmid
pCEP-rColXVII-B. This plasmid encodes 956 amino acid residues and is
identical to rColXVII-A except for a 15-amino acid deletion (NC1
domain) at the C-terminal end.
To prepare the expression plasmid for rColXVII-C (amino acid residues
527-808), the cDNA was amplified with sense primer
5'-TTCGCTAGCTGAGGAGGTGAGGAAGCTG-3' (nucleotides 1573 to 1590) and the
antisense primer 5'-ACGCGTCGACTCAGATCTTGCCTGGAG-3' (nucleotides 2516 to
2529). The NheI-SalI fragment from this product was ligated into pCEP-Pu, resulting in plasmid pCEP-rColXVII-C. This
plasmid encodes a 282-amino acid residue polypeptide comprising 40 residues of the NC16a domain (
The expression plasmid for rColXVII-D (amino acid residues 1188-1497)
was generated by cDNA amplification using the sense primer
5'-TTCGCTAGCTCCAGGCAATGTGTGGTCCAGCATC-3' (nucleotides 3670 to 3693) and
the antisense primer 5'-ACGCGTCGACTCACGGCTTGACAGCAATACTTCTTC-3' (nucleotides 4574 to 4596). The NheI-SalI
fragment was ligated into pCEP-Pu, resulting in plasmid
pCEP-rColXVII-D. This plasmid encodes a 310-amino acid polypeptide
spanning from the NC1 to the NC6 domain.
Generation of Recombinant Cell Clones and Production of
Conditioned Medium--
Human embryonic kidney 293-EBNA cells
(Invitrogen) were cultivated in Dulbecco's modified Eagle's medium
(Life Technologies) containing 10% fetal calf serum, 0.25 mg/ml G418
(Calbiochem), 0.1 mg/ml penicillin/streptomycin (Biochrom KG), and 2 mM L-glutamine (Biochrom KG). The pCEP-rColXVII
plasmids (25 µg) were separately transfected into 293-EBNA cells (one
million cells/10 cm2 culture dish) using a calcium
phosphate precipitation method (14). After a selection with 0.5 µg/ml
puromycin (Calbiochem), the transfected cells were grown to confluence,
washed twice with phosphate-buffered saline, and switched to serum-free
medium containing 50 µg/ml ascorbic acid (Sigma), freshly prepared.
The media were collected every 24 h, cooled, centrifuged to remove
cellular debris, supplemented with 1 mM protease inhibitor
Pefablock (Roth), and frozen at -80 °C until further use.
Purification of Recombinant Collagen Type XVII
Subdomains--
The serum-free media (2-3 liters) containing
recombinant fragments of rColXVII-A and rColXVII-B were concentrated to
~80 ml by ultrafiltration and dialyzed against 20 mM
Tris-HCl, pH 8.8, at 4 °C. The material was passed over an anion
exchange column (HiTrapQ, 5 ml; Amersham Pharmacia Biotech)
equilibrated in the same buffer and subsequently eluted with a
linear 0-0.4 M NaCl gradient. Fractions containing the
recombinant fragments were concentrated by ultrafiltration to ~0.5 ml
and passed over a Superose 6 column (30 ml; Amersham Pharmacia Biotech)
equilibrated in 50 mM Tris-HCl, pH 8.6, 150 mM
NaCl at a flow rate of 0.3 ml/min. Fractions containing recombinant
fragments were detected by SDS gel electrophoresis and Western blotting.
The serum-free cell culture media (2-3 liters) containing recombinant
fragments of rColXVII-C and rColXVII-D were concentrated to ~70 ml,
dialyzed against 50 mM sodium acetate, pH 4.8, at 4 °C,
passed over a cation exchange column (HiTrapS; 5 ml; Amersham Pharmacia
Biotech) equilibrated in the same buffer, and eluted with a linear
0-0.4 M NaCl gradient. Fractions containing the recombinant fragments were concentrated to ~0.5 ml and passed over a
Superose 12 column (30 ml; Amersham Pharmacia Biotech) equilibrated in
50 mM sodium acetate, pH 4.8, 200 mM NaCl.
Fractions were collected, and recombinant fragments were identified as
described above.
