Shedding of Collagen XVII/BP180 in Skin Depends on Both ADAM10 and ADAM9

Collagen XVII is a transmembrane collagen and the major autoantigen of the autoimmune skin blistering disease bullous pemphigoid. Collagen XVII is proteolytically released from the membrane, and the pathogenic epitope harbors the cleavage site for its ectodomain shedding, suggesting that proteolysis has an important role in regulating the function of collagen XVII in skin homeostasis. Previous studies identified ADAMs 9, 10, and 17 as candidate collagen XVII sheddases and suggested that ADAM17 is a major sheddase. Here we show that ADAM17 only indirectly affects collagen XVII shedding and that ADAMs 9 and 10 are the most prominent collagen XVII sheddases in primary keratinocytes because (a) collagen XVII shedding was not stimulated by phorbol esters, known activators of ADAM17, (b) constitutive and calcium influx-stimulated shedding was sensitive to the ADAM10-selective inhibitor GI254023X and was strongly reduced in Adam10^−/− cells, (c) there was a 55% decrease in constitutive collagen XVII ectodomain shedding from Adam9^−/− keratinocytes, and (d) H₂O₂ enhanced ADAM9 expression and stimulated collagen XVII shedding in skin and keratinocytes of wild type mice but not of Adam9^−/− mice. We conclude that ADAM9 and ADAM10 can both contribute to collagen XVII shedding in skin with an enhanced relative contribution of ADAM9 in the presence of reactive oxygen species. These results provide critical new insights into the identity and regulation of the major sheddases for collagen XVII in keratinocytes and skin and have implications for the treatment of blistering diseases of the skin.Collagen XVII (also called BP180 or BPAG2) is a hemidesmosomal adhesion component in the skin and mucosa and belongs to the emerging group of collagenous transmembrane proteins (1). This type II oriented transmembrane protein is involved in the molecular pathology of human skin diseases. Mutations in the COL17A1 gene are associated with junctional epidermolysis bullosa, a genetic skin blistering disease (2). Patients with bullous pemphigoid and related autoimmune bullous dermatoses have tissue-bound and circulating autoantibodies targeting collagen XVII (3). Structural and functional changes of collagen XVII play an important role in these diseases, although the molecular pathology is not yet fully understood. The collagen XVII consists of three 180-kDa α1 (XVII) chains, each with an intracellular N-terminal domain, a short transmembrane stretch, and a flexible extracellular C-terminal ectodomain with collagenous (Col)² subdomains that are interrupted by short non-collagenous (NC) sequences. The human and murine collagen XVII molecules differ in size and in the number of the Col and NC domains. Human collagen XVII consists of 1497 amino acid residues with 15 Col and 16 NC domains, whereas the murine form, which is 86% identical (4), consists of 1433 amino acid residues with 13 Col and 14 NC domains. In humans the extracellular linker domain NC16A between the plasma membrane and the Col15 domain is functionally important because it is believed to play a role in both ectodomain shedding and in the proper folding of the triple helical structure of collagen XVII (5–7).Our previous studies revealed two forms of collagen XVII, the 180-kDa membrane-anchored form and the soluble 120-kDa form. The latter represents the extracellular collagenous ectodomain, which is released by cleavage by membrane-anchored metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family (8). The shed ectodomain of collagen XVII is very stable in vivo and in vitro. In wound scratch assays, both addition of the purified soluble ectodomain or overexpression of ADAMs suppressed cell motility (8), indicating that the ectodomain has a role in regulating keratinocyte-matrix interactions. In the context of the known functions of collagen XVII as an adhesion molecule, its shedding could therefore regulate its functions in keratinocyte migration, differentiation, and proliferation.ADAMs are also involved in the release of several other type I or type II transmembrane proteins and are considered to be critical regulators of epidermal growth factor receptor signaling, tumor necrosis factor α release, and Notch signaling to name a few examples (9, 10). Previously ADAM9, ADAM10, and ADAM17 had been identified as potential sheddases for collagen XVII in keratinocytes by overexpression in cell-based assays (8). Moreover Adam17^−/− keratinocytes had 50% diminished collagen XVII shedding, which was interpreted to suggest that ADAM17 represents an important, if not the major, physiological collagen XVII sheddase (8). The major goal of the current study was to further explore the contribution of ADAM17 and other candidate sheddases to the release of collagen XVII from primary keratinocytes and mouse skin. The identification of the major collagen XVII sheddases and their regulation is critical for understanding the role of collagen XVII shedding in the pathogenesis of skin diseases.     

