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1.
Food Chem ; 329: 126775, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512387

RESUMO

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.


Assuntos
Colágeno Tipo I/química , Hidroxiprolina/química , Prolina/química , Serina/química , Animais , Dicroísmo Circular , Peixes , Desnaturação Proteica , Temperatura
2.
Proc Natl Acad Sci U S A ; 117(21): 11450-11458, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385162

RESUMO

Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments-each containing the respective PEDF-binding region (residues 930 to 938)-are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link-susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner.


Assuntos
Colágeno Tipo I/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Serpinas/química , Serpinas/metabolismo , Sítios de Ligação , Dicroísmo Circular , Colágeno Tipo I/química , Cristalografia por Raios X , Dissulfetos/química , Lisina/química , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Transdução de Sinais , Análise Espaço-Temporal
3.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 55(2): 135-138, 2020 Feb 09.
Artigo em Chinês | MEDLINE | ID: mdl-32074678

RESUMO

Establishing a stable resin-dentin hybrid layer is an effective method to improve the adhesion durability of the restoration. The biomodification of dentin by cross-linkers can enhance the mechanical properties of collagen and resistance to enzymatic hydrolysis while, inhibiting the process of demineralization and promoting the remineralization of dentin, which has the potential clinical applicability of preventing dental caries and improving adhesive property. This review summarizes the biomodification of dentin type Ⅰ collagen by different cross-linkers.


Assuntos
Colágeno Tipo I/química , Colagem Dentária , Adesivos Dentinários , Dentina/química , Cárie Dentária/prevenção & controle , Humanos , Teste de Materiais , Cimentos de Resina
4.
Nat Chem Biol ; 16(4): 423-429, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31907373

RESUMO

The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.


Assuntos
Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Colágeno/química , Sequência de Aminoácidos , Aminoácidos , Colágeno/metabolismo , Biologia Computacional/métodos , Humanos , Modelos Moleculares , Peptídeos/química , Conformação Proteica
5.
Nat Prod Res ; 34(1): 53-62, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30821504

RESUMO

The study focuses on the understanding, at molecular level, the mechanism of interaction between protein and flavonoids. Collagen and catechin interactions were investigated by NMR in solution and solid state. The effect of catechin on the stability of collagen to oxidation was also explored. Collagen was treated with two concentrations of catechin solutions. Oxidation was carried out by incubation of collagen solution with three oxidation systems: Fe(II)/H2O2, Cu(II)/H2O2, and NaOCl/H2O2. The effects of oxidation systems were evaluated by high resolution 1 D and 2 D proton spectroscopy and solid state NMR (13C CP MAS) experiments. Interactions between collagen and catechin preferentially occur between catechin B ring and the amino acids Pro and Hyp of collagen. Results showed that both iron and copper oxidation systems were able to interact with collagen by site specific attack. Moreover, catechin protects collagen proline from oxidation by metal/H2O2 systems, preventing copper and iron approach to collagene molecule;this behaviour was more evident for the copper/H2O2 system.


Assuntos
Catequina/metabolismo , Colágeno Tipo I/metabolismo , Peróxido de Hidrogênio/química , Sítios de Ligação , Catequina/química , Catequina/farmacologia , Colágeno Tipo I/química , Flavonoides/química , Flavonoides/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metais/química , Oxirredução , Estabilidade Proteica/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismo
6.
Colloids Surf B Biointerfaces ; 185: 110597, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675640

RESUMO

Conferring orientational order to biological hydrogels constitutes a fruitful strategy for the guided growth of cells. The ability of anisotropic magnetic particles to align along an external magnetic field appears as a particularly, yet poorly explored, strategy to achieve such an orientation in 3D. For this purpose, silica rods coated with magnetite nanoparticles were prepared. When dispersed in a collagen type I solutions, they could be aligned along the magnetic field generated by two plate magnets within five minutes, such an alignment being preserved during hydrogel formation. Both magnetic and rheological measurements evidenced that different structures could be obtained in the absence of the magnetic field and when it was applied parallel or perpendicular to the hydrogel surface. These variations in rods organization also impacted the growth of 2D cultures of Normal Human Dermal Fibroblasts, which was attributed to the higher affinity of the cells for type I collagen compared to silica. These composites have a clear potential as biomaterials associating cell guidance and drug delivery.