Electrophoresis and Western Blot Analysis--
SDS gel
electrophoresis (15) and Western blot analysis (16) were performed
according to standard procedures. 6% polyacrylamide gels were used for
rColXVII-A and -B, and 10% polyacrylamide gels were used for
rColXVII-C and -D. Polyclonal rabbit antiserum NC16a was used 1:1000
diluted for rColXVII-A, -B, and -C (17), and polyclonal chicken
antiserum Col17ecto-1 (5) was used 1:20-diluted for rColXVII-D. The
antisera were a generous gift of Dr. L. Bruckner-Tuderman. Goat-anti
rabbit (1:1000; Dako) or goat anti-chicken (1:100; Dako) antibodies
conjugated to horseradish peroxidase were used as secondary antibodies.
Circular Dichroism Measurements--
Purified recombinant
rColXVII polypeptides were adjusted to a concentration of 20 µg/ml
and dialyzed against 0.05% acetic acid. Far UV circular dichroism
spectra (190-260 nm) were recorded in a 1-cm quartz cuvette on a Jasco
J-715 A spectropolarimeter equipped with a temperature controller. The
molar ellipticities were calculated on the basis of a mean residue
molar mass of 96 g/mol. Thermal transition curves were recorded at a
fixed wavelength (221 nm) by raising the temperature linearly at a rate
of 30 °C/h using a Gilford temperature programmer. No deviation from
the melting profiles were observed with extended temperature gradients. The degree of triple helicity at various temperatures was calculated by
setting the 221-nm signal at 20 °C to 1 (maximal amount of triple
helicity) and at 45 °C to zero (completely denatured collagen).
Low Angle Rotary Shadowing Electron Microscopy--
rColXVII
polypeptides were dialyzed against a solution of 50% glycerol in
0.05% acetic acid for 16 h at 4 °C. Samples were sprayed onto
freshly cleaved mica using an air brush. The droplets on the mica were
dried at room temperature at 10 Analysis of Hydroxylation and Glycosylation--
For amino acid
analyses, purified recombinant rColXVII polypeptides were hydrolyzed
with 6 N HCl for 24 h at 110 °C. Samples were
analyzed on an amino acid analyzer (Biochrom 20, Amersham Pharmacia
Biotech) using ninhydrin for post-column color development.
For determination of glucosylgalactosylhydroxylysine and
galactosylhydroxylysine samples, up to 1 mg of purified rColXVII-C was
hydrolyzed in 1 ml of 2 N KOH for 24 h at 110 °C.
After hydrolysis, 100 µl of glacial acetic acid and 150 µl of 70%
perchloric acid were added, mixed, and centrifuged for 10 min at 14,000 rpm. The supernatant was decanted and lyophilized. Lyophilized samples were re-dissolved in 1 ml of water and passed over a CF1 (Whatman) column to remove the majority of amino acids. Eluates were lyophilized and analyzed on an amino acid analyzer (Biochrom 20, Amersham Pharmacia Biotech).
N-terminal Sequence Analysis--
Purified native or
pepsin-digested rColXVII-C and -D was subjected to SDS gel
electrophoresis and transferred onto a Mini Pro Blott membrane (Applied
Biosystems). Protein bands were visualized by Coomassie Blue staining
and identified by comparison with immunoblotted material. Relevant
protein bands were excised and loaded directly onto a Procise 494 protein sequencer (Applied Biosystems) for N-terminal sequencing.
Enzyme Digests--
For assessment of the domain structure and
stability of collagen XVII, purified recombinant rColXVII polypeptides
were subjected to treatment by various enzymes. Collagenase
treatment was used to release collagenous peptides, and pepsin
treatment was used to remove protease-sensitive non-collagenous
regions. Trypsin digestion was used to determine the melting
temperature of the rColXVII fragments A and C.
Digestion of purified polypeptides with highly purified bacterial
collagenase (Sigma) was performed with 40 units/ml enzyme in 0.2 M NH4HCO3, freshly prepared, at
37 °C for 2 h (18). For pepsin digestion, 100 µl of rColXVII
polypeptides (~25 µg) were acidified by dialysis against 0.05%
acetic acid and incubated with 1 µg/ml pepsin (Roche Diagnostics) at
4 °C for 24 h (19). After neutralization with saturated Tris
solution, samples were separated by SDS gel electrophoresis followed by
Western blotting using anti-NC16a and anti Col17ecto-1 antisera.
