From the Department of Dermatology, University of
Münster, D-48149 Münster, Germany, the
§ Department of Dermatology, University of Hamburg, D-22587
Hamburg, Germany, and the ¶ Central Laboratory of Electron
Microscopy, University of Zürich,
CH-8091 Zürich, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
COL7A1 gene mutations cause dystrophic epidermolysis bullosa, a skin blistering disorder. The phenotypes result from defects of collagen VII, the major component of the anchoring fibrils at the dermo-epidermal junction; however, the molecular mechanisms underlying the phenotypes remain elusive. We investigated naturally occurring COL7A1 mutations and showed that some, but not all, glycine substitutions in collagen VII interfered with biosynthesis of the protein in a dominant-negative manner. Three point mutations in exon 73 caused glycine substitutions G2006D, G2034R, and G2015E in the triple helical domain of collagen VII and interfered with its folding and secretion. Confocal laser scanning studies and semiquantitative immunoblotting determined that dystrophic epidermolysis bullosa keratinocytes retained up to 2.5-fold more procollagen VII within the rough endoplasmic reticulum than controls. Limited proteolytic digestions of mutant procollagen VII produced aberrant fragments and revealed reduced stability of the triple helix. In contrast, the glycine substitution G1519D in another segment of the triple helix affected neither procollagen VII secretion nor anchoring fibril function and remained phenotypically silent. These data demonstrate that collagen VII presents a remarkable exception among collagens in that not all glycine substitutions within the triple helix exert dominant-negative interference and that the biological consequences of the substitutions probably depend on their position within the triple helix.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anchoring fibrils attach the epidermal basement membrane of the skin to the underlying dermal connective tissue (1, 2). They represent polymers of collagen VII, a large homotrimeric protein with a central triple helix and flanking amino- and carboxyl-terminal globular domains. Epidermal keratinocytes synthesize and secrete collagen VII as a triple helical precursor (procollagen VII) into the extracellular matrix. After secretion, procollagen VII undergoes proteolytic trimming to collagen VII (3) and assembles to polymers (1). This is a multistep process during which collagen VII monomers first form disulfide-bonded antiparallel dimers and then laterally aggregate into anchoring fibrils, which interact with laminin 5 to secure the dermo-epidermal adhesion (1, 4). Further stabilization of anchoring fibrils and presumably of intermolecular aggregates is achieved through cross-linking by transglutaminase-2 (5). Anchoring fibrils are functionally deficient in hereditary dystrophic epidermolysis bullosa (DEB),1 a heterogeneous group of bullous skin disorders (for reviews, see Refs. 6 and 7) with mechanically induced blistering and scarring of the skin. In the most severe forms of the disease, both collagen VII protein and anchoring fibrils are absent from the skin (8), whereas in milder forms, collagen VII is expressed, but the morphology of the anchoring fibrils may be altered (7, 9).
Mutations in COL7A1 encoding collagen VII have been disclosed in both recessive and dominant DEB subtypes (10-17). In recessive subtypes, homozygous or compound heterozygous mutations leading to premature termination codons underlie very severe skin blistering and scarring (18), whereas homozygous or compound heterozygous missense mutations cause milder phenotypic manifestations. In dominant DEB (DDEB), only about a dozen mutations have been identified, most of them causing substitution of a glycine in the triple helical domain of collagen VII (12, 17, 19, 20). However, despite a growing number of known collagen VII mutations, the biological consequences of these mutations and the pathogenic pathways from the gene defect to dermo-epidermal tissue separation in the skin have remained elusive.
Glycine substitution mutations in other collagen genes underlie heritable connective tissue diseases, such as osteogenesis imperfecta, chondrodysplasias, certain subtypes of Ehlers-Danlos syndrome, or Alport's syndrome (for reviews, see Refs. 21-24). These mutations cause pathologic phenotypes through dominant-negative interference. Therefore, the prediction was that glycine substitution mutations in COL7A1 had similar effects. Surprisingly, however, molecular genetic analyses of a number of DEB families disclosed several compound heterozygous glycine substitution mutations in COL7A1 that did not cause a pathologic phenotype in obligate carriers (20).
