scholarly journals The bspA Locus of Lactobacillus fermentum BR11 Encodes an l-Cystine Uptake System

1999 ◽  
Vol 181 (7) ◽  
pp. 2192-2198 ◽  
Author(s):  
Mark S. Turner ◽  
Tonia Woodberry ◽  
Louise M. Hafner ◽  
Philip M. Giffard
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Binding Proteins ◽  
Type I Collagen ◽  
Lactobacillus Reuteri ◽  
Binding Protein ◽  
Lactobacillus Fermentum ◽  
Extracellular Matrix Protein ◽  
Type I ◽  
Uptake System

ABSTRACT BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, fromLactobacillus reuteri. Compared with the database ofEscherichia coli proteins, BspA is most similar to thel-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take upl-cystine. Uptake rates of l-glutamine,l-histidine, and l-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of L. fermentum BR11 with l-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed. L. fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.

scholarly journals Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein.

1995 ◽  
Vol 131 (2) ◽  
pp. 539-549 ◽  
Author(s):  
S L Dallas ◽  
K Miyazono ◽  
T M Skerry ◽  
G R Mundy ◽  
L F Bonewald
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Staining Pattern ◽  
Binding Protein ◽  
Type I ◽  
Tgf Beta ◽  
Fetal Rat ◽  
Structural Matrix

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.

scholarly journals Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials

2020 ◽  
Author(s):  
Marimikel Charrier ◽  
Maria Teresa Orozco-Hidalgo ◽  
Nicholas Tjahjono ◽  
Dong Li ◽  
Sara Molinari ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Caulobacter Crescentus ◽  
Extracellular Protein ◽  
Extracellular Matrix Protein ◽  
High Yield ◽  
Type I ◽  
Self Healing ◽  
Protein Matrix ◽  
Living Materials

ABSTRACTThe bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions, but require exogenously added material or have limited programmability. Here we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of elastin-like polypeptide (ELP) hydrogel fused to Supercharged SpyCatcher (SC(-)). This biopolymer was secreted at levels of 60 mg/L, an unprecedented level of biopolymer secretion by a gram-negative bacterium. The ELP domain was swapped with either a crosslinkable variant of ELP or resilin-like polypeptide, demonstrating this system is flexible. The SC(-)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag peptides via its engineered Surface-layer. Our work develops protein design rules for Type I secretion in C. crescentus, and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward towards the formation of cohesive engineered living materials.IMPORTANCEEngineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy intensive processing or have limited tunability. We propose a bacterially-synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step towards understanding the necessary parameters to engineering living cells to autonomously construct ELMs.

scholarly journals Cloning and Characterization of a GC-Box Binding Protein, G10BP-1, Responsible for Repression of the Rat Fibronectin Gene

1998 ◽  
Vol 18 (8) ◽  
pp. 4772-4782 ◽  
Author(s):  
Eri Oda ◽  
Kenna Shirasuna ◽  
Mitsuhiro Suzuki ◽  
Kuniko Nakano ◽  
Takuma Nakajima ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Extracellular Matrix ◽  
Matrix Protein ◽  
Binding Protein ◽  
Cell Surface Receptor ◽  
Expression Level ◽  
Extracellular Matrix Protein ◽  
Cortical Actin ◽  
Cell Extracts ◽  
Gc Box

ABSTRACT Fibronectin (FN) is an extracellular matrix protein that connects the extracellular matrix to intracellular cortical actin filaments through binding to its cell surface receptor, α5β1, a member of the integrin superfamily. The expression level of FN is reduced in most tumor cells, facilitating their anchorage-independent growth by still unclarified mechanisms. The cDNA clone encoding G-rich sequence binding protein G10BP-1, which is responsible for repression of the rat FN gene, was isolated by using a yeast one-hybrid screen with the G10 stretch inserted upstream of the HIS3 and lacZgene minimal promoters. G10BP-1 comprises 385 amino acids and contains two basic regions and a putative zipper structure. It has the same specificity of binding to three G-rich sequences in the FN promoter and the same size as the G10BP previously identified in adenovirus E1A- and E1B-transformed rat cells. Expression of G10BP-1 is cell cycle regulated; the level was almost undetectable in quiescent rat 3Y1 cells but increased steeply after growth stimulation by serum, reaching a maximum in late G1. Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the level decreased steeply during G1-to-S progression. This down regulation was strictly dependent on the downstream GC box (GCd), and base substitutions within GCd abolished the sensitivity of the promoter to G10BP-1. In contrast, the level of Sp1, which competes with G10BP for binding to the G-rich sequences, was constant throughout the cell cycle, suggesting that the concentration of G10BP-1 relative to that of Sp1 determines the expression level of the FN gene. Preparation of glutathione S-transferase pulldowns of native proteins from the cell extracts containing exogenously or endogenously expressed G10BP-1, followed by Western blot analysis, showed that G10BP-1 forms homodimers through its basic-zipper structure.