For testing the triple-helical conformation with trypsin (20), the
purified samples (~20 µg/ml) were dialyzed against 100 mM Tris-HCl, pH 7.4, 0.4 M NaCl and preheated
for 5 min at each desired temperature between 20 and 46 °C (2 °C
steps). An aliquot of 10 µl was removed, cooled quickly to 20 °C,
and treated with 10 µl of trypsin (1 mg/ml; Sigma) for 2 min.
Reactions were stopped by adding 10 µl of soybean trypsin inhibitor
(5 mg/ml; Sigma) to each individual sample. The incubated samples were
analyzed by SDS gel electrophoresis followed by Western blotting with
specific antibodies. Intensities of the immunoblotted bands were
quantified with Gel-Pro-Analyzer (Media Cybernetics).
Production and Characterization of Recombinant Col XVII
Subdomains--
To investigate triple-helix formation and stability of
the collagen type XVII ectodomain, we have produced several recombinant subfragments of the ectodomain in mammalian 293 cells. These subdomains included the full-length ectodomain rColXVII-A (positions 527-1497), a
C-terminal truncated form thereof, rColXVII-B (positions 527-1482), the largest collagenous stretch, C15 (positions 527-808), and a short
C-terminal subdomain rColXVII-D (positions 1188-1497) spanning the NC6
to the NC1 domain. These recombinant fragments are schematically
summarized in Fig. 1. Each of the
constructs were designed with a heterologous signal peptide to enable
secretion into the cell culture medium and proper post-translational
modifications. The recombinant cell clones produced each subdomain in
quantities of 1-2 µg of protein/ml medium/day. Ion exchange
chromatography followed by gel filtration chromatography resulted in
highly purified recombinant subdomains with apparent molecular masses
of 101 kDa (rColXVII-A), 96 kDa (rColXVII-B), 30 kDa (rColXVII-C), and
33 kDa (rColXVII-D), which corresponded well with the calculated values
(Fig. 2). Interestingly, the collagenous
domain fragment rColXVII-C migrates as a broad band, similar to what
has been published recently for a corresponding construct (21). No
multimers have been observed after electrophoresis by SDS gel
electrophoresis (Fig. 2). N-terminal sequencing of rColXVII-C, which
N-terminally starts at the same position as rColXVII-A and -B,
demonstrated the expected N-terminal sequence (APLAMA) but also some
minor N-terminal truncations of 2-5 amino acid residues (Table
I). Sequencing of the C-terminal
construct rColXVII-D resulted in a sequence (APGNVWSSIS) that was three
amino acid residues shorter than expected (Table I). These results
indicate that the sequence APLA, which represents an artificial
sequence due to the expression strategy (see "Experimental
Procedures"), is not stable in the recombinant constructs. The
authentic collagen type XVII sequences, however, were stable.
The rColXVII-A and rColXVII-C polypeptide were further analyzed by
amino acid analysis. Within the limits of error, the amino acid
compositions determined correlated well with the expected compositions
calculated from the cDNA (Table II).
For rColXVII-A, about 30% of the total prolyl and 34% of the total
lysyl residues were hydroxylated. For rColXVII-C, about 29% of the
total prolyl and 36% of the total lysyl residues were hydroxylated
(Table II). Some of these residues in rColXVII-C were also further
modified by the attachment of a small but significant number of mono-
and disaccharides (0.01 monosaccharide and 0.17 disaccharide per
molecule; Table II).
Pepsin and Collagenase Degradation of Recombinant Col XVII
Subdomains--
Limited enzymatic digestion with pepsin converted
rColXVII-A into a ~30-kDa fragment via an intermediate protein of the
apparent molecular mass of 58 kDa (Fig.
3, panel A, lane
2), whereas digestion with bacterial collagenase completely
degraded this recombinant ectodomain under the experimental conditions.