Coiled Coils Ensure the Physiological Ectodomain Shedding of Collagen XVII

α-Helical coiled coils, frequent protein oligomerization motifs, are commonly observed in vital proteins. Here, using collagen XVII as an example, we provide evidence for a novel function of coiled coils in the regulation of ectodomain shedding. Transmembrane collagen XVII, an epithelial cell surface receptor, mediates dermal-epidermal adhesion in the skin, and its dysfunction is linked to human skin blistering diseases. The ectodomain of this collagen is constitutively shed from the cell surface by proteinases of a disintegrin and metalloprotease family; however, the mechanisms regulating shedding remain elusive. Here, we used site-specific mutagenesis to target the coiled-coil heptad repeats within the juxtamembranous, extracellular noncollagenous 16th A (NC16A) domain of collagen XVII. This resulted in a substantial increase of ectodomain shedding, which was not mediated by disintegrin and metalloproteases. Instead, conformational changes induced by the mutation(s) unmasked a furin recognition sequence that was used for cleavage. This study shows that apart from their functions in protein oligomerization, coiled coils can also act as regulators of ectodomain shedding depending on the biological context.     

Collagenous transmembrane proteins: collagen XVII as a prototype.

Collagenous transmembrane proteins are an emerging group of biologically versatile molecules which function as both cell surface receptors and matrix molecules. The seven group members have interesting structural similarities: they are integral membrane proteins in type II orientation and have one or more collagenous domains in the extracellular C-terminus; interspersed by non-collagenous stretches which confer structural flexibility to the ectodomain. A conserved coiled-coil sequence (linker domain) immediately adjacent to the extracellular face of the cell membrane presumably serves as a nucleus for trimerization and triple-helix folding of each collagen. Intriguingly, the ectodomains of at least some of these molecules are proteolytically shed from the cell surface, releasing a shorter form of the collagen into the extracellular matrix. Collagenous transmembrane proteins are expressed in many different tissues and cells, and are involved in a broad spectrum of biological functions, reaching from epithelial and neural cell adhesion, and epithelial-mesenchymal interactions during morphogenesis to host defense against microbial agents. Several group members are involved in the molecular pathology of genetic and acquired human diseases including epidermolysis bullosa, ectodermal dysplasia, bullous pemphigoid or Alzheimer disease. An extensively investigated member is collagen XVII, a keratinocyte surface protein, which attaches the epidermis to the basement membrane in the skin. In this review, the structure and functions of the currently known collagenous transmembrane proteins are summarized and, as a 'prototype' of the group, collagen XVII and its biology and pathophysiology are delineated.     

Transmembrane collagen XVII,an epithelial adhesion protein,is shed from the cell surface by ADAMs

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identified the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP-3, but not by inhibitors of other protease classes or by TIMP-2. This profile implicated MMPs or ADAMs as candidate sheddases. MMP-2, MMP-9 and MT1-MMP were excluded, but TACE, ADAM-10 and ADAM-9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE-deficient cells shedding was significantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.     

