Dataset Information

Flagellar display of bone-protein-derived peptides for studying peptide-mediated biomineralization.

ABSTRACT: A bacterial flagellum is self-assembled primarily from thousands of flagellin (FliC), a protein subunit. A foreign peptide can be fully displayed on the surface of the flagellum through inserting it into every constituent protein subunit. To shed light on the role of bone proteins during the nucleation of hydroxyapatite (HAP), representative domains from type I collagen, including part of the N-,C-terminal, N-,C-zone around the hole zone and an eight repeat unit Gly-Pro-Pro (GPP8) sequence similar to the central sequence of type I collagen, were separately displayed on the surface of the flagella. Moreover, eight negatively charged, contiguous glutamic acid residues (E8) and two other characteristic sequences derived from a representative noncollagenous protein called bone sialoprotein (BSP) were also displayed on flagella. After being incubated in an HAP supersaturated precursor solution, flagella displaying E8 or GPP8 sequences were found to be coated with a layer of HAP nanocrystals. Very weak or no nucleation was observed on flagella displaying other peptides being tested. We also found that calcium ions can induce the assembly of the negatively charged E8 flagella into bundles mimicking collagen fibers, followed by the formation of HAP nanocrystals with the crystallographic c axis preferentially aligned with long axis of flagella, which is similar to that along the collagen fibrils in bone. This work demonstrates that because of the ease of the peptide display on flagella and the self-assembly of flagella, flagella can serve as a platform for studying biomineralization and as a building block to generate bonelike biomaterials.

SUBMITTER: Li D

PROVIDER: S-EPMC3508360 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Flagellar display of bone-protein-derived peptides for studying peptide-mediated biomineralization.

Li Dong D Newton Salete M C SM Klebba Philip E PE Mao Chuanbin C

Langmuir : the ACS journal of surfaces and colloids 20121114 47

A bacterial flagellum is self-assembled primarily from thousands of flagellin (FliC), a protein subunit. A foreign peptide can be fully displayed on the surface of the flagellum through inserting it into every constituent protein subunit. To shed light on the role of bone proteins during the nucleation of hydroxyapatite (HAP), representative domains from type I collagen, including part of the N-,C-terminal, N-,C-zone around the hole zone and an eight repeat unit Gly-Pro-Pro (GPP8) sequence simil ...[more]

PMID: 23148645

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Project description:PURPOSE:Osteosarcoma represents the most common malignant primary bone tumor in childhood; however, the survival rate has remained unchanged for the past 20 years. To improve existing diagnosis and treatment methods and broaden the spectrum of imaging agents that can be used for early detection and assessment of tumor response to therapy, we performed a phage display-based screening for peptide sequences that bind specifically to osteosarcoma cells. EXPERIMENTAL DESIGN:From the Ph.D.-12 phage display peptide library composed of 2.7 x 10(9) different displayed peptides, one peptide was enriched after four rounds of in vitro selection in 143B osteosarcoma tumor cells with 293T human embryonic kidney cells as a control. Both the peptide and the phage clone displaying the peptide were conjugated with fluorescent dyes for in vitro cell and ex vivo tumor tissue stainings. The peptide was further labeled with (18)F for positron emission tomography imaging studies. Cell uptake and efflux and ex vivo biodistribution were also done with (18)F-labeled osteosarcoma specific peptide. RESULTS:ASGALSPSRLDT was the dominant sequence isolated from biopanning and named as OSP-1. OSP-1 shares a significant homology with heparinase II/III family protein, which binds and reacts with heparan sulfate proteoglycans. The fluorescence staining showed that FITC-OSP-1-phage or Cy5.5-OSP-1 had high binding with a panel of osteosarcoma cell lines, much less binding with UM-SCC1 human head and neck squamous cell carcinoma cells, and almost no binding with 293T cells, whereas the scrambled peptide OSP-S had virtually no binding to all the cell lines. (18)F-OSP-1 had significantly higher accumulation in 143B tumor cells both in vitro and in vivo than (18)F-OSP-S. (18)F-OSP-1 also had higher uptake in 143B tumors than in UM-SCC-1 tumors. CONCLUSIONS:Our data suggest that OSP-1 peptide is osteosarcoma specific, and the binding site of OSP-1 might be related to heparan sulfate proteoglycans. Appropriately labeled OSP-1 peptide has the potential to serve as a novel probe for osteosarcoma imaging.

Project description:BACKGROUND:Signal transduction events often involve transient, yet specific, interactions between structurally conserved protein domains and polypeptide sequences in target proteins. The identification and validation of these associating domains is crucial to understand signal transduction pathways that modulate different cellular or developmental processes. Bioinformatics strategies to extract and integrate information from diverse sources have been shown to facilitate the experimental design to understand complex biological events. These methods, primarily based on information from high-throughput experiments, have also led to the identification of new connections thus providing hypothetical models for cellular events. Such models, in turn, provide a framework for directing experimental efforts for validating the predicted molecular rationale for complex cellular processes. In this context, it is envisaged that the rational design of peptides for protein-peptide binding studies could substantially facilitate the experimental strategies to evaluate a predicted interaction. This rational design procedure involves the integration of protein-protein interaction data, gene ontology, physico-chemical calculations, domain-domain interaction data and information on functional sites or critical residues. RESULTS:Here we describe an integrated approach called "PeptideMine" for the identification of peptides based on specific functional patterns present in the sequence of an interacting protein. This approach based on sequence searches in the interacting sequence space has been developed into a webserver, which can be used for the identification and analysis of peptides, peptide homologues or functional patterns from the interacting sequence space of a protein. To further facilitate experimental validation, the PeptideMine webserver also provides a list of physico-chemical parameters corresponding to the peptide to determine the feasibility of using the peptide for in vitro biochemical or biophysical studies. CONCLUSIONS:The strategy described here involves the integration of data and tools to identify potential interacting partners for a protein and design criteria for peptides based on desired biochemical properties. Alongside the search for interacting protein sequences using three different search programs, the server also provides the biochemical characteristics of candidate peptides to prune peptide sequences based on features that are most suited for a given experiment. The PeptideMine server is available at the URL: http://caps.ncbs.res.in/peptidemine.

Dataset Information

Flagellar display of bone-protein-derived peptides for studying peptide-mediated biomineralization.

Publications

Flagellar display of bone-protein-derived peptides for studying peptide-mediated biomineralization.

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