ABSTRACT
Shell matrix proteins have important roles in the biomineralization of shells. In this study, we isolated and identified a novel shell matrix protein gene, hic7, from the mussel Hyriopsis cumingii. The cDNA of hic7 was 459 bp long, including a 240-bp open reading frame. It encoded a 79 amino acid-long protein, with amino acids 1-19 constituting the signal peptide. The resulting hic7 is rich in cysteine (16.5%). After removing the signal peptide, the molecular weight was 8.85 kDa and the theoretical isoelectric point was 6.34, indicating that hic7 is a weakly acidic shell matrix protein. Hic7 is mainly expressed in the mantle tissue of H. cumingii. In situ hybridization showed hic7 signals at the edge and dorsal region of the mantle outer fold, indicating that it is related to the formation of the prismatic and nacreous layer of the shell. RNA interference indicated that when hic7 was inhibited by 80%, the crystal morphology of the prism and nacre layers of the shell were irregular and disordered. In addition, the expression of hic7 during the early development of the pearl sac indicated that it has an important role in the transformation of calcium carbonate crystals from a disordered to an orderly deposition pattern. These results suggest that matrix protein hic7 take part in constructing the framework of crystal nucleation and regulating the calcium carbonate crystal morphology of the nacreous and prismatic layers of shells and pearls.
Subject(s)
Animal Shells/growth & development , Extracellular Matrix Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nacre/biosynthesis , Unionidae/growth & development , Amino Acid Sequence , Animals , Base Sequence , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/isolation & purification , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/isolation & purification , Unionidae/genetics , Unionidae/metabolismABSTRACT
Despite the poor regenerative capacity of the adult central nervous system (CNS) in mammals, two distinct regions, subventricular zone (SVZ) and the subgranular zone (SGZ), continue to generate new functional neurons throughout life which integrate into the pre-existing neuronal circuitry. This process is not fixed but highly modulated, revealing many intrinsic and extrinsic mechanisms by which this performance can be optimized for a given environment. The capacity for self-renewal, proliferation, migration, and multi-lineage potency of neural stem cells (NSCs) underlines the necessity of controlling stem cell fate. In this context, the native and local microenvironment plays a critical role, and the application of this highly organized architecture in the CNS has been considered as a fundamental concept in the generation of new effective therapeutic strategies in tissue engineering approaches. The brain extracellular matrix (ECM) is composed of biomacromolecules, including glycosaminoglycans, proteoglycans, and glycoproteins that provide various biological actions through biophysical and biochemical signaling pathways. Herein, we review predominantly the structure and function of the mentioned ECM composition and their regulatory impact on multiple and diversity of biological functions, including neural regeneration, survival, migration, differentiation, and final destiny of NSCs.
Subject(s)
Extracellular Matrix Proteins/pharmacology , Extracellular Matrix/chemistry , Glycosaminoglycans/pharmacology , Neural Stem Cells/drug effects , Proteoglycans/pharmacology , Regeneration/drug effects , Biomimetic Materials/isolation & purification , Biomimetic Materials/pharmacology , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Movement/drug effects , Extracellular Matrix Proteins/isolation & purification , Glycosaminoglycans/isolation & purification , Humans , Neural Stem Cells/cytology , Neurogenesis/drug effects , Neurogenesis/physiology , Proteoglycans/isolation & purificationABSTRACT
Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein's function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin's central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin's known receptors, calculating the KD of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.
