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

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 ␣ 1 I, ␣ 2 I, and ␣ 10 I domains and cell spreading assays with ␣ 1 ␤ 1 -and ␣ 2 ␤ 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 ␣ 5 ␤ 1 and ␣ V ␤ 1 integrins, and it could be blocked by RGD peptides. The collagen ␣-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three ␣-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.The collagens are a family of extracellular matrix proteins (1). Among the 19 different collagen subtypes that have been identified, types XIII and XVII are the only transmembrane proteins (2, 3). Type XVII collagen (BP180) is hemidesmosomal transmembrane protein that is mutated in junctional epidermolysis bullosa and is targeted by autoantibodies in blistering skin diseases (4 -6). It occurs in two forms: as a full-length transmembrane protein and as a distinct, soluble ectodomain that is shed from the cell surface by a furin-mediated, proteolytic process (7,8). This collagen possesses intracellular and transmembrane domains that are of 560 and 23 amino acids in length, respectively. In addition, it has an extracellular domain of 914 amino acids that contains multiple noncollagenous interruptions, dividing it into 15 collagenous subdomains (3). The longest of these subdomains, COL15, consists of 242 amino acids (residues 567-808 of collagen XVII). Thus, it is much larger than any of the other collagenous domains, which vary from 14 to 45 residues in length (3).Most collagen subtypes can be recognized by a group of integrin-type cell adhesion receptors. Although all collagenreceptor integrins share the common ␤ 1 in...

Section: Discussionmentioning

confidence: 99%

The Cell Adhesion Domain of Type XVII Collagen Promotes Integrin-mediated Cell Spreading by a Novel Mechanism

Nykvist¹,

Tasanen²,

Viitasalo³

et al. 2001

“…Generally, fibrillar collagens and type IV collagen require the presence of globular sequences C-terminal to the triple-helical domain to initiate chain registration (3-5, 32). However, trimerization of type XII collagen is dependent on specific post-translational modifications of the collagen domain (33), whereas chain association of the membraneassociated collagen, type XIII, occurs in the N-terminal region (34). Refolding experiments on collagen type III indicated that interchain disulfide bridges at the C terminus of the triple helix were sufficient to function as a nucleus for the refolding of the triple helix (6).…”

Section: Cooperativity Exists Between the Iia Nh 2 -Propeptide Collagmentioning

confidence: 99%

Trimerization of the Amino Propeptide of Type IIA Procollagen Using a 14-Amino Acid Sequence Derived from the Coiled-Coil Neck Domain of Surfactant Protein D

McAlinden¹,

Crouch²,

Bann

et al. 2002

The folding of a collagen triple helix usually requires the presence of additional sequences that contribute to the association and correct alignment of the collagen chains. We recently reported that the C-terminal neck and lectin domains of a collagenous C-type lectin, rat pulmonary surfactant protein D (SP-D), are sufficient to drive the trimerization of a heterologous type IIA procollagen amino propeptide sequence. However, the conformation of the resulting trimeric IIA propeptide and the specific contributions of the SP-D sequence to trimerization were not elucidated. In the present study, we show that trimerization of the fusion protein is associated with correct folding of the collagen helix within the IIA propeptide domain (as assessed by circular dichroism) and that the constituent chains are hydroxylated. Chemical cross-linking and analytical ultracentrifugation showed that the IIA amino-propeptide retains its trimeric configuration even after proteolytic removal of the SP-D domains. By contrast, IIA amino-propeptides synthesized without fusion to the neck or lectin domains are assembled exclusively as monomers. To localize the trimerization sequence, mutant chimeric cDNA constructs were designed containing premature termination codons within the coiled-coil neck domain. A short, 14-amino acid sequence corresponding to the first two heptad repeats of the neck domain was sufficient to drive the trimeric association of the IIA amino-propeptide ␣-chains. However, deletion of the collagen domain resulted in the secretion of monomers. These studies demonstrate that two heptad repeats are sufficient for trimeric association of the propeptide but indicate that cooperative interactions between the coiled-coil and collagen domains are required for the formation of a stable helix.The type IIA NH 2 -propeptide is encoded by eight exons; the translated protein consists of a short globular domain, a 69-amino acid von Willebrand factor type C cysteine-rich domain, a minor collagen triple-helical domain containing 26 GXY repeats, and a short telopeptide domain, which links the minor collagen domain to the major collagen triple helix (1, 2). Trimerization of most fibrillar collagens is dependent on the globular COOH-propeptide for the recognition and association of the three polypeptide chains resulting in registered nucleation of triple helix formation in a zipper-like fashion from the C to N terminus (3-7). Functions proposed for procollagen NH 2 -propeptides include the regulation of collagen fibrillogenesis (8) and a feedback control of net collagen biosynthesis (9 -11). Recently, it was proposed by our laboratory (12) and by others (13) that the NH 2 -propeptide of type IIA procollagen regulates growth factor activity in the extracellular matrix. Trimeric assembly of fibrillar NH 2 -propeptides affects protein valency and stability, which appear important for function in vivo. This emphasizes the importance of a procollagen COOH-propeptide, or indeed other protein domains with similar function, to drive this trime...

