Andrzej Fertala

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates.

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

Direct quantification of the flexibility of type I collagen monomer

Collagens are the most abundant structural proteins found in the extracellular matrix of vertebrates. Knowledge of the mechanical behavior of collagen monomers is essential for understanding the mechanical properties of collagen fibrils that constitute the main architectural framework of skin, bone, cartilage, and other connective tissues. In this study, the flexibility of type I collagen monomer was studied by stretching type I collagen monomers directly. The force-extension relationship was measured and analyzed by fitting the data into a worm-like chain elasticity model. The persistence length of collagen I monomer was determined to be 14.5 nm and the contour length was 309 nm. The results confirm that type I collagen monomer is flexible rather than rigid, rod-like molecule. Such flexibility may possibly be a consequence of the micro-unfolding of discrete domains of single collagen molecule.

Mapping critical sites in collagen II for rational design of gene-engineered proteins for cell-supporting materials

Collagen II is the most abundant protein of cartilage and forms a network of fibrils extended by proteoglycans that enables cartilage to resist pressure. The surface of the collagen fibril serves as a platform for the attachment of collagen IX, growth factors, and cells. In this study we examined the mechanism of the interaction of chondrocytes with recombinant versions of procollagen II, in which one of the four blocks of 234 amino acids that define repeating D periods of the collagen triple helix has been deleted. Analysis of the attachment of chondrocytes to collagen II variants with deleted D periods indicated that the collagen II monomer contains randomly distributed sites critical for cell binding. However, as was shown by spreading and migration assays, the D4 period, which is between residues 703 to 936, contains amino acids critical for cell motility. We also showed that binding, spreading, and migration of chondrocytes through three-dimensional nanofibrillar collagenous matrices are controlled by an interaction of the collagen triple helix with beta1 integrins. The results of this study provide a basis for the rational design of a scaffold containing genetically engineered collagen with a high density of specific sites of interaction.

Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents

Stroke is the leading cause of long-term disability and the third leading cause of death in the United States. While most research thus far has focused on acute stroke treatment and neuroprotection, the exploitation of endogenous brain self-repair mechanisms may also yield therapeutic strategies. Here, we describe a distinct type of stroke treatment, the naturally occurring extracellular matrix fragment of perlecan, domain V, which we found had neuroprotective properties and enhanced post-stroke angiogenesis, a key component of brain repair, in rodent models of stroke. In both rat and mouse models, Western blot analysis revealed elevated levels of perlecan domain V. When systemically administered 24 hours after stroke, domain V was well tolerated, reached infarct and peri-infarct brain vasculature, and restored stroke-affected motor function to baseline pre-stroke levels in these multiple stroke models in both mice and rats. Post-stroke domain V administration increased VEGF levels via a mechanism involving brain endothelial cell α5β1 integrin, and the subsequent neuroprotective and angiogenic actions of domain V were in turn mediated via VEGFR. These results suggest that perlecan domain V represents a promising approach for stroke treatment.

Extracellular matrix protein 1 inhibits the activity of matrix metalloproteinase 9 through high‐affinity protein/protein interactions

Abstract:  Extracellular matrix protein 1 (ECM1), an approximately 85‐kDa glycoprotein with broad tissue distribution, harbors mutations in lipoid proteinosis (LP), a heritable disease characterized by reduplication of basement membranes and hyalinization of dermis, associated with neurologic disorders. The mechanisms leading from ECM1 mutations to LP phenotype are unknown. In this study, we explored ECM1 protein‐protein interactions utilizing yeast two‐hybrid genetic screen of human placental library, which identified nine interacting proteins, including matrix metalloproteinase 9 (MMP9). The interactions were confirmed by β‐galactosidase assay with isolated clones and by co‐immunoprecipitation which narrowed the interacting segment in ECM1 to the C‐terminal tandem repeat 2 (amino acids 236–361). This peptide segment also inhibited MMP9 activity in a gelatin‐based ELISA assay. We propose that ECM1‐mediated reduction in MMP9 proteolytic activity may have relevance to pathogenesis of LP.

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses.

Structural determinants of the selectivity of KTS‐disintegrins for the α1β1 integrin

KTS‐disintegrins are a subfamily of short monomeric disintegrins that are potent and selective inhibitors of α1β1 integrin. The amino acid sequence of the new KTS‐disintegrin, viperistatin, differs from previously characterized obtustatin in three residues at position 24 (within the integrin binding loop), 38 (hydrophobic core) and 40 (C‐terminal region). Noteworthy, viperistatin is about 25‐fold more potent than obtustatin inhibiting the binding of this integrin to collagen IV. Synthetic peptides representing the full‐length of integrin‐binding loops of these disintegrins showed that the Leu24/Arg substitution appears to be partly responsible for the increased inhibitory activity of viperistatin over obtustatin.

Recombinant Procollagen II: Deletion of D Period Segments Identifies Sequences That Are Required for Helix Stabilization and Generates a Temperature-sensitive N-Proteinase Cleavage Site

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 °C as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3 °C lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3 °C lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen N-proteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 °C that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the α1(II) chain.

Collagen fibril formation; a new target to limit fibrosis

We present a concept for reducing formation of fibrotic deposits by inhibiting self-assembly of collagen molecules into fibrils, a main component of fibrotic lesions. Employing monoclonal antibodies that bind to the telopeptide region of a collagen molecule, we found that blocking telopeptide-mediated collagen/collagen interactions reduces the amount of collagen fibrils accumulated in vitro and in keloid-like organotypic constructs. We conclude that inhibiting extracellular steps of the fibrotic process provides a novel approach to limit fibrosis in a number of tissues and organs.

Assembly in Vitroof Thin and Thick Fibrils of Collagen II from Recombinant Procollagen II THE MONOMERS IN THE TIPS OF THICK FIBRILS HAVE THE OPPOSITE ORIENTATION FROM MONOMERS IN THE GROWING TIPS OF COLLAGEN I FIBRILS

Human type II procollagen was prepared in a recombinant system and cleaved to pC-collagen II by procollagen N-proteinase. The pC-collagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase at 37°C. Electron microscopy of the fibrils demonstrated that, at the early stages of fibril assembly, very thin fibrils were formed. As the system approached equilibrium over 7–12 h, however, the thin fibrils were largely but not completely replaced by thick fibrils that had diameters of about 240 nm and a distinct D-period banding pattern. One typical fibril was photographed and analyzed in its entirety. The fibril was 776 D-periods (52 μm) long. It had a central shaft with a uniform diameter that was about 516 D-periods long and two tips of about 100 D-periods each. Most of the central shaft had a symmetrical banding pattern flanked by two transition regions of about 30 D-periods each. Measurements by scanning transmission electron microscopy demonstrated that the mass per unit length from the tips to the shafts increased linearly over approximately 100 D-periods from the fibril end. The linear increase in mass per unit length was consistent with previous observations for collagen I fibrils and established that the tips of collagen II also had a near paraboloidal shape. However, the orientation of monomers in the tips differed from the tips of collagen I fibrils in that the C termini instead of the N termini were directed toward the tips. The thin fibrils that were present at early stages of assembly and at equilibrium were comparable to the collagen II fibrils seen in embryonic tissues and probably represented intermediates on the pathway of thick fibrils formation. The results indicated that the molecular events in the self-assembly of collagen II fibrils are apparently similar to those in self-assembly of collagen I fibrils, but that there are also important differences in the structural information contained in collagen I and collagen II monomers.