Leptospira Immunoglobulin-Like Protein B Interacts with the 20th Exon of Human Tropoelastin Contributing to Leptospiral Adhesion to Human Lung Cells.

Leptospira immunoglobulin-like protein B (LigB), a surface adhesin, is capable of mediating the attachment of pathogenic leptospira to the host through interaction with various components of the extracellular matrix (ECM). Human tropoelastin (HTE), the building block of elastin, confers resilience and elasticity to lung, and other tissues. Previously identified Ig-like domains of LigB, including LigB4 and LigB12, bind to HTE, which is likely to promote Leptospira adhesion to lung tissue. However, the molecular mechanism that mediates the LigB-HTE interaction is unclear. In this study, the LigB-binding site on HTE was further pinpointed to a N-terminal region of the 20th exon of HTE (HTE20N). Alanine mutants of basic and aromatic residues on HTE20N significantly reduced binding to the LigB. Additionally, HTE-binding site was narrowed down to the first ß-sheet of LigB12. On this binding surface, residues F1054, D1061, A1065, and D1066 were critical for the association with HTE. Most importantly, the recombinant HTE truncates could diminish the binding of LigB to human lung fibroblasts (WI-38) by 68%, and could block the association of LigA-expressing L. biflexa to lung cells by 61%. These findings should expand our understanding of leptospiral pathogenesis, particularly in pulmonary manifestations of leptospirosis.

Anti-elastin antibodies and elastin turnover in normal pregnancy and recurrent pregnancy loss.

PROBLEM: The aim of this study was to investigate elastin turnover and autoimmunity in patients with a history of recurrent pregnancy loss (RPL) and during normal pregnancy. METHOD OF STUDY: Anti-alpha-elastin and anti-tropoelastin IgG and IgM antibodies were measured by a home-made ELISA in serum samples of 60 medically and obstetrically normal pregnant women, classified to three trimester groups, 18 female patients with RPL and 18 healthy non-pregnant women with a history of successful pregnancies. One way analyses of variance and Least Significant Difference method were used for a statistical analysis. RESULTS: Anti-alpha-elastin IgG autoantibodies were significantly decreased in the third trimester pregnant women. IgM anti-alpha-elastin autoantibodies were significantly decreased in all pregnancy groups compared with the controls. Synthesis/degradation ratio of elastin was significantly increased in the third trimester pregnancy group, suggesting decreased elastin degradation during this period of pregnancy. Comparing the RPL patients with the healthy non-pregnant controls showed a significantly increased anti-alpha-elastin IgG antibody and significantly decreased synthesis/degradation ratio in the patient's group, suggesting increased elastin degradation in RPL. CONCLUSION: Elastin degradation is decreased during normal pregnancy. Increased anti-elastin IgG antibodies may contribute to the pathogenesis of pregnancy losses.

Elastic fiber macro-assembly is a hierarchical, cell motion-mediated process.

Elastic fibers are responsible for the extensibility and resilience of many vertebrate tissues, and improperly assembled elastic fibers are implicated in a number of human diseases. It was recently demonstrated that in vitro, cells first secrete tropoelastin into a punctate pattern of globules. To study the dynamics of macroassembly, that is, the assembly of the secreted tropoelastin globules into elastic fibers, we utilized long-term time-lapse immunofluorescence imaging and a tropoelastin p Timer fusion protein, which shifts its fluorescence spectrum over time. Pulse-chase immunolabeling of the fibroblast-like RFL-6 cells demonstrates that tropoelastin globules aggregate in a hierarchical manner, creating progressively larger fibrillar structures. By analyzing the correlation between cell and extracellular matrix movements, we show that both the aggregation process and shaping the aggregates into fibrillar form is coupled to cell motion. We also show that the motion of non-adjacent cells becomes more coordinated as the physical size of elastin-containing aggregates increases. Our data imply that the formation of elastic fibers involves the concerted action and motility of multiple cells.

Heat modulation of tropoelastin, fibrillin-1, and matrix metalloproteinase-12 in human skin in vivo.

