Self-assembling multimeric integrin  5 1 ligands for cell attachment and spreading

Kreiner, M.; Li, Z.; Beattie, James; Kelly, Sharon M.; Mardon, Helen J.; Walle, Christopher F. van der

doi:10.1093/protein/gzn032

Cited by 18 publications

(24 citation statements)

References 39 publications

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“…The calculated, theoretical sedimentation coefficients, computed for the bead-models, are summarised in Table 1 6 S). However, the existence of a pentameric species for FIII9'-10-trimer is inconsistent with the gel filtration experiments previously performed [12]. An alternative hypothesis is that this species is a homodimer of two FIII9'-10-trimeric species, interacting either via the polyhistidine tags [22] or an inter-oligomer disulphide bond via oxidation of two C-terminal cysteines.…”

Section: Variables Affecting the Oligomerisation State Of The Ligandsmentioning

confidence: 60%

“…Three chimeras were made: FIII9'-10-dimer, FIII9'-10-trimer and FIII9'-10-tetramer, whose construction and expression have been described previously [12]. General chemical reagents were sourced from Sigma-Aldrich, UK, at analytical standard.…”

Section: Methodsmentioning

confidence: 99%

“…Nevertheless, the oligomeric ligands in this study appear to have stability at least equivalent to the wild type GCN4 peptide. Importantly, the ligands would therefore remain in their oligomeric form over the range of concentrations used in cell adhesion and spreading assays [12].…”

Section: The Ligands Self-assemble To Form Stable Oligomersmentioning

confidence: 99%

“…However, the intensity of the α-helical band will be compromised by the unusual positive CD signal of FIII9'-10 at 226 nm, representing aromatic contributions in the far-UV [29]. (CD scans for the ligands in the far and near UV regions in denaturing and nondenaturing buffer have been described previously [12].) To avoid contribution from FIII9'-10 aromatic residues in the far-UV, all experiments were performed in the presence of 6 M GdnHCl.…”

Section: The Oligomerised Ligands Are Further Stabilised By Disulphidmentioning

confidence: 99%

“…In contrast, we have designed much smaller and more flexible, clustered α5β1 integrin ligands consisting of: the 9 th -10 th type III FN domain pair (FIII9'-10, a L1408P mutant with increased conformational stability and cell adhesion activity [10]), a spacer derived from the IgGhinge (previously shown to be sufficient to prevent steric clashes between bound integrins [11]) and five heptad repeats based on the GCN4 leucine zipper (with isoleucine and leucine variously placed in positions a and d to promote di-, tri-and tetramerisation [4]). For controlled immobilisation of these chimeras to a substrate or surface a unique C-terminal cysteine was introduced to facilitate either selective biotinylation via a thioether bond, followed by binding to avidin-coated surfaces, or direct covalent binding via the free thiol to gold surfaces [12].…”

Section: Introductionmentioning

confidence: 99%

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Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands

Kreiner

Byron

Domingues

et al. 2009

Biophysical Chemistry

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Please cite this article as: Michaela Kreiner, Olwyn Byron, Diana Domingues, Christopher F. van der Walle, Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands, Biophysical Chemistry (2009Chemistry ( ), doi: 10.1016Chemistry ( /j.bpc.2009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractSynthetic oligomeric integrin α5β1 ligands, specifically immobilised to surfaces, facilitate increased fibroblast cell spreading compared with that associated with the monomer. These ligands consist of a N-terminal fibronectin domain pair, a spacer and a di-, tri-or tetrameric coiled coil. However, itis not yet clear what effect fusion of the fibronectin domains has on the predicted oligomerisation of the coiled coils. Using analytical ultracentrifugation we show that the predicted tetrameric and trimeric coiled coils facilitate a corresponding ligand oligomerisation with half-dissociation at 0.7 and 0.2 µM, respectively. In contrast, the predicted dimeric coiled coil formed both dimers and trimers. Under non-reducing conditions, the unique C-terminal thiol-facilitated inter-oligomer dimerisation of the trimeric species, generating hexameric ligands. Disulphide bonding also increased helical stability during thermal unfolding. The work allows the cellular response to these clustered integrin α5β1 ligands to be more accurately interpreted, and has wider implications with respect to the utility of coiled coils as tools to facilitate protein oligomerisation.

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Section: Variables Affecting the Oligomerisation State Of The Ligandsmentioning

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Section: The Ligands Self-assemble To Form Stable Oligomersmentioning

confidence: 99%

Section: The Oligomerised Ligands Are Further Stabilised By Disulphidmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands

Kreiner

Byron

Domingues

et al. 2009

Biophysical Chemistry

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Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin‐Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration

Karimi

O’Connor

Qiao

et al. 2018

Adv Healthcare Materials

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Material systems that exhibit tailored interactions with cells are a cornerstone of biomaterial and tissue engineering technologies. One method of achieving these tailored interactions is to biofunctionalize materials with peptide ligands that bind integrin receptors present on the cell surface. However, cell biology research has illustrated that both integrin binding and integrin clustering are required to achieve a full adhesion response. This biophysical knowledge has motivated researchers to develop material systems biofunctionalized with nanoscale clusters of ligands that promote both integrin occupancy and clustering of the receptors. These materials have improved a wide variety of biological interactions in vitro including cell adhesion, proliferation, migration speed, gene expression, and stem cell differentiation; and improved in vivo outcomes including increased angiogenesis, tissue healing, and biomedical device integration. This review first introduces the techniques that enable the fabrication of these nanopatterned materials, describes the improved biological effects that have been achieved, and lastly discusses the current limitations of the technology and where future advances may occur. Although this technology is still in its nascency, it will undoubtedly play an important role in the future development of biomaterials and tissue engineering scaffolds for both in vitro and in vivo applications.

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Perfusion culture enhanced human endometrial stromal cell growth in alginate‐multivalent integrin α5β1 ligand scaffolds

Li¹,

Kreiner²,

Edrada-Ebel³

et al. 2011

J Biomedical Materials Res

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A method to functionalize alginate by introducing monomeric or self-assembling (tetrameric) fibronectin (FN) domains is described, leading to a functional scaffold, which is used for three dimensional (3D) culture of human endometrial stromal cells (EnSCs). EnSCs encapsulated in the functional alginate were cultured under perfusion using the TissueFlex® platform, a multiple parallel microbioreactor system for 3D cell culture. The effect of the novel scaffold and the effect of perfusion were examined. Cell viability, proliferation, and extracellular matrix (ECM) deposition were determined and the results compared with those obtained with cells encapsulated in non-functionalized alginate, and also those without perfusion. Staining for focal adhesions and actin showed maximal cell adhesion only for alginate-tetrameric FN scaffolds under perfusion, associated with a significant increase in cell number over 7 days culture; in contrast to poor cell adhesion and a decrease in cell number for non-functionalized alginate scaffolds (irrespective of perfused/static culture) and 3D static culture (irrespective of the scaffold). Conjugation of alginate to FN was an absolute requirement to attenuate the loss of cell metabolic activity over 7 days culture. ECM deposition for blank alginate and alginate-monomeric FN was similar, but increased around 2-fold and 3-fold for alginate-tetrameric FN under static and perfusion culture, respectively. It is concluded that the requirement for EnSC engagement with multivalent integrin α5β1 ligands and perfused culture are both essential as a first step toward endometrial tissue engineering.

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Self-assembling multimeric integrin 5 1 ligands for cell attachment and spreading

Cited by 18 publications

References 39 publications

Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands

Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands

Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin‐Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration

Perfusion culture enhanced human endometrial stromal cell growth in alginate‐multivalent integrin α5β1 ligand scaffolds

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