ABSTRACT
Carbohydrates play important and diverse roles in the fundamental processes of life. We have established a method for accurately and a large-scale synthesis of functional carbohydrates with diverse properties using a unique enzymatic method. Furthermore, various artificial glycan-conjugated molecules have been developed by adding these synthetic carbohydrates to macromolecules and to middle- and low-molecular-weight molecules with different properties. These glycan-conjugated molecules have biological activities comparable to or higher than those of natural compounds and present unique functions. In this review, several synthetic glycan-conjugated molecules are taken as examples to show design, synthesis, and function.
Subject(s)
Antiviral Agents/chemical synthesis , Biological Factors/chemical synthesis , Glycoconjugates/chemical synthesis , Polysaccharides/chemistry , Sialoglycoproteins/chemical synthesis , Animals , Antiviral Agents/pharmacology , Biological Factors/pharmacology , Carbohydrate Conformation , Cheminformatics/methods , Cross-Linking Reagents/chemistry , Drug Design , Glycoconjugates/pharmacology , Glycosides/chemistry , Glycosylation , Humans , Nanoparticles/chemistry , Orthomyxoviridae/drug effects , Orthomyxoviridae/growth & development , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Sialoglycoproteins/pharmacologyABSTRACT
BACKGROUND: Individuals with cerebral palsy (CP) experience bone loss due to impaired weight bearing. Despite serious complications, there is no standard medication. OBJECTIVE: To develop a new pharmacological agent, we performed a series of studies. The primary aim was to develop an animal model of CP to use our target medication based on transcriptome analysis of individuals with CP. The secondary aim was to show the therapeutic capability of collagen-binding peptide (CBP) in reversing bone loss in the CP mouse model. METHODS: A total of 119 people with CP and 13 healthy adults participated in the study and 140 mice were used for the behavioral analysis and discovery of therapeutic effects in the preclinical study. The mouse model of CP was induced by hypoxic-ischemic brain injury. Inclusion and exclusion criteria were established for CBP medication in the CP mouse model with bone loss. RESULTS: On the basis of clinical outcomes showing insufficient mechanical loading from non-ambulatory function and that underweight mainly affects bone loss in adults with CP, we developed a mouse model of CP with bone loss. Injury severity and body weight mainly affected bone loss in the CP mouse model. Transcriptome analysis showed SPP1 expression downregulated in adults with CP who showed lower bone density than healthy controls. Therefore, a synthesized CBP was administered to the mouse model. Trabecular thickness, total collagen and bone turnover activity increased with CBP treatment as compared with the saline control. Immunohistochemistry showed increased immunoreactivity of runt-related transcription factor 2 and osteocalcin, so the CBP participated in osteoblast differentiation. CONCLUSIONS: This study can provide a scientific basis for a promising translational approach for developing new anabolic CBP medication to treat bone loss in individuals with CP.
Subject(s)
Bone Density , Bone Resorption/prevention & control , Cerebral Palsy , Peptide Fragments/pharmacology , Sialoglycoproteins/pharmacology , Animals , Cerebral Palsy/drug therapy , Disease Models, Animal , Humans , Mice , Weight-BearingABSTRACT
Sialylation is involved in receptor-ligand interactions, communication between cells, and host-pathogen interactions and it is involved in the ability of glycoproteins of bovine milk to inhibit the influenza A virus (IAV). The present paper describes a simple and efficient method to isolate sialoglycoproteins from bovine milk using serotonin-magnetic particle conjugates. Then, the isolated glycoproteins were analysed by lectin blotting and LC-MS/MS. The N-glycans on isolated glycoproteins were characterized by MALDI-TOF/TOF-MS. The role of the isolated sialoglycoproteins against IAV was validated in vitro. As a result, there were 91 proteins and 17 sialylated N-glycans to be identified. The isolated proteins have ability to inhibit attachment of IAV mimics to MDCK cells. However, the role of inhibition was abolished when the sialic acid moieties were destroyed. This method could provide useful information for the large-scale production of sialoglycoproteins from bovine milk against IAV.