Similar data were obtained for the C-terminally truncated rColXVII-B
(Fig. 3, panel B). The 30-kDa pepsin fragments corresponded
to the size and the immunoreactivity of rColXVII-C (collagen XVII
domain C15), indicating that this domain adopts a stable triple-helical
conformation in rColXVII-A and -B and, thus, resists degradation by
pepsin. Recombinant rColXVII-C itself was also resistant to limited
degradation with pepsin, whereby a slightly (~ 3 kDa) faster
electrophoretic mobility of the pepsin-treated material was observed
(Fig. 3, panel C). N-terminal sequencing of the
pepsin-treated rColXVII-C revealed a N-terminally truncation of 32 amino acid residues, corresponding to parts of the non-collagenous
Ultrastructural Analysis of Recombinant rColXVII
Subdomains--
To obtain information of the molecular shape, the
recombinant rColXVII subdomains were analyzed by electron microscopy
after rotary shadowing (Fig. 4). The
full-length ectodomain rColXVII-A displayed a long extended shape with
a globular portion on one end (Fig. 4A). Measurement of the
lengths of individual particles revealed 3 groups of about 60-70 nm,
130-140 nm, and 240-250 nm. A length of 130-140 nm corresponds well
with expected values for the ectodomain (22). A length of 240-250 nm
may represent dimers that occur through lateral alignment, whereas
shorter fragments may derive from proteolytic cleavage of the
polypeptide. The globular domain at one end is about 11-12 nm in
diameter. The widths of the rotary-shadowed molecules (~ 4 nm) are
consistent with the width of triple-helical collagen molecules,
accounting for about 2 nm for the platinum coat. The molecules often
adopt relatively rigid conformations including some wave-like regions
but without the presence of sharp kinks or bends.
Rotary-shadowed molecules of rColXVII-C display an extended thread-like
shape with a uniform length distribution of 63 ± 5 nm (Fig.
4B). The extended shape and the width of the molecules clearly indicate a triple-helical conformation of rColXVII-C. No
globular regions nor obvious kinks were observed. Since this construct
starts at the same amino position as rColXVII-A, we conclude that the
globular domain in rColXVII-A represents the C-terminal end of the molecule.
The C-terminal rColXVII-D always resulted in globular particles of
about 10-12 nm in diameter, with some resemblance to the globular
domain at the C-terminal end of rColXVII-A (Fig. 4C). No
extended regions, suggestive for triple-helical collagenous domains, were observed. Since rColXVII-D contains five collagenous regions (C1-C5), we concluded that this recombinant construct was not
able to adopt triple-helical conformations and instead assembled into a
globular pepsin-sensitive aggregate (see also Fig. 3D,
lane 2).
Conformation and Stability of the Recombinant Collagen XVII
Fragments--
To further investigate structural aspects, the
recombinant subdomains rColXII-A, -B, and -C were analyzed by spectral
and thermal circular dichroism analyses. For rColXVII-C,a spectrum typical for a collagen triple helix with a maximum at 221 nm was observed (Fig. 5A). Purified
molecules that include non-collagenous domains (rColXII-A and -B)
demonstrated shoulders at 221 nm (Fig. 5A). These shoulders
did not reach values of positive ellipticities typical for triple
helices. However, when rColXVII-A (Fig. 5A) or -B (not
shown) were treated with pepsin to remove the non-collagenous regions
before analysis, typical maxima at 221 nm were observed. These data
demonstrate that pepsin-resistant portions of rColXVII-A or -B adopt a
triple-helical conformation. We concluded that in intact rColXVII-A or
-B the ellipticities originating from non-collagenous regions
interfered with those originating from collagenous triple-helical regions to produce shoulders instead of maxima at 221 nm.
Thermal denaturation profiles of the recombinant fragments were
recorded as a decrease of ellipticity at 221 nm (Fig. 5B). Polypeptide rColXVII-C demonstrated a melting temperature of about 35 °C and a melting range from 30 to 40 °C. Full-length
ectodomain rColXVII-A melted at about 41 °C, with a melting range of
35 °C to 46 °C. After treatment of rColXVII-A with pepsin, a
biphasic melting curve was observed. The first phase of this profile
resembles the denaturation profile of rColXVII-C and represents likely
the intact C15 triple-helical domain in the proteolytic degradation mixture. The second phase demonstrated a somewhat higher melting temperature of about 38 °C and probably represents domain C15 plus
additional C-terminal regions corresponding to the 58-kDa fragment
obtained after limited pepsin degradation of rColXVII-A (Fig.