A short sequence in the N-terminal region is required for the trimerization of type XIII collagen and is conserved in other collagenous transmembrane proteins

The recombinant transmembrane protein type XIII collagen is shown to reside on the plasma membrane of insect cells in a 'type II' orientation. Expressions of deletion constructs showed that sequences important for the association of three alpha1(XIII) chains reside in their N- rather than C-terminal portion. In particular, a deletion of residues 63-83 immediately adjacent to the transmembrane domain abolished the formation of disulfide-bonded trimers. The results imply that nucleation of the type XIII collagen triple helix occurs at the N-terminal region and that triple helix formation proceeds from the N- to the C-terminus, in opposite orientation to that of the fibrillar collagens. Interestingly, a sequence homologous to the deleted residues was found at the same plasma membrane-adjacent location in other collagenous transmembrane proteins, suggesting that it may be a conserved association domain. The type XIII collagen was secreted into insect cell medium in low amounts, but this secretion was markedly enhanced when the cytosolic portion was lacking. The cleavage occurred in the non-collagenous NC1 domain after four arginines and was inhibited by a furin protease inhibitor.     

Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal disassembly

Bullous pemphigoid antigen 180 (BP180)/type XVII collagen is a transmembrane hemidesmosomal protein. Previously, we demonstrated that the collagenous ectodomain of BP180 can be cleaved within the extracellular non-collagenous (NC) 16A domain adjacent to the cell membrane and released from the cell surface. Here, we report that the BP180 cleavage is mediated by a membrane-associated metalloprotease expressed in epithelial cells. A tissue inhibitor of metalloprotease 1 (TIMP-1), but not TIMP-2, like the synthetic metalloprotease inhibitor KB-R8301, significantly reduced the cleavage. Within epithelial cells cultured for more than 36 h past confluency, antibodies to BP180 showed a reduced hemidesmosomal staining. Observed for the first time, addition of KB-R8301 to the cell culture preserved this staining. To examine the effect of the extracellular cleavage of BP180 on molecular interactions among hemidesmosomal components, we eliminated its collagenous extracellular portion, except for the NC16A domain, by collagenase digestion. Interestingly, this collagenase treatment caused partial disassembly of hemidesmosomal components in cultured human keratinocytes. Moreover, a monoclonal antibody specific for the cleaved extracellular fragment detected a unique tissue distribution of the fragment that might reflect an association of the cleavage process with the mitotic activity of epithelial tissues. Our observations demonstrate that the cleavage of BP180 occurring within the NC16A domain is mediated by a membrane-associated metalloprotease and suggest a possible involvement of the cleavage in hemidesmosomal disassembly.     

The role of disulfide bonds and alpha-helical coiled-coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins

Type XIII collagen is a type II transmembrane protein with three collagenous (COL1-3) and four noncollagenous domains (NC1-4). The human alpha1(XIII) chain contains altogether eight cysteine residues. We introduced point mutations to six of the most N-terminal cysteine residues, and we show here that the two cysteines 117 and 119 at the end of the N-terminal noncollagenous domain (NC1) are responsible for linking the three alpha1(XIII) chains together by means of interchain disulfide bonds. In addition, the intracellular and transmembrane domains have an impact on trimer formation, whereas the cysteines in the transmembrane domain and the COL1, the NC2, and the C-terminal NC4 domains do not affect trimer formation. We also suggest that the first three noncollagenous domains (NC1-3) harbor repeating heptad sequences typical of alpha-helical coiled-coils, whereas the conserved NC4 lacks a coiled-coil probability. Prevention of the coiled-coil conformation in the NC3 domain is shown here to result in labile type XIII collagen molecules. Furthermore, a new subgroup of collagenous transmembrane proteins, the Rattus norvegicus, Drosophila melanogaster, and Caenorhabditis elegans colmedins, is enlarged to contain also Homo sapiens collomin, and Pan troglodytes, Mus musculus, Tetraodon nigroviridis, and Dano rerio proteins. We suggest that there is a structurally varied group of collagenous transmembrane proteins whose biosynthesis is characterized by a coiled-coil motif following the transmembrane domain, and that these trimerization domains appear to be associated with each of the collagenous domains. In the case of type XIII collagen, the trimeric molecule has disulfide bonds at the junction of the NC1 and COL1 domains, and the type XIII collagen-like molecules (collagen types XXIII and XXV) and the colmedins are similar in that they all have a pair of cysteines in the same location. Moreover, furin cleavage at the NC1 domain can be expected in most of the proteins.     