Subject(s)
Cell Adhesion Molecules, Neuronal/chemistry , Cell Adhesion Molecules, Neuronal/metabolism , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Multimerization , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Cell Adhesion Molecules, Neuronal/isolation & purification , Extracellular Matrix Proteins/isolation & purification , HEK293 Cells , Humans , Nerve Tissue Proteins/isolation & purification , Neurons/cytology , Neurons/metabolism , Protein Binding , Protein Structure, Quaternary , Reelin Protein , Serine Endopeptidases/isolation & purification , Signal TransductionABSTRACT
Dentin sialophosphoprotein (DSPP), which expresses and synthesizes in odontoblasts of dental pulp, is a critical protein for normal teeth mineralization. Originally, DSPP was identified as a dentin-specific protein. In 2010, DSPP was also found in femoral head cartilage, and it is still unclear what roles DSPP play in femoral head cartilage formation, growth, and maintenance. To reveal biological functions of DSPP in the femoral head cartilage, we examined Dspp null mice compared with wild-type (WT) mice to observe DSPP expression as well as localization in WT mice and to uncover differences of femoral head cartilage, bone morphology, and structure between these two kinds of mice. Expression data demonstrated that DSPP had heterogeneous fragments, expressed in each layer of femoral head cartilage and subchondral bone of WT mice. Dspp null mice exhibited a significant reduction in the thickness of femoral head cartilage, with decreases in the amount of proliferating cartilage cells and increases in apoptotic cells. In addition, the subchondral bone mineralization decreased, and the expressions of vessel markers (vascular endothelial growth factor [VEGF] and CD31), osteoblast markers (Osterix and dentin matrix protein 1 [DMP1]), osteocyte marker (sclerostin [SOST]), and osteoclast marker (tartrate-resistant acid phosphatase [TRAP]) were remarkably altered. These indicate that DSPP deletion can affect the proliferation of cartilage cells in the femoral head cartilage and endochondral ossification in subchondral bone. Our data clearly demonstrate that DSPP plays essential roles in the femoral head cartilage growth and maintenance and subchondral biomineralization.
Subject(s)
Calcification, Physiologic , Cartilage/metabolism , Extracellular Matrix Proteins/metabolism , Femur Head/metabolism , Phosphoproteins/metabolism , Sialoglycoproteins/metabolism , Animals , Cartilage/cytology , Cell Proliferation , Extracellular Matrix Proteins/deficiency , Extracellular Matrix Proteins/isolation & purification , Femur Head/cytology , Mice , Mice, Knockout , Phosphoproteins/deficiency , Phosphoproteins/isolation & purification , Sialoglycoproteins/deficiency , Sialoglycoproteins/isolation & purificationABSTRACT
Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF-PVR and RV donor samples, as was collagen content and cross-linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine-rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.
Subject(s)
Extracellular Matrix/genetics , Gene Expression , Myocytes, Cardiac/physiology , Myofibrils/physiology , Tetralogy of Fallot/genetics , Ventricular Function, Right/genetics , Adolescent , Adult , Child , Collagen/analysis , Down-Regulation , Extracellular Matrix Proteins/isolation & purification , Female , Gene Expression Profiling/methods , Heart Valve Prosthesis Implantation , Humans , Male , Middle Aged , Muscle Contraction/physiology , Polymerase Chain Reaction , Pulmonary Valve/surgery , Pulmonary Valve Insufficiency/surgery , RNA, Messenger/metabolism , Small Leucine-Rich Proteoglycans/metabolism , Tetralogy of Fallot/surgery , Up-Regulation , Young AdultABSTRACT
Here, we have identified a novel matrix protein, named PfX, from the pearl oyster Pinctada fucada, and investigated the effects of recombinant PfX protein on calcium carbonate crystallization. The expression of PfX was spatially concentrated in the mantle tissue and gill, the former of which is responsible for the formation of shell structures. The shell notching assay showed a PfX expression response during injured shell repair and regeneration, suggesting the potential involvement of this matrix protein in shell biomineralization. Further, an in vitro crystallization assay showed that PfX could alter the CaCO3 morphologies of both calcite and aragonite polymorphs. Correspondingly, a binding assay indicated that PfX has strong binding affinity for CaCO3 crystals, especially aragonite. Further, the protein's calcite binding capacity increased obviously when particular crystal faces were induced. In addition, PfX conjugated with fluorescent dye cyanine-5 (cy5) was preferentially distributed on rough crystal faces instead of the smooth and common (1 0 4) faces of calcite during the crystallization. These results suggest that matrix protein PfX might regulate CaCO3 morphology via selective binding and inhibit the growth of certain crystal faces, providing new clues for understanding biomineralization mechanisms in mollusk.