Subject(s)
Influenza A virus/drug effects , Milk/chemistry , Sialoglycoproteins/isolation & purification , Sialoglycoproteins/pharmacology , Animals , Chromatography, Liquid , Glycoproteins/pharmacology , Hydrogen-Ion Concentration , Influenza A virus/metabolism , Lectins/metabolism , N-Acetylneuraminic Acid/metabolism , Polysaccharides/chemistry , Serotonin/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
Recent reports highlight the potential tumorigenic role of Dentin Sialophosphoprotein (DSPP) and its cognate partner Matrix Metalloproteinase 20 (MMP-20) in Oral Squamous Cell Carcinomas (OSCCs). However, the function/mechanism of these roles is yet to be fully established. The present study aimed to investigate the effects of DSPP and MMP20 silencing on specific proteins involved in oral cancer cell adhesion, angiogenesis, metastasis, and epithelial-mesenchymal transition (EMT). Stable lines of DSPP/MMP20 silenced OSCC cell line (OSC2), previously established via lentiviral-mediated shRNA transduction, were analyzed for the effects of DSPP, MMP20, and combined DSPP-MMP20 silencing on MMP2, MMP9, integrins αvß3 and αvß6, VEGF, Kallikerin- 4,-5,-8,-10, E-cadherin, N-cadherin, Vimentin, met, src, snail, and Twist by Western blot. Results show a significant decrease (p < 0.05) in the expression of MMP2, MMP9, integrin αvß3, αvß6, VEGF, Kallikerins -4, -5, -8, -10, N-cadherin, vimentin met, src, snail and twist following DSPP and MMP20 silencing, individually and in combination. On the other hand, the expression of E-cadherin was found to be significantly increased (p < 0.05). These results suggest that the tumorigenic effect of DSPP and MMP20 on OSC2 cells is mediated via the upregulation of the genes involved in invasion, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT).
Subject(s)
Extracellular Matrix Proteins/pharmacology , Matrix Metalloproteinase 20/metabolism , Phosphoproteins/pharmacology , Sialoglycoproteins/pharmacology , Squamous Cell Carcinoma of Head and Neck/metabolism , Cadherins/metabolism , Carcinoma, Squamous Cell/pathology , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Epithelial-Mesenchymal Transition/genetics , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Neoplastic/genetics , Head and Neck Neoplasms/pathology , Humans , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 20/genetics , Matrix Metalloproteinase 9/metabolism , Mouth Neoplasms/pathology , Neoplasm Invasiveness , Neoplasm Metastasis/genetics , Neovascularization, Pathologic/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Squamous Cell Carcinoma of Head and Neck/geneticsABSTRACT
Helicobacter pylori (H. pylori) is a primary etiologic factor in gastric diseases. Sulglycotide is a glycopeptide derived from pig duodenal mucin. Esterification of its carbohydrate chains with sulfate groups creates a potent gastroprotective agent used to treat various gastric diseases. We investigated the inhibitory effects of sulglycotide on adhesion and inflammation after H. pylori infection in human gastric adenocarcinoma cells (AGS cells). H. pylori reference strain 60190 (ATCC 49503) was cultured on Brucella agar supplemented with 10% bovine serum. Sulgylcotide-mediated growth inhibition of H. pylori was evaluated using the broth dilution method. Inhibition of H. pylori adhesion to AGS cells by sulglycotide was assessed using a urease assay. Effects of sulglycotide on the translocation of virulence factors was measured using western blot to detect cytotoxin-associated protein A (CagA) and vacuolating cytotoxin A (VacA) proteins. Inhibition of IL-8 secretion was measured using enzyme-linked immunosorbent assay (ELISA) to determine the effects of sulglycotide on inflammation. Sulglycotide did not inhibit the growth of H. pylori, however, after six and 12 hours of infection on AGS cells, H. pylori adhesion was significantly inhibited by approximately 60% by various concentrations of sulglycotide. Sulglycotide decreased H. pylori virulence factor (CagA and VacA) translocation to AGS cells and inhibited IL-8 secretion. Sulglycotide inhibited H. pylori adhesion and inflammation after infection of AGS cells in vitro. These results support the use of sulglycotide to treat H. pylori infections.