β1 integrin-extracellular matrix protein interaction modulates the migratory response to chemokine stimulation

2001 ◽  
Vol 79 (4) ◽  
pp. 399-407 ◽  
Author(s):  
Priti S Shenoy ◽  
Shashi Uniyal ◽  
Kohei Miura ◽  
Christopher McColl ◽  
Tamas Oravecz ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Collagen Type ◽  
Cell Movement ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Extracellular Matrix Protein ◽  
Type I ◽  
Migratory Response ◽  
Cc Chemokine Receptor 5

It is well established that chemokines have a major role in the stimulation of cell movement on extracellular matrix (ECM) substrates. However, it is also clear that ECM substrates may influence the ability of cells to undergo migration. Using the migration chamber method, we assessed the migratory response of human embryonic kidney-293 (HEK) transfectant cells expressing the CC chemokine receptor 5 (CCR5) (HEK-CCR5) to stimulation by chemokines (macrophage inflamatory protein (MIP)-1α, MIP-1β, and regulated on activation normal-T cell expressed and secreted (RANTES)) on ECM substrates (collagen type I and fibronectin). Using filters coated with collagen (20 µg/mL), results showed that the chemokines differed in their ability to elicit cell movement according to the order MIP-1β > RANTES [Formula: see text] MIP-1α. In contrast, using filters coated with fibronectin (20 µg/mL), all three chemokines were similar in their ability to stimulate migration of HEK-CCR5 cells. In addition, the migratory response with respect to the concentrations of ECM substrates appeared biphasic; thus, chemokine-stimulated cell movement was inhibited at high ECM concentrations (100 µg/mL). To determine the involvement of β1 integrins, results showed that the migratory response to chemokine stimulation on collagen was largely inhibited by monoclonal antibody (mAb) to α2β1; however, complete inhibition required a combination of mAbs to α1β1 and α2β1. In comparison, migration on fibronectin was inhibited by mAb to α3β1 and α5β1. Our results suggest that the migratory response to CCR5 stimulation may vary quantitatively with both the CCR5 ligand (MIP-1α, MIP-1β, and RANTES), as well as the nature and concentration of the ECM substrate involved.Key words: chemokines, integrins, cell movement, extracellular matrix proteins, CCR5.

Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

1987 ◽  
Vol 65 (12) ◽  
pp. 1031-1038 ◽  
Author(s):  
Anat Lev ◽  
Paul C. Holland
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Binding Proteins ◽  
Type I Collagen ◽  
De Novo ◽  
Binding Protein ◽  
Apparent Molecular Weight ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Binding

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin–Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen–Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63 000 – 72 000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.

scholarly journals BMP1 and TLL1 Are Required for Maintaining Periodontal Homeostasis

2017 ◽  
Vol 96 (5) ◽  
pp. 578-585 ◽  
Author(s):  
J. Wang ◽  
D. Massoudi ◽  
Y. Ren ◽  
A.M. Muir ◽  
S.E. Harris ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Type I Collagen ◽  
Alveolar Bone ◽  
Significant Loss ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Dentin Matrix Protein ◽  
Morphogenetic Protein ◽  
Periodontal Breakdown

