Biomedical subjects

Andrzej Fertala

Publications and source records attributed to Andrzej Fertala.

At least 19 recordsLinked to original sources

PubMed2007

Y-position cysteine substitution in type I collagen (alpha1(I) R888C/p.R1066C) is associated with osteogenesis imperfecta/Ehlers-Danlos syndrome phenotype.

The most common mutations in type I collagen causing types II-IV osteogenesis imperfecta (OI) result in substitution for glycine in a Gly-Xaa-Yaa triplet by another amino acid. We delineated a Y-position substitution in a small pedigree with a combined OI/Ehlers-Danlos Syndrome (EDS) phenotype, characterized by moderately decreased DEXA z-score (-1.3 to -2.6), long bone fractures, and large-joint hyperextensibility. Affected individuals have an alpha1(I)R888C (p.R1066C) substitution in one COL1A1 allele. Polyacrylamide gel electrophoresis (PAGE) of [(3)H]-proline labeled steady-state collagen reveals slight overmodification of the alpha1(I) monomer band, much less than expected for a substitution of a neighboring glycine residue, and a faint alpha1(I) dimer. Dimers form in about 10% of proband type I collagen. Dimer formation is inefficient compared to a possible 25%, probably because the SH-side chains have less proximity in this Y-position than when substituting for a glycine. Theoretical stability calculations, differential scanning calorimetry (DSC) thermograms, and thermal denaturation curves showed only weak local destabilization from the Y-position substitution in one or two chains of a collagen helix, but greater destabilization is seen in collagen containing dimers. Y-position collagen dimers cause kinking of the helix, resulting in a register shift that is propagated the full length of the helix and causes resistance to procollagen processing by N-proteinase. Collagen containing the Y-position substitution is incorporated into matrix deposited in culture, including immaturely and maturely cross-linked fractions. In vivo, proband dermal fibrils have decreased density and increased diameter compared to controls, with occasional aggregate formation. This report on Y-position substitutions in type I collagen extends the range of phenotypes caused by nonglycine substitutions and shows that, similar to X- and Y-position substitutions in types II and III collagen, the phenotypes resulting from nonglycine substitutions in type I collagen are distinct from those caused by glycine substitutions.

Adult↗

PubMed2006

Molecular basis of organization of collagen fibrils.

The collagen fibrils are formed by self-assembly of individual collagen molecules, but the mechanism that drives their orderly packing during fibril formation is not clearly defined. To identify structural determinants critical for the D-periodic alignment of collagen molecules we employed three sets of genetically engineered collagen II variants: (i) a set in which domains corresponding to the specific D periods have been purposely deleted, (ii) a set of collagen variants consisting of tandem repeats of a specific D period, and (iii) a set lacking definite fragments of the D4 period. All collagen variants were analyzed for their ability to assemble into D-periodic fibrils. Even though all genetically engineered collagen variants differ significantly from the wild-type collagen II, most of them were able to form filamentous structures. The D-periodic banding pattern, an indication of the staggered arrangement of collagen monomers, however, occurred only when the D1, D4, and D0.4 domains of interacting collagen monomers could potentially cluster together to form a triad through telopeptide-mediated binding. Our results identify a critical step in the formation of collagenous matrices and provide experimental evidence for the active involvement of the N-terminal and C-terminal regions of fibrillar collagens in this process.

Amino Acids↗

PubMed2006

High-affinity binding of the NC1 domain of collagen VII to laminin 5 and collagen IV.

Anchoring functions of collagen VII depend on its ability to form homotypic fibrils and to bind to other macromolecules to form heterotypic complexes. Biosensor-based binding assays were employed to analyze the kinetics of the NC1 domain-mediated binding of collagen VII to laminin 5, collagen IV, and collagen I. We showed that collagen VII interacts with laminin 5 and collagen IV with a Kd value of 10(-9) M. In contrast, the NC1-mediated binding to collagen I was weak with a Kd value of 10(-6) M. Binding assays also showed that the NC1 domain utilizes the same region to bind to both laminin 5 and collagen IV. We postulate that the ability of the NC1 domains to bind with high affinities to laminin 5 and collagen IV facilitates stabilization of the structure of the basement membrane itself and that the NC1-collagen I interaction may be less important for stabilization of the dermal-epidermal junction.

Binding Sites↗

PubMed2006

Testing the utility of rationally engineered recombinant collagen-like proteins for applications in tissue engineering.

Collagens are attractive proteins as materials for tissue engineering. Over the last decade, significant progress has been made in developing technologies for large-scale production of native-like human recombinant collagens. Yet, the rational design of customized collagen-like proteins for smart biomaterials to enhance the quality of engineered tissues has not been explored. We mapped the D4 domain of human collagen II as most critical for supporting migration of chondrocytes and used this information to genetically engineer a collagen-like protein consisting of tandem repeats of the D4 domain (mD4 collagen). This novel collagen has been utilized to fabricate a scaffold for support of chondrocytes. We determined superior qualities of cartilaginous constructs created by chondrocytes cultured in scaffolds containing the mD4 collagen in comparison to those formed by chondrocytes cultured in bare scaffolds or those coated with wild-type collagen II. Our results are a first attempt to rationally engineer collagen-like proteins with characteristics tailored for specific needs of cartilage engineering and provide a basis for rational engineering of similar proteins for a variety of biomedical applications.

Cartilage↗

PubMed2006

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

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 beta-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.

Biosensing Techniques↗

PubMed2005

Identifying the SPARC binding sites on collagen I and procollagen I by atomic force microscopy.

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein associated with the extracellular matrix (ECM). It has been found that the production of collagen I is a requisite for the association of SPARC with ECM, and studies with SPARC-null mice indicate that SPARC plays a role in modifying the structure of collagen fibers. It is not known, however, whether SPARC interacts with the collagen I precursor, procollagen I. In this study, the binding of SPARC to collagen I and procollagen I was verified by surface plasmon resonance. The SPARC-binding sites on collagen I and procollagen I were identified by directly visualizing their complexes using tapping-mode atomic force microscopy (TM-AFM). The characteristic chain end feature in collagen I is not readily detected by AFM, so unambiguous location of the binding sites relative to the C- or N-termini is difficult. In contrast, procollagen I, with its large globular C-propeptide, permits easy identification of the C-terminus. Histograms were constructed and compared based on the distances of the bound SPARC to the C-terminus of procollagen I and to the closest end of collagen I. There is a broad distribution of SPARC binding sites on procollagen I with the most preferred binding region located approximately 1/3 from the C-terminus. Characterization of the SPARC-binding sites on collagen I and procollagen I provides useful information for further understanding of the functional implications of their interactions.

Animals↗

PubMed2005

Single amino acid substitutions in the C-terminus of collagen II alter its affinity for collagen IX.

The structural integrity of cartilage depends on the presence of extracellular matrices (ECM) formed by heterotypic fibrils composed of collagen II, collagen IX, and collagen XI. The formation of these fibrils depends on the site-specific binding between relatively small regions of interacting collagen molecules. Single amino acid substitutions in collagen II change the physicochemical and structural characteristics of those sites, thereby leading to an alteration of intermolecular collagen II/collagen IX interaction. Employing a biosensor to study interactions between R75C, R789C or G853E collagen II mutants and collagen IX, we demonstrated significant changes in the binding affinities. Moreover, analyses of computer models representing mutation sites defined exact changes in physicochemical characteristics of collagen II mutants. Our study shows that changes in collagen II/collagen IX affinity could represent one of the steps in a cascade of changes occurring in the ECM of cartilage as a result of single amino acid substitutions in collagen II.

Amino Acid Substitution↗

PubMed2005

Guilty by association: some collagen II mutants alter the formation of ECM as a result of atypical interaction with fibronectin.

Among the structural components of extracellular matrices (ECM) fibrillar collagens play a critical role, and single amino acid substitutions in these proteins lead to pathological changes in tissues in which they are expressed. Employing a biologically relevant experimental model consisting of cells expressing R75C, R519C, R789C, and G853E procollagen II mutants, we found that the R789C mutation causing a decrease in the thermostability of collagen not only alters individual collagen molecules and collagen fibrils, but also has a negative impact on fibronectin. We propose that thermolabile collagen molecules are able to bind to fibronectin, thereby altering intracellular and extracellular processes in which fibronectin takes part, and we postulate that such an atypical interaction could change the architecture of the ECM of affected tissues in patients harboring mutations in genes encoding fibrillar collagens.

Cell Line, Tumor↗

PubMed2005

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2.

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellular matrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin-1D, measured by biosensor assay, revealed a K(d) of 5.71 x 10(-8) M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.

Amino Acid Substitution↗

PubMed2004

The D2 period of collagen II contains a specific binding site for the human discoidin domain receptor, DDR2.