Assuntos
Colágeno Tipo I/química , Fibroblastos/citologia , Hidrogéis/química , Nanopartículas de Magnetita/química , Nanotubos/química , Dióxido de Silício/química , Pele/citologia , Células Cultivadas , Humanos
7.
Mar Drugs ; 17(10)2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569390

RESUMO

Collagen is widely used in the pharmaceutical, tissue engineering, nutraceutical, and cosmetic industries. In this study, acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the skin of red stingray, and its physicochemical and functional properties were investigated. The yields of ASC and PSC were 33.95 ± 0.7% and 37.18 ± 0.71% (on a dry weight basis), respectively. ASC and PSC were identified as type I collagen by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis, possessing a complete triple helix structure as determined by UV absorption, Fourier transform infrared, circular dichroism, and X-ray diffraction spectroscopy. Contact angle experiments indicated that PSC was more hydrophobic than ASC. Thermal stability tests revealed that the melting temperature of PSC from red stingray skin was higher than that of PSC from duck skin, and the difference in the melting temperature between these two PSCs was 9.24 °C. Additionally, both ASC and PSC were functionally superior to some other proteins from terrestrial sources, such as scallop gonad protein, whey protein, and goose liver protein. These results suggest that PSC from red stingray skin could be used instead of terrestrial animal collagen in drugs, foods, cosmetics, and biological functional materials, and as scaffolds for bone regeneration.


Assuntos
Colágeno Tipo I/química , Proteínas de Peixes/química , Rajidae , Pele/química , Ácidos/química , Animais , Regeneração Óssea , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/isolamento & purificação , Colágeno Tipo I/toxicidade , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/toxicidade , Teste de Materiais , Camundongos , Células NIH 3T3 , Pepsina A/química , Estabilidade Proteica , Solubilidade , Tecidos Suporte/química , Testes de Toxicidade , Difração de Raios X
8.
Comput Methods Programs Biomed ; 182: 105056, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31542705

RESUMO

BACKGROUND AND OBJECTIVE: During the last years different model solutions were proposed for solving cell forces under different conditions. The solution relies on a deformation field that is obtained under cell relaxation with a chemical cocktail. Once the deformation field of the matrix is determined, cell forces can be computed by an inverse algorithm, given the mechanical properties of the matrix. Most of the Traction Force Microscopy (TFM) methods presented so far relied on a linear stress-strain response of the matrix. However, the mechanical response of some biopolymer networks, such as collagen gels is more complex. In this work, we present a numerical method for solving cell forces on non-linear materials. METHODS: The proposed method relies on solving the inverse problem based on an iterative optimization. The objective function is defined by least-square minimization of the difference between the target and the current computed deformed configuration of the cell, and the iterative formulation is based on the solution of several direct mechanical problems. The model presents a well-posed discretized inverse elasticity problem in the absence of regularization. The algorithm can be easily implemented in any kind of Finite Element (FE) code as a sequence of different standard FE analysis. RESULTS: To illustrate the proposed iterative formulation we apply the theoretical model to some illustrative examples by using real experimental data of Normal Human Dermal Fibroblast cells (NHDF) migrating inside a 2 mg/ml collagen-based gel. Different examples of application have been simulated to test the inverse numerical model proposed and to investigate the effect of introducing the correct cell properties onto the obtained cell forces. The algorithm converges after a small number of iterations, generating errors of around 5% for the tractions field in the cell contour domain. The resulting maximum traction values increased by 11% as a consequence of doubling the mechanical properties of the cell domain. CONCLUSIONS: With the results generated from computations we demonstrate the application of the algorithm and explain how the mechanical properties of both, the cell and the gel, domains are important for arriving to the correct results when using inverse traction force reconstruction algorithms, however, have only a minor effect on the resulting traction values.