Subject(s)
Bacterial Adhesion , Helicobacter Infections , Helicobacter pylori , Sialoglycoproteins , Animals , Bacterial Adhesion/drug effects , Bacterial Proteins , Cattle , Helicobacter Infections/drug therapy , Helicobacter pylori/drug effects , Humans , Inflammation/drug therapy , Sialoglycoproteins/pharmacology , SwineABSTRACT
OBJECTIVE: The objective of this study was to investigate the effects of dentin phosphoprotein (DPP) on lipopolysaccharide-induced inflammatory responses of macrophages in vitro. DESIGN: Wildtype and mutant recombinant dentin phosphoprotein (rDPP) proteins were generated using a mammalian expression system. Macrophages, phorbol 12-myristate 13-acetate-differentiated THP-1 cells, were stimulated with lipopolysaccharide in the absence or presence of rDPP proteins. After the 24-hr incubation, the inflammatory gene expression levels were examined by quantitative reverse-transcription polymerase chain reaction and the amount of secreted TNF-α protein was evaluated by enzyme-linked immunosorbent assay. Furthermore, the subcellular localization of exogenously added rDPP was examined by immunocytochemistry, and the direct binding of rDPP to lipopolysaccharide was quantified by solid-phase binding assay. RESULTS: rDPP dose-dependently reduced the expression of lipopolysaccharide-induced inflammatory genes, such as TNFα, IL-1ß, and IL-8, and TNF-α protein secretion from the macrophages. Furthermore, mutant rDPP having a shortened serine/aspartic acid-rich repeats (SDrr) was also able to inhibit lipopolysaccharide-induced inflammatory responses of macrophages. rDPP was localized adjacent to the cellular membrane rather than in the cytoplasm, and rDPP was able to bind to lipopolysaccharide. These results suggested that rDPP inhibited lipopolysaccharide-induced inflammatory responses by binding to lipopolysaccharide. CONCLUSIONS: In addition to the well-known functions of DPP for dentin mineralization that depend on the SDrr, we demonstrated that DPP possesses anti-inflammatory effects on lipopolysaccharide-stimulated macrophages that are independent of the SDrr.
Subject(s)
Dentin , Extracellular Matrix Proteins , Macrophage Activation , Phosphoproteins , Sialoglycoproteins , Animals , Aspartic Acid , Dentin/immunology , Extracellular Matrix Proteins/pharmacology , Inflammation , Lipopolysaccharides , Phosphoproteins/pharmacology , Serine , Sialoglycoproteins/pharmacology , Tumor Necrosis Factor-alphaABSTRACT
Objective: Several studies demonstrated that sulglycotide has anti-inflammatory and anti-cancer effects. However, the effect of sulglycotide is limited to gastric mucosal tissues and cells and underlying molecular mechanisms are not clear. This study estimated the effect of sulglycotide on lipopolysaccharide (LPS)-induced inflammatory responses in the macrophage cell line, RAW 264.7 and elucidated the molecular mechanisms. Materials and methods: The inhibitory effect of sulglysotide on LPS-induced oxidative stress and inflammatory reactions were determined by Immunofluorescence staining, ELISA, Western blotting and RT-PCR. Results: Our results show that sulglycotide has the ability to inhibit inflammatory mediators and cytokine production as well as reactive oxygen species (ROS) generation. This effect can be the result from regulating the activation of nuclear factor-kappa B (NF-κB) through blocking mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Conclusions: These results indicate that sulglycotide could be an anti-inflammatory and anti-oxidative compound that may be a useful candidate for treatment of inflammatory diseases.
Subject(s)
Inflammation/chemically induced , Inflammation/drug therapy , Lipopolysaccharides/pharmacology , Macrophages/drug effects , NF-kappa B/metabolism , Sialoglycoproteins/pharmacology , Signal Transduction/drug effects , Animals , Anti-Inflammatory Agents/pharmacology , Cell Line , Cytokines/metabolism , Inflammation/metabolism , MAP Kinase Signaling System/drug effects , Macrophages/metabolism , Mice , RAW 264.7 Cells , Reactive Oxygen Species/metabolismABSTRACT
In this study, we explore whether the pro-osteogenic effects of sialoglycoprotein from Carassius auratus eggs (Ca-SGP) involve mesenchymal stem cells (MSCs). Ovariectomized osteoporotic mice treated with Ca-SGP had increased bone formation and reduced bone marrow adipose tissue. As MSCs are common progenitors of osteoblasts and adipocytes, we isolated MSCs from Ca-SGP-treated mice and found that they tended to differentiate into osteoblasts over adipocytes confirmed by Alizarin red and Oil red O staining. This change was seen at the gene and protein level. To further explore the effect of Ca-SGP on MSCs, we isolated MSCs from healthy mice and treated them with Ca-SGP in vitro. We discovered that Ca-SGP promoted MSC differentiation to osteoblasts. In addition, Ca-SGP promoted osteogenesis and reduced the fat in marrow cavity of adolescent mice. For the first time, our results demonstrate that Ca-SGP promotes osteogenesis via stimulating MSCs to commit to osteoblasts. Graphical Abstract á .