In an attempt to establish genotype-phenotype relationships and to elucidate the different biological consequences in COL7A1 mutations, we identified glycine substitution mutations in COL7A1 exons 44 and 73 and investigated the biological and clinical phenotypes of the mutations. Whereas a glycine substitution mutation in exon 44 remained biologically and clinically silent, three glycine substitution mutations in exon 73 interfered with the biosynthesis and function of collagen VII in a dominant-negative manner.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Probands-- The diagnosis of DDEB (6, 7) in probands 1-3 was based on 1) the pedigree consistent with dominant inheritance; 2) history of mechanically induced skin blistering and scarring since infancy; 3) clinical observations of skin blisters, scarring, milia, and nail dystrophy at trauma-exposed body sites, such as hands, feet, and knees; 4) electron microscopic findings of dermo-epidermal tissue separation below the lamina densa of the skin basement membrane and/or paucity and altered morphology of the anchoring fibrils; 5) antigen mapping (25) of blistered skin regions that revealed structural antigens of the dermo-epidermal junction at the blister roof; and 6) positive immunofluorescence staining of collagen VII using different domain-specific antibodies. In family 1, the affected individuals included the 48-year-old male propositus (1-1) and his 8-year-old son (1-2). A biochemical and morphological study on collagen VII expression in vitro of proband 1-1 was published earlier (26), at a time when the genetic basis of DDEB was not yet known. In family 2, the proband was a 64-year-old male (2-1). His father and brother were deceased, but were reported to have had similar blistering tendency. No other family member had a skin disease or a genetic disorder. The affected members of family 3 included the 7-year-old proband (3-1), his 5-year-old brother (3-2), the 36-year-old mother (3-3), the 33-year-old aunt (3-4), and the 65-year-old grandfather (3-5). The father and grandfather of 3-5 as well as 13 other relatives in five generations reportedly suffered from similar mechanically induced skin blistering. Proband 4 was the clinically unaffected father of a compound heterozygous epidermolysis bullosa child with two different glycine substitution mutations in COL7A1.2
Mutation Detection-- Genomic DNA was isolated from peripheral blood or cultured cells using the Easy-DNATM kit (Invitrogen, Leek, The Netherlands) according to the manufacturer's instructions. Balanced primer pairs were used for PCR amplification of exons directly from genomic DNA (GenBankTM accession number L23982), and the products were examined for heteroduplex formation by conformation-sensitive gel electrophoresis (27). Heteroduplex bands were detected in the amplimers corresponding to exon 73. Primers used for amplification of exon 73 were as follows: sense primer, 5'-GGGTGTAGCTGTACAGCCAC-3' (nucleotides 23399-23419); and antisense primer, 5'-CCCTCTTCCCTCACTCTCCT-3' (nucleotides 23684-23704) (20). For PCR, 100 ng of genomic DNA were used as template, and amplification conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 45 s, 60 °C for 45 s, and 72 °C for 45 s in a Perkin-Elmer 9600 thermal cycler. The size of the PCR product was 286 base pairs. The PCR products were sequenced by an automated sequencer (Genome Express, Grenoble, France). The detection of the mutation in exon 44 has been described elsewhere.2
Keratinocyte Cultures-- Keratinocytes obtained by trypsinization of skin biopsies were cultured in serum-free, low calcium keratinocyte growth medium (28) supplemented with bovine pituitary extract and epidermal growth factor (keratinocyte growth medium, Life Technologies, Inc.). Prior to analyses, cells at an early passage received 50 µg/ml L-ascorbate for 48 h (28, 29).
Antibodies and Immunofluorescence Staining--
The polyclonal
antibodies to the triple helical and carboxyl-terminal (NC-2) domains
of human collagen VII were produced as described (3, 30). The
monoclonal antibody LH-7.2 to the amino-terminal (NC-1) domain of
collagen VII was a kind gift from Dr. I. Leigh (London Hospital,
London) (31). The monoclonal antibody to protein-disulfide isomerase
and the fluorescein isothiocyanate-labeled anti-rabbit antibodies were
purchased from Dako (Glostrup, Denmark). The Texas Red-labeled
polyclonal anti-mouse antibodies were obtained from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA). For
immunofluorescence (IF) staining, subconfluent cells on coverslips were
permeabilized and fixed with absolute methanol at 20 °C and
incubated at room temperature with the first antibody overnight and the
second antibody for 1 h (26). Preparations were mounted in Mowiol
(Hoechst, Hoechst, Germany) and examined using an inverted confocal
scanning microscope (IRBE, Leitz, or an LSM 410, Carl Zeiss,
Oberkochen, Germany) combined with two HeNe lasers (543/633 nm) and one
argon laser (488 nm) for multicolor fluorescence.