Mutations in bone morphogenetic protein 1 (BMP1) in humans or deletion of BMP1 and related protease tolloid like 1 (TLL1) in mice lead to osteogenesis imperfecta (OI). Here, we show progressive periodontal defects in mice in which both BMP1 and TLL1 have been conditionally ablated, including malformed periodontal ligament (PDL) (recently shown to play key roles in normal alveolar bone formation), significant loss in alveolar bone mass ( P < 0.01), and a sharp reduction in cellular cementum. Molecular mechanism studies revealed a dramatic increase in the uncleaved precursor of type I collagen (procollagen I) and a reduction in dentin matrix protein 1 (DMP1), which is partially responsible for defects in extracellular matrix (ECM) formation and mineralization. We also showed a marked increase in the expression of matrix metallopeptidase 13 (MMP13) and tartrate-resistant acid phosphatase (TRAP), leading to an acceleration in periodontal breakdown. Finally, we demonstrated that systemic application of antibiotics significantly improved the alveolar bone and PDL damage of the knockdown phenotype, which are thus shown to be partially secondary to pathogen-induced inflammation. Together, identification of the novel roles of BMP1 and TLL1 in maintaining homeostasis of periodontal formation, partly via biosynthetic processing of procollagen I and DMP1, provides novel insights into key contributions of the extracellular matrix environment to periodontal homeostasis and contributes toward understanding of the pathology of periodontitis.

scholarly journals Fabrication of low-cost micropatterned polydimethyl-siloxane scaffolds to organise cells in a variety of two-dimensioanl biomimetic arrangements for lab-on-chip culture platforms

2017 ◽  
Vol 8 ◽  
pp. 204173141774150 ◽  
Author(s):  
Lidia Escutia-Guadarrama ◽  
Genaro Vázquez-Victorio ◽  
David Martínez-Pastor ◽  
Brenda Nieto-Rivera ◽  
Marcela Sosa-Garrocho ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Type I Collagen ◽  
Microcontact Printing ◽  
Low Cost ◽  
Extracellular Matrix Protein ◽  
Type I ◽  
Human Hepatoma Cells ◽  
Lab On Chip ◽  
Chip Technology ◽  
On Chip

We present the rapid-prototyping of type I collagen micropatterns on poly-dimethylsiloxane substrates for the biomimetic confinement of cells using the combination of a surface oxidation treatment and 3-aminopropyl triethoxysilane silanisation followed by glutaraldehyde crosslinking. The aim of surface treatment is to stabilise microcontact printing transfer of this natural extracellular matrix protein that usually wears out easily from poly-dimethylsiloxane, which is not suitable for biomimetic cell culture platforms and lab-on-chip applications. A low-cost CD-DVD laser was used to etch biomimetic micropatterns into acrylic sheets that were in turn replicated to poly-dimethylsiloxane slabs with the desired features. These stamps were finally inked with type I collagen for microcontact printing transfer on the culture substrates in a simple manner. Human hepatoma cells (HepG2) and rat primary hepatocytes, which do not adhere to bare poly-dimethylsiloxane, were successfully seeded and showed optimal adhesion and survival on simple protein micropatterns with a hepatic cord geometry in order to validate our technique. HepG2 cells also proliferated on the stamps. Soft and stiff poly-dimethylsiloxane layers were also tested to demonstrate that our cost-effective process is compatible with biomimetic organ-on-chip technology integrating tunable stiffness with a potential application to drug testing probes development where such cells are commonly used.

scholarly journals Functional Analysis of the Collagen Binding Proteins of Streptococcus parasanguinis FW213

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Yi-Ywan M. Chen ◽  
Pei-Hua Tsai ◽  
Zong-Sian Ye ◽  
Yu-Wen Huang ◽  
Hui-Ru Shieh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Binding Proteins ◽  
Type I Collagen ◽  
Galleria Mellonella ◽  
Opportunistic Pathogen ◽  
Type I ◽  
Collagen Binding ◽  
Content Type ◽  
Extracellular Matrix Molecules ◽  
Streptococcus Parasanguinis