Subject(s)
Adipocytes/cytology , Cell Differentiation/drug effects , Goldfish , Mesenchymal Stem Cells/drug effects , Osteoblasts/cytology , Osteogenesis/drug effects , Sialoglycoproteins/pharmacology , Animals , Bone and Bones/cytology , Cells, Cultured , Disease Models, Animal , Mesenchymal Stem Cells/cytology , Mice, Inbred C57BL , Sialoglycoproteins/isolation & purificationABSTRACT
Dentin phosphoprotein (DPP) is a major component of the dentin matrix playing crucial role in hydroxyapatite deposition during bone mineralization, making it a prime candidate for the design of novel materials for bone and tooth regeneration. The bioactivity of DPP-derived proteins is controlled by the phosphorylation and dephosphorylation of the serine residues. Here an enzyme-responsive peptide nanofiber system inducing biomineralization is demonstrated. It closely emulates the structural and functional properties of DPP and facilitates apatite-like mineral deposition. The DPP-mimetic peptide molecules self-assemble through dephosphorylation by alkaline phosphatase (ALP), an enzyme participating in tooth and bone matrix mineralization. Nanofiber network formation is also induced through addition of calcium ions. The gelation process following nanofiber formation produces a mineralized extracellular matrix like material, where scaffold properties and phosphate groups promote mineralization. It is demonstrated that the DPP-mimetic peptide nanofiber networks can be used for apatite-like mineral deposition for bone regeneration.
Subject(s)
Biomimetic Materials , Bone Regeneration/drug effects , Calcification, Physiologic/drug effects , Extracellular Matrix Proteins , Nanofibers/chemistry , Peptides , Phosphoproteins , Sialoglycoproteins , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Cell Line, Tumor , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/pharmacology , Humans , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Phosphoproteins/chemistry , Phosphoproteins/pharmacology , Sialoglycoproteins/chemistry , Sialoglycoproteins/pharmacologyABSTRACT
Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. ß-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.
Subject(s)
Gadus morhua/metabolism , Osteogenesis/drug effects , Ovum/chemistry , Sialoglycoproteins/pharmacology , Tibia/growth & development , Amino Acid Motifs , Animals , Cell Line , Cell Proliferation/drug effects , Chromatography, Gel , Chromatography, Ion Exchange , Fish Proteins/chemistry , Fish Proteins/genetics , Fish Proteins/pharmacology , Mice , Molecular Weight , Phosphorus/analysis , Rats , Sialoglycoproteins/chemistry , Sialoglycoproteins/genetics , Spectroscopy, Fourier Transform Infrared , Tibia/chemistry , Tibia/drug effectsABSTRACT
In recent years, rapid development of tissue engineering technology provides possibilities for the construction of artificial tissues or organs. In construction of engineered kidneys, researchers used native decellularized extracellular matrix (ECM) as the scaffolds to recellularization. However, thrombosis has been a great issue that hinders the progress of transplantation in vivo. In this study, heparin was immobilized to the collagen part of decellularized scaffold with collagen-binding peptide (CBP). Through the anticoagulant and endothelial cell reperfusion experiments, it can be demonstrated that the heparinized scaffolds absorbed less platelets and red blood cells which can effectively reduce the formation of thrombosis. Moreover, it is conducive to long-term adhesion of endothelial cells which is important for the formation of subsequent vascularization. Taken together, our results reveal that the whole kidney can be modified by CBP-heparin composite to reduce the thrombosis and provide the better conditions for neovascularization.
Subject(s)
Cellular Microenvironment , Endothelial Cells/metabolism , Heparin/metabolism , Kidney/metabolism , Thrombosis/metabolism , Tissue Scaffolds , Animals , Anticoagulants/metabolism , Anticoagulants/pharmacology , Cells, Cultured , Endothelial Cells/drug effects , Extracellular Matrix/metabolism , Heparin/pharmacology , Humans , Kidney/cytology , Kidney/ultrastructure , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Platelet Adhesiveness , Rats, Sprague-Dawley , Sialoglycoproteins/metabolism , Sialoglycoproteins/pharmacology , Thrombosis/prevention & control , Tissue Engineering/methodsABSTRACT
A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2 â 3)- and α(2 â 6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a-c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.