Assuntos
Análise de Elementos Finitos , Microscopia/métodos , Algoritmos , Colágeno Tipo I/química , Humanos , Hidrogéis/química
9.
J Mater Sci Mater Med ; 30(9): 107, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31512084

RESUMO

In the present study, collagen hydrogel containing naringin was fabricated, characterized and used as the scaffold for peripheral nerve damage treatment. The collagen was dissolved in acetic acid, naringin added to the collagen solution, and cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide powder (EDC; 0.10 mM) to form the hydrogel. The microstructure, swelling behavior, biodegradation, and cyto/hemocompatibility of the fabricated hydrogels were assessed. Finally, the healing efficacy of the prepared collagen hydrogel loaded with naringin on the sciatic nerve crush injury was assessed in the animal model. The characterization results showed that the fabricated hydrogels have a porous structure containing interconnected pores with the average pore size of 90 µm. The degradation results demonstrated that about 70% of the primary weight of the naringin loaded hydrogel had been lost after 4 weeks of storage in PBS. The in vitro study showed that the proliferation of Schwann cells on the collagen/naringin hydrogel was higher than the control group (tissue culture plate) at both 48 and 72 h after cell seeding and even significantly higher than pure collagen 72 h after cell seeding (*p < 0.005, **p < 0.001). The animal study implied that the sciatic functional index reached to -22.13 ± 3.00 at the end of 60th days post-implantation which was statistically significant (p < 0.05) compared with the negative control (injury without the treatment) (-82.60 ± 1.06), and the pure collagen hydrogel (-59.80 ± 3.20) groups. The hot plate latency test, the compound muscle action potential, and wet weight-loss of the gastrocnemius muscle evaluation confirmed the positive effect of the prepared hydrogels on the healing process of the induced nerve injury. In the final, the histopathologic examinations depicted that the collagen/naringin hydrogel group reduced all the histological changes induced from the nerve injury and showed more resemblance to the normal sciatic nerve, with well-arranged fibers and intact myelin sheath. The overall results implied that the prepared collagen/naringin hydrogel can be utilized as a sophisticated alternative to healing peripheral nerve damages.


Assuntos
Colágeno Tipo I/química , Flavanonas/farmacologia , Regeneração Tecidual Guiada/métodos , Hidrogéis/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Animais , Células Cultivadas , Colágeno Tipo I/farmacologia , Flavanonas/química , Humanos , Hidrogéis/química , Masculino , Teste de Materiais , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/terapia , Ratos , Ratos Wistar , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/fisiologia , Nervo Isquiático/efeitos dos fármacos , Tecidos Suporte/química , Cicatrização/efeitos dos fármacos
10.
Biomater Sci ; 7(11): 4519-4535, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31436263

RESUMO

Macromolecular crowding is used by tissue engineers to accelerate extracellular matrix assembly in vitro, however, most mechanistic studies focus on the impact of crowding on collagen fiber assembly and largely ignore the highly abundant provisional matrix protein fibronectin. We show that the accelerated collagen I assembly as induced by the neutral crowding molecule Ficoll is regulated by cell access to fibronectin. Ficoll treatment leads to significant increases in the amount of surface adherent fibronectin, which can readily be harvested by cells to speed up fibrillogenesis. FRET studies reveal that Ficoll crowding also upregulates the total amount of fibronectin fibers in a low-tension state through upregulating fibronectin assembly. Since un-stretched fibronectin fibers have more collagen binding sites to nucleate the onset of collagen fibrillogenesis, our data suggest that the Ficoll-induced upregulation of low-tension fibronectin fibers contributes to enhanced collagen assembly in crowded conditions. In contrast, chemical cross-linking of fibronectin to the glass substrate prior to cell seeding prevents early force mediated fibronectin harvesting from the substrate and suppresses upregulation of collagen I assembly in the presence of Ficoll, even though the crowded environment is known to drive enzymatic cleavage of procollagen and collagen fiber formation. To show that our findings can be exploited for tissue engineering applications, we demonstrate that the addition of supplemental fibronectin in the form of an adsorbed coating markedly improves the speed of tissue formation under crowding conditions.


Assuntos
Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Colágeno Tipo I/química , Matriz Extracelular/química , Fibronectinas/química , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Polimerização , Engenharia Tecidual
11.
Mar Drugs ; 17(8)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394862

RESUMO

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5-29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.