“…In accordance with a previous report (18), CLAC formed dimers and trimers even though the splice variant expressed in this study lacks parts of the sequences of the COL3 and NC4 domains (residues 589 -640, according to the numbering of GenBank TM /EBI accession number AF293341). Multimer formation has also been observed with a COL3 and NC4 domain-truncated collagen XIII protein, indicating that the C-terminal domains of these collagens are not critical for chain association (40). The majority of CLAC isolated from human AD brain is in a monomeric or dimeric form; a trimeric form is rarely observed.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Alzheimer's Disease-associated CLAC Protein and Identification of an Amyloid β-Peptide-binding Site

Söderberg

Kakuyama

Möller

et al. 2005

Amyloid ␤-peptide (A␤) deposition into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Other proteins co-deposit with A␤ in plaques, and one recently identified amyloidassociated protein is the collagen-like Alzheimer amyloid plaque component CLAC. It is not known how CLAC deposition affects A␤ plaque genesis and the progress of the disease. Here, we studied the in vitro properties of CLAC purified from a mammalian expression system. CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin, and is glycosylated. Purified CLAC was also used to investigate the interaction between CLAC and A␤. Using a solid-phase binding assay, we show that CLAC bound with a similar affinity to aggregates formed by A␤-(1-40) and A␤-(1-42) and that the interaction was impaired by increasing salt concentrations. An 8-residue-long sequence located in non-collagenous domain 2 of CLAC was found to be crucial for the interaction with A␤. These findings may be useful for future therapeutic interventions aimed at finding compounds that modulate the binding of CLAC to A␤ deposits.Alzheimer's disease (AD) 1 is a progressive neurodegenerative disorder characterized by selective neuronal loss associated with intracellular neurofibrillary tangle formation and extracellular amyloid plaques (1). The major constituents of the amyloid plaques are fibrils formed from the 40 -42-residue amyloid ␤-peptide (A␤) (2). A␤ is generated from the type I transmembrane Alzheimer amyloid precursor protein by two consecutive proteolytic cleavages. The aspartyl protease ␤-secretase BACE generates the N terminus of A␤, whereas the C terminus results from the proteolytic action of the ␥-secretase complex (3). Data from genetic and biochemical studies suggest that accumulation of the longer and more amyloidogenic species of A␤, A␤42, is a primary event in the development of AD (4). For instance, autosomal dominant forms of early-onset familial AD appear to result from an increased production of A␤42 (5), and total levels of brain A␤42 correlate with cognitive decline in AD (6).Several proteins other than A␤ have been shown by immunohistochemical methods to be associated with AD amyloid plaques (7). Some of these plaque-associated proteins, e.g. apolipoprotein J (8, 9), apolipoprotein E (10), and ␣ 1 -antichymotrypsin (11), modulate peptide aggregation. In addition, constituents of the extracellular brain matrix, e.g. the heparan sulfate proteoglycans (12-14), laminin (15, 16), and collagen type IV (17), accumulate in amyloid plaques and bind to A␤. The appearance of some of these plaque-associated proteins may be indicative of a local inflammatory response to the amyloid, and others may promote A␤ aggregation or stabilize the amyloid plaques. Recently, a novel plaque-associated protein, CLAC (collagen-like Alzheimer amyloid plaque component), was identified using antibodies raised against insoluble amyloid fr...