An outcome-defining role for the triple-helical domain in regulating collagen-I assembly.

Collagens are the foundational component of diverse tissues, including skin, bone, cartilage, and basement membranes, and are the most abundant protein class in animals. The fibrillar collagens are large, complex, multidomain proteins, all containing the characteristic triple helix motif. The most prevalent collagens are heterotrimeric, meaning that cells express at least two distinctive procollagen polypeptides that must assemble into specific heterotrimer compositions. The molecular mechanisms ensuring correct heterotrimeric assemblies are poorly understood - even for the most common collagen, type-I. The longstanding paradigm is that assembly is controlled entirely by the ~30 kDa globular C-propeptide (C-Pro) domain. Still, this dominating model for procollagen assembly has left many questions unanswered. Here, we show that the C-Pro paradigm is incomplete. In addition to the critical role of the C-Pro domain in templating assembly, we find that the amino acid sequence near the C terminus of procollagen's triple-helical domain plays an essential role in defining procollagen assembly outcomes. These sequences near the C terminus of the triple-helical domain encode conformationally stabilizing features that ensure only desirable C-Pro-mediated trimeric templates are committed to irreversible triple-helix folding. Incorrect C-Pro trimer assemblies avoid commitment to triple-helix formation thanks to destabilizing features in the amino acid sequences of their triple helix. Incorrect C-Pro assemblies are consequently able to dissociate and search for new binding partners. These findings provide a distinctive perspective on the mechanism of procollagen assembly, revealing the molecular basis by which incorrect homotrimer assemblies are avoided and setting the stage for a deeper understanding of the biogenesis of this ubiquitous protein.

Static magnetic field effects on the secondary structure and elasticity of collagen molecules; a possible biophysical approach to treat keratoconus.

Type I collagen is among the major extracellular proteins that play a significant role in the maintenance of the cornea's structural integrity and is essential in cell adhesion, differentiation, growth, and integrity. Here, we investigated the effect of 300 mT Static Magnetic Field (300 mT SMF) on the structure and molecular properties of acid-solubilized collagens (ASC) isolated from the rat tail tendon. The SMF effects at molecular and atomic levels were investigated by various biophysical approaches like Circular Dichroism Spectropolarimetery (CD), Fourier Transform Infrared Spectroscopy (FTIR), Zetasizer light Scattering, and Rheological assay. Exposure of isolated type I collagen to 300 mT SMF retained its triple helix. The elasticity of collagen molecules and the keratoconus (KCN) cornea treated with SMF decreased significantly after 5 min and slightly after 10, 15, and 20 min of treatments. The exposure to 300 mT SMF shifted the Amid I bond random coil to antiparallel wave number from 1647 to 1631 cm-1. The pH of the 300 mT SMF treated collagen solution increased by about 25 %. The treatment of the KCN corneas with 300 mT SMF decreased their elasticity significantly. The promising results of the effects of 300 mT SMF on the collagen molecules and KCN cornea propose a novel biophysical approach capable of manipulating the collagen's elasticity, surface charges, electrostatic interactions, cross binding, network formation and fine structure. Therefore, SMF treatment may be considered as a novel non-invasive, direct, non-chemical and fast therapeutic and manipulative means to treat KCN cornea where the deviated physico-chemical status of collagen molecules cause deformation.

Self-Assembly of Cyclic-Bending Collagen Fibrils by Polyimide Films.

The cyclic-bending morphologies of the fibrils formed by the self-assembly of type I collagen (Col) are closely related to the mechanisms of various diseases. Therefore, studies that allow the self-assembly of Col molecules to form cyclic-bending fibrils in vitro are vitally important. In this study, we successfully achieved the cyclic-bending shapes (specifically, a regular hexagonal shape) of Col molecules by controlling the steric structures of polyimide (PI) molecular chains through the film formation process. Specifically, when a single layer of PI film was baked, the PI molecular chains within the film bent in the direction parallel to the substrate surface plane. Repeating the layering and baking processes resulted in 3D structures of the PI molecular chains, which were oriented in the direction perpendicular to the substrate surface plane. This three-dimensional bending would result from the PI molecular chain interactions between the upper and lower layers. When the Col molecules were reacