Biomedical subjects

D R Keene

Publications and source records attributed to D R Keene.

At least 37 records · Page 2Linked to original sources

PubMed1998

An alpha2(I) glycine to aspartate substitution is responsible for the presence of a kink in type I collagen in a lethal case of osteogenesis imperfecta.

Type I collagen synthesized by cultured skin fibroblasts was analyzed biochemically and molecularly to characterize the defect in a patient affected by lethal Osteogenesis Imperfecta. The SDS-Urea-PAGE of procollagen and collagen revealed a broad alpha1(I) band, a normal alpha2(I) and another alpha2(I) band migrating equidistant between alpha1 and alpha2. When synthesized in the presence of alphaalpha'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylation, procollagen and collagen of media and cell layers contained both normal and slower alpha2(I), but only normal alpha1(I). The persistence of the two forms of alpha2(I) chains suggested a mutation in a COL1A2 gene. CNBr cleavage of collagen yielded overmodified alpha1(I) CB3 and CB7 peptides and delayed migration of the alpha2(I) CB3-5 peptide. A delayed CB3-5 was also found after alpha,alpha'-dipyridyl treatment. These data localized the mutation between aa 353 and 551 in alpha2(I) (CB3-5). Sequencing the subcloned alleles in this region revealed a G-->A transition at nt 1671 in one allele, changing Gly 421 to Asp in an alpha2(I) chain. The mutation was demonstrated to occur on the paternally derived allele, using a common C-->A polymorphism at alpha2(I) nt 1585 and by the presence of a rare variant, Arg618-->Gln (Phillips et al., 1990), in the paternal genomic DNA and the proband's mutant allele. Procollagen processing was normal. The Tm of the slow alpha2(I) collagen was 2 degrees C lower than the control, indicating decreased triple helix stability. Mutant collagen was incorporated in the extracellular matrix deposited by cultured fibroblasts. The dramatic delay in alpha2(I) electrophoretic mobility must be induced by the Gly-->Asp substitution, since the Arg-->Gln variant causes only mild electrophoretic delay. Substantial delay in gel mobility even in the absence of overmodification suggested the presence of a kink in the mutated alpha2(I) chains. Rotary shadowing electron microscopy of secreted fibroblast procollagen confirmed the presence of a kink in the region of the helix containing the glycine substitution. The kinking of the collagen helix occurs in the absence of dimer formation. Kinking may interfere with normal helix folding, as well as with the interactions of collagen fibrils with the collagenous and non-collagenous extracellular matrix proteins.

Amino Acid Substitution↗

PubMed1998

Type VI microfilaments interact with a specific region of banded collagen fibrils in skin.

Immunolocalization studies demonstrate that Type VI collagen forms a flexible network that interweaves among collagen fibrils in the dermis of skin as well as in other loose connective tissues. Although binding of Type VI collagen with other matrix components has been suggested, no structural evidence supporting these studies has been reported. In this study, we demonstrate that Type VI microfilaments consistently crossbanded collagen fibrils near the "d" band, indicating that the interaction of Type VI collagen with banded fibrils is not passive. This "d" band is also the location of the binding domain of decorin to banded fibrils, suggesting that decorin mediates the interaction of Type VI microfilaments with banded fibers. Examination of the architecture of the Type VI network in a decorin nullizygous mouse demonstrates a continuance of this specific interaction, indicating that the association is not entirely dependent on the presence of decorin. At least one other component, whose identity is uncertain, persists near the "d" band, which may also serve to mediate the attachment of Type VI collagen to collagen fibrils.

Actin Cytoskeleton↗

PubMed1997

The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia.