Extracellular phosphorylation of collagen XVII by ecto-casein kinase 2 inhibits ectodomain shedding

Ecto-phosphorylation is emerging as an important mechanism to regulate cellular ligand interactions and signal transduction. Here we show that extracellular phosphorylation of the cell surface receptor collagen XVII regulates shedding of its ectodomain. Collagen XVII, a member of the novel family of collagenous transmembrane proteins and component of the hemidesmosomes, mediates adhesion of the epidermis to the dermis in the skin. The ectodomain is constitutively shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family, mainly by tumor necrosis factor-alpha converting enzyme (TACE). We used biochemical, mutagenesis, and structural modeling approaches to delineate mechanisms controlling ectodomain cleavage. A standard assay for extracellular phosphorylation, incubation of intact keratinocytes with cell-impermeable [gamma-(32)P]ATP, led to collagen XVII labeling. This was significantly diminished by both broad-spectrum extracellular kinase inhibitor K252b and a specific casein kinase 2 (CK2) inhibitor. Collagen XVII peptides containing a putative CK2 recognition site were phosphorylated by CK2 in vitro, disclosing Ser(542) and Ser(544) in the ectodomain as phosphate group acceptors. Phosphorylation of Ser(544) in vivo and in vitro was confirmed by immunoblotting of epidermis and HaCaT keratinocyte extracts with phosphoepitope-specific antibodies. Functionally, inhibition of CK2 kinase activity or mutation of the phosphorylation acceptor Ser(544) to Ala significantly increased ectodomain shedding, whereas overexpression of CK2alpha inhibited cleavage of collagen XVII. Structural modeling suggested that the phosphorylation of serine residues prevents binding of TACE to its substrate. Thus, extracellular phosphorylation of collagen XVII by ecto-CK2 inhibits its shedding by TACE and represents novel mechanism to regulate adhesion and motility of epithelial cells.     

Shedding of collagen XVII ectodomain depends on plasma membrane microenvironment

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-alpha-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MbetaCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.     

Shedding of membrane type matrix metalloproteinase 5 by a furin-type convertase: a potential mechanism for down-regulation

The shedding of membrane-associated proteins has been recognized as a regulatory mechanism to either up-regulate or down-regulate cellular functions by releasing membrane-bound growth factors or removing ectodomains of adhesion molecules and receptors. We have reported previously that the ectoenzyme of membrane type matrix metalloproteinase 5 (MT5-MMP) is shed into extracellular milieu (Pei, D. (1999) J. Biol. Chem. 274, 8925-8932). Here we present evidence that MT5-MMP is shed by a furin-type convertase activity in the trans-Golgi network. Among proteinase inhibitors screened, only decanoyl-Arg-Val-Lys-Arg-chloromethylketone, a known inhibitor for furin-type convertases, blocked the shedding of MT5-MMP in a dose-dependent manner. As expected, decanoyl-Arg-Val-Lys-Arg-chloromethylketone also prevented the activation of MT5-MMP, raising the possibility that the observed shedding could be autolytic. However, an active site mutant devoid of any catalytic activity, is also shed efficiently, thus ruling out the autolytic pathway. The shedding cleavage was subsequently mapped to the stem region immediately upstream of the transmembrane domain, where a cryptic furin recognition site, (545)RRKERR, was recognized. Indeed, MT5-MMP and furin are co-localized in the trans-Golgi network and the shed species could be detected inside the cells. Furthermore, deletion mutations removing this cryptic site prevented MT5-MMP from shedding. The resulting mutants express a gain-of-function phenotype by mediating more robust activation of proMMP-2 than the wild type molecule. Thus, shedding provides a potential mechanism to regulate proteolytic activity of membrane-bound MMPs.     

Properties of the collagen type XVII ectodomain. Evidence for n- to c-terminal triple helix folding

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 deltaNC16a to NC1, rColXVII-B is similar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains deltaNC16a 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 ( approximately 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 degrees C (rColXVII-A), 39 degrees C (rColXVII-B), and 35 degrees 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.     

