REVIEW Type VII Collagen, Anchoring Fibrils, and Epidermolysis Bullosa

Robert E. Burgeson Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, U.S.A.

The anchoring fibrils at the dermal-epidermal junction have ally into unstaggered arrays that are the anchoring fibrils . been well characterized as ultrastructural entities. From their This arrangement allows for the protrusion of large globular appearance, it was proposed that they fortified the attach­ domains (NC-1) from both ends of the fibrils. The aggre­ ment of the epidermis to the dermis. This hypothesized gated triple-helical domains extend into the papillary dermis function was strengthened by observations indicating that and entrap fibrous dermal components. The NC-l domains the anchoring fibrils were abnormal, diminished, or absent are believed to interact with components of the basement from individuals with dystrophic epidermolysis bullosa. membrane and thus to mediate the attachment of the base­ Therefore, characterization of the molecular constituents of ment membrane to the dermis. This model predicts that mu­ the anchoring fibrils and their interactions with other base­ tations in the type VII collagen gene that prevent the secre­ ment membrane and dermal components might lead to iden­ tion of the molecule will be the most devastating, whereas tification of the gene defects underlying at least some forms mutations in the regions encoding the globular domains may of epidermolysis bullosa. Type VII collagen was identified as show more variable phenotype. Ultimately, understanding the protein component of anchoring fibrils in 1986. Since the function of type VII collagen at the molecular level will then, the major characteristics of the molecule have been be the key to devising strategies to moderate the pathophysi­ described. These are consistent with a model wherein se­ ology of dystrophic epidermolysis bullosa. ] Invest Dermatol creted type VII collagen molecules form disulfide-bond sta­ 101:252-255,1993 bilized antiparallel dimers. The dimers then condense later-

he purpose of this brief review is to summarize the polypeptides of about 150 kDa that could not be separated by any current status of studies of type VII collagen and its applied technology, suggesting that all three subunits were derived function as the anchoring fibril protein. I will try to from the same gene product. This estim.ate compares well with the indicate those aspects of past studies that have with­ predicted amino acid sequence of the a1(VII) chain, which indicates stood the test of time during the ten years since type that the domain is a composite of a cartilage matrix protein (CMP) TVII was first reported, and try to make corrections where more homology, followed by nine fibronectin type III (FNIII) repeats and recent studies contradict past findings. I will also try to point out a von Willebrand factor (vWF) repeat [5-8] (Fig 2). The sequence certain aspects of type VII collagen function that are based upon of the complete amino terminus is not yet published. The data correlations or modeling, which mayor may not be correct in detail, suggest that the C-terminallarge globule of NC-1 joining the indi­ as well as those that are based upon solid structural information. It vidual arms is contributed by the second vWF repeat, while the will be very interesting to observe the modifications of these con­ extension of the arms results from the series of FNIII repeats. The cepts that will inevitably occur as characterization of the molecular CMP repeat may coincide with the N-terminal globule sometimes defects underlying dystrophic epidermolysis bullosa (DEB) become observed by rotary shadowing. increasingly abundant. We originally assigned the NC-1 domain to the C-terminus of At this point, as a result of the initial biochemical analyses and the a1(VII) chain [3]. This was based upon a comparison of the subsequent primary structure prediction, the structure of the type amino acid sequence determined for the intact triple-helical domain VII molecule is quite well understood. The amino terminus con­ with the sequence of the P1 pepsin fragment, which was identified tains the large NC-1 domain [1- 6]. Rotary shadowed images (Fig by electron microscopy to be involved in dimer formation. These 1) of this domain examined by transmission electron microscopy peptides both produced the same sequence, indicating that the P1 appear as three apparently identical extended structures joined by fragment was at the amino terminus of the triple-helical domain and disulfide bonds at their C-terminus within a large globule. In some therefore P2, which was adjacent to the NC-1 domain, must be at micrographs, the N-tennini appear to end in smaller globules, but the C-terrninus. Predictions of amino acid sequence from analysis of most often the individual arms appear as elongated structures in­ the cDNA of a1 (VII) [7] has proved this to be incorrect. Presum­ creasing in thickness from the C- to the N-terminus [2]. Biochemi­ ably, the sequence ofP1 we reported actually derived from contam­ cal analysis of the NC-1 domain subunit chains described three inating P2 pep tides that produced the dominant sequence signal because the N-terminus ofp1 was probably blocked by spontaneous Reprint requests to: Dr. Robert E. Burgeson, Cutaneous Biology Research cyclization. The occurrence of NC-1 at the amino terminus is far Center, Massachusetts General Hospital, Harvard Medical School, Building more consistent with the structures of other collagen types. The 149, 13th Street, Charlestown, MA 02129. large non-triple-helical domains of types IV, XII, and XIV colla­ Abbreviations: CMP, cartilage matrix protein; vWF, von Willebrand gens occur at the amino terminus. It is also consistent with the factor. generally accepted concept that the triple-helical domain of colla-

0022-202X/93/S06.00 Copyright © 1993 by The Society for Investigative Dermatology, Inc.