Photoaged skin contains elastotic materials in the upper reticular dermis. This phenomenon is commonly known as solar elastosis. In this study, we investigated the effects of heat on the expression of tropoelastin and fibrillin-1, two main components of elastic fibers, and on matrix metalloproteinase (MMP)-12, the most active MMP against elastin, in human skin in vivo. Heat was found to increase tropoelastin mRNA and protein expression in the epidermis and in the dermis. Fibrillin-1 mRNA and protein expression were increased by heat in the epidermis, but were decreased in the dermis. We found that pre-treatment of skin with N-acetyl cysteine or genistein for 24 h prior to heat treatment inhibited the heat-induced expression of tropoelastin, but not of fibrillin-1. These data indicate that reactive oxygen species may play a role in tropoelastin expression by heat, but not in fibrillin-1 expression. We also found that heat treatment increases MMP-12 mRNA and protein expression in human skin. Our results suggest that the abnormal production of tropoelastin and fibrillin by heat in human skin and that their degradation by various MMP, such as MMP-12, may contribute to the accumulation of elastotic material in photoaged skin.

Abnormalities of serum antielastin antibodies in connective tissue diseases.

BACKGROUND: Antibodies (Abs) to alpha-elastin (elastin breakdown product) and tropoelastin (elastin precursor) are found in the serum of all human subjects and correlate with their respective serum peptide levels; however, peptide levels vary with age and some disease states. This study was undertaken to determine if serum elastin Abs, peptides, and elastin metabolism were altered in autoimmune diseases by detecting a changing ratio of serum anti-alpha:tropoelastin Ab levels. METHODS: Serum from patients with a variety of connective tissue diseases, including 28 with systemic lupus erythematosus (SLE), 24 with scleroderma, 18 with rheumatoid arthritis (RA), 10 with polymyositis, and 39 with vasculitis, was compared with serum from 19 age-matched healthy subjects for levels of antitropoelastin and anti-alpha-elastin Abs. RESULTS: We found an increase in IgG anti-alpha-elastin and a decrease in antitropoelastin Abs in the sera of patients with scleroderma (p < .02 and .00005) and SLE (p < .006 and .011). There was also a marked increase in anti-alpha-elastin Abs in patients with polyarteritis nodosa (p < .0005) and decreases in antitropoelastin Abs in patients with RA (p < .05), polymyositis (p < .01), and a variety of other vasculidities (p < .0003). CONCLUSIONS: Abnormal variations in elastin metabolism may be detected in several connective tissue diseases by measuring ratios of alpha- and tropoelastin IgG Abs as markers of elastin degradation and synthesis.

Predictive estimation of protein linear epitopes by using the program PEOPLE.

A single small segment (sequence recognition) or domain (conformation recognition) of a protein could act as an antigen (antigenic determinant) vs an antibody. Epitopes of the first kind being a continuous segment along the sequence (linear), generally bent with a typical non-ordered structure (turns and/or loops), can be predicted from the only knowledge of the primary structure. After reviewing the different algorithms, we present PEOPLE (Predictive Estimation Of Protein Linear Epitopes) which uses combined prediction methods, taking into account the basic fundamental properties corresponding to what should be an ideal epitope: bent (secondary structure mainly beta-turns), surface accessible, hydrophilic and mobile and/or flexible. Four classes of basic biophysical parameters are considered for the determination of an antigenic index AG - secondary structure; hydrophilicity; surface accessibility; flexibility. The AG index is finally defined as a linear combination of the four class profiles. Typical applications are presented.

Antibody raised to AKAAAKAAAKA sequence on tropoelastin recognizes tropoelastin but not mature crosslinked elastin: A new tool in metabolic and structural studies of elastogenesis.