3A, lane 2). The denaturation profile of
rColXVII-B was, as expected, very similar to the results obtained with
rColXVII-A, with a range of melting temperature between 34 and
46 °C. After pepsin degradation of rColXVII-B, a biphasic melting
curve was observed, and the melting temperature decreased from 39 to
34 °C (data not shown).
The results obtained by circular dichroism analysis were further
confirmed by using a temperature-dependent trypsin
digestion assay (20). In this test, denaturation of the triple-helical regions of rColXVII-A and -C was measured at various temperatures by
monitoring the degradation of the protein by trypsin. The results of
this assay are shown in Fig. 6. Melting
temperatures of about 43 °C for rColXVII-A (Fig. 6A) and
of about 33 °C for rColXVII-C (Fig. 6B) are in good
agreement with data obtained by circular dichroism (Fig.
5B). The rColXVII-A is not degraded by trypsin at
temperatures below ~35 °C, although many non-collagenous domains containing arginine and lysine residues are present in this
polypeptide. This result indicates that the non-collagenous domains are
protected against proteolytic attack by trypsin under the conditions
applied.
Collagen XVII is a transmembrane component of hemidesmosomes and
other cell-matrix interfaces. Although the function of the collagen
XVII intracellular region has been the focus of intensive analyses
(23-25), little is known about structure and function of the
ectodomain. Here we report on the structural analysis of recombinantly
expressed fragments of the collagen type XVII ectodomain.
We have recombinantly expressed four constructs composing various
regions of the collagen XVII ectodomain: the full-length ectodomain
(rColXVII-A), the ectodomain lacking the short C-terminal NC1 domain
(rColXVII-B), the longest collagenous region C15 plus 40 N-terminal
amino acid residues from the NC16a domain (rColXVII-C), and a
C-terminal fragment spanning NC1 to NC6 (rColXVII-D). These recombinant
polypeptides were expressed in mammalian 293 cells, a system that
previously has been reported to produce correctly folded recombinant
collagen XVII constructs (21). All recombinant constructs were designed
with a heterologous sequence for a signal peptide to ensure that the
recombinant polypeptides pass through the endoplasmic reticulum for
post-translational modification. The secreted polypeptides were
purified to homogeneity from the cell culture medium by conventional
chromatography procedures.
The structure of the recombinant collagen XVII fragments were
extensively studied. Analysis of circular dichroism spectra and limited
enzymatic digestion with pepsin and trypsin clearly demonstrate that
the large fragments rColXVII-A and -B and the much shorter fragment
rColXVII-C adopt triple-helical conformations. Based on hydrodynamic
properties of the ectodomain, recombinantly expressed in a transient
system with COS cells, the ectodomain of collagen XVII has been
described as elongated, and a trimeric assembly of Rotary-shadowed images of rColXVII-A demonstrated extended molecules
with a globular domain at one end. The molecules demonstrated several
regions of flexibility but no kinks. These results are in agreement
with data from sedimentation studies suggesting more flexibility than a
rigid rod (7). Electron micrographs from rColXVII-C, the long
collagenous stretch, also revealed a long extended shape of 63 nm.
Although the molecules appeared relatively rigid, some curved molecules
were observed. Based on the absence of a globular domain in rColXVII-C,
we can now unambiguously assign the globular domain in rColXVII-A to
the C-terminal end, since both fragments start at the same N-terminal
position. This C-terminal globular domain was either not present or
only occasionally present in other studies (6). In this regard it is
interesting that rColXVII-D, corresponding to the C-terminal end,
ultrastructurally appeared as a globular module of about 10-12 nm in
diameter that resembled the globular domain of rColXVII-A. These
results are suggestive that this globular domain represents the fold of
the five C-terminal collagenous domains C1-C5 interrupted by six
non-collagenous regions. However, no evidence for a triple-helical
conformation of rColXVII-D was obtained by circular dichroism analysis
or by enzyme resistance experiments. These data may indicate that
fragment rColXVII-D is not able to adopt its native conformation and
rather aggregates to a globular structure.
