- Email: [email protected]
Characterization of the C_terminus of mature elastin
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
strips was decreased when compared to WT controls. Although of lesser magnitude than the changes in ELN-deficient animals, the findings were none-the-less concordant. Thus, the data indicate that both quantitative and qualitative defects of ELN alter lung mechanical properties in a similar manner. doi:10.1016/j.matbio.2006.08.052
30 Initial mechanisms in fibrillin-1 assembly E. El-Hallous b, K. Tiedemann a, D. Hubmacher a, A. Weiss d, T. Sasaki c, E.C. Davis a, D.P. Reinhardt a a
University of Lübeck, Germany McGill University, Montreal, QC, Canada c Max-Planck-Institute of Biochemistry, Martinsried, Germany d University of Sydney, Australia b
Recombinant constructs of fibrillin-1 were produced in a mammalian expression system. The recombinant polypeptides were purified to homogeneity. Biochemical and ultrastructural characterization of these polypeptides indicate correct folding. Self-interaction assays of the recombinant polypeptides established that the first four N-terminal domains and the last three EGF domains of fibrillin-1 contain the entire or at least part of the self-assembly epitopes. The shape of the N-terminal region of fibrillin-1 is curved, a property which may be important for self-assembly. The N-to C-terminal self-assembly was inhibited by heparin/heparan sulfate demonstrating that the inhibition of fibrillin-1 network formation by heparin/ heparan sulfate published previously acts on the self-assembly level. Inhibition by NaCl demonstrated that the initial phases of self-assembly are of ionic nature. However, once when the fibrillin-1 complex has formed it cannot be dissociated by either high concentrations of NaCl or heparin/heparan sulfate. These data demonstrate that after the initial formation of hydrophilic interactions, other forces come into play. N-ethyl maleimide which is highly reactive with sulfhydryl groups was not able to interfere with the assembly process while the reducing agent dithiothreitol abolished self assembly. These results demonstrate that fibrillin-1 self-assembly at this early stage does not include a potential free cysteine reacting with another one. doi:10.1016/j.matbio.2006.08.053
31 MAGP-1: A novel member of the matricellular protein family J.S. Weinberg, C.C. Werneck, R.P. Mecham
S19
Department of Cell Biology and Physiology, Washington University School of Medicine Microfibril-associated glycoprotein-1 (MAGP-1) is an integral component of the 10–12 nm elastic fiber microfibril. As yet, no structural role has been determined for MAGP-1, although it has been localized to a prominent feature of the microfibril, the “bead”. To investigate whether MAGP-1 has biological function we have taken two approaches: we have produced recombinant full-length bovine MAGP-1 to be used in cell biological assays and have generated MAGP-1 deficient mice. We have thus far discovered two effects of recombinant MAGP-1 on fibroblasts. Soluble MAGP-1 can decrease adhesion of cells plated in the presence of serum and immobilized MAGP-1, while not by itself an adhesive substrate, can facilitate adhesion to fibronectin. Preliminarly results in the mouse have shown two healing defects in MAGP-1 deficient mice: they have delayed clot formation in response to endothelial damage and delayed healing of excisional skin wounds. Both the cell biological and organismal phenotypes found in these studies are reminiscent of findings with matricellular proteins such as thrombospondin-1 and -2 and SPARC. Therefore we suggest that MAGP-1 in the extracellular matrix has matricellular function. doi:10.1016/j.matbio.2006.08.054
32 Characterization of the C-terminus of mature elastin T.J. Broekelmann, C.H. Ciliberto, J.E. Wagenseil, A. Shifren, R.P. Mecham Cell biology and Physiology, Washington University Medical School, Saint Louis MO, United States A 25 residue synthetic peptide from the carboxyl-terminus of tropoelastin contains a specific cell interactive site capable of promoting adhesion with a wide variety of cell types. Heparan sulfate or chondroitin sulfate proteoglycans on the cell surface are sufficient to promote adhesion to this peptide independent of integrins. To determine if this region of tropoelastin exists in mature elastin we developed an antibody to the C-terminal peptide. The antibody is specific in western blots and only reacts with fragments that contain the C-terminus. By ELISA the antibody reacts with the bovine exon-36 peptide but not an exon35 peptide. This antibody recognizes soluble elastin fragments released after pepsin digestion of crude ligamentum nuchae and neutral salt purified elastin but not elastin purified by alkaline extraction. Amino acid analysis of performic acid oxidized neutral salt elastin shows proper levels of cystiene characteristic of exon 36. These results indicate exon 36 can be detected in mature elastin but can be lost with alkaline treatments. The addition of biotin labeled C-terminal peptide followed by visualization with alexa-