We have identified a novel missense mutation in a pseudoachondroplasia (PSACH) patient in one of the type III repeats of cartilage oligomeric matrix protein (COMP). Enlarged lamellar rough endoplasmic reticulum vesicles were shown to contain accumulated COMP along with type IX collagen, a cartilage-specific component. COMP was secreted and assembled normally into the extracellular matrix of tendon, demonstrating that the accumulation of COMP in chondrocytes was a cell-specific phenomenon. We believe that the intracellular storage of COMP causes a nonspecific aggregation of cartilage-specific molecules and results in a cartilage matrix deficient in required structural components leading to impaired cartilage growth and maintenance. These data support a common pathogenetic mechanism behind two clinically related chondrodysplasias, PSACH and multiple epiphyseal dysplasia.

Achondroplasia↗

PubMed1997

Type VI collagen anchors endothelial basement membranes by interacting with type IV collagen.

Type VI collagen filaments are found associated with interstitial collagen fibers, around cells, and in contact with endothelial basement membranes. To identify type VI collagen binding proteins, the amino-terminal domains of the alpha1(VI) and alpha2(VI) chains and a part of the carboxyl-terminal domain of the alpha3(VI) chain were used as bait in a yeast two-hybrid system to screen a human placenta library. Eight persistently positive clones were identified, two coding the known matrix proteins fibronectin and basement membrane type IV collagen and the rest coding new proteins. The amino-terminal domain of alpha1(VI) was shown to interact with the carboxyl-terminal globular domain of type IV collagen. The specificity of this interaction was further studied using the yeast two-hybrid system in a one-on-one format and confirmed by using isolated protein domains in immunoprecipitation, affinity blots, and enzyme-linked immunosorbent assay-based binding studies. Co-distribution of type VI and type IV collagens in human muscle was demonstrated using double labeling immunofluorescent microscopy and immunoelectron microscopy. The strong interaction of type VI collagen filaments with basement membrane collagen provided a possible molecular pathogenesis for the heritable disorder Bethlem myopathy.

Adult↗

PubMed1997

Immunodissection of the connective tissue matrix in human skin.

Much of what has been learned of the components and structure of human skin over the past few years has been accomplished with the aid of antibody technology. Antibodies are used in techniques such as affinity chromatography to isolate individual molecules and by immunofluorescence and immunoelectron microscopy to identify each of those molecules as components of specific macromolecular assemblies present within the dermis. This manuscript is meant not as a review of technique but instead as a summary of recent progress made in the understanding of dermal matrix architecture.

Actin Cytoskeleton↗

PubMed1997

Laminin 5 binds the NC-1 domain of type VII collagen.

Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

Antigens, Surface↗

PubMed1997

Calcium determines the shape of fibrillin.

Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s20,w0 = 5.1 +/- 0.1 in the Ca2+ form and s20,w0 = 6.2 +/- 0.1 in the Ca2+-free form. Calculations based on these results and the corresponding molecular mass predicted a shortening of fibrillin by approximately 25% and an increase in width of approximately 13-17% upon removal of Ca2+. These observations were confirmed by analysis of Ca2+-loaded and Ca2+-free rotary shadowed fibrillin molecules. Analysis of recombinant fibrillin-1 subdomain rF17, consisting primarily of an array of 12 Ca2+-binding epidermal growth factor (cbEGF)-like repeats, by analytical ultracentrifugation and rotary shadowing further confirmed Ca2+-dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contribution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be approximately 3 nm in the Ca2+ form. Ca2+-free forms demonstrated a decrease of 20-30% in length, indicating significant structural changes of these motifs when they occur in tandem. Circular dichroism measurements of rF17 in the presence and absence of Ca2+ indicated secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here suggest a flexible structure for the Ca2+-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca2+ form.

Calcium↗

PubMed1997

Skeletal development in transgenic mice expressing a mutation at Gly574Ser of type II collagen.