For rColXVII-C, comprising 81 Gly-X-Y (including one Gly-Ser-Gly)
repeats plus 40 additional N-terminal amino acid residues from the
NC16a domain, clear evidence for triple-helical conformation was
obtained by circular dichroism analysis and limited proteolytic degradation. Thermal denaturation profiles demonstrated a melting temperature of 35 °C, which did not change upon incubation with pepsin. Recently, Tasanen and co-workers (21) recombinantly expressed a
similar fragment composing the C15 domain (residues 567-808) in
293-EBNA cells. This fragment lacks the 40 residues originating from
the NC16a domain but is otherwise identical to rColXVII-C. Although
this recombinant fragment contained a higher amount of hydroxylated
proline compared with rColXVII-C (74% of the eligible prolyl residues
versus 53% in rColXVII-C), the transition curve revealed a
significant lower melting temperature (26.5 °C; Ref. 21). This
comparison suggest that the 40 additional residues (positions 527-566)
N-terminal to the C15 domain contribute somehow to the stability of the
C15 domain, even after these residues are removed after pepsin
treatment. We suggest that the three Triple helix formation of fibril-forming collagens starts at the
C-terminal end of the molecule (8, 27). Collagen XVII is a type II
transmembrane protein with an intracellular N and an extracellular C
terminus. It has been suggested that unlike other collagens, the
membrane-bound collagens fold from the N- to the C-terminal end (28).
The data presented here support this hypothesis for several reasons as
follows. (i) The deletion of the C-terminal NC1 domain does not abolish
triple-helix formation of rColXVII-B. (ii) The small C-terminal
fragment rColXVII-D, without the context of more N-terminal sequences,
does not adopt a triple-helical structure despite the presence of
several collagenous stretches. (iii) The N-terminal C15 collagenous
domain can be recombinantly expressed as a triple-helical polypeptide,
although it lacks the entire C-terminal portion of the ectodomain. (iv) The stability of this polypeptide increases when N-terminal amino acid
residues from the NC16a domain are present.
NC16a to NC1, rColXVII-B is similar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains
NC16a to C15, and a small C-terminal polypeptide rColXVII-D comprises domains NC6 to NC1. Amino acid analysis of rColXVII-A and -C demonstrated prolyl and lysyl hydroxylation with
ratios for hydroxyproline/proline of 0.4 and for hydroxylysine/lysine of 0.5. A small proportion of the hydroxylysyl residues in rColXVII-C (~3.3%) was glycosylated. Limited pepsin and trypsin degradation assays and analyses of circular dichroism spectra clearly demonstrated a triple-helical conformation for rColXVII-A, -B, and -C, whereas the
C-terminal rColXVII-D did not adopt a triple-helical fold. These
results were further substantiated by electron microscope analyses,
which revealed extended molecules for rColXVII-A and -C, whereas
rColXVII-D appeared globular. Thermal denaturation experiments revealed
melting temperatures of 41 °C (rColXVII-A), 39 °C (rColXVII-B),
and 35 °C (rColXVII-C). In summary, our data suggest that triple
helix formation in the ectodomain of ColXVII occurs with an N- to
C-terminal directionality.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(XVII) chains and as a 120-kDa soluble form. The latter
corresponds to the extracellular domain and is presumably released from
the cell surface by furin-mediated proteolytic processing (5). In some
instances, an even shorter fragment with ~90-100 kDa has been
observed (6).
-chains (8). A higher content of hydroxyproline
residues in the C-terminal region of the amino acid sequence of
fibrillar collagens has been considered as evidence that the C terminus
is the locus of initiation of triple helix formation (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NC16a) and the C15 domain.
6 mm Hg for
12 h in a vacuum coater (Edwards 306). The dried specimens were
rotary-shadowed with platinum using an electron gun positioned at 6°
to the mica surface and then coated with a film of carbon generated by
an electron gun positioned at 90° to the mica surface. The replica
were floated on distilled water and collected on formvar-coated grids.
The replicas were examined on a Zeiss 109 transmission electron
microscope. Length and diameter of molecules and aggregates were
determined with the Scion Image program (Scion Image Corporation).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of recombinant
subdomain constructs of human collagen XVII (BP 180). The
extracellular domain consists of a series of 15 collagenous domains (C
1 through C 15, solid vertical boxes) and stretches of
non-collagenous domains (NC 1 through NC16a, horizontal
bars). The rColXVII-A construct extends from nucleotide 1683 to
4596 (amino acid positions 527-1497). rColXVII-B construct extends
from nucleotide 1683 to 4551 (amino acid positions 527-1482).
rColXVII-C construct extends from nucleotide 1683 to 2529 (amino acid
positions 527-808). rColXVII-D construct extends from nucleotide 3670 to 4596 (amino acid positions 1188-1497). The total numbers of amino
acid residues for each of the constructs are indicated. GH,
globular head. TM, transmembrane region; Nt,
nucleotide; aa, amino acids.