S20
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
488 labeled avidin in cell culture revealed an extracellular matrix pattern that co-localized with fibrillin I. Thus the C-terminus is capable of targeting to the matrix. We are currently using the antibody to the C-terminus and the biotin lableled C-terminal peptide for localization studies in tissue. doi:10.1016/j.matbio.2006.08.055
33 MAGP-1 and hemostasis C.C. Werneck, C.P. Vicente, T.J. Broekelmann, J.S. Weinberg, R.A. Pierce, D.M. Tollefsen, R.P. Mecham Washington University School of Medicine, Saint Louis, MO 63110, United States Microfibril-associated glycoprotein-1 (MAGP-1), a small extracellular glycoprotein (∼31KD), is one of the major components of elastin-containing microfibrils present along the arterial wall. Ross et al. have showed that microfibrils are capable of supporting platelet adhesion under dynamic shear conditions suggesting that microfibrillar components might be involved in hemostasis. To verify if MAGP-1 plays a role in this process we submitted MAGP-1KO mice to a photochemical injury thrombosis assay. In this assay, the endothelium from carotid is denuded leading to thrombus formation and vessel occlusion. The occlusion time obtained for MAGP1KO mice was almost double that of wild-type siblings. The coagulation cascade pathways, platelet number and function are normal. We were not able to detect MAGP-1 inside the platelets and MAGP-1 is incapable of activating platelets by itself or modulating their response in the presence of known agonists. MAGP-1 injection in deficient mice 15 minutes before starting the photochemical assay rescues the normal occlusion time. During earlier steps of thrombus formation, von Willebrand factor (vWF) has an important role binding to the exposed sub-endothelial components making platelets slow down, bind and become activated. We showed that vWF interacts with MAGP-1. Our current hypothesis is that MAGP-1 becomes exposed at the moment of the injury and might be working as an anchor, binding vWF and making the thrombus firm and compact at the site of injury. The mechanisms through which MAGP-1 are involved in thrombus formation are still under investigation. doi:10.1016/j.matbio.2006.08.056
34 ECM protein fibulin-5 and development of atherosclerosis S. Hacker a, P. Boucher b, J. Herz b, H. Yanagisawa
a
a
Molecular Biology, UT Southwestern Medical Center, Dallas, TX, United States b Molecular Genetics, UT Southwestern Medical Center, Dallas, TX, United States Atherosclerosis is a complex process involving endothelial cell dysfunction, production of superoxides, and activation of vascular smooth muscle cells (SMCs). The migration and proliferation of SMC and influx of macrophages and cholesterol molecules produces inflammation and damage to the elastic laminae. We have previously reported that mice deficient for the fibulin-5 gene (fbln5−/−) develop disorganized elastic laminae and fbln5−/− vessels display an exaggerated remodeling response to mechanical injury. We hypothesized that fbln5−/− mice would develop more severe atherosclerotic lesions due to the loss of intact elastic laminae. Fbln5−/− mice were mated to low-density lipoprotein receptor-null (ldlr−/−) mice and the progression of atherosclerotic lesions was compared between ldlr−/− and fbln5−/−; ldlr−/− (DKO) mice. Although we observed a significant increase in plasma cholesterol level in DKO mice after 8 weeks of high cholesterol diet, no difference was observed after 16 weeks of diet. Unexpectedly, whole-mount sudan IV staining revealed that DKO mice developed less severe atherosclerotic lesions compared to the ldlr−/− mice. Modest activation of the PDGFß receptormediated signaling pathway was observed in both genotypes. MMP2 activity was comparable between ldlr−/− and DKO mice. In contrast, MMP9 activity and SOD3 levels were altered in DKO mice. These data suggest that elastic laminae do not play a protective role in development of atherosclerosis, however, the cellular function of fibulin-5 needs to be further examined. doi:10.1016/j.matbio.2006.08.057
35 Fibulin-4 function in zebrafish A.B. Maxfield a, V. Hucthagowder a, J.D. Gitlin a, E.M. Joseph b, Z. Urban a a
Departments of Pediatrics and Genetics, Washington University School of Medicine, St. Louis, MO 63110, United States b Massachusetts General Hospital, Charlestown, MA 02129, United States Recently, a homozygous mutation in fibulin-4 has been linked to a severe connective-tissue disease. To elucidate the molecular mechanisms of this novel disease, a zebrafish model organism was chosen for study. In silico analysis of the zebrafish genome demonstrated that human FBLN4 has two paralogues in the zebrafish genome, fbln4a and fbln4b. Whole mount in situ hybrization analysis of zebrafish fbln4a and fbln4b displays a dynamic expression pattern during embryogenesis associated with the segmentation of the lateral mesoderm. Expression of fibulin 4a is then detectable in the bulbus arteriosus (outflow tract)