The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism

Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.     

Shedding of collagen XXIII is mediated by furin and depends on the plasma membrane microenvironment

Collagen XXIII belongs to the class of type II orientated transmembrane collagens. A common feature of these proteins is the presence of two forms of the molecule: a membrane-bound form and a shed form. Here we demonstrate that, in mouse lung, collagen XXIII is found predominantly as the full-length form, whereas in brain, it is present mostly as the shed form, suggesting that shedding is tissue-specific and tissue-regulated. To analyze the shedding process of collagen XXIII, a cell culture model was established. Mutations introduced into two putative proprotein convertase cleavage sites showed that altering the second cleavage site inactivated much of the shedding. This supports the idea that furin, a major physiological protease, is predominantly responsible for shedding. Furthermore, our studies indicate that collagen XXIII is localized in lipid rafts in the plasma membrane and that ectodomain shedding is altered by a cholesterol-dependent mechanism. Moreover, newly synthesized collagen XXIII either is cleaved inside the Golgi/trans-Golgi network or reaches the cell surface, where it becomes protected from processing by being localized in lipid rafts. These mechanisms allow the cell to regulate the amounts of cell surface-bound and secreted collagen XXIII.     

Collagenous sequence governs the trimeric assembly of collagen XII

A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.     

Type XIII collagen and some other transmembrane collagens contain two separate coiled-coil motifs,which may function as independent oligomerization domains

Type XIII collagen is a homotrimeric transmembrane collagen composed of a short intracellular domain, a single membrane-spanning region, and an extracellular ectodomain with three collagenous domains (COL1-3) separated by short non-collagenous domains (NC1-4). Several collagenous transmembrane proteins have been found to harbor a conserved sequence next to their membrane-spanning regions, and in the case of type XIII collagen this sequence has been demonstrated to be important for chain association. We show here that this 21-residue sequence is necessary but not sufficient for NC1 association. Furthermore, the NC1 association region was predicted to form an alpha-helical coiled-coil structure, which may already begin at the membrane-spanning region, as is also predicted for the related collagen types XXIII and XXV. Interestingly, a second coiled-coil structure is predicted to be located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV. It is found experimentally that the absence of the NC1 coiled-coil domain leads to a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1, whereas the COL2 and COL3 domains are correctly folded. We suggest that the NC1 coiled-coil domain is important for association of the N-terminal part of the type XIII collagen alpha chains, whereas the NC3 coiled-coil domain is implicated in the association of the C-terminal part of the molecule. All in all, we propose that two widely separated coiled-coil domains of type XIII and related collagens function as independent oligomerization domains participating in the folding of distinct areas of the molecule.     

Cleavage and oligomerization of gliomedin, a transmembrane collagen required for node of ranvier formation

Gliomedin, which has been implicated as a major player in genesis of the nodes of Ranvier, contains two collagenous domains and an olfactomedin-like domain and belongs to the group of type II transmembrane collagens that includes collagens XIII and XVII and ectodysplasin A. One characteristic of this protein family is that constituent proteins can exist in both transmembrane and soluble forms. Recently, gliomedin expressed at the tips of Schwann cell microvilli was found to bind axonal adhesion molecules neurofascin and NrCAM in interactions essential for Na(+)-channel clustering at the nodes of Ranvier in myelinating peripheral nerves. Interestingly, exogenously added olfactomedin domain was found to have the same effect as intact gliomedin. Here we analyze the tissue form of gliomedin and demonstrate that the molecule not only exists as full-length gliomedin but also as a soluble form shed from the cell surface in a furin-dependent manner. In addition, gliomedin can be further proteolytically processed by bone morphogenetic protein 1/Tolloid-like enzymes, resulting in release of the olfactomedin domain from the collagen domains. Interestingly, the later cleavage induces formation of higher order, insoluble molecular aggregates that may play important roles in Na(+)-channel clustering.     

Assembly of the surfactant protein SP-A. Deletions in the globular domain interfere with the correct folding of the molecule.