The human discoidin domain receptors (DDRs), DDR1 and DDR2, are expressed widely and, uniquely among receptor tyrosine kinases, activated by the extracellular matrix protein collagen. This activation is due to a direct interaction of collagen with the DDR discoidin domain. Here, we localised a specific DDR2 binding site on the triple-helical region of collagen II. Collagen II was found to be a much better ligand for DDR2 than for DDR1. As expected, DDR2 binding to collagen II was dependent on triple-helical collagen and was mediated by the DDR2 discoidin domain. Collagen II served as a potent stimulator of DDR2 autophosphorylation, the first step in transmembrane signalling. To map the DDR2 binding site(s) on collagen II, we used recombinant collagen II variants with specific deletions of one of the four repeating D periods. We found that the D2 period of collagen II was essential for DDR2 binding and receptor autophosphorylation, whereas the D3 and D4 periods were dispensable. The DDR2 binding site on collagen II was further defined by recombinant collagen II-like proteins consisting predominantly of tandem repeats of the D2 or D4 period. The D2 construct, but not the D4 construct, mediated DDR2 binding and receptor autophosphorylation, demonstrating that the D2 period of collagen II harbours a specific DDR2 recognition site. The discovery of a site-specific interaction of DDR2 with collagen II gives novel insight into the nature of the interaction of collagen II with matrix receptors.

Binding Sites↗

PubMed2004

Structural determinants of the selectivity of KTS-disintegrins for the alpha1beta1 integrin.

KTS-disintegrins are a subfamily of short monomeric disintegrins that are potent and selective inhibitors of alpha1beta1 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.

Amino Acid Motifs↗

PubMed2004

Single amino acid substitutions in procollagen VII affect early stages of assembly of anchoring fibrils.

Procollagen VII is a homotrimer of 350-kDa pro-alpha1(VII) chains, each consisting of a central collagenous domain flanked by the noncollagenous N-terminal NC1 domain and the C-terminal NC2 domain. After secretion from cells, procollagen VII molecules form anti-parallel dimers with a C-terminal 60-nm overlap. Characteristic alignment of procollagen VII monomers forming a dimer depends on site-specific binding between the NC2 domain and the triple-helical region adjacent to Cys-2634 of the interacting procollagen VII molecules. Formation of the intermolecular disulfide bonds between Cys-2634 and either Cys-2802 or Cys-2804 is promoted by the cleavage of the NC2 domain by procollagen C-proteinase. By employing recombinant procollagen VII variants harboring G2575R, R2622Q, or G2623C substitutions previously disclosed in patients with dystrophic epidermolysis bullosa, we studied how these amino acid substitutions affect intermolecular interactions. Binding assays utilizing an optical biosensor demonstrated that the G2575R substitution increased affinity between mutant molecules. In contrast, homotypic binding between the R2622Q or G2623C molecules was not detected. In addition, kinetics of heterotypic binding of all analyzed mutants to wild type collagen VII were different from those for binding between wild type molecules. Moreover, solid-state binding assays demonstrated that R2622Q and G2623C substitutions prevent formation of stable assemblies of procollagen C-proteinase-processed mutants. These results indicate that single amino acid substitutions in procollagen VII alter its self-assembly and provide a basis for understanding the pathomechanisms leading from mutations in the COL7A1 gene to fragility of the dermal-epidermal junction seen in patients with dystrophic forms of epidermolysis bullosa.

Amino Acid Substitution↗

PubMed2004

Thermostability gradient in the collagen triple helix reveals its multi-domain structure.

A triple-helical conformation and stability at physiological temperature are critical for the mechanical and biological functions of the fibril-forming collagens. Here, we characterized the role of consecutive domains of collagen II in stabilizing the triple helix. Analysis of melting temperatures of genetically engineered collagen-like proteins consisting of tandem repeats of the D1, D2, D3 or D4 collagen II periods revealed the presence of a gradient of thermostability along the collagen molecule with thermolabile N-terminal domains and thermostable C-terminal domains. These results imply a multi-domain character of the collagen triple helix. Assays of thermostabilities of the Arg75Cys and Arg789Cys collagen II mutants suggest that, in contrast to the thermostable domains, the thermolabile domains are able to accommodate amino acid substitutions without altering the thermostability of the entire collagen molecule.

Blotting, Western↗

PubMed2004

Stretching type II collagen with optical tweezers.

Collagens are the most abundant proteins of vertebrates and they provide mechanical and supportive functions in a wide range of connective tissues. Knowledge of the mechanical properties of single collagen molecules is essential in studying the self-assembly of collagen, the interaction between cells and extracellular matrix, the etiology of tissue degeneration and mechanism of regeneration, and the relationship between the structures and mechanical properties of tissues. Here we stretched single type II collagen molecules in neutral pH solution using optical tweezers. The molecular parameters of collagen were obtained by fitting force-extension curves into worm-like chain elasticity model. The molecule length of type II collagen monomer was 295.8 nm. The persistence length of type II collagen monomer was 11.2 nm. These observations indicate that collagen molecules are flexible rather than rigid rod molecules at neutral pH solution.

Collagen Type II↗

PubMed2004

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils.