Subject(s)
Calcium Carbonate/chemistry , Calcium Carbonate/metabolism , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Pinctada/metabolism , Animals , Chitin/chemistry , Chitin/metabolism , Cloning, Molecular , Crystallization , Extracellular Matrix Proteins/isolation & purification , Gene Expression , Protein Binding , Recombinant Proteins , Sequence Analysis, DNA , Spectrum Analysis , Structure-Activity RelationshipABSTRACT
Chronic venous disease (CVD) is the most common reported chronic condition in the United States, affecting more than 25 million Americans. Regardless of its high occurrence, current therapeutic options are far from ideal due to their palliative nature. For best treatment outcomes, challenging cases of chronic venous insufficiency (CVI) are treated by repair or replacement of venous valves. Regrettably, the success of venous valve transplant is dependent on the availability of autologous venous valves and hindered by the possibility of donor site complications and increased patient morbidity. Therefore, the use of alternative tissue sources to provide off-the-shelf venous valve replacements has potential to be extremely beneficial to the field of CVI. This manuscript demonstrates the capability of producing off-the-shelf fully functional venous valved extracellular matrix (ECM) scaffold conduits from bovine saphenous vein (SV), using an antigen removal (AR) method. AR ECM scaffolds maintained native SV structure-function relationships and associated venous valves function. Conversely, SDS decellularization caused significant changes to the collagen and elastin macromolecular structures, resulting in collagen fibril merging, elimination of fibril crimp, amalgaming collagen fibers and fragmentation of the inner elastic lamina. ECM changes induced by SDS decellularization resulted in significant venous valve dysfunction. Venous valved conduits generated using the AR approach have potential to serve as off-the-shelf venous valve replacements for CVI. STATEMENT OF SIGNIFICANCE: Retention of the structure and composition of extracellular matrix (ECM) proteins within xenogeneic scaffolds for tissue engineering is of crucial importance, due to the undeniable effect ECM proteins can impose on repopulating cells and function of the resultant biomaterial. This manuscript demonstrates that alteration or elimination of ECM proteins via commonly utilized decellularization approach results in complete disruption of venous valve function. Conversely, retention of the delicate ECM structure and composition of native venous tissue, using an antigen removal tissue processing method, results in preservation of native venous valve function.
Subject(s)
Antigens, Surface/isolation & purification , Extracellular Matrix/metabolism , Tissue Scaffolds/chemistry , Venous Valves/metabolism , Animals , Antigens, Surface/chemistry , Cattle , Chemical Fractionation , Collagen/metabolism , Elastin/metabolism , Extracellular Matrix/drug effects , Extracellular Matrix/ultrastructure , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/isolation & purification , Humans , Rabbits , Saphenous Vein/drug effects , Saphenous Vein/metabolism , Saphenous Vein/ultrastructure , Sodium Dodecyl Sulfate/chemistry , Tissue Engineering/methods , Venous Valves/drug effects , Venous Valves/ultrastructureABSTRACT
Understanding molecular mechanisms and cellular metabolism in varied plant processes necessitates knowledge of the expressed proteins and their subcellular distribution. Spatial partitioning of organelles generates an enclosed milieu for physiochemical reactions designed and tightly linked to a specific organelle function. Of which, extracellular matrix (ECM)/cell wall (CW) is a dynamic and chemically active compartment. The ECM proteins are organized into complex structural and functional networks involved in several metabolic processes, including carbon and nitrogen metabolism. Organellar proteomics aim for comprehensive identification of resident proteins that rely on the isolation of highly purified organelle free from contamination by other intracellular components. Extraction and isolation of plant ECM proteins features key caveats due to the lack of adjoining membrane, the presence of a polysaccharide-protein network that traps contaminants, and the existence of high phenolic content. Furthermore, due to diverse biochemical forces, including labile, weakly bound and strongly bound protein in the protein-polysaccharide matrix different elution procedures are required to enrich ECM proteins. Here, we describe a method that allows efficient fractionation of plant ECM, extraction of ECM proteins and protein profiling from variety of crop plants, including rice, chickpea and potato. This method can easily be adapted to other plant species for varied experimental conditions.