Protein Extraction and Immunoblot Analyses-- The culture medium and the cell layer were extracted separately. The medium was precipitated with ethanol in the presence of proteinase inhibitors (28), and the cell layer was extracted with a neutral buffer containing 0.1 M NaCl, 0.020 M Tris-HCl, pH 7.4, 1% Nonidet P-40, and a mixture of proteinase inhibitors (32). The samples were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting using domain-specific collagen VII antibodies (3). Semiquantitative immunoassays were used to determine relative amounts of collagen VII in the cells and the medium (28).
Proteolytic Digestions--
The stability of procollagen VII
from control and DDEB keratinocytes was analyzed by limited pepsin or
sequential pepsin/trypsin digestions. For a limited pepsin digestion of
procollagen VII, cell extracts were acidified with glacial acetic acid
to a final concentration of 0.1 M, and the samples were
incubated with 10 µg/ml pepsin (Fluka, Deisenhofen, Germany) at
5 °C for 2 h (1, 30). After neutralization with 0.5 M Hepes, pH 8.0, the samples were either directly
precipitated with ethanol at 20 °C overnight or treated with 10 µg/ml trypsin (Sigma, Deisenhofen, Germany) for 2 min at different
temperatures (33). The reaction was stopped by adding soybean trypsin
inhibitor (Sigma) to a final concentration of 10 µg/ml (34). Proteins
were precipitated with ethanol, redissolved in sample buffer, separated
by SDS-polyacrylamide gel electrophoresis, electrotransferred to
nitrocellulose, and analyzed by immunoblotting using domain-specific
collagen VII antibodies (3, 28).
Electron Microscopy-- Electron microscopy of biopsy samples of intact normal appearing skin from probands 1-1 and 3-1 was performed using standard methods as described (7, 17).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of Mutations--
Direct sequencing of the 286-base
pair PCR product of exon 73 disclosed point mutations in all probands
(Fig. 1). The affected members of family
1 were heterozygous for a 6017GA transition (G2006D), family 2 for a
6100G
A transition (G2034R), and family 3 for a 6044G
A transition
(G2015E). The sequence variations did not represent neutral
polymorphisms since they were not found in healthy first-degree
relatives or in 100 normal chromosomes by dideoxynucleotide sequencing.
Mutation G2034R was recently also identified in a Japanese
epidermolysis bullosa patient (35). Proband 4 was heterozygous for a
4556G
A transition in exon 44 (G1519D), as described
elsewhere.2
|
Effect of the Mutations on Procollagen VII Production in Keratinocytes in Vitro-- Immunofluorescence staining with antibodies to collagen VII demonstrated a weak intracellular granular staining in normal control keratinocytes (Fig. 2) that colocalized with protein-disulfide isomerase, a resident protein in the endoplasmic reticulum (data not shown). In contrast, two of the three DDEB keratinocyte strains (1-1 and 2-1) revealed a clearly increased intracellular collagen VII content as evidenced by a strong IF signal (Fig. 2 and Table I). A certain proportion of 1-1 (26) and 2-1 keratinocytes exhibited confluent intracellular granular patches. These were consistent with accumulation of collagen VII in dilated cisternae of the rough endoplasmic reticulum as evidenced by colocalization with protein-disulfide isomerase (data not shown). Use of domain-specific antibodies against the NC-1, NC-2, and triple helical domains demonstrated that the retained material consisted of procollagen VII molecules. These observations were in concert with previous investigations on proband 1-1 keratinocytes, which accumulated collagen VII in the rough endoplasmic reticulum as shown by IF and immunoelectron microscopy (26). In 3-1 cells, only a slight increase was observed.