Skeletal development of transgenic mice with a type II collagen mutation was analyzed and compared with wild-type littermates. The single base substitution in Col2a1 resulted in a glycine to serine mutation within the helical domain and corresponded to one previously identified in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:4583-4587). Skeletal staining of embryos from 14.5 through 18.5 days of gestation demonstrated a dwarf phenotype in the transgenic embryos, most notably short limb bones and vertebral column that was first detected at 15.5 days post-coitus. In addition to the reduced length, the extent of ossification was less in the transgenic mice. The architecture of the long bone growth plate was abnormal in the transgenic tissue, in particular there was no discernible proliferative zone. There were few stacks of characteristically flattened cells and the overall length of the growth plate in the mutant embryos was reduced. At the ultrastructural level, there were fewer collagen fibrils present in the transgenic mouse cartilage compared to that of wild-type littermates. Ultrastructural localization of collagen types II, IX and XI revealed a similar pattern between the transgenic and wild-type pups, suggesting that the collagen fibrils present in the matrix of littermates with both phenotypes had a similar composition. Skeletal analysis and cartilage histochemistry indicated that effect of the type II collagen mutation was to reduce the density of the collagen fibrils within the cartilage matrix which was associated with delayed bone formation and resulted in a short-limbed phenotype.

Animals↗

PubMed1997

Type II collagen pro-alpha-chains containing a Gly574Ser mutation are not incorporated into the cartilage matrix of transgenic mice.

The biochemical consequences of a type II procollagen mutation that contained a Gly574Ser amino acid substitution were analyzed in a transgenic mouse strain. The mutation correlated with one previously characterized in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al., 1992), and resulted in a similar shortlimbed phenotype. There were fewer collagen fibrils present in the transgenic cartilage and reduced immunofluorescence of cartilage matrix using a type II collagen antibody. Pepsin-extracted collagen from transgenic mouse embryo cartilage was analyzed electrophoretically and indicated less type II as well as type XI collagen compared to their wild-type littermates. A pulse-chase experiment was performed to evaluate the biosynthesis and fate of type II collagen. Chondrocytes isolated from transgenic tissue synthesized fewer stable molecules, resulting in decreased secretion of the procollagen chains. By amino acid sequence analysis of the type II collagen peptides from cartilage of transgenic mouse embryos, serine was not detected at residue 574, the site mutated in the transgene. Based on sequence data, we believe that the molecules incorporated into collagen fibrils of the extracellular matrix, while fewer in number, were composed of normal alpha 1(II) chains.

Amino Acid Sequence↗

PubMed1997

Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome.

Aortic aneurysm and dissection account for about 2% of all deaths in industrialized countries; they are also components of several genetic diseases, including Marfan syndrome (MFS). The vascular phenotype of MFS results from mutations in fibrillin-1 (FBN1), the major constituent of extracellular microfibrils. Microfibrils, either associated with or devoid of elastin, give rise to a variety of extracellular networks in elastic and non-elastic tissues. It is believed that microfibrils regulate elastic fibre formation by guiding tropo-elastin deposition during embryogenesis and early post-natal life. Hence, vascular disease in MFS is thought to result when FBN1 mutations preclude elastic fibre maturation by disrupting microfibrillar assembly. Here we report a gene-targetting experiment in mice that indicates that fibrillin-1 microfibrils are predominantly engaged in tissue homeostasis rather than elastic matrix assembly. This finding, in turn, suggests that aortic dilation is due primarily to the failure by the microfibrillar array of the adventitia to sustain physiological haemodynamic stress, and that disruption of the elastic network of the media is a secondary event.

Aortic Dissection↗

PubMed1997

Fibrillin-1 in human cartilage: developmental expression and formation of special banded fibers.

The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites.

Adolescent↗

PubMed1996

Fibrillin-1 and fibulin-2 interact and are colocalized in some tissues.