Subject(s)
Cicer/metabolism , Electrophoresis, Gel, Two-Dimensional/methods , Extracellular Matrix Proteins/metabolism , Extracellular Matrix/metabolism , Proteome/metabolism , Proteomics/methods , Chromatography, Liquid , Cicer/enzymology , Extracellular Matrix Proteins/isolation & purification , Microscopy , Seedlings/enzymology , Seedlings/metabolism , Tandem Mass Spectrometry , WorkflowABSTRACT
Due the proteins from bone remains are highly resistant to pass of time and environmental conditions, they could tell us about the events that probably happened in the past. In the forensic and physical anthropology context, burnt bone remains are one of the most common pieces of recovered evidence and, generally, they are associated with funerary practices, criminal scenes or massive catastrophic events. In the present study, bone pieces of pigs were calcined at different calcination temperatures, and proteins were searched using biochemical, immunochemical and ultrastructure visualization under these experimentally conditions. For this purpose, it was successfully developed a non-demineralizing protein extraction method from burnt bone remains and the use of specific antibodies permitted the identification of different extracellular matrix and intracellular proteins. While collagen proteins type I and IV were identified and detected under middle and high calcination temperatures (300°C and 600°C); cytoskeletal proteins as actin, tubulin and, the microtubule associated protein Tau, were found under calcination process, even up high calcination temperatures. Under ultrastructural analysis, fibrous materials with a classical disposition of collagens were observed even at high calcination temperatures of the burnt bone remains. The protein identification and characterization in burnt bones as performed in present studies, is clearly demonstrating that using specific strategies for protein characterizations it is possible to found protein biomarkers in burnt bone remains and this strategy could be useful for forensic and anthropological purposes.
Subject(s)
Bone and Bones/chemistry , Cytoskeletal Proteins/isolation & purification , Extracellular Matrix Proteins/isolation & purification , Fires , Animals , Antibodies/analysis , Biomarkers/chemistry , Blotting, Western , Bone Demineralization Technique , Bone and Bones/pathology , Collagen/ultrastructure , Cytoskeletal Proteins/immunology , Electrophoresis , Extracellular Matrix Proteins/immunology , Forensic Pathology/methods , Humans , Microscopy, Electron, Scanning , Swine , TemperatureABSTRACT
BACKGROUND: Olfactomedin-1 (Olfm1; also known as Noelin or Pancortin) is a highly-expressed secreted brain and retina protein and its four isoforms have different roles in nervous system development and function. Structural studies showed that the long Olfm1 isoform BMZ forms a disulfide-linked tetramer with a V-shaped architecture. The tips of the Olfm1 "V" each consist of two C-terminal ß-propeller domains that enclose a calcium binding site. Functional characterisation of Olfm1 may be aided by new biochemical tools derived from these core structural elements. RESULTS: Here we present the production, purification and structural analysis of three novel monomeric, dimeric and tetrameric forms of mammalian Olfm1 for functional studies. We characterise these constructs structurally by high-resolution X-ray crystallography and small-angle X-ray scattering. The crystal structure of the Olfm1 ß-propeller domain (to 1.25 Å) represents the highest-resolution structure of an olfactomedin family member to date, revealing features such as a hydrophilic tunnel containing water molecules running into the core of the domain where the calcium binding site resides. The shorter Olfactomedin-1 isoform BMY is a disulfide-linked tetramer with a shape similar to the corresponding region in the longer BMZ isoform. CONCLUSIONS: These recombinantly-expressed protein tools should assist future studies, for example of biophysical, electrophysiological or morphological nature, to help elucidate the functions of Olfm1 in the mature mammalian brain. The control over the oligomeric state of Olfm1 provides a firm basis to better understand the role of Olfm1 in the (trans-synaptic) tethering or avidity-mediated clustering of synaptic receptors such as post-synaptic AMPA receptors and pre-synaptic amyloid precursor protein. In addition, the variation in domain composition of these protein tools provides a means to dissect the Olfm1 regions important for receptor binding.