ABSTRACT Streptococcus parasanguinis is a dominant isolate of dental plaque and an opportunistic pathogen associated with subacute endocarditis. As the expression of collagen binding proteins (CBPs) could promote the establishment of S. parasanguinis in the host, the functions of three putative CBP-encoding loci, Spaf_0420, Spaf_1570, and Spaf_1573, were analyzed using isogenic mutant strains. It was revealed that S. parasanguinis FW213 bound effectively to fibronectin and type I collagen, but the strain’s affinity for laminin and type IV collagen was quite low. By using various deletion derivatives, it was found that these three loci mediated the binding of S. parasanguinis to multiple extracellular matrix molecules, with type I collagen as the common substrate. Derivative strains with a deletion in any of the three loci expressed reduced binding to trypsin-treated swine heart valves. The deletion of these loci also reduced the viable count of S. parasanguinis bacteria within macrophages, especially the loss of Spaf_0420, but only strains with deletions in Spaf_0420 and Spaf_1570 expressed reduced virulence in the Galleria mellonella larva model. The deletion of Spaf_1570 and Spaf_1573 affected mainly the structure, but not the overall mass, of biofilm cultures in a flow cell system. Thus, CBPs are likely to be more critical for the initial colonization of S. parasanguinis on host tissues during the development of endocarditis. IMPORTANCE Bacteria generally can utilize multiple adhesins to establish themselves in the host. We found that Streptococcus parasanguinis, a dominant oral commensal and an opportunistic pathogen for subacute endocarditis, possesses at least three collagen-binding proteins that enable S. parasanguinis to successfully colonize damaged heart tissues and escape innate immune clearance. The binding specificities of these three proteins for extracellular matrix molecules differ, although all three proteins participate in biofilm formation by S. parasanguinis. The “multiligand for multisubstrate” feature of these adhesins may explain the high adaptability of this microbe to different tissue sites.

scholarly journals Gelsolin Contributes to the Motility of A375 Melanoma Cells and this Activity is Mediated by the Fibrous Extracellular Matrix Protein Profile

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1848
Author(s):  
Ewa Mazurkiewicz ◽  
Aleksandra Makowiecka ◽  
Ewa Mrówczyńska ◽  
Iryna Kopernyk ◽  
Dorota Nowak ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Melanoma Cell ◽  
Matrix Protein ◽  
Protein Profile ◽  
Melanoma Cells ◽  
Actin Binding ◽  
Extracellular Matrix Protein ◽  
Collagen Type Iv ◽  
Type I ◽  
A375 Melanoma

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)—an actin-binding protein—levels in melanoma cell lines and GSN’s importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin’s BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN’s influence on parameters associated with cells’ ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin’s BM.

Load-induced regulation of tendon homeostasis by SPARC, a genetic predisposition factor for tendon and ligament injuries

2021 ◽  
Vol 13 (582) ◽  
pp. eabe5738
Author(s):  
Tao Wang ◽  
Andrea Wagner ◽  
Renate Gehwolf ◽  
Wenjin Yan ◽  
Fabian S. Passini ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Type I Collagen ◽  
Botulinum Toxin A ◽  
Three Dimensional ◽  
Extracellular Matrix Protein ◽  
Matricellular Protein ◽  
Type I ◽  
Ligament Injuries ◽  
Ribosomal S6 Kinase

Tendons and tendon interfaces have a very limited regenerative capacity, rendering their injuries clinically challenging to resolve. Tendons sense muscle-mediated load; however, our knowledge on how loading affects tendon structure and functional adaption remains fragmentary. Here, we provide evidence that the matricellular protein secreted protein acidic and rich in cysteine (SPARC) is critically involved in the mechanobiology of tendons and is required for tissue maturation, homeostasis, and enthesis development. We show that tendon loading at the early postnatal stage leads to tissue hypotrophy and impaired maturation of Achilles tendon enthesis in Sparc−/− mice. Treadmill training revealed a higher prevalence of spontaneous tendon ruptures and a net catabolic adaptation in Sparc−/− mice. Tendon hypoplasia was attenuated in Sparc−/− mice in response to muscle unloading with botulinum toxin A. In vitro culture of Sparc−/− three-dimensional tendon constructs showed load-dependent impairment of ribosomal S6 kinase activation, resulting in reduced type I collagen synthesis. Further, functional calcium imaging revealed that lower stresses were required to trigger mechanically induced responses in Sparc−/− tendon fascicles. To underscore the clinical relevance of the findings, we further demonstrate that a missense mutation (p.Cys130Gln) in the follistatin-like domain of SPARC, which causes impaired protein secretion and type I collagen fibrillogenesis, is associated with tendon and ligament injuries in patients. Together, our results demonstrate that SPARC is a key extracellular matrix protein essential for load-induced tendon tissue maturation and homeostasis.

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