Microfibrils 10-12 nm in diameter are found in elastic and non-elastic tissues with fibrillin as a major component. Little is known about the supramolecular structure of these microfibrils and the protein interactions it is based on. To identify protein binding ligands of fibrillin-1, we tested binding of recombinant fibrillin-1 peptides to different extracellular matrix proteins in solid phase assays. Among the proteins tested, only fibulin-2 showed significant binding to rF11, the N-terminal half of fibrillin-1, in a calcium-dependent manner. Surface plasmon resonance demonstrated high affinity binding with a Kd = 56 nM. With overlapping recombinant fibrillin-1 peptides, the binding site for fibulin-2 was narrowed down to the N terminus of fibrillin-1 (amino acid positions 45-450). Immunofluorescence in tissues demonstrated colocalization of fibrillin and fibulin-2 in skin, perichondrium, elastic intima of blood vessels, and kidney glomerulus. Fibulin-2 was not present in ocular ciliary zonules, tendon, and the connective tissue around kidney tubules and lung alveoli, which all contain fibrillin. Immunogold labeling of fibulin-2 on microfibrils in skin was found preferentially at the interface between microfibrils and the amorphous elastin core, suggesting that in vivo the interaction between fibrillin-1 and fibulin-2 is regulated by cellular expression and deposition as well as by protein-protein interactions.

Amino Acid Sequence↗

PubMed1996

Conserved neuron promoting activity in Drosophila and vertebrate laminin alpha1.

Drosophila S2 cells were transfected with constructs that code for two portions of the Drosophila laminin alpha chain. Construct recalphaL coded for domains III, I/II, and G of laminin alpha. Construct recalphaS coded for only the COOH-most 12% of the I/II domain and the G domain. The corresponding polypeptides were isolated and characterized from the culture media. The recalphaL chain partly formed disulfide-linked heterotrimers with the endogenously produced beta and gamma laminin chains. Like normal Drosophila laminin, a substrate coating of either recalphaL or recalphaS supported neuron differentiation and neurite extension of primary Drosophila embryo cell cultures. However, at the same low concentrations, only Drosophila laminin-1, but neither recalphaL nor recalphaS supported myogenesis in these cultures. Previously, an overlapping set of dodecapeptides that covered a region of the murine laminin alpha1 chain similar to recalphaS had been synthesized and tested for cell culture support properties (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). The Drosophila laminin alpha homologues of the six most active vertebrate dodecapeptides were now synthesized and tested as substrates for differentiation of primary Drosophila embryo cells. Peptides that contained either the Drosophila sequence SIKVGV or the murine homologue, SIKVAV, provided support for neurite extension.

Amino Acid Sequence↗

PubMed1996

Type IX collagen NC1 domain peptides can trimerize in vitro without forming a triple helix.

Synthetic peptides of the three chains of type IX collagen consisting of the carboxyl-terminal end of the COL1 domain and the complete NC1 domain were characterized by circular dichroism spectroscopy and analyzed for their ability to assemble into trimers. In vitro association and oxidation result in disulfide-linked oligomers as shown by molecular sieve chromatography and SDS-polyacrylamide electrophoresis. Whereas the individual peptides show a tendency to self-associate, when an equimolar amount of the three peptides was oxidized, a heterotrimer of the three chains was observed. This heterotrimer is recognized by a monoclonal antibody against the disulfide-linked NC1 domain of chicken type IX collagen, indicating the correct formation of the disulfide bonds. Circular dichroism measurements show that under the association conditions used, a triple helix does not form between the chains. These results indicate that these peptides contain all the necessary information for chain selection and assembly.

Amino Acid Sequence↗

PubMed1996

Fibrillin-1: organization in microfibrils and structural properties.

To investigate the microfibrillar organization and structural properties of fibrillin-1, we produced overlapping recombinant peptides in human cells which altogether span the fibrillin-1 molecule. The peptides were purified under non-denaturing conditions and extensive characterization indicated correct folding. The purified proteins were used to map monoclonal antibodies 26, 69 and 201. The binding sites are located at the N-terminal end between amino acid residues 45 and 450 (mAb 26), 451 and 909 (mAb 201) and at the C-terminal end between residues 2093 and 2871 (mAb 69). Immunolocalization of these antibodies to extended beaded structures (microfibrils) demonstrated that the N- and C-terminal ends of fibrillin-1 are located in proximity and on opposite sides of the beads, and more central parts of the molecule are located between the beads. Each epitope is present once between each bead. These data allow two possible models for the organization of fibrillin in microfibrils. However, comparison of distances between antibody binding sites on the recombinant peptides and labeling events in tissue suggests that fibrillin molecules are compacted within their tissue form as microfibrils. Additional analysis of the recombinant peptides provide new information regarding the eight-cysteine motif, a novel domain present in fibrillins and TGF beta binding proteins, and suggest that fibrillins are processed at their N-and C-terminal ends.