Assuntos
Colágeno Tipo I/química , Colágenos Associados a Fibrilas/química , Proteínas de Peixes/química , Pele/química , Takifugu/metabolismo , Tetraodontiformes/metabolismo , Ácidos/química , Aminoácidos/química , Animais , Ligação de Hidrogênio , Pepsina A/química , Rios , Solubilidade , Temperatura
12.
Biophys Chem ; 253: 106224, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31351390

RESUMO

Self-assembly of rat tail collagen type I was investigated by means of turbidity measurements and molecular dynamics simulations. Turbidity curves collected at different pH values show that the rate of aggregation was not linear in dependence from pH, with the fastest kinetics at pH 5.0 and the lowest at neutral pH. MD simulations were carried out on two regions with different hydropathicity, monitoring the aggregation of up to four staggered tropocollagen fragments at different ionic strength. At physiological conditions, association of lowly charged regions occurs more easily than for highly charged ones, the latter seeming to aggregate in a sequential way. The first contacts indicate for both regions that the driving force is hydrophobic, the electrostatic contribution becoming relevant at short distance. The direct inter-tropocollagen H-bonds confirm that fibrillogenesis is driven by loss of surface water from the monomers and involves in large percentage hydroxyproline residues. Low ionic strength dynamics leads to the formation of incorrect assemblies, driven by not shielded pairwise charge interactions.


Assuntos
Colágeno Tipo I/síntese química , Simulação de Dinâmica Molecular , Animais , Colágeno Tipo I/química , Ligação de Hidrogênio , Ratos , Espectrofotometria Ultravioleta , Cauda/química
13.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 54(7): 475-480, 2019 Jul 09.
Artigo em Chinês | MEDLINE | ID: mdl-31288328

RESUMO

Objective: To observe the effect of adenosine triphosphate (ATP) phosphorylation on type Ⅰ collagen mineralization and explore the role of small molecule compound ATP in biomimetic mineralization. Methods: Fourier transform infrared spectroscopy (FT-IR) was used to analyze the phosphorylation of collagen molecules by different concentrations (0, 25, 50, 100 mmol/L) of ATP. The concentration of 50 mmol/L ATP was chosen to construct the phosphorylated collagen mineralization model. Transmission electron microscopy (TEM) was used to observed the ultrastructure of mineralized collagen and the collagen mineralization rate was further calculated by ImageJ software. The surface morphology of the collagen gel ATP group and the control group was observed by scanning electron microscopy (SEM) and the elemental analysis was performed by using an X-ray energy spectrometer. The artificial demineralized dentin samples were mineralized for 2 days and 4 days to compare the effect of ATP on dentin remineralization by SEM. Results: FT-IR analysis showed that the formation of new peaks at wavenumbers of 642, 818, and 902 cm(-1) indicated that ATP can phosphorylate type Ⅰ collagen. Through TEM and SEM observation, the mineralization degree of type Ⅰ collagen and demineralized dentin pretreated with 50 mmol/L ATP were significantly higher than that of the control group. Compared with the control group [(31.65±1.62)%], the mineralization rate of collagen in the ATP group [(100±0)%] was significantly increased after 2 days of mineralization (P<0.05). Conclusions: ATP phosphorylation can effectively promote the mineralization process of type Ⅰ collagen.


Assuntos
Trifosfato de Adenosina , Materiais Biomiméticos , Colágeno Tipo I , Trifosfato de Adenosina/farmacologia , Materiais Biomiméticos/química , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Colágeno Tipo I/ultraestrutura , Dentina/química , Dentina/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fosforilação , Espectroscopia de Infravermelho com Transformada de Fourier
14.
Biomater Sci ; 7(8): 3480-3488, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31282511