strips was decreased when compared to WT controls. Although of lesser magnitude than the changes in ELN-deficient animals, the findings were none-the-less concordant. Thus, the data indicate that both quantitative and qualitative defects of ELN alter lung mechanical properties in a similar manner. doi:10.1016/j.matbio.2006.08.052
30 Initial mechanisms in fibrillin-1 assembly E. El-Hallous b, K. Tiedemann a, D. Hubmacher a, A. Weiss d, T. Sasaki c, E.C. Davis a, D.P. Reinhardt a a
University of Lübeck, Germany McGill University, Montreal, QC, Canada c Max-Planck-Institute of Biochemistry, Martinsried, Germany d University of Sydney, Australia b
Recombinant constructs of fibrillin-1 were produced in a mammalian expression system. The recombinant polypeptides were purified to homogeneity. Biochemical and ultrastructural characterization of these polypeptides indicate correct folding. Self-interaction assays of the recombinant polypeptides established that the first four N-terminal domains and the last three EGF domains of fibrillin-1 contain the entire or at least part of the self-assembly epitopes. The shape of the N-terminal region of fibrillin-1 is curved, a property which may be important for self-assembly. The N-to C-terminal self-assembly was inhibited by heparin/heparan sulfate demonstrating that the inhibition of fibrillin-1 network formation by heparin/ heparan sulfate published previously acts on the self-assembly level. Inhibition by NaCl demonstrated that the initial phases of self-assembly are of ionic nature. However, once when the fibrillin-1 complex has formed it cannot be dissociated by either high concentrations of NaCl or heparin/heparan sulfate. These data demonstrate that after the initial formation of hydrophilic interactions, other forces come into play. N-ethyl maleimide which is highly reactive with sulfhydryl groups was not able to interfere with the assembly process while the reducing agent dithiothreitol abolished self assembly. These results demonstrate that fibrillin-1 self-assembly at this early stage does not include a potential free cysteine reacting with another one. doi:10.1016/j.matbio.2006.08.053
31 MAGP-1: A novel member of the matricellular protein family J.S. Weinberg, C.C. Werneck, R.P. Mecham
S19
Department of Cell Biology and Physiology, Washington University School of Medicine Microfibril-associated glycoprotein-1 (MAGP-1) is an integral component of the 10–12 nm elastic fiber microfibril. As yet, no structural role has been determined for MAGP-1, although it has been localized to a prominent feature of the microfibril, the “bead”. To investigate whether MAGP-1 has biological function we have taken two approaches: we have produced recombinant full-length bovine MAGP-1 to be used in cell biological assays and have generated MAGP-1 deficient mice. We have thus far discovered two effects of recombinant MAGP-1 on fibroblasts. Soluble MAGP-1 can decrease adhesion of cells plated in the presence of serum and immobilized MAGP-1, while not by itself an adhesive substrate, can facilitate adhesion to fibronectin. Preliminarly results in the mouse have shown two healing defects in MAGP-1 deficient mice: they have delayed clot formation in response to endothelial damage and delayed healing of excisional skin wounds. Both the cell biological and organismal phenotypes found in these studies are reminiscent of findings with matricellular proteins such as thrombospondin-1 and -2 and SPARC. Therefore we suggest that MAGP-1 in the extracellular matrix has matricellular function. doi:10.1016/j.matbio.2006.08.054
32 Characterization of the C-terminus of mature elastin T.J. Broekelmann, C.H. Ciliberto, J.E. Wagenseil, A. Shifren, R.P. Mecham Cell biology and Physiology, Washington University Medical School, Saint Louis MO, United States A 25 residue synthetic peptide from the carboxyl-terminus of tropoelastin contains a specific cell interactive site capable of promoting adhesion with a wide variety of cell types. Heparan sulfate or chondroitin sulfate proteoglycans on the cell surface are sufficient to promote adhesion to this peptide independent of integrins. To determine if this region of tropoelastin exists in mature elastin we developed an antibody to the C-terminal peptide. The antibody is specific in western blots and only reacts with fragments that contain the C-terminus. By ELISA the antibody reacts with the bovine exon-36 peptide but not an exon35 peptide. This antibody recognizes soluble elastin fragments released after pepsin digestion of crude ligamentum nuchae and neutral salt purified elastin but not elastin purified by alkaline extraction. Amino acid analysis of performic acid oxidized neutral salt elastin shows proper levels of cystiene characteristic of exon 36. These results indicate exon 36 can be detected in mature elastin but can be lost with alkaline treatments. The addition of biotin labeled C-terminal peptide followed by visualization with alexa-