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.

Binding Sites↗

PubMed2004

Differential structural properties and expression patterns suggest functional significance for multiple mouse desmoglein 1 isoforms.

The four isoforms of desmosomal cadherin desmogleins (Dsg1-4) are expressed in epithelial tissues in a differentiation-specific manner. Extensive sequencing of the human genome has revealed only one copy of the Dsg1 gene. However, we recently cloned two novel additional mouse Dsg1 genes, Dsg1-beta and -gamma, which flank the original Dsg1-alpha on chromosome 18. Sequence conservation between the Dsg1 isoforms diverged significantly at exon 11, particularly in the region that encodes for the extracellular anchoring (EA) domains. Computational analysis revealed very low hydrophilic potential of the Dsg1-gamma EA compared with the corresponding sequences of Dsg1-alpha and -beta, suggesting that the Dsg1-gamma EA domain may have a stronger affinity to the cell membrane. We generated antibodies using synthetic peptides or recombinant proteins localized within the EA domains. These antibodies were tested for their specificity and were then used to demonstrate expression of Dsg1 isoforms in various tissues. In the epidermis, all Dsg1 isoforms were differentially expressed in the differentiating cell layers. In the hair follicle, all Dsg1 isoforms were present throughout the entire process of its development and cycling but the expression of Dsg1 isoforms is subject to significant hair cycle-dependent changes. Dsg1-beta and -gamma, but not Dsg1-alpha, were detected in the sebaceous gland epithelium and the stratified epithelium of the stomach. Finally, Dsg1-alpha and Dsg1-beta, but not Dsg1-gamma, are proteolytically cleaved by exfoliative toxin A. These results suggest that the developmental complexity of mouse tissues, including skin and hair, may play a significant role in the evolutionary driving force to maintain multiple Dsg1 genes in mouse.

Amino Acid Sequence↗

PubMed2003

Procollagen VII self-assembly depends on site-specific interactions and is promoted by cleavage of the NC2 domain with procollagen C-proteinase.

Procollagen VII is a homotrimer of 350-kDa proalpha1(VII) chains. Each chain has a central collagenous domain flanked by a noncollagenous amino-terminal NC1 domain and a carboxy-terminal NC2 domain. After secretion from cells, procollagen VII molecules form antiparallel dimers with a 60 nm overlap. These dimers are stabilized by disulfide bonds formed between cysteines present in the NC2 domain and cysteines present in the triple-helical domain. Electron microscopy has provided direct evidence for the existence of collagen VII dimers, but the dynamic process of dimer formation is not well understood. In the present study, we tested the hypothesis that, during dimer formation, the NC2 domain of one procollagen VII molecule specifically recognizes and binds to the triple-helical region adjacent to Cys-2625 of another procollagen VII molecule. We also investigated the role of processing of the NC2 domain by the procollagen C-proteinase/BMP-1 in dimer assembly. We engineered mini mouse procollagen VII variants consisting of intact NC1 and NC2 domains and a shortened triple helix in which the C-terminal region encompassing Cys-2625 was either preserved or substituted with the region encompassing Cys-1448 derived from the N-terminal part of the triple-helical domain. The results indicate that procollagen VII self-assembly depends on site-specific interactions between the NC2 domain and the triple-helical region adjacent to Cys-2625 and that this process is promoted by the cleavage of the NC2 by procollagen C-proteinase/BMP1.

Amino Acid Sequence↗

PubMed2003

Biochemical composition and histologic structure of the forearm interosseous membrane.

PURPOSE: The purpose of this study was to determine the structure and composition of the forearm interosseous membrane (IOM). METHODS: The IOM of 12 cadaver forearms was fixed in formalin. After fixation 5 individual IOM fiber bundles per arm were separated by dissection, excised, and processed with hematoxylin-eosin, trichrome, and Verhoff-vanGeison stains. Nine additional fresh forearms were dissected and 5 IOM fiber bundles per arm were analyzed using the hydroxyproline assay. Bundles were evaluated at ulnar, central, and radial locations. RESULTS: Histologic analysis of the IOM bundles obtained from the 12 fixed forearms showed an abundance of collagen in the main bundle central location (84% +/- 7.8%). A progressive increase in collagen was noted from distal to proximal bundles (r =.72). The hydroxyproline assay of collagen content of the main IOM bundle's central location from the 9 additional fresh forearms was 99.3% +/- 16.5%. There was no difference between bundles or location (power = 0.25 and 0.46). CONCLUSIONS: We found that the IOM possesses a large collagen content arranged in fibrillar structures surrounded by elastin. Collagen was abundant in the proximal bundles and decreased in the distal bundles.

Aged↗