Subject(s)
Antiviral Agents/pharmacology , Hemagglutination/drug effects , Orthomyxoviridae/drug effects , Sialoglycoproteins/pharmacology , Sialyltransferases/chemistry , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Binding, Competitive , Bombyx , Carbohydrate Sequence , Cloning, Molecular , Dogs , Erythrocytes/drug effects , Erythrocytes/virology , Gene Expression , Hemagglutination Inhibition Tests , Hemolymph/chemistry , Horses , Humans , Neuraminic Acids/chemistry , Nucleopolyhedroviruses/genetics , Nucleopolyhedroviruses/metabolism , Polyglutamic Acid/analogs & derivatives , Polyglutamic Acid/chemistry , Polyglutamic Acid/metabolism , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sialoglycoproteins/biosynthesis , Sialoglycoproteins/chemistry , Sialyltransferases/genetics , Sialyltransferases/metabolism , beta-Galactoside alpha-2,3-SialyltransferaseABSTRACT
The anti-osteoporotic effect of sialoglycoprotein isolated from the eggs of Carassius auratus (Ca-SGP) has previously been demonstrated in vivo. However, the mechanism by which this macromolecular substance regulates bone metabolism remains unclear. Given the correlation between gut microbiota and the homeostasis of bone metabolism, the current study applied real-time PCR and 16S rRNA high-throughput sequencing to investigate the influence of Ca-SGP on gut microbiota in ovariectomized rats. Real-time PCR analysis showed that Ca-SGP treatment significantly reversed the increase of Escherichia coli and Bacteroides fragilis, and the decrease of Clostridium leptum, Faecalibacterium prausnitzii, and Lactobacillus induced by ovariectomy. Subsequent high-throughput sequencing of 16S rRNA further demonstrated that Ca-SGP not only partly recovered the alterations of gut microbiota to baseline levels in OVX rats, but also significantly increased the relative abundance of Lactobacillus, which may support the protection of bone metabolism. The present study may contribute towards our understanding of the mechanism by which Ca-SGP seemingly preserves bone mass.
Subject(s)
Gastrointestinal Microbiome/drug effects , Goldfish/physiology , Ovum/chemistry , Sialoglycoproteins/pharmacology , Animals , Bacteria/drug effects , Female , Gene Expression Regulation/drug effects , Ovariectomy , Random Allocation , Rats , Sialoglycoproteins/chemistryABSTRACT
Remineralization of dentin during dental caries is of considerable clinical interest. Dentin matrix protein 1 (DMP1) is a non-collagenous calcium-binding protein that plays a critical role in biomineralization. In the present study, we tested if peptides derived from DMP1 can be used for dentin remineralization. Peptide pA (pA, MW = 1.726 kDa) and peptide pB (pB, MW = 2.185), containing common collagen-binding domains and unique calcium-binding domains, were synthesized by solid-phase chemistry. An extreme caries lesion scenario was created by collagenase digestion, and the biomineral-nucleating potential of these peptides was ascertained when coated on collagenase-treated dentin matrix and control, native human dentin matrix under physiological levels of calcium and phosphate. Scanning electron microscopy analysis suggests that peptide pB was an effective nucleator when compared with pA. However, a 1:4 ratio of pA to pB was determined to be ideal for dentin remineralization, based on hydroxyapatite (HA) morphology and calcium/phosphorus ratios. Interestingly, HA was nucleated on collagenase-challenged dentin with as little as 20 min of 1:4 peptide incubation. Electron diffraction confirmed the presence of large HA crystals that produced a diffraction pattern indicative of a rod-like crystal structure. These findings suggest that DMP1-derived peptides may be useful to modulate mineral deposition and subsequent formation of HA when exposed to physiological concentrations of calcium and phosphate.
