Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells

Wachi, Hiroshi; Sato, Fumiaki; Murata, Hayato; Nakazawa, Junji; Starcher, Barry; Seyama, Yousuke

doi:10.1016/j.clinbiochem.2005.04.006

Cited by 29 publications

(35 citation statements)

References 30 publications

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“…In the present study, we demonstrated the importance of domain 36 of tropoelastin for elastic fiber assembly using our established in vitro model, 15) which enables the quantitative comparison of various tropoelastin molecules.…”

Section: )mentioning

confidence: 99%

“…15) In order to generate the C-terminal mutants, bovine elastin gene templates were amplified using the primers listed in Table 1 (BTEex2S was commonly used, all mutant, anti-sense primers were used: C750/755-AS for C750/755 A, RK-AS for ΔRKRK and Ex35AS for Δ36). These products were inserted into a bacterial expression pTrcHis-TOPO vector (Invitrogen).…”

Section: Purification Of Recombinant Tropoelastin Moleculesmentioning

confidence: 99%

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Domain 36 of Tropoelastin in Elastic Fiber Formation

Nonaka

Sato

Wachi

2014

Biological & Pharmaceutical Bulletin

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Elastic fiber assembly is a complex stepwise process involving multiple different proteins and enzymes. Domain 36, encoded by the last exon of the elastin gene, is recognized to be an important domain for deposition onto microfibrils, an essential step in elastic fiber assembly. However, the role of domain 36 in elastic fiber assembly has not been clarified. Here, we utilized our established in vitro assembly model to identify the importance of domain 36 for the assembly process. Our results showed that the lack of domain 36 in bovine tropoelastin results in deficient elastic fiber assembly. A similar result was obtained with the point mutation of two cysteine residues and the deletion of the Lysine-Arginine-Lysine-Arginine (RKRK) sequence in domain 36. Double immunofluorescence of tropoelastin and fibrillin-1, a main component of microfibrils, demonstrated reduced localization of these mutant tropoelastin molecules on fibrillin-1 fibers. Moreover, the binding affinity of these mutants to fibrillin-1 and microfibril-associated glycoprotein (MAGP) was significantly decreased. These data indicate that domain 36 of tropoelastin facilitates elastic fiber assembly by interacting with microfibrils via two cysteine residues and the RKRK sequence.

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Section: )mentioning

confidence: 99%

Section: Purification Of Recombinant Tropoelastin Moleculesmentioning

confidence: 99%

Domain 36 of Tropoelastin in Elastic Fiber Formation

Nonaka

Sato

Wachi

2014

Biological & Pharmaceutical Bulletin

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“…13) In the present study, we investigated the elastic fiber formation of the frameshift-mutated tropoelastin (fmTE) using an in vitro model of elastic fiber assembly and biochemical analysis for the deposition of tropoelastin, the cross-linked amino acids, and the molecular interactions between tropoelastin and scaffold proteins such as fibulin-5 or fibrillin-1 (FBN-1).…”

Section: )mentioning

confidence: 99%

“…The bacteria were treated with isopropyl-β-D-thiogalactoside (IPTG) to induce the expression of recombinant tropoelastin and were then purified as described previously. 13) Purified nTE and fmTE were verified by Western blot analysis. Immunofluorescence, Semi-Quantitative ELISA, and Determination of Cross-Linked Amino Acids into the Cell Matrix ---The methods used for the immunofluorescence study, ELISA, and determination of the desmosine content have been described previously.…”

Section: )mentioning

confidence: 99%

“…Immunofluorescence, Semi-Quantitative ELISA, and Determination of Cross-Linked Amino Acids into the Cell Matrix ---The methods used for the immunofluorescence study, ELISA, and determination of the desmosine content have been described previously. 13) For the immunofluorescence assay, ARPE-19 cells were plated at a density of 5 × 10 4 cells on eight-well LabTek chamber slides (Nunc no. 177445, Fisher Scientific, Pittsburgh, PA, U.S.A.) in dulbecco's modified eagle medium (DMEM) supplemented with L-glutamine and penicillin/streptomycin and 10% cosmic calf serum (CCS).…”

Section: )mentioning

confidence: 99%

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Biochemical Analysis of Elastic Fiber Formation with a Frameshift-Mutated Tropoelastin (fmTE) at the C-Terminus of Tropoelastin

Sato

Wachi

Starcher

et al. 2006

JOURNAL OF HEALTH SCIENCE

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Elastic fibers play an important role in characteristic elastic properties of tissues such as skin, lungs, ligaments, and blood vessels. Many of the disorders related to elastic fibers are due to errors in assembly, yet the basic mechanisms of this process remain unclear. Cutis laxa, which is caused by an elastin gene mutation, includes a rare and heterogeneous group of the disorders characterized by lax, inelastic skin. To investigate whether elastic fiber assembly insufficiency is a possible factor in cutis laxa, we compared normal tropoelastin (nTE) and frameshift-mutated tropoelastin (fmTE) from a patient with autosomal dominant cutis laxa using an in vitro elastic fiber assembly model, in which purified recombinant tropoelastin was added to the culture medium. Assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Immunofluorescence microscopy indicated an approximate 50% decrease in the deposition of fmTE in the extracelluar matrix, in contrast to that of nTE. The amount of cross-linked amino acids unique to mature insoluble elastin in the fmTE assembly was also decreased by approximately 20%. In addition, we clarified that the molecular interaction between fmTE and the amino-terminal domain of fibrillin-1 or full-length fibulin-5 was significantly decreased. Our results suggest that the defect of fmTE fiber assembly is due, at least in part, to decreased molecular interactions between tropoelastin and the microfibrillar components fibulin-5 and fibrillin.

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New insights into the pathogenesis of autosomal‐dominant cutis laxa with report of fiveELNmutations

et al. 2011

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Autosomal dominant cutis laxa (ADCL) is characterized by a typical facial appearance and generalized loose skin folds, occasionally associated with aortic root dilatation and emphysema. We sequenced exons 28-34 of the ELN gene in 5 probands with ADCL features and found 5 de novo heterozygous mutations: c.2296_2299dupGCAG (CL-1), c.2333delC (CL-2), c.2137delG (CL-3), c.2262delA (monozygotic twin CL-4 and CL-5) and c.2124del25 (CL-6). Four probands (CL-1, -2, -3, -6) presented with progressive aortic root dilatation. CL-2 and CL-3 also had bicuspid aortic valves. CL-2 presented with severe emphysema. Electron microscopy revealed elastic fiber fragmentation and diminished dermal elastin deposition. RT-PCR studies showed stable mutant mRNA in all patients. Exon 32 skipping explains a milder phenotype in patients with exon 32 mutations. Mutant protein expression in fibroblast cultures impaired deposition of tropoelastin onto microfibril-containing fibers, and enhanced tropoelastin coacervation and globule formation leading to lower amounts of mature, insoluble elastin. Mutation-specific effects also included endoplasmic reticulum stress and increased apoptosis. Increased pSMAD2 staining in ADCL fibroblasts indicated enhanced transforming growth factor beta (TGFβ) signaling. We conclude that ADCL is a systemic disease with cardiovascular and pulmonary complications,

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Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells

Cited by 29 publications

References 30 publications

Domain 36 of Tropoelastin in Elastic Fiber Formation

Domain 36 of Tropoelastin in Elastic Fiber Formation

Biochemical Analysis of Elastic Fiber Formation with a Frameshift-Mutated Tropoelastin (fmTE) at the C-Terminus of Tropoelastin

New insights into the pathogenesis of autosomal‐dominant cutis laxa with report of fiveELNmutations

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