252 VOL. 101, NO.3 SEPTEMBER 1993 COLLAGEN. ANCHORlNG HBRlLS. EPIDERMOLYSIS BULLOSA 253

• I ! • I • ~ I • I .' \. • ~ 2 X . I • I I • Figure 1. Rotary shadowed image of the type VII molecule synthesized and secreted from human keratinocytes in culture. The image shows the 424- Anchting run -long triple helical domain and the tow terminal globular domains. The Fibrils amino-terminal NC-l domain is particularly large with eac h subunit a chain contributing the structure of an extended arm. The NC-2 domain is consid­ Figure 3_ Hypothetical model for th e assembly of anchoring fibrils from erabl y smaller and appears as a single globule. Micrograph courtesy of type VII collagen. Type VII collagen is secreted as a monomer. Monomers Douglas Keene, Shriners Hospital for Crippled Children, Portland, Oregon. then dimerizc by a 60-nm overlap of the C-terminal ends. The stagger of the ovedap is believed to be specified by an interaction of NC-2 with a specific re~lOn of the tnple heltx. The .overlap allows alignment of opposing cysteine reSidues, whIch then form disulfide bonds that stabilize the dimer. After gens fold intracellularly from .the C-terminus. It has always bee.n disulfide bond formation. the NC-2 domains are proteolytically removed. difficult to reconCIle the foldtng of type VII collagen WIth tillS The dllners then condense 111 an unstaggered lateral array as anchoring mechanism when it was believed that the independently folded fibrils. arms of the NC-1 domain somehow nucleated triple-helix assem­ bly. single gene product, al(VII). This prediction derives from the fail­ The triple-helical domain of a1(VII) is unusually long (424 nm, ure of biochemical methods to fractionate the whole type VII chains 170 kDa) and flexible [1 ,9]. The flexibility of the domain is due to or the P1 and P2 peptides into more than one chromatograrhic or the presence of a large discontinuity in the triple-helical structure at e1ectrophore.tic fraction. This is supported by the finding 0 single the junction of the major pepsin fragments P1 and P2. Shorter mRNA species cncodlllg type VII -like sequences [7]- Further sup­ disruption of the Gly-X-Y motif specifying helical structure occur port derives from the mapping of all DEB mutations to the same throughout the length of the domain. genomic region [11 - 15]. However, there are several observations The C-terrninal NC-2 domain forms a small globule visualized that urge caution in fully accepting this conclusion. Attempts to by rotary shadowing [10] (Fig 1). Biochemical estimates of the characterize the NC-2 domain always showed two species: one with molecular mass of this domain predicted masses of 32 and 34 kDa. an electrophoretic migration consistent with a mass of 32 kDa, This is in considerable excess of the cDNA prediction. The reason another with a predicted mass of 34 kDa. This heterogeneity has for this discrepancy may eventually be explained by post-transla­ never been understood. Secondly, protein sequencing of CNBr tional glycosylation (which has yet to be characterized), or by sec­ fragments of the triple-helical domain by Dr. Ivan Rocos, and of ondary structures that are resistant to sodium dodecylsulfate dena­ NC-1 by Dr. Louise Rosenbaum when the work was done in Port­ turation and hold the polypeptide in an extended conformation, land, identified a number of peptide sequences that have not been slowing its migration through electrophoretic gels. found in the reported cDNA predictions. The percentage of uni­ It is ass umed that the type VII collagen molecule is composed of a dentified sequences t~ sequences'p'l~ced within al(VII) is signifi­ cant, and consistent With the pOSSlblltty that another type VII chain may exist. The DEB linkage studies do not rule out this possibility if a second type VII gene is closely linked to COL7A1. Alternatively, NC-1 Trlple-Hollcal Domain NC-2 these observations could have trivial explanations. In any case, those directly working with type VII collagen might bear this possibility II Illor~nus in