Subject(s)
Dentin/drug effects , Extracellular Matrix Proteins/pharmacology , Phosphoproteins/pharmacology , Tooth Remineralization , Biomimetic Materials/pharmacology , Calcium/pharmacology , Calcium-Binding Proteins/pharmacology , Collagen Type I/metabolism , Crystallography , Dental Caries/metabolism , Dental Caries/pathology , Dentin/metabolism , Dentin/ultrastructure , Durapatite/metabolism , Humans , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Peptide Fragments/pharmacology , Phosphates/pharmacology , Protein Binding , Sialoglycoproteins/pharmacology , Time Factors , X-Ray DiffractionABSTRACT
The aim of this study is to determine the effects of the combination of recombinant human BMP-2 (rh-BMP-2) and dentin sialoprotein (rh-DSP) on growth and differentiation in human cementoblasts and determine the underlying signal transduction mechanism. Compared to treatment of cementoblasts with either rh-BMP-2 or rh-DSP alone, the combination of rh-BMP-2 and rh-DSP synergistically increased cell growth, ALP activity, nodule formation and expression of differentiation markers. The differentiation-promoting effect was also observed in periodontal ligament cells and an osteoblastic cell line. Likewise, combination of rh-DSP and rh-BMP-2 increased BMP-2 mRNA expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. The expression levels of α2ß1 integrin and RhoA, as well as the phosphorylation status of FAK and Akt, were increased by the combination of rh-BMP-2 and rh-DSP in a time-dependent manner. In addition, rh-BMP-2 and rh-DSP enhanced expression of Wnt ligands, ß-catenin activation and GSK-3ß phosphorylation, all of which were inhibited by the Wnt receptor antagonist DKK1. Furthermore, treatment with rh-DSP plus rh-BMP-2 resulted in phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 and also induced the nuclear translocation of the NF-κB p65 subunit, which was blocked by noggin. This study demonstrates for the first time that rh-DSP and rh-BMP-2 act synergistically, enhancing each other's ability to stimulate cementoblastic cell growth and differentiation in vitro via autocrine BMP, integrin, Wnt/ß-catenin, MAP kinase and NF-κB pathways. These results support the therapeutic potential of a combination strategy for aiding periodontal regeneration.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Dental Cementum/cytology , Dental Cementum/drug effects , Extracellular Matrix Proteins/pharmacology , Phosphoproteins/pharmacology , Sialoglycoproteins/pharmacology , Transforming Growth Factor beta/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Dental Cementum/metabolism , Humans , Periodontal Ligament/cytology , Periodontal Ligament/drug effects , Periodontal Ligament/metabolism , Recombinant Proteins/pharmacology , Signal Transduction/drug effectsABSTRACT
Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-ß activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-ß receptor inhibitor. We then purified TGF-ß-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-ß isoform bound to DPP and DSP was identified as being TGF-ß1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-ß1 (CF-hTGF-ß1) with TGF-ß-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-ß1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-ß1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-ß to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-ß1. We conclude that both DPP and DSP help retain TGF-ß1 activity in porcine dentin.
Subject(s)
Dentin/chemistry , Extracellular Matrix Proteins/pharmacology , Phosphoproteins/pharmacology , Sialoglycoproteins/pharmacology , Transforming Growth Factor beta1/drug effects , Alkaline Phosphatase/drug effects , Animals , Cells, Cultured , Chromatography, Ion Exchange , Chromatography, Liquid , Chromatography, Reverse-Phase , Extracellular Matrix Proteins/analysis , Humans , Periodontal Ligament/cytology , Periodontal Ligament/enzymology , Phosphoproteins/analysis , Protein Binding , Protein Isoforms/analysis , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Recombinant Proteins , Sialoglycoproteins/analysis , Swine , Transforming Growth Factor beta1/analysisABSTRACT
Dentin phosphophoryn is nature's most acidic protein found predominantly in the dentin extracellular matrix. Its unique amino acid composition containing Asp-Ser (DS)-rich repeats makes it highly anionic. It has a low isoelectric point (pI 1.1) and, therefore, tends to be negatively charged at physiological pH. Phosphophoryn is normally associated with matrix mineralization as it can bind avidly to Ca(2+). It is well known that several macromolecules present in the extracellular matrix can be internalized and localized to specific intracellular compartments. In this study we demonstrate that dentin phosphophoryn (DPP) is internalized by several cell types via a non-conventional endocytic process. Utilizing a DSS polypeptide derived from DPP, we demonstrate the repetitive DSS-rich domain facilitates that endocytosis. As a proof-of-concept, we further demonstrate the use of this polypeptide as a protein delivery vehicle by delivering the osteoblast transcription factor Runx2 to the nucleus of mesenchymal cells. The functionality of the endocytosed Runx2 protein was demonstrated by performing gene expression analysis of Runx2 target genes. Nuclear localization was also demonstrated with the fusion protein DSS-Runx2 conjugated to quantum dots in two- and three-dimensional culture models in vitro and in vivo. Overall, we demonstrate that the DSS domain of DPP functions as a novel cell-penetrating peptide, and these findings demonstrate new opportunities for intracellular delivery of therapeutic proteins and cell tracking in vivo.