Tropoelastin, which is secreted from the cell in a soluble form, contains specific alanine rich repeat domains that are destined to form covalent desmosine and isodesmosine crosslinks in mature insoluble elastin. We raised a monospecific polyclonal antibody to a AKAAAKAAAKA synthetic peptide (AKA) which represents this alanine rich region of tropoelastin. The antibody was reactive with the original peptide antigen and purified tropoelastin, but not with mature crosslinked elastin isolated from several animal species. Conditioned media from chick aorta smooth muscle cells in culture reacted in an ELISA with the AKA antibody, but only in the presence of BAPN to block the conversion of the epsilon-amino groups to aldehydes. Immunofluorescence demonstrated that the AKA antibody decorated newly deposited tropoelastin assembled in fine fibrils in matrix produced by cultured human skin fibroblasts. EM-immunogold specifically localized this antibody to the immature elastic fibers present in fetal sheep ductus arteriosus. Moreover, immunohistochemistry demonstrated that the antibody recognized nonpolymerized tropoelastin assembled on the periphery of elastic fibers in the aorta of chicks raised on copper deficient and BAPN containing diets. These studies demonstrate that this new anti-tropoelastin antibody can be used as a useful tool to investigate elastin metabolism where it is important to distinguish between tropoelastin and mature crosslinked elastin.

Epitope specificity of monoclonal and polyclonal antibodies to human elastin.

Polyclonal (pAb) and monoclonal (mAb) anti-human aorta elastin antibodies were reacted with a series of overlapping hexapeptides along the human tropoelastin sequence covering exons 2-7 and 23-36 from the N-terminus to the C-terminus, advancing 1 amino acid residue each time. ELISA indicated reactive epitopes. mAb A2.1 recognized sequences containing Ala-Lys, mAb G8.1, A7.1 and pAb, hydrophobic sequences. None of them reacted with the hexapeptide VGVAPG, or with desmosine or isodesmosine. pAb L85 reacted with a His-containing sequence coded in exon 26A. pAb kappaE(L), kappaE(S) and L85 reacted with the Cys-containing sequence of exon 36. A synthetic 14-residue peptide containing the three proximal tyrosines coded in exon 13 did not react with any of the antisera tested. It appears therefore that the most frequently recognized epitopes are hydrophobic sequences. One polyclonal antibody detected several isoforms of tropoelastin in the medium of cultured vascular smooth muscle cells. Monoclonal and polyclonal antibodies stained elastic fibers on tissue sections, suggesting that the epitopes recognized are available on the native fibers for reaction with the antibodies.

Functional domains on elastin and microfibril-associated glycoprotein involved in elastic fibre assembly.

Studies in vitro suggest that the C-terminus of tropoelastin mediates elastin polymerization through an interaction with microfibril-associated proteins. In this study we have used cultured auricular chondrocytes as a model system to examine whether this interaction is critical for elastic fibre formation in vivo. Auricular chondrocytes, which deposit an abundant elastic fibre matrix, were cultured in the presence of Fab fragments of antibodies directed against the C-terminus (CTe) or an N-terminal domain (ATe) of tropoelastin. Immunofluorescent staining of the extracellular matrix deposited by the cells showed that the CTe antibody inhibited the deposition of elastin without affecting microfibril structure. Cells grown under identical conditions in the presence of ATe, however, formed fibres that stained normally for both elastin and microfibril proteins. Chondrocytes cultured in the presence of microfibril-associated glycoprotein (MAGP):21-35, an antibody directed against a domain near the N-terminus of MAGP, did not organize tropoelastin into fibres. However, immunostaining for MAGP and fibrillin revealed normal microfibrils. In agreement with the immunofluorescence staining patterns, fewer elastin-specific cross-links, indicative of insoluble elastin, were detected in the extracellular matrix of cells cultured in the presence of CTe. The medium from these cultures, however, contained more soluble elastin, consistent with an antibody-induced alteration of elastin assembly but not its synthesis. Northern analysis of antibody-treated and control cultures substantiated equivalent levels of tropoelastin mRNA. These results confirm that the C-terminus of tropoelastin interacts with microfibrils during the assembly of elastic fibres. Further, the results suggest that the interaction between tropoelastin and microfibrils might be mediated by a domain involving the N-terminal half of MAGP.