mind. ccc 6c 39 amino acid a 1 (VII) is synthesized by keratinocytes [2,4,16- 22] and to a lesser "hlng e" roglon Kunltz extent by dermal mesenchymal cells [4,20].171 vitro, the mesenchy­ modulo cartilage nino von mal contribution is incorporated into the basement membrane made matri x consecullve Wiliebrand by dermal equivalents (Marinkovich, Nishiyama, and Burgeson, protein flbroneclln faclor A (CMP) III domains domain unpublished observations). After secretion, the type VII molecules dimerize [3] (Fig 3). Subsequently, the dimers become disulfide Figure 2_ The domain structure of type VII collagen predicted from cDNA bonded, but this crosslink is not made in cell culture under standard sequencing. The diagram indicates the relative positions of the CMP, type conditions, even though non-covalently stabilized dimers are ob­ III fibronectin , and vWF homologies within NC-1. As shown, nearly one served i71 vitro. We have assumed that dimerization is catalyzed by half of the transcript encodes non-triple-helical structure. The positions of cysteinyl residues are indicated (c) in clusters towards the C-terrrunus where binding of the NC-2 domain to a specific region of the triple-helix. the are likely to stabilize the type VII dimers. Additional cysteine clusters This assumption is based upon the observation that the dimers occur at the NC-l - triple helical junction. These stabilize the structure of formed i71 vitro retain the NC-2 domains on the C-terminii of the the central globule of NC-1. The illustration is courtesy of Drs. Angela molecules; therefore it is not necessary that NC-2 be removed to Christiano and Jouni Vitto, Jefferson Medical School, Philadelphia, Penn­ obtain dimer formation. Also, we have never been able to form sylvania. dimers i71 vitro from isolated triple-helical domains or from P1 254 BURGESON THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Epithelial cell degradation. However, mutations that interrupt the binding capaci­ \ I / ties of the NC-l subdomains are also likely, and these might result in some of the more mild forms of the disease. Hybrid mutant/ type IV wild-type molecules could exist, exhibiting partial ligand binding lamina densa",.jI!8I11~ __il •• due to the predicted trivalency of N C-1. The predicted relationship of type VII collagen to DEB is now well documented. The absence of anchoring fibrils from the base­ ment membrane zone of patients with severe generalized recessive epidermolysis bullosa has been a characteristic of the disease. Lack of type VII collagen correlates with the same condition [17,21,28_ 34]. Type VII has also been identified intracellularly within kerati­ nocytes in some patients [16,21], suggesting that a mutation may prevent secretion. COL7Al has now been localized to the human gene locus 3p21.3 [7,35,36]. Use of observed polymorphisms [35] has allowed the demonstration oflinkage of dominant and recessive epidermolys~s bullosa t~ the tyfe VII gene [11-15]. The consist­ ency of the hnkage studies of al forms of DEB to type COL7 Al is surprising. It has been generally predicted that some of the defects Figure 4. Drawing of the proposed anchoring fibril network. As shown, resulting in anchoring fibril defects would be secondary to other anchoring fibrils originate within the lamina densa and extend perpendicu­ gene defects. The possibility that anchoring fibrils were degraded larly into the upper regions of the papillary dermis where they condense with the ends of other anchoring fibrils and with ubiquitous basement due to aberrant synthesis or activation of collagenase or another membrane components to form anchoring plaques. Additional anchoring protease is a particularly attractive possibility [37]. The type VII fibrils bridges these plaques with plaques deeper in the papillary dermis. The molecule has been shown to be a potential substrate for a variety of extended network thus formed is capable of entrapping large numbers of degradative enzymes [38]. The linkage studies also suggest that type dermal fibrous