S20
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
488 labeled avidin in cell culture revealed an extracellular matrix pattern that co-localized with fibrillin I. Thus the C-terminus is capable of targeting to the matrix. We are currently using the antibody to the C-terminus and the biotin lableled C-terminal peptide for localization studies in tissue. doi:10.1016/j.matbio.2006.08.055
33 MAGP-1 and hemostasis C.C. Werneck, C.P. Vicente, T.J. Broekelmann, J.S. Weinberg, R.A. Pierce, D.M. Tollefsen, R.P. Mecham Washington University School of Medicine, Saint Louis, MO 63110, United States Microfibril-associated glycoprotein-1 (MAGP-1), a small extracellular glycoprotein (∼31KD), is one of the major components of elastin-containing microfibrils present along the arterial wall. Ross et al. have showed that microfibrils are capable of supporting platelet adhesion under dynamic shear conditions suggesting that microfibrillar components might be involved in hemostasis. To verify if MAGP-1 plays a role in this process we submitted MAGP-1KO mice to a photochemical injury thrombosis assay. In this assay, the endothelium from carotid is denuded leading to thrombus formation and vessel occlusion. The occlusion time obtained for MAGP1KO mice was almost double that of wild-type siblings. The coagulation cascade pathways, platelet number and function are normal. We were not able to detect MAGP-1 inside the platelets and MAGP-1 is incapable of activating platelets by itself or modulating their response in the presence of known agonists. MAGP-1 injection in deficient mice 15 minutes before starting the photochemical assay rescues the normal occlusion time. During earlier steps of thrombus formation, von Willebrand factor (vWF) has an important role binding to the exposed sub-endothelial components making platelets slow down, bind and become activated. We showed that vWF interacts with MAGP-1. Our current hypothesis is that MAGP-1 becomes exposed at the moment of the injury and might be working as an anchor, binding vWF and making the thrombus firm and compact at the site of injury. The mechanisms through which MAGP-1 are involved in thrombus formation are still under investigation. doi:10.1016/j.matbio.2006.08.056
34 ECM protein fibulin-5 and development of atherosclerosis S. Hacker a, P. Boucher b, J. Herz b, H. Yanagisawa
a
a
Molecular Biology, UT Southwestern Medical Center, Dallas, TX, United States b Molecular Genetics, UT Southwestern Medical Center, Dallas, TX, United States Atherosclerosis is a complex process involving endothelial cell dysfunction, production of superoxides, and activation of vascular smooth muscle cells (SMCs). The migration and proliferation of SMC and influx of macrophages and cholesterol molecules produces inflammation and damage to the elastic laminae. We have previously reported that mice deficient for the fibulin-5 gene (fbln5−/−) develop disorganized elastic laminae and fbln5−/− vessels display an exaggerated remodeling response to mechanical injury. We hypothesized that fbln5−/− mice would develop more severe atherosclerotic lesions due to the loss of intact elastic laminae. Fbln5−/− mice were mated to low-density lipoprotein receptor-null (ldlr−/−) mice and the progression of atherosclerotic lesions was compared between ldlr−/− and fbln5−/−; ldlr−/− (DKO) mice. Although we observed a significant increase in plasma cholesterol level in DKO mice after 8 weeks of high cholesterol diet, no difference was observed after 16 weeks of diet. Unexpectedly, whole-mount sudan IV staining revealed that DKO mice developed less severe atherosclerotic lesions compared to the ldlr−/− mice. Modest activation of the PDGFß receptormediated signaling pathway was observed in both genotypes. MMP2 activity was comparable between ldlr−/− and DKO mice. In contrast, MMP9 activity and SOD3 levels were altered in DKO mice. These data suggest that elastic laminae do not play a protective role in development of atherosclerosis, however, the cellular function of fibulin-5 needs to be further examined. doi:10.1016/j.matbio.2006.08.057
35 Fibulin-4 function in zebrafish A.B. Maxfield a, V. Hucthagowder a, J.D. Gitlin a, E.M. Joseph b, Z. Urban a a
Departments of Pediatrics and Genetics, Washington University School of Medicine, St. Louis, MO 63110, United States b Massachusetts General Hospital, Charlestown, MA 02129, United States Recently, a homozygous mutation in fibulin-4 has been linked to a severe connective-tissue disease. To elucidate the molecular mechanisms of this novel disease, a zebrafish model organism was chosen for study. In silico analysis of the zebrafish genome demonstrated that human FBLN4 has two paralogues in the zebrafish genome, fbln4a and fbln4b. Whole mount in situ hybrization analysis of zebrafish fbln4a and fbln4b displays a dynamic expression pattern during embryogenesis associated with the segmentation of the lateral mesoderm. Expression of fibulin 4a is then detectable in the bulbus arteriosus (outflow tract)