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Transcriptome-based systematic identification of extracellular matrix proteins.

ABSTRACT: Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs.

SUBMITTER: Manabe R

PROVIDER: S-EPMC2529034 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Transcriptome-based systematic identification of extracellular matrix proteins.

Manabe Ri-ichiroh R Tsutsui Ko K Yamada Tomiko T Kimura Mina M Nakano Itsuko I Shimono Chisei C Sanzen Noriko N Furutani Yutaka Y Fukuda Tomohiko T Oguri Yasuko Y Shimamoto Keiko K Kiyozumi Daiji D Sato Yuya Y Sado Yoshikazu Y Senoo Haruki H Yamashina Shohei S Fukuda Shiro S Kawai Jun J Sugiura Nobuo N Kimata Koji K Hayashizaki Yoshihide Y Sekiguchi Kiyotoshi K

Proceedings of the National Academy of Sciences of the United States of America 20080829 35

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computationa ...[more]

PMID: 18757743

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Project description:We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.

Project description:1. Investigations were designed to identify the proteins which characterize the ameloblast phenotype, and to determine to what extent these extracellular-matrix proteins were degraded as a function of enamel matrix mineralization and maturation. 2. The identification of enamel proteins was based on comparisons between the electrophoretic patterns of enamel-containing and non-enamel-containing matrix extracts isolated from specific regions within 26-day embryonic New Zealand White rabbit incisor and molar tooth organs. 3. Since enamel proteins become mineralized on secretion, matrix specimens were demineralized in cold 5% (w/v) trichloroacetic acid, extracted with buffered 6M-urea and reduced with mercaptoethanol, and then the solubilized proteins were fractionated by urea/polyacrylamide-gel electrophoresis. 4. Three enamel-specific electrophoretic components were identified in newly secreted enamel-matrix specimens and this number increased as a function of mineralization and maturation. 5. Antibodies were prepared against embryonic rabbit extracellular matrix containing enamel. Comparison between immunoelectrophoretic patterns demonstrated that two of the three enamel components were antigenic. 6. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate was used to identify four enamel proteins of mol.wts. (1) 65 000 (2) 58000 (3) 22 000 and (4) 20 000, localized within enamel matrix. Enamel proteins (1) and (3) were phosphorylated, whereas (2) and (4) did not contain detectable phosphate. Labelled proline, leucine, tryptophan and glucosamine were incorporated into each of the four enamel proteins extracted from tooth explants incubated in the presence of radioactive precursors for 6 h. Whereas four proteins were identified in newly secreted enamel matrix, the concentrations of high-molecular-weight proteins (1) and (2) were found to decrease and the number (greater than 10) and concentration of low-molecular-weight polypeptides increased as a function of advanced enamel-matrix mineralization and maturation.

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Transcriptome-based systematic identification of extracellular matrix proteins.

Publications

Transcriptome-based systematic identification of extracellular matrix proteins.

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