elements, thereby securing the lamina densa to the dermis. VII is the only component of the anchoring fibril. This is very unexpected. Although the banding pattern of the segment long spacing fibers made from type VII collagen triple-helical domain do fragments. Based upon this assumption it is logical to predict that account for most, if not all, of the centrosymmetric banding seen in mutations in NC-2 or in the region of triple-helix about 60 nm anchoring fibrils, and type VII is the major structural component of N-terminal to NC-2 could prevent dimer formation, resulting in a the fibrils, there are indications that other molecules may be present non-functional anchoring fibril network. It will be very interesting in the structures. The antigens recognized by the monoclonal anti­ to see if this prediction holds. bodies AF-l and AF-2 [39] are prime candidates. The epitopes rec­ The anchoring fibril network (Fig 4) has been described as pri­ ognized by these antibodies have been localized to the anchoring mary fibrils originating in the lamiria densa of the basement mem­ fibrils, but have not been found within the type VII molecule. This brane..'looping into the, upper' regions of the papillary dermis and discrepancy remains a mystery, and the AF-l and AF-2 antigens reinserting into the lamina densa. The loops surround and entrap have not been further characterized. However, the possibility that the fibrous dermal elements, securing the basement membrane to additional components contribute to anchoring fibril structure and the dermis. We described a secondary network [23,24] in which function remains. anchoring fibrils originating in the lamina densa extend perpendic­ The linkage studies firmly tie type VII collagen defects to the ularly into the dermis and insert into amorphous elements contain­ dystrophic phenotype. Further support for this close association ing type IV collagen and probably laminin, and perhaps other ubiq­ derives for studies of the acquired form of DEB. The autoantibodies uitous basement membrane components. We called these structures characteristic of this condition specifically recognize the NC-l do­ anchoring plaques. The acceptance of these structures has been con­ main of the type VII molecule [40-44]. The autoimmunity appears troversial; however, none of our subsequent experience with ultra­ to be associated with the class II HLA allele [45]. structural visualization of the upper papillary de'rmis contradicts this Very recently, the first mutation in CO L7 A 1 has been identified original conclusion. A ~imilar arrangement of these molecules has as the cause of recessive DEB within one kindred [46]. The single been reported in corncil [25]' , base transmutation encodes a methionine to lysine substitution, near The formation of the anchoring fibril network requires the NC-l the NC-2-triple-helixjunction. The mutation lies within a highly domain to interact with constituents of the lamina densa and the conserved region containing cysteine residues that are likely to be anchoring plaques. Solid-phase binding studies indicated that iso­ involved in the process of type VII dimerization. lated NC-l could bind type IV collagen and laminin [26] (and kalinin/K-Iaminin; Burgeson, unpublished), suggesting that the as­ sembly and stability of the anchoring fibril network is dependent Preparatioll oJthis review was sigl'l!Jicalltly aided by Ms, Cilldy Clatterbllck and Mr. upon the interactions of basement membrane components with Martill McDo,lOugh, I gratefully ackrlOwledge tile cOlltributions oj Drs, Allgela N C-l . The molecular mechanisms of these potential interactions Cllristiallo and JOllni Uitto wllo provided Fig 2 and allowed the citation oJliteratu Te have not been further characterized. Potential interactions of the ill press, a"d tile contribution oj the micrographs oj Fig 1 by Mr. DOllglas KeerJe. NC-l domains with keratinocyte basal surface transmembrane pro­ REFERENCES teins has never been documented. 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