Increased serum levels of anti-elastin antibodies in patients with Peyronie's disease.

The cause of Peyronie's disease is unknown. Immunological mechanisms in the pathogenesis have been previously suggested. Antibodies to elastin are present in all individuals. However, abnormal serum levels of anti-tropoelastin (reflecting elastin synthesis) and anti-alpha-elastin (reflecting elastin destruction) are seen in a variety of autoimmune diseases. We show that patients with Peyronie's disease have higher levels of antibodies to tropoelastin (p < 0.047) and alpha-elastin (p < 0.012) than age-matched controls, suggesting an increase in elastin synthesis and breakdown, respectively. These findings suggest the presence of autoimmune mechanisms in the pathogenesis of Peyronie's disease, which may have future diagnostic and therapeutic implications.

Peptide sequences selected by BA4, a tropoelastin-specific monoclonal antibody, are ligands for the 67-kilodalton bovine elastin receptor.

A 67-kDa cell-surface elastin/laminin receptor is expressed by fetal bovine ligamentum nuchae fibroblasts and neutrophils. Two hexapeptides, VGVAPG and PGAIPG, contained within hydrophobic domains of tropoelastin are binding sites for this receptor. Studies of recombinant tropoelastin proteins and synthetic peptides demonstrated that a monoclonal antibody, BA4, recognized peptide sequences similar to those recognized by the 67-kDa receptor. Taking advantage of this similarity, an "epitope library" containing random hexapeptides was screened with BA4. Four BA4-selected peptides (VGAMPG, VGMAPG, VGSLPG, and VGLSPG) were synthesized; studies of fibroblast and neutrophil migration support the hypothesis that these peptides are ligands of the 67-kDa receptor present on ligamentum nuchae fibroblasts and neutrophils. Two additional, physically similar tropoelastin peptides,AGAIPG and PGAVGP, were also identified as peptide ligands, and hence potential binding sites within tropoelastin, of the elastin receptor. These data suggest that the 67-kDa elastin/laminin receptor may interact with a wide range of structurally similar peptides containing amino acid substitutions involving small nonpolar and uncharged amino acids.

Serum anti-tropo:anti-alpha-elastin antibody ratio assessing elastin turnover in scleroderma.

Serum antibodies to native (tropo) and denatured (alpha) elastins appear to correlate with the production and breakdown respectively of elastic tissue. Elastin may be degraded as a part of autoimmune diseases. This possibility was tested by measuring IgG antibodies to tropo- and alpha-elastins by ELISA in the sera of 111 patients with a variety of connective tissue diseases compared with 18 healthy individuals. Anti-alpha-elastin antibodies were significantly higher in sera from 18 scleroderma patients than from healthy controls (p less than 0.008). Conversely, anti-tropoelastin antibody levels for scleroderma patients (p less than 0.03) and for patients with a variety of other connective tissue diseases (p less than 0.02) were lower than in healthy controls. Low antibody levels to native elastin and high levels of antibodies to denatured elastin suggest a low synthesis: degradation ratio for elastin in scleroderma. Scleroderma may be a unique model for elastin turnover because of its heretofore unrecognized accelerated elastolysis.

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.

Predictions of the secondary structure and antigenicity of human and bovine tropoelastins.

Secondary structure and antigenicity predictive methods have been applied to the sequences of human and bovine tropoelastins in order to have some insight into the molecular structure of its insoluble counterpart, i.e., elastin. For both tropoelastins, all the predictions yielded 11 major regions, in which the pleated conformation was predominant, separated by 10 strong helical segments of various lengths located within alanyl rich regions of the chains. The overall conformations of human and bovine tropoelastins were estimated to contain 18 +/- 5% alpha-helices, 63 +/- 17% beta-sheets, 13 +/- 13% beta-turns and 6 +/- 6% random coil. For both tropoelastins, antigenicity predictions indicated the presence of seven synthetic decapeptides corresponding to continuous linear epitopes of the molecule. Some of the predicted epitopes are located in the same regions in both species while others are not. These predictions have allowed us to propose an alpha/beta conformation for tropoelastin. Therefore this extracellular matrix macromolecule might be more structured (10 helical segments for about 18% of the overall structure) than previously suggested.