Subject(s)
Extracellular Matrix Proteins , Glycoproteins , Neurobiology/methods , Animals , Binding Sites , Brain/cytology , Brain/metabolism , Crystallography, X-Ray , Extracellular Matrix Proteins/biosynthesis , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/isolation & purification , Glycoproteins/biosynthesis , Glycoproteins/chemistry , Glycoproteins/isolation & purification , HEK293 Cells , Humans , Mice , Protein Isoforms/biosynthesis , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , TransfectionABSTRACT
The extracellular matrix (ECM) is a complex assembly of hundreds of proteins that constitutes the scaffold of multicellular organisms. In addition to providing architectural and mechanical support to the surrounding cells, it conveys biochemical signals that regulate cellular processes including proliferation and survival, fate determination, and cell migration. Defects in ECM protein assembly, decreased ECM protein production or, on the contrary, excessive ECM accumulation, have been linked to many pathologies including cardiovascular and skeletal diseases, cancers, and fibrosis. The ECM thus represents a potential reservoir of prognostic biomarkers and therapeutic targets. However, our understanding of the global protein composition of the ECM and how it changes during pathological processes has remained limited until recently.In this mini-review, we provide an overview of the latest methodological advances in sample preparation and mass spectrometry-based proteomics that have permitted the profiling of the ECM of now dozens of normal and diseased tissues, including tumors and fibrotic lesions.
Subject(s)
Extracellular Matrix Proteins/analysis , Extracellular Matrix/chemistry , Proteomics , Animals , Extracellular Matrix Proteins/isolation & purification , Fibrosis , Humans , Mass Spectrometry , Neoplasms/chemistryABSTRACT
Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates ß cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal ß cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal ß cell maturation.
Subject(s)
Extracellular Matrix Proteins/genetics , Pancreas/metabolism , Proteoglycans/genetics , Proteomics , Collagen/genetics , Collagen/metabolism , Extracellular Matrix Proteins/isolation & purification , Humans , Hydrogels/chemistry , Proteoglycans/isolation & purificationABSTRACT
The extracellular matrix of the bone and dentin contains several non-collagenous proteins (NCPs). One category of NCPs is termed the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family, which includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), etc. These proteins have abundant phosphoserines, aspartic acids, and glutamic acids. In this protocol, we describe the extraction of NCPs from the bone and dentin matrices using guanidine-HCl/EDTA and the isolation of polyanionic SIBLINGs from NCPs using ion-exchange fast protein liquid chromatography (FPLC) to separate the differentially charged proteins into different fractions through a gradient elution by NaCl.
Subject(s)
Bone and Bones/chemistry , Chromatography, Ion Exchange/methods , Dentin/chemistry , Extracellular Matrix Proteins/isolation & purification , Integrin-Binding Sialoprotein/isolation & purification , Osteopontin/isolation & purification , Phosphoproteins/isolation & purification , Sialoglycoproteins/isolation & purification , Animals , Chromatography, Ion Exchange/instrumentation , Edetic Acid/chemistry , Equipment Design , Extracellular Matrix/chemistry , Guanidine/chemistry , Hydrochloric Acid/chemistry , Mice , Sodium Chloride/chemistry , Ultrafiltration/instrumentation , Ultrafiltration/methodsABSTRACT
The organic material in developing dentin is 90% type I collagen and 10% non-collagenous proteins. The key to understanding dentin biomineralization is to study how these proteins collectively precipitate and organize hydroxyapatite crystals. The first step in characterizing the proteins within a mineralizing matrix is to efficiently extract and isolate the essential molecular participants and elucidate their structural and biochemical properties. In this study, we expanded previous approaches to develop an improved strategy for the extraction of extracellular matrix proteins from the dentin of developing teeth. Proteins in dentin powder were sequentially extracted in the order Tris-guanidine buffer, HCl-formic acid solution, acetic acid-NaCl solution, Tris-NaCl buffer, and a second Tris-guanidine buffer. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by gelatin or casein zymography, and by Western blot analysis using dentin sialoprotein (DSP)- or dentin glycoprotein (DGP)-specific antibodies. This approach was used to purify assorted porcine dentin non-collagenous proteins.