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Fig. 2.
Purification of recombinant extracellular
subdomains of human collagen type XVII. rColXVII polypeptides were
purified, separated by SDS-gel electrophoresis, and stained with
Coomassie Blue. A and B, rColXVII-A and
rColXVII-B, respectively; control medium from nontransfected 293-EBNA
cells (lane 1), protein pattern after HiTrapQ anion exchange
chromatography (lane 2), and Superose 6 gel filtration
chromatography (lane 3). C and D,
rColXVII-C and rColXVII-D, respectively; control medium from
nontransfected 293-EBNA cells (lane 1), protein pattern
following HiTrapS cation exchange chromatography (lane 2),
and Superose 12 gel filtration chromatography (lane 3). The
relatively broad protein band in panel C (lane 3)
is probably due to heterogeneity in glycosylation or in the amino acid
sequence at the N-terminal end. Positions of globular marker proteins
are indicated in kDa.
N-terminal sequence analysis of rColXVII-C and -D
indicates the cleavage site between the
signal peptide and the mature polypeptide. The amino acid residues APLA
at the N terminus of each construct are a result of the cloning
strategy. The first authentic amino acid residues from the
1(XVII)
polypeptide and their corresponding positions are indicated in bold
face. Gly-X-Y repeats are underlined.
Amino acid and glycosylation analysis of rColXVII-A and -C
NC16a domain (Table I). Accordingly, the Gly-X-Y repeats of
rColXVII-C were not degraded by pepsin. These results suggest a
triple-helical conformation for rColXVII-C and indicate that more
C-terminally located domains are not required for triple helix
formation of domain C15. Treatment of the C-terminal rColXVII-D with
pepsin resulted in a complete degradation, indicating the absence of a
stabilizing triple-helical conformation (Fig. 3, panel D).
Controls with bacterial collagenase demonstrated complete degradation
of all recombinant polypeptides (Fig. 3, lanes 3).
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Fig. 3.
Proteolytic fragmentation of the recombinant
extracellular subdomains of collagen XVII. Purified rColXVII
polypeptides (lane 1) were digested with either 1 µg/ml
pepsin (P; lane 2) at 4 °C for 24 h or
with bacterial collagenase (C; lane 3) at
37 °C for 2 h. Detection of the proteolytic fragments was
performed with specific antibodies after Western blotting (see
"Experimental Procedures"). The panel labels correspond
to the individual recombinant subdomains rColXVII-A to -D. The position
of marker proteins are indicated in kDa.
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Fig. 4.
Shape and length of recombinant
polypeptides. Electron microscope images of rotary shadowed
molecules of rColXVII-A (A), rColXVII-C (B), and
rColXVII-D (C). Histograms of measured lengths (rColXVII-A
and -C) or diameter (rColXVII-D) of the recombinant polypeptides are
shown to the right of each micrograph. Measurements are plotted as
numbers of measurements in 10-nm windows (rColXVII-A) or 2-nm windows
(rColXVII-C and -D). Bars represent 150 nm.
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Fig. 5.
Far UV circular dichroism spectra of
recombinant collagen XVII subdomains. A, circular
dichroism spectra of rColXVII-A (dotted line), rColXVII-A
treated with pepsin (dashed line), rColXVII-C (solid
line), and rColXVII-D (dots and dashes).
Ellipticity is plotted as a function of wavelength. B,
thermal denaturation profiles of rColXVII polypeptides determined at
221 nm as a function of temperature. The signal at 20 °C was set to
1 and represents the maximal amount of triple helicity in the
polypeptides, and the signal at 45 °C was set to 0 and represents
completely denatured triple-helical regions. rColXVII-A (dotted
line), rColXVII-A treated with pepsin (dashed line),
rColXVII-B (dash-dot-dash line), rColXVII-C (solid
line), rColXVII-C treated with pepsin (dash-dot-dot-dash
line).