The C-terminal non-collagenous domain of the surfactant glycoprotein SP-A was shown to be essential for its correct folding and assembly, as judged by the secretion of various deletion mutants transiently expressed in COS cells. A deletion mutant coding for this domain was successfully secreted while the expression of the collagenous domain only did not lead to any detectable secretion. Deletion mutants lacking small parts of the non-collagenous domain interfered more or less with the correct folding and assembly of the molecule, thus either reducing or inhibiting the secretion. These data suggest that three prefolded non-collagenous domains register and act as a nucleation center for the folding of the collagenous triple helix which proceeds in a zipper-like fashion towards the N-terminus.     

Folding of collagen IV

Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44 degrees C with a midpoint at 37 degrees C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25 degrees C following complete unfolding at 50 degrees C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple-helix folding.     

Cloning of the human type XVII collagen gene (COL17A1), and detection of novel mutations in generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal variant of junctional epidermolysis bullosa (JEB). Previous findings have suggested that type XVII collagen is the candidate gene for mutations in this disease. We now have cloned the entire human type XVII collagen gene (COL17A1) and have elucidated its intron-exon organization. The gene comprises 56 distinct exons, which span approximately 52 kb of the genome, on the long arm of chromosome 10. It encodes a polypeptide, the alpha1(XVII) chain, consisting of an intracellular globular domain, a transmembrane segment, and an extracellular domain that contains 15 separate collagenous subdomains, the largest consisting of 242 amino acids. We also have developed a strategy to identify mutations in COL17A1 by use of PCR amplification of genomic DNA, using primers placed on the flanking introns. The PCR products are scanned for sequence variants by heteroduplex analysis using conformation-sensitive gel electrophoresis and then are subjected to direct automated sequencing. We have identified several intragenic polymorphisms in COL17A1, as well as mutations, in both alleles, in two Finnish families with GABEB. The probands in both families showed negative immunofluorescence staining with an anti-type XVII collagen antibody. In one family, the proband was homozygous for a 5-bp deletion, 2944del5, which resulted in frameshift and a premature termination codon of translation. The proband in the other family was a compound heterozygote, with one allele containing the 2944del5 mutation and the other containing a nonsense mutation, Q1023X. These results expand the mutation database in different variants of JEB, and they attest to the functional importance of type XVII collagen as a transmembrane component of the hemidesmosomes at the dermal/epidermal junction.     

Type XIII collagen forms homotrimers with three triple helical collagenous domains and its association into disulfide-bonded trimers is enhanced by prolyl 4-hydroxylase

Type XIII collagen is a type II transmembrane protein predicted to consist of a short cytosolic domain, a single transmembrane domain, and three collagenous domains flanked by noncollagenous sequences. Previous studies on mRNAs indicate that the structures of the collagenous domain closest to the cell membrane, COL1, the adjacent noncollagenous domain, NC2, and the C-terminal domains COL3 and NC4 are subject to alternative splicing. In order to extend studies of type XIII collagen from cDNAs to the protein level we have produced it in insect cells by means of baculoviruses. Type XIII collagen alpha chains were found to associate into disulfide-bonded trimers, and hydroxylation of proline residues dramatically enhanced this association. This protein contains altogether eight cysteine residues, and interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the two cysteine residues in NC4 are likely to form intrachain bonds. Pepsin and trypsin/chymotrypsin digestions indicated that the type XIII collagen alpha chains form homotrimers whose three collagenous domains are in triple helical conformation. The thermal stabilities (T(m)) of the COL1, COL2, and COL3 domains were 38, 49 and 40 degrees C, respectively. The T(m) of the central collagenous domain is unusually high, which in the light of this domain being invariant in terms of alternative splicing suggests that the central portion of the molecule may have an important role in the stability of the molecule. All in all, most of the type XIII collagen ectodomain appears to be present in triple helical conformation, which is in clear contrast to the short or highly interrupted triple helical domains of the other known collagenous transmembrane proteins.