Mapping of binding sites for monoclonal antibodies to chick tropoelastin by recombinant DNA techniques.

A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin.

Polyclonal antibodies to tropoelastin and the specific detection and measurement of tropoelastin in vitro.

Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays.

Immunohistochemical detection of intracellular tropoelastin: an assay for elastin production and its use in the detection and assessment of elastogenic factors.

Techniques are described for visualizing intracellular tropoelastin at the light level using immunofluorescence and immunogold techniques. Best results were obtained with B5 fixative on cells permeabilized with acetone. Using either formaldehyde or paraformaldehyde for fixation (instead of B5) resulted in both less reproducible and less intense intracellular staining, and permeabilization of the cells with ethanol resulted in relatively high background staining compared with that obtained with cold (-20 degrees C) acetone. Intracellular tropoelastin was seen most prominently in the perinuclear region, and the intensity of staining agreed with the reported rate of tropoelastin synthesis as assayed by enzyme-linked immunosorbent assay (ELISA) and RNA hybridization studies. The applicability of the intracellular staining technique for studying the elastin phenotype was tested by demonstrating increases in both the number of positive cells and in the intensity of elastin staining in cells treated with smooth muscle elastogenic factor (SMEF), an elastogenic factor known to stimulate elastin production.

Ultrastructural immunocytochemical localization of elastin in normal human trabecular meshwork.

Previous studies have suggested that hydrophobic moieties within the aqueous outflow channels might interact with certain aqueous components to retard outflow. While elastin is among the most hydrophobic proteins in the trabecular meshwork, it reacts poorly with conventional ultrastructural staining methods, so its potential role in regulating outflow could not be assessed. It was our goal to specifically localize elastin ultrastructurally using polyclonal antibodies against alpha elastin and its soluble precursor, tropoelastin. Human aorta served as a positive control. Preadsorption of the primary antibodies or their substitution with either normal rabbit serum or Tris buffer resulted in negligible labelling. With either antibody, only the electron-lucent elements in the center of elastic fibers of the trabecular meshwork were labelled, indicating that only these elements truly represent elastin. The pattern of elastin distribution within these fibers is most consistent with that found in tendons elsewhere in the body.

Characterization of elastin protein and mRNA from salmonid fish (Oncorhynchus kisutch).

1. Elastin was isolated from the bulbus arteriosus of a salmonid fish. Monoclonal and polyclonal antibodies, elicited against a CNBr digest of this protein, immunoprecipitated a polypeptide of Mr 43,000 from fish cell culture medium. 2. Cell-free translation of salmon poly A+ RNA produced a protein of approximately 43 kD that was immunoprecipitated with anti-elastin antibodies. The corresponding mRNA had an approximate Mr of 2 kb. 3. Despite similarities in amino acid composition, the differences in Mr between mammalian and salmon mRNA and protein suggest a divergence of fish and higher vertebrate elastins from an earlier ancestral gene.

Fine mapping of tropoelastin-derived components in the aorta of developing chick embryo.

Affinity-purified antitropoelastin antibodies have been used to localize tropoelastin-derived components in aortas from chick embryos of different age by immunoelectron microscopy. Staining in the matrix is first noted at day 3 associated with irregular bundles of filaments resembling microfibrils, in the absence of amorphous elastin deposits. Amorphous material, which rapidly accumulates at later stages, is heavily labelled, while surrounding microfibrils are only poorly labelled. By contrast, a more intense staining of microfibrils persists in regions in which amorphous material is not morphologically evident. These observations indicate that the initial accumulation of elastin requires microfibrils, while the two components are not in close association in the subsequent growth of the amorphous core of the fibre. Intracellular staining is evident in the secretory apparatus of the cell and in peripheral large vesicles. Differentiated cells also show regions of close contact with elastic fibres in which immunological staining for elastin is very close to the cell membrane.