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Fig. 6.
Temperature-dependent trypsin
digestion assay of rCol XVII-A (A) and rCol XVII-C
(B). Samples were pre-heated to discrete
temperatures in 2 °C steps from 20 to 46 °C and then incubated
with trypsin. Degradation was monitored by Western blotting after gel
electrophoresis (upper panels). Note that rColXII-A and -C
were only degraded at elevated temperatures. The intensity of the bands
were quantified by densitometry. The protein amounts in arbitrary units
are plotted as a function of temperature (lower
panels).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chains was
expected from sedimentation analysis (7). Furthermore, limited trypsin
digestion of authentic collagen XVII suggested a triple-helical
conformation of the ectodomain (5). In our studies, we analyzed for the
first time the recombinant ectodomain of collagen XVII, represented by
rColXVII-A and -B, by circular dichroism. The typical maxima for triple
helices at 221 nm were less prominent as compared with "pure"
collagenous proteins. However, when rColXVII-A and -B were digested
with pepsin, the typical maxima at 221 nm could be observed. These
results indicate that the signals from triple-helical regions and
non-triple-helical regions interfere, providing an explanation for the
reduced maxima at 221 nm. Thermal transition curves recorded at 221 nm
showed a melting temperature of 41 °C for rColXVII-A, demonstrating
a stable triple-helical conformation at physiological temperatures. These data are in good agreement with data obtained by a trypsin protection assay of the collagen XVII ectodomain from ex
vivo preparations (5). Since the C-terminally truncated version rColXVII-B of the recombinant ectodomain displayed very similar circular dichroism spectra and transition profiles, we conclude that
the NC1 domain (position 1483-1497) at the C-terminal end does not
have a major impact on the stability of the ectodomain. Furthermore,
these results indicate that the NC1 domain is apparently not necessary
for the formation of the triple-helical conformation. Comparison of
thermal denaturation profiles of rColXVII-A with -C revealed a higher
(6 °C) melting temperature for rColXVII-A. These results suggest
that at least some of the collagenous domains C1-C14 adopt
triple-helical conformations (see also below). Even the non-collagenous
domains interspersed between C1-C14 may form relatively tight
structures or are stabilized by the contiguous collagenous domains,
since rColXVII-A was completely resistant to trypsin up to 35 °C,
although a fair number of basic residues resides in this region.
-chains in the NC16a domain
interact with each other to allow the formation of a proper stagger
within the C15 domain that otherwise would not form. This would provide
an explanation of why pepsin-trimmed rColXVII-C shows a much higher
melting temperature as compared with the recombinant C15 domain
expressed without the portion of the NC16a domain (21). In this light
it is interesting that a stretch of three heptad repeats preceding the
Gly-X-Y repeats show a probability to form a coiled-coil structure when
analyzed by the COILS program (26). It has previously been hypothesized that 39 amino acid residues (positions 471-509) close to the
transmembrane region can form a three-stranded coiled-coil that may
play a role in assembly (7). Analysis of this region with the COILS
program results in a very high probability to form coiled-coils.
However, in fragment rColXVII-C, this region is not included. It may be possible that in vivo both regions may play a role in
aligning the
-chains in a proper stagger.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. L. Bruckner-Tuderman and Dr. T. Tasanen for the generous gift of antibodies to the NC16a domain of
collagen XVII, Dr. E. Kohfeldt for the pCEP-Pu expression vector, and
Dr. G. J. Giudice for the collagen XVII cDNA. We acknowledge
the excellent technical assistance of G. Baines and Karin Wiemann.
M. P. Marinkovich acknowledges the Office of Research and
Development, Veterans Affairs Palo Alto Health Care System, and the
Dermatology Foundation.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grants DFG-No-147-6 (to H. N.), and Re 1021/4-1 (to D. P. R.), and National Institutes of Health Grant P01-AR 44012 (to M. P. M.).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 scholarship from the Zoological Department, Faculty of Science, University of Mansura, Mansura, Egypt.
¶ Recipient of a Volkswagen Foundation stipend for his stay on sabbatical leave in Stanford.
To whom correspondence should be addressed. Tel.:
49-451-500-4083; Fax: 49-451-500-3637; E-mail:
notbohm@molbio.mu-luebeck.de.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M008709200
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