Jyrki Heino

Selective Binding of Collagen Subtypes by Integrin α1I, α2I, and α10I Domains

Four integrins, namely α1β1, α2β1, α10β1, and α11β1, form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their α subunit. We have produced the human integrin α10I domain as a recombinant protein to reveal its ligand binding specificity. In general, α10I did recognize collagen types I–VI and laminin-1 in a Mg2+-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When α10I was tested together with the α1I and α2I domains, all three I domains seemed to have their own collagen binding preferences. The integrin α2I domain bound much better to fibrillar collagens (I–III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin α1I had the opposite binding pattern. The integrin α10I domain was similar to the α1I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the α1I and α2I domains, we modeled the structure of the α10I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the αI domains, and their binding to types I, IV, and VI collagen was tested. In the α2I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the α1I and α10I domains is oppositely charged (Arg-218). The mutation D219R in the α2I domain changed the ligand binding pattern to resemble that of the α1I and α10I domains and, vice versa, the R218D mutation in the α1I and α10I domains created an α2I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.

A Peptide Inhibiting the Collagen Binding Function of Integrin α2I Domain

Integrin α2 subunit forms in the complex with the β1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special “inserted” domain (I domain) in the integrin α2 subunit. Venom from a pit viper,Bothrops jararaca, has been shown to inhibit the interaction of platelet α2β1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant rα2I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrin-like domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit rα2I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant α2I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids, arginine-lysine-lysine (RKK), is essential for rα2I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to α2I domain in the absence of Mg2+, suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed α2β1integrin, and the attachment was inhibited by anti-integrin antibodies.

Increased Expression of β6-Integrin in Skin Leads to Spontaneous Development of Chronic Wounds

Integrin alphavbeta6 is an epithelial cell-specific receptor that is not normally expressed by resting epithelium but its expression is induced during wound healing. The function of alphavbeta6-integrin in wound repair is not clear. In the present study, we showed that beta6-integrin expression was strongly up-regulated in the epidermis in human chronic wounds but not in different forms of skin fibrosis. To test whether increased beta6-integrin expression plays a role in abnormal wound healing we developed four homozygous transgenic mouse lines that constitutively expressed human beta6-integrin in the epithelium. The mice developed normally and did not show any histological abnormalities in the skin. The rate of experimental skin wound closure was unaltered and the wounds healed without significant scar formation. However, during breeding program 16.1 to 27.0% of transgenic mice developed spontaneous, progressing fibrotic chronic ulcers. None of the wild-type animals developed these lesions. The chronic lesions had areas with severe fibrosis and numerous activated macrophages and fibroblasts expressing transforming growth factor (TGF)-beta. The level of TGF-beta1 was significantly increased in the lesions as compared with normal skin. The findings suggest that increased alphavbeta6-integrin in keratinocytes plays an active part in abnormal wound healing possibly through a mechanism involving increased activation of TGF-beta.

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.

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 α1I, α2I, and α10I domains and cell spreading assays with α1β1- and α2β1-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 α5β1 and αVβ1integrins, and it could be blocked by RGD peptides. The collagen α-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three α-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.

Jararhagin-derived RKKH Peptides Induce Structural Changes in α1I Domain of Human Integrin α1β1

Integrin α1β1 is one of four collagen-binding integrins in humans. Collagens bind to the αI domain and in the case of α2I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In α2I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound “open” conformation of α2I differs significantly from the “closed” conformation seen in the structure of apo-α2I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to α1I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of α1I with Mg2+ and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-α1I (“closed” conformation) and a model structure of the α1I (“open” conformation) based on the closely related structure of α2I reveals that the binding site is partially blocked to ligands by Glu255 and Tyr285 in the “closed” structure, whereas in the “open” structure helix C is unwound and these residues are shifted, and the “RKKH” peptides fit well when docked. The “open” conformation of α2I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of α1I and shown experimentally for α1I using a fluorescent hydrophobic probe.

“RKKH” Peptides from the Snake Venom Metalloproteinase ofBothrops jararaca Bind Near the Metal Ion-dependent Adhesion Site of the Human Integrin α2 I-domain

Integrin α1β1and α2β1 are the major cellular receptors for collagen, and collagens bind to these integrins at the inserted I-domain in their α subunit. We have previously shown that a cyclic peptide derived from the metalloproteinase domain of the snake venom protein jararhagin blocks the collagen-binding function of the α2 I-domain. Here, we have optimized the structure of the peptide and identified the site where the peptide binds to the α2 I-domain. The peptide sequence Arg-Lys-Lys-His is critical for recognition by the I-domain, and five negatively charged residues surrounding the “metal ion-dependent adhesion site” (MIDAS) of the I-domain, when mutated, show significantly impaired binding of the peptide. Removal of helix αC, located along one side of the MIDAS and suggested to be involved in collagen-binding in these I-domains, does not affect peptide binding. This study supports the notion that the metalloproteinase initially binds to the α2 I-domain at a location distant from the active site of the protease, thus blocking collagen binding to the adhesion molecule in the vicinity of the MIDAS, while at the same time leaving the active site free to degrade nearby proteins, the closest being the β1 subunit of the α2β1cell-surface integrin itself.