Fibrillin-1: Organization in Microfibrils and Structural Properties

Reinhardt, Dieter P.; Keene, Douglas R.; Corson, Glen M.; Pöschl, Ernst; Bächinger, Hans Peter; Gambee, Jay E.; Sakai, Lynn Y.

doi:10.1006/jmbi.1996.0237

Cited by 235 publications

(247 citation statements)

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“…Recombinant fibrillin-1 peptides were purified from culture supernatants under non-denaturing conditions and were considered >95% homogeneous based on their appearance in SDS polyacrylamide gel electrophoresis. The correct structure was verified by Edman degradation, glycosylation analysis, rotary shadowing, analysis of cysteines, and by functional characterization, including Ca 2+ binding and binding of monoclonal antibodies [19].…”

Section: Recombinant Production Of Human Fibrillin-1 Fragmentsmentioning

confidence: 95%

“…The construction of expression vectors from human fibrillin-1 cDNA, the transfection of human fibrosarcoma HT1080 or embryonic kidney 293 cells and the purification and functional characterization of the recombinant proteins have been described in detail elsewhere [19]. Recombinant fibrillin-1 peptides were purified from culture supernatants under non-denaturing conditions and were considered >95% homogeneous based on their appearance in SDS polyacrylamide gel electrophoresis.…”

Section: Recombinant Production Of Human Fibrillin-1 Fragmentsmentioning

confidence: 99%

“…These human fibrillin-1 peptides were produced in a mammalian expression system and purified under non-denaturing conditions [19]. Extensive characterization indicated correct folding [19]. Using an experimental approach already applied to characterize cell-adhesive and integrin-binding functions of fibulins [20], laminin [21] and collagen VI [22], we demonstrate the ability of cells to adhere to fibrillin-1 via an RGD motif located in the fourth 8-cysteine domain.…”

mentioning

confidence: 98%

“…necessitates the use of buffers containing denaturing agents and cannot separate fibrillin-1 and fibrillin-2 [18], we chose to use recombinant human fibrillin-1 polypeptides for a study of possible cell-adhesive functions. These human fibrillin-1 peptides were produced in a mammalian expression system and purified under non-denaturing conditions [19]. Extensive characterization indicated correct folding [19].…”

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confidence: 99%

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Cell adhesion and integrin binding to recombinant human fibrillin‐1

et al. 1996

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Fibrillin-1 is a major constituent of tissue microfibrfls that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell-adhesive functions of this protein, we used recombinant human fibrlllin-1 polypeptides produced in a mammalian expression system in cell attachment and solid-phase integrin binding assays. necessitates the use of buffers containing denaturing agents and cannot separate fibrillin-1 and fibrillin-2 [18], we chose to use recombinant human fibrillin-1 polypeptides for a study of possible cell-adhesive functions. These human fibrillin-1 peptides were produced in a mammalian expression system and purified under non-denaturing conditions [19]. Extensive characterization indicated correct folding [19]. Using an experimental approach already applied to characterize cell-adhesive and integrin-binding functions of fibulins [20], laminin [21] and collagen VI [22], we demonstrate the ability of cells to adhere to fibrillin-1 via an RGD motif located in the fourth 8-cysteine domain. In addition we identify integrin aV[33 as a major cellular receptor for this site.

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Section: Recombinant Production Of Human Fibrillin-1 Fragmentsmentioning

confidence: 95%

Section: Recombinant Production Of Human Fibrillin-1 Fragmentsmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

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Cell adhesion and integrin binding to recombinant human fibrillin‐1

et al. 1996

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“…These large, modular glycoproteins are highly homologous, consisting of repeating calcium-binding epidermal growth factor-like domains interspersed between unique 8-cysteine domains. The fibrillins are thought to create the core structure of microfibrils through a head-totail alignment of monomer bundles (6). Ultrastructurally, microfibrils are characterized by a "beads on a string" appearance, with fibrillin molecules contributing to both the bead and interbead regions.…”

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confidence: 99%

Posttranslational Modifications of Microfibril Associated Glycoprotein-1 (MAGP-1)

et al. 2001

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Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components.To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

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Calcium‐Binding EGF ‐Like Domains

Boswell¹,

Kurniawan²,

Downing³

2004

Handbook of Metalloproteins

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Epidermal growth factor‐like (EGF) domains are one of the more abundant modules found in functionally diverse extracellular proteins. The domain is characterized by six cysteine residues, which normally disulphide bond in a 1–3, 2–4, 5–6 pattern. A particular subset (cbEGF) contains the consensus sequence [DEQN]‐x‐[DEQN] 2 ‐x m ‐[DN]*‐x n ‐[YF] (where m and n are variables and * indicates possible β‐hydroxylation) associated with the ability to bind calcium. At present, cbEGFs have three main functional roles, as a spacer unit, in protein–protein interactions, and in structural stabilization. High‐resolution structures of cbEGF domains have identified a common fold, comprising a major and a minor double‐stranded β‐hairpin, stabilized by the three consensus disulphide bonds. A single calcium ion binds to the amino terminus and is coordinated by a pentagonal bipyramidal arrangement of oxygen atoms. Each individual cbEGF domain contains a calcium‐binding site, whose affinity may be considerably enhanced by N‐terminal linkage to another domain. A number of inherited diseases caused by missense mutations within cbEGFs highlight the importance of the domain.

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Fibrillin-1: Organization in Microfibrils and Structural Properties

Cited by 235 publications

References 0 publications

Cell adhesion and integrin binding to recombinant human fibrillin‐1

Cell adhesion and integrin binding to recombinant human fibrillin‐1

Posttranslational Modifications of Microfibril Associated Glycoprotein-1 (MAGP-1)

Calcium‐Binding EGF ‐Like Domains

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