Subject(s)
Cell-Penetrating Peptides/metabolism , Extracellular Matrix Proteins/metabolism , Phosphoproteins/metabolism , Sialoglycoproteins/metabolism , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Animals , Cell Line , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/pharmacology , Core Binding Factor Alpha 1 Subunit/chemistry , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Endocytosis/drug effects , Endocytosis/physiology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/pharmacology , Humans , Hydrogen-Ion Concentration , Mice , Phosphoproteins/genetics , Phosphoproteins/pharmacology , Protein Structure, Tertiary , Sialoglycoproteins/genetics , Sialoglycoproteins/pharmacologyABSTRACT
To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p < 0.05). Cell viability was also significantly increased by rhDPP at 5 days (p < 0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.
Subject(s)
Cell Differentiation/drug effects , Dental Pulp/cytology , Extracellular Matrix Proteins/biosynthesis , Odontoblasts/physiology , Phosphoproteins/biosynthesis , Sialoglycoproteins/biosynthesis , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Calcification, Physiologic , Cell Adhesion , Cell Proliferation , Cell Survival/drug effects , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Escherichia coli , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/isolation & purification , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/pharmacology , Gene Expression , Humans , Odontoblasts/enzymology , Odontoblasts/metabolism , Phosphoproteins/genetics , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , Phosphoproteins/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Sialoglycoproteins/isolation & purification , Sialoglycoproteins/pharmacologyABSTRACT
Dentin sialoprotein (DSP) is cleaved from dentin sialophosphoprotein (DSPP) and most abundant dentinal non-collagenous proteins in dentin. DSP is believed to participate in differentiation and mineralization of cells. In this study, we first constructed recombinant human DSP (rhDSP) in Escherichia coli (E. coli) and investigated its odontoblastic differentiation effects on human dental pulp cells (hDPCs). Cell adhesion activity was measured by crystal violet assay and cell proliferation activity was measured by MTT assay. To assess mineralization activity of rhDSP, Alizarin Red S staining was performed. In addition, the mRNA levels of collagen type Ð (Col Ð), alkaline phosphatase (ALP), and osteocalcin (OCN) were measured due to their use as mineralization markers for odontoblast-/osteoblast-like differentiation of hDPCs. The obtained rhDSP in E. coli was approximately identified by SDS-PAGE and Western blot. Initially, rhDSP significantly enhanced hDPCs adhesion activity and proliferation (p<0.05). In Alizarin Red S staining, stained hDPCs increased in a time-dependent manner. This odontoblastic differentiation activity was also verified through mRNA levels of odontoblast-related markers. Here, we first demonstrated that rhDSP may be an important regulatory ECM in determining the hDPCs fate including cell adhesion, proliferation, and odontoblastic differentiation activity. These findings indicate that rhDSP can induce growth and differentiation on hDPCs, leading to improve tooth repair and regeneration.
Subject(s)
Dental Pulp/drug effects , Escherichia coli/genetics , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/pharmacology , Phosphoproteins/metabolism , Phosphoproteins/pharmacology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Sialoglycoproteins/metabolism , Sialoglycoproteins/pharmacology , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Collagen Type I/genetics , Collagen Type I/metabolism , Dental Pulp/cytology , Extracellular Matrix Proteins/genetics , Humans , Odontoblasts , Osteocalcin/genetics , Osteocalcin/metabolism , Phosphoproteins/genetics , Recombinant Proteins/genetics , Sialoglycoproteins/geneticsABSTRACT
Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor γ. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a novel CBP could be a useful candidate for regenerating bone and treating osteoporosis, which result from an imbalance in osteogenesis and adipogenesis differentiation.