Subject(s)
Blotting, Western/methods , Dentin/chemistry , Electrophoresis, Polyacrylamide Gel/methods , Extracellular Matrix Proteins/isolation & purification , Glycoproteins/isolation & purification , Phosphoproteins/isolation & purification , Sialoglycoproteins/isolation & purification , Tooth/chemistry , Acetic Acid/chemistry , Animals , Dentin/growth & development , Formates/chemistry , Guanidine/chemistry , Swine , Tooth/growth & development , Tromethamine/chemistryABSTRACT
Ligaments are prone to injury and degeneration in humans and animals, however the healing potential of ligament is poor and current treatment options ineffective. Stem cell-based therapies hold potential for treatment of ligament injuries. This study aimed to characterize a ligament progenitor cell (LPC) population and to identify specific niche components which could promote the survival and function of LPCs. LPCs were isolated from canine cranial cruciate ligament and characterized for clonogenicity, multipotency and marker expression. The extracellular matrix (ECM) composition was characterized by the novel application of a metabolic labeling and mass spectrometry technique. LPCs demonstrated clonogenicity, multipotency, and stem cell marker expression. A number of different collagens, glycoproteins, and proteoglycans were identified in the LPC niche using proteomics. Metabolic labeling of cells demonstrated unique turnover profiles for distinct ECM protein groups, indicating the importance of certain niche components for LPC survival and function. The newly synthesized niche components identified in this study could be exploited to aid identification of LPCs and to promote their survival and function for potential ligament repair strategies.
Subject(s)
Anterior Cruciate Ligament/cytology , Extracellular Matrix Proteins/genetics , Stem Cell Niche/genetics , Stem Cells/cytology , Animals , Anterior Cruciate Ligament/transplantation , Cell Lineage/genetics , Collagen/genetics , Collagen/metabolism , Colony-Forming Units Assay , Dogs , Extracellular Matrix/genetics , Extracellular Matrix Proteins/isolation & purification , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental/genetics , Humans , Liver/metabolism , Proteoglycans/genetics , Stem Cells/metabolismABSTRACT
The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite. Biomineralization is elaborately controlled and involves several macromolecules, especially matrix proteins, but little is known about the regulatory mechanisms. The matrix protein Shematrin-2, expression of which peaks in the mantle tissues and in the shell components of the pearl oyster Pinctada fucata, has been suggested to be a key participant in biomineralization. Here, we expressed and purified Shematrin-2 from P. fucata and explored its function and transcriptional regulation. An in vitro functional assay revealed that Shematrin-2 binds the calcite, aragonite, and chitin components of the shell, decreases the rate of calcium carbonate deposition, and changes the morphology of the deposited crystal in the calcite crystallization system. Furthermore, we cloned the Shematrin-2 gene promoter, and analysis of its sequence revealed putative binding sites for the transcription factors CCAAT enhancer-binding proteins (Pf-C/EBPs) and nuclear factor-Y (NF-Y). Using transient co-transfection and reporter gene assays, we found that cloned and recombinantly expressed Pf-C/EBP-A and Pf-C/EBP-B greatly and dose-dependently up-regulate the promoter activity of the Shematrin-2 gene. Importantly, Pf-C/EBP-A and Pf-C/EBP-B knockdowns decreased Shematrin-2 gene expression and induced changes in the inner-surface structures in prismatic layers that were similar to those of antibody-based Shematrin-2 inhibition. Altogether, our data reveal that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B up-regulate the expression of the matrix protein Shematrin-2 during shell formation in P. fucata, improving our understanding of the transcriptional regulation of molluscan shell development at the molecular level.
Subject(s)
Animal Shells/chemistry , Calcification, Physiologic/genetics , Extracellular Matrix Proteins/genetics , Animal Shells/growth & development , Animals , CCAAT-Binding Factor/genetics , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-beta/genetics , Calcium Carbonate/metabolism , Extracellular Matrix Proteins/isolation & purification , Extracellular Matrix Proteins/physiology , HEK293 Cells , Humans , Phylogeny , Pinctada/chemistry , Pinctada/growth & development , Transcriptional ActivationABSTRACT
Microcontact printing has become a versatile soft lithography technique used to produce molecular micro- and nano-patterns consisting of a large range of different biomolecules. Despite intensive research over the last decade and numerous applications in the fields of biosensors, microarrays and biomedical applications, the large-scale implementation of microcontact printing is still an issue. It is hindered by the stamp-inking step that is critical to ensure a reproducible and uniform transfer of inked molecules over large areas. This is particularly important when addressing application such as cell microarray manufacturing, which are currently used for a wide range of analytical and pharmaceutical applications. In this paper, we present a large-scale and multiplexed microcontact printing process of extracellular matrix proteins for the fabrication of cell microarrays. We have developed a microfluidic inking approach combined with a magnetic clamping technology that can be adapted to most standard substrates used in biology. We have demonstrated a significant improvement of homogeneity of printed protein patterns on surfaces larger than 1 cm2 through the control of both the flow rate and the wetting mechanism of the stamp surface during microfluidic inking. Thanks to the reproducibility and integration capabilities provided by microfluidics, we have achieved the printing of three different adhesion proteins in one-step transfer. Selective cell adhesion and cell shape adaptation on the produced patterns were observed, showing the suitability of this approach for producing on-demand large-scale cell microarrays.