RESUMO

A hyperglycemic condition like diabetes in patients renders them with an increased risk of developing breast cancer. Hyperglycemia and ageing increase the non-enzymatic glycosylation (glycation) of nearly all proteins in our body including collagen type I, which is an important extracellular matrix (ECM) component. This results in the formation of advanced glycated end products (AGEs), which can form covalent crosslinks in collagen fibers and change the overall architecture and stiffness of the matrix. In this study we have used MDA-MB-231 breast cancer cells to study the interaction of tumor cells with glycated collagen and have explored the role of matrix architecture and RAGE-mediated signaling in cellular behavior. We mimicked the non-enzymatic glycation of protein by treating collagen I with glucose or ribose and found that crosslinking due to AGEs induces collagen fiber bundling and an increase in pore size and stiffness of the matrix. We also observed that AGE formation triggers AGE-RAGE signaling playing a role in the morphology and migration of cells. Furthermore, our study suggests an interplay of the pore size of the collagen matrix and RAGE mediated signaling in 3D invasion of cells and our findings demonstrate that the effect of the AGE-RAGE interaction is more pronounced than that of an altered matrix architecture. This study has helped us develop a 3D system using glycated collagen to study the effects of pathological conditions such as diabetes on extracellular matrix proteins, which may have downstream effects on cell behavior and dysfunction.


Assuntos
Colágeno Tipo I/metabolismo , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Colágeno Tipo I/química , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Humanos , Estresse Oxidativo , Estrutura Secundária de Proteína , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Ribose/metabolismo , Transdução de Sinais
15.
Methods Mol Biol ; 1934: 127-144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256377

RESUMO

Fibrillar type I collagen is the most abundant structural protein in most tissues and organs. One of the unique and functionally important characteristics of collagen is sequential posttranslational modifications of lysine (Lys) residues. In the endoplasmic reticulum, hydroxylation of specific Lys occurs producing 5-hydroxylysine (Hyl). Then, to the 5-hydroxyl group of Hyl, a single galactose unit can be attached to form galactosyl-Hyl (Gal-Hyl) and further glucose can be added to Gal-Hyl to form glucosylgalactosyl-Hyl (GlcGal-Hyl). These are the only two O-linked glycosides found in mature type I collagen. It has been shown that this modification is critically involved in a number of biological and pathological processes likely through its regulatory roles in collagen fibrillogenesis, intermolecular cross-linking, and collagen-cell interaction. Recently, with the advances in molecular/cell biology and analytical chemistry, the molecular mechanisms of collagen glycosylation have been gradually deciphered, and the type and extent of glycosylation at the specific molecular loci can now be quantitatively analyzed. In this chapter, we describe quantitative analysis of collagen glycosylation by high-performance liquid chromatography (HPLC) and semiquantitative, site-specific analysis by HPLC-tandem mass spectrometry.


Assuntos
Colágeno Tipo I/química , Aminoácidos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Colágeno Tipo I/metabolismo , Glicosilação , Hidrólise , Hidroxilisina/química , Hidroxilisina/metabolismo , Espectrometria de Massas , Domínios Proteicos , Processamento de Proteína Pós-Traducional
16.
J Genet ; 98(2)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31204718

RESUMO

Mutations in several genes, including SERPRINF1 and COL1A1, have been associated with the development of osteogenesis imperfecta (OI). Here, we reported the co-occurrence of a rare heterozygous variant (c.167C>G p.Ala56Gly) in SERPRINF1 and a novel heterozygous mutation (c.1634G>A p.Gly545Asp) in COL1A1 in a foetus with a severe form of OI. Bioinformatics modelling revealed that the effect of the mutation on SPERINF1 is neutral. In contrast, the mutation in COL1A1 is deleterious. It is predicted to cause distortion of the α (1) chain of the type I collagen and results in structural instability of the protein. Therefore, a novel dominant variant of COL1A1 likely underlies the severe foetal pathology observed, although we do not exclude the possibility that the heterozygous mutations in SERPINF and COL1A1 may interact and co-ordinately cause pathogenesis. This novel COL1A1 mutation is recommended to be included in the diagnostic panels for OI.


Assuntos
Colágeno Tipo I/genética , Proteínas do Olho/genética , Heterozigoto , Mutação , Fatores de Crescimento Neural/genética , Osteogênese Imperfeita/genética , Serpinas/genética , Alelos , Sequência de Aminoácidos , Instabilidade Cromossômica , Colágeno Tipo I/química , Biologia Computacional/métodos , Análise Mutacional de DNA , Evolução Molecular , Proteínas do Olho/química , Feminino , Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Modelos Moleculares , Fatores de Crescimento Neural/química , Osteogênese Imperfeita/diagnóstico , Fenótipo , Gravidez , Conformação Proteica , Serpinas/química , Relação Estrutura-Atividade , Ultrassonografia
17.
Sci Adv ; 5(6): eaaw4991, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206025

RESUMO

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.