Actin Cytoskeleton↗

PubMed1996

Stiparin: a glycoprotein from sea cucumber dermis that aggregates collagen fibrils.

The interactions between collagen fibrils in many echinoderm connective tissues are rapidly altered by the secretions of resident neurosecretory cells. Recent evidence has suggested that a secreted protein is responsible for the interactions that lead to an increase in tissue stiffness (Trotter and Koob, 1995). Structurally intact collagen fibrils have been isolated from such a connective tissue- the dermis of the sea cucumber Cucumaria frondosa- and used in an assay in vitro to identify a protein that binds to them and causes them to aggregate. This protein has been purified by anion-exchange and molecular sieve chromatography. It is eluted from a MonoQ column at approximately 0.55 M NaCl. Its isoelectric point is 5.2. It elutes from a Superose-6 column in a position corresponding to a molecule with a Stokes radius of 11.5 nm. Its native molecular weight estimated from sedimentation equilibrium analysis under non-denaturing conditions is 375,000, and its monomer molecular weight, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, is approximately 350,000. Sedimentation velocity measurements indicated for the native molecule a sedimentation coefficient of 11 x 10(-13)s, a diffusion coefficient of 3.274 x 10(-7) cm2s-1, and a frictional ratio of 1.95, which corresponds to a prolate ellipsoid of revolution with an axial ratio of 19. The highly asymmetric structure suggested by the above correlated well with the images obtained by transmission electron microscopy following rotary shadowing, which revealed a flexible structure approximately 125 nm long. Based on its ability to aggregate collagen fibrils, this protein has been named "stiparin," from the Latin stipare, "to pack together."

Animals↗

PubMed1996

Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment.

Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (alpha3beta1gamma1) and a novel laminin isotype we have termed laminin 7 (alpha3beta2gamma1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.

Adult↗

PubMed1996

LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kDa anchoring filament protein synthesized by epidermal cells.

This study characterizes a novel basement membrane component that is the target of autoantibodies in patients with linear IgA bullous dermatosis. Tissue surveys showed that this protein localized to the epidermal side of 1 M NaCl split skin and to basement membranes in cornea, oral mucosa, esophagus, intestine, kidney collecting ducts, ureter, bladder, urethra, and thymus, but was absent in lung, blood vessels, skeletal muscle, and nerve. Monoclonal antibody 123, which recognizes this protein, induced dermal-epidermal separation of human skin in situ, and this protein was found, by immunoelectron microscopy, to localize exclusively to anchoring filaments. This protein was secreted as as a 120-kDa peptide from primary cultures of keratinocytes as determined by radioimmunoprecipitation. Monoclonal antibody 123 recognized this protein as a 120-kDa band from conditioned cell culture medium and a 97-kDa band from human skin extracts as shown by immunoblot. Serum from five patients with the autoimmune blistering disorder linear IgA bullous dermatosis specifically recognized bands of 120 and 97 kDa from culture medium and skin extracts, respectively, that were of identical electrophoretic migration to the bands recognized by monoclonal antibody 123. In summary, this study characterizes a novel anchoring filament protein that is the target of linear IgA bullous dermatosis autoantibodies. Because monoclonal antibody 123 induces blistering of human skin, we hypothesize that this protein functions to maintain dermal-epidermal cohesion and that autoantibodies in this disease are themselves pathogenic. We propose LAD-1 as the name for this protein.

Animals↗