Subject(s)
Extracellular Matrix Proteins/isolation & purification , Microfluidic Analytical Techniques/methods , Printing/instrumentation , Tissue Array Analysis/instrumentation , Biosensing Techniques , Cell Adhesion/genetics , Cell Shape/genetics , Extracellular Matrix Proteins/chemistryABSTRACT
Capturing cell-secreted extracellular matrix (ECM) proteins through cooperative binding with high specificity and affinity is an important function of native tissue matrices during both tissue homeostasis and repair. However, while synthetic hydrogels, such as those based on poly(ethylene glycol) (PEG), are often proposed as ideal materials to deliver human mesenchymal stem cells (hMSCs) to sites of injury to enable tissue repair, they do not have this capability-a capability that would enable cells to actively remodel their local extracellular microenvironment and potentially provide the required feedback control for more effective tissue genesis. In this work, we detail a methodology that engenders poly(ethylene glycol) (PEG)-based two-dimensional substrates and three-dimensional porous hydrogels with the ability to capture desired extracellular matrix (ECM) proteins with high specificity. This "encoded" ECM protein capture is achieved by decorating the PEG-based materials with protein binding peptides (PBPs) synthesized to be specific in their binding of fibronectin, laminin, and collagen I, which are not only the most omnipresent ECM proteins in human tissues but, as we confirmed, are also secreted to differing extents by hMSCs under in vitro maintenance conditions. By encapsulating hMSCs into these PBP-functionalized hydrogels, and culturing them in protein-free maintenance media, we demonstrate that these PBPs not only actively recruit targeted ECM proteins as they are secreted from hMSCs but also retain them to much higher levels compared to nonfunctionalized gels. This novel approach thus enables the fabrication of encoded surfaces and hydrogels that capture cell-secreted proteins, with high specificity and affinity, in a programmable manner, ready for applications in many bioengineering applications, including bioactive surface coatings, bioassays, stem cell culture, tissue engineering, and regenerative medicine.
Subject(s)
Extracellular Matrix Proteins , Hydrogels/chemistry , Mesenchymal Stem Cells/metabolism , Peptides/chemistry , Polyethylene Glycols/chemistry , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/isolation & purification , Extracellular Matrix Proteins/metabolism , Humans , Mesenchymal Stem Cells/chemistry , Mesenchymal Stem Cells/cytologyABSTRACT
Functional studies of extracellular proteins are often performed using coimmunoprecipitation without purified proteins. However, in order to exclude unspecific reactions of contaminants and for quantitative analysis of specific functions, it is necessary to use purified proteins. It is usually very difficult, however, to purify sufficient amounts of reasonably pure extracellular matrix proteins from tissue samples, but the recombinant expression and purification of proteins in eukaryotic expression systems including insect cells and mammalian cells has proven an alternative powerful method. In this chapter, we describe the expression and purification of recombinant fibulins, but the methods can be also used for other extracellular proteins.
Subject(s)
Calcium-Binding Proteins/isolation & purification , Cell Culture Techniques/methods , Chromatography, Affinity/methods , Extracellular Matrix Proteins/isolation & purification , Animals , Calcium-Binding Proteins/chemistry , Cell Culture Techniques/instrumentation , Chromatography, Affinity/instrumentation , Extracellular Matrix Proteins/chemistry , HEK293 Cells , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Transfection/instrumentation , Transfection/methodsABSTRACT
AIM: To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY: Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS: High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS: A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.