Assuntos
Reabsorção Óssea/genética , Calcificação Fisiológica/genética , Osteogênese/genética , Osteoprotegerina/genética , Tecidos Suporte , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Reabsorção Óssea/prevenção & controle , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Técnicas de Cocultura , Colágeno Tipo I/química , Colágeno Tipo I/farmacologia , Reagentes para Ligações Cruzadas/química , Expressão Gênica , Glicosaminoglicanos/química , Glicosaminoglicanos/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Cultura Primária de Células , Engenharia Tecidual , Transgenes
18.
ACS Appl Mater Interfaces ; 11(25): 22152-22163, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31194504

RESUMO

Natural nerve tissue is composed of nerve bundles with multiple aligned assembles, and matrix electroconductivity is beneficial to the transmission of intercellular electrical signals, or effectively deliver external electrical cues to cells. Herein, aiming at the biomimetic design of the extracellular matrix for neurons, we first synthesized electroconductive polypyrrole (PPy) nanoparticles with modified hydrophilicity to improve their uniformity in collagen hydrogel. Next, cell-laden collagen-PPy hybrid hydrogel microfibers with highly oriented microstructures were fabricated via a microfluidic chip. The hydrogel microfibers formed a biomimetic three-dimensional microenvironment for neurons, resulting from the native cell adhesion domains, oriented fibrous structures, and conductivity. The oriented fibrous microstructures enhanced neuron-like cells aligning with fibers' axon; the matrix conductivity improved cell extension and upregulated neural-related gene expression; moreover, external electrical stimulation further promoted the neuronal functional expression. This mechanism was attributed to the electroconductive matrix and its delivered electrical stimulation to cells synergistically upregulated the expression of an L-type voltage-gated calcium channel, resulting in an increase in the intracellular calcium level, which in turn promoted neurogenesis. This approach has potential in constructing the biomimetic microenvironment for neurogenesis.


Assuntos
Hidrogéis/química , Nanopartículas/química , Neurogênese/fisiologia , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Colágeno Tipo I/química , Condutividade Elétrica , Imunofluorescência , Neurogênese/genética , Células PC12 , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Água/química
19.
Macromol Rapid Commun ; 40(15): e1900127, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31136037

RESUMO

Injectable hydrogels are considered important to realize safe and effective minimally invasive therapy. Although animal-derived natural polymers are well studied, they typically lack injectability and fail to eliminate the potential risks of immunogenic reactions or unknown pathogen contamination. Despite extensive research activities to explore ideal injectable hydrogels, such state-of-the-art technology remains inaccessible to non-specialists. In this article, the design of a new injectable hydrogel platform that can be extemporaneously prepared from commercially available animal-component-free materials is described. The hydrogels can be prepared simply by mixing mutually reactive aqueous solutions without necessitating specialized knowledge or equipment. Their solidification time can be adjusted by choosing proper buffer conditions from immediate to an extended period of time, that is, few or several tens of minutes depending on the concentration of polymeric components, which not only provides injectability, but enables 3D encapsulation of cells. Mesenchymal stromal/stem cells can be encapsulated and cultured in the hydrogels at least for 2 weeks by traditional cell culture techniques, and retrieved by collagenase digestion with cell viability of approximately 80%. This hydrogel platform accelerates future cell-related research activities.


Assuntos
Técnicas de Cultura de Células , Colágeno Tipo I/metabolismo , Colagenases/metabolismo , Hidrogéis/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Sobrevivência Celular , Colágeno Tipo I/química , Colagenases/química , Humanos , Hidrogéis/química , Células-Tronco Mesenquimais/metabolismo
20.
Biochim Biophys Acta Mol Basis Dis ; 1865(9): 2210-2223, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31055083

RESUMO

Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ±â€¯PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.


Assuntos
Colágeno Tipo I/genética , Retículo Endoplasmático/metabolismo , Pró-Colágeno/metabolismo , Varredura Diferencial de Calorimetria , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Microscopia de Fluorescência , Mutação de Sentido Incorreto , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Estrutura Terciária de Proteína
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