|
|
|
|
Mutations in Exon 73 Cause Reduced Stability of Mutant Procollagen VII against Proteolytic Digestion-- To assess the stability of the mutant procollagen VII retained in the cells, the protein was extracted from control and DEB keratinocytes and probed by limited pepsin digestion or a sequential pepsin/trypsin treatment (33). Since collagen VII is very sensitive to proteolysis (1), a low enzyme concentration and a reaction temperature were chosen for the pepsin digestion. Under these conditions, the globular NC-1 and NC-2 domains were removed from procollagen VII. The triple helical domain mostly resisted digestion, except for a partial cleavage into the P1 and P2 fragments (Fig. 3B) at the site of the hinge region in the center of the helical domain, as described by Burgeson (1). In the case of mutant 3-1, pepsin treatment led to a fragment pattern similar to that of controls, but mutant 2-1 yielded a double band of the C-terminal P1 fragment, reflecting decreased stability of the triple helix in mutant procollagen VII (Fig. 3B). In addition, the cell extracts were subjected to a combined, limited pepsin/trypsin digestion (Fig. 3C). To define conditions under which the triple helix of normal collagen VII loses its resistance to this combined proteolytic digestion, the trypsin incubations were carried out at different temperatures. In controls, the triple helical fragment lost its stability around 35 °C. Subsequently, the stability of normal and mutant collagen VII was compared at 33 °C for the trypsin digestion. No significant difference between pepsin digestion alone and the pepsin/trypsin combination was observed in controls at 33 °C, i.e. both the triple helical region and the P1 fragment were visualized on immunoblots (Fig. 3C). In contrast, collagen VII from 2-1 cells was less stable. As shown in Fig. 3C (lane 6), the triple helical fragment was completely digested, and the double band of the P1 fragment appeared weaker. These observations demonstrate that the triple helix of the mutant collagen VII molecules from 2-1 cells is less stable against proteolysis, suggesting that the glycine substitution causes abnormal, incomplete folding of the collagen. Cell extracts of probands 1-1 and 1-2 were not available for these analyses.
Abnormalities of Anchoring Fibrils in the Skin-- The ultrastructure of anchoring fibrils in DDEB skin was altered. Electron microscopic examination of the dermo-epidermal basement membrane zone in 1-1 and 3-1 skin (Fig. 5) showed a reduced number of anchoring fibrils as compared with normal control skin (Fig. 5a). In 1-1, patches of some normal appearing and some slender, narrow anchoring fibrils along the dermo-epidermal junction were observed (Fig. 5b), but long stretches of the basement membrane zone were devoid of fibrils. In 3-1, only rudimentary fibril-like structures could be discerned (Fig. 5c).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since early studies on folding of fibrillar collagens with long uninterrupted triple helices, i.e. collagens I-III, had shown that the presence of a glycine in every third amino acid position in the polypeptide is a prerequisite for formation and secretion of a stable triple helix (for review, see Ref. 23), it has been generally assumed that glycine substitutions prevent adequate folding of all collagens by dominant-negative interference. We now demonstrate that collagen VII presents an exception to this rule in that not all glycine substitutions lead to abnormal folding of the molecule. We show that naturally occurring glycine substitutions in the segment encoded by exon 73, but not by exon 44, of COL7A1 interfere with secretion of procollagen VII by keratinocytes in vitro and affect the function of the anchoring fibrils in the skin.
Since all four mutations in this study led to substitution of a Gly
residue by a larger amino acid, the initial prediction was that
procollagen VII molecules containing one, two, or three mutant
pro-1(VII) polypeptide chains undergo slow and/or inadequate triple
helix folding, delayed secretion, intracellular accumulation, and
degradation. This phenomenon, coined "protein suicide" (23), has
been shown to apply to disorders of collagen genes including COL1A1, COL1A2, COL2A1, and
COL3A1 (21-24, 34, 36). In the case of a homotrimeric
collagen, with equal amounts of the normal and mutant chains
synthesized, seven-eighths of the trimeric molecules contain at least
one mutant pro-
-chain, and only one-eighth consists solely of normal
polypeptides. The normal molecules, and presumably some mutant ones,
are secreted into the extracellular matrix. Protein suicide can be
regarded as a protective mechanism since incorporation of abnormally
folded molecules into fibrillar polymers destabilizes the
suprastructures and renders them ultrastructurally abnormal and
functionally inadequate. The present findings with collagen VII mutants
G2006D, G2034R, and G2015E on intracellular accumulation of procollagen
VII and secretion of only a fraction of the molecules are consistent
with the above hypothesis. They also explain the ultrastructural
observations of a reduced number and abnormal appearance of anchoring
fibrils in the probands' skin (Fig. 5). In concurrence, a quantitative
ultrastructural study showed that, in DDEB skin, the number of
anchoring fibrils was reduced to 0-30% of normal (37).
In view of the above considerations, the finding that glycine substitution G1519D did not interfere with folding and secretion of procollagen VII was unexpected. The evaluation of procollagen VII synthesis and secretion in keratinocytes of proband 4 was comparable to that of controls, an observation consistent with the lack of a clinical phenotype in the proband. The mutation obviously causes pathologic consequences only in combination with another gene defect, as exemplified by the daughter of proband 4. The child was compound heterozygous for G1519D and a second maternal glycine substitution and presented with skin blistering at birth.2 Clinical and genetic evidence of "silent" glycine substitution mutations in COL7A1 has been found in some DEB families, but no biochemical studies on these have been reported. Such mutations include G1782R, G2749R, G2569R, G2653R, and G2674R (20); G2575R (15, 38); G1982W, G2025A, and G2049E (38); G1347R (39); and G2671V (40).
The reasons why these glycine substitutions remain clinically silent when combined with a normal allelic product remain unclear at present. One reason may be the location of the substituted glycine within the collagen VII molecule and the degree of abnormal folding it causes. The exon 73 mutations described here cause glycine substitutions in close vicinity of the non-collagenous hinge region in the center of the long triple helix (Fig. 6). The segment encoded by the exon contains amino acid residues 1994-2060 as part of a 35-triplet stretch of -Gly-X-Y- that is flanked by non-collagenous sequences of 39 and 6 amino acid residues in length (Fig. 6). In the middle of the 35 triplets, an imperfection in the form of an additional single leucine residue occurs. The mutated glycine residues within the 35-triplet segment are evolutionarily highly conserved in human, mouse, and hamster (41, 42), suggesting the functional importance of these amino acids. Substitution of such glycine residues in this short, imperfectly collagenous segment may lead to a greater destabilization than glycine substitutions within a long uninterrupted collagenous segment or close to the N or C terminus. Most silent substitutions are located close to either the N- or C-terminal ends of the triple helix or in the middle of long uninterrupted segments of -Gly-X-Y- repeats, like the G1519G substitution investigated here. Gly-1519 is located within a long uninterrupted stretch of the collagen VII triple helix, within the P2 fragment (Fig. 6). These long helix stretches may be more stable than the segment encoded by exon 73 next to the hinge region, and the substitution of one glycine may not abolish the function of the molecule.
|
Among other collagen genes, a silent glycine substitution mutation has
been identified in the COL11A2 gene encoding the
2-chain of collagen XI (43). In recessive
osteochondrodysplasia, a substitution of arginine for glycine in the
2(XI)-chain caused a severe phenotype in homozygous individuals, but
remained silent in heterozygous carriers. Although no protein chemical
or other phenotypic studies were performed, the prediction was that the
position of the glycine substitution within the collagen XI triple
helix was important. The silent glycine substitution was localized
close to the amino terminus of the molecule and may have allowed
incorporation of mutated molecules into collagen fibrils and permitted
some residual function. Like collagen VII, collagen XI is not a
classical fibrillar collagen consisting of uninterrupted
-Gly-X-Y- repeat sequences, but has an
interrupted triple helix and globular domains. Therefore, the rules
found for the effects of glycine substitutions in fibrillar collagens
may not apply without exception to other collagen types, and it is
likely that further examples of mutations causing unanticipated biological and clinical phenotypes will be found when new defects in
other collagen genes are disclosed.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Margit Schubert, Michaela Floeth, and Andrea Wissel for expert technical assistance and Drs. Jouni Uitto and Leena Pulkkinen for valuable advice. We are grateful to Drs. Mathias Tschödrich-Rotter and Reiner Peters for support of the confocal laser scanning studies.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants Br 1475/1-2 and Br 1475/2-2 (to L. B.-T.) and Grants M.Ra 447/3-1 and M.Ra 447/3-2 (to M. R.) from the Deutsche Forschungsgemeinschaft and by European Union Contract BMH-4-97-2559.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.
To whom correspondence should be addressed: Dept. of
Dermatology, University of Münster, Von-Esmarch-Strasse 56, 48149 Münster, FRG. Tel.: 49-251-83-565-35; Fax: 49-251-83-565-34;
E-mail: tuderma{at}uni-muenster.de.
1 The abbreviations used are: DEB, dystrophic epidermolysis bullosa; DDEB, dominant DEB; PCR, polymerase chain reaction; IF, immunofluorescence.
2 N. Hammami-Hauasli, M. Raghunath, W. Küster, and L. Bruckner-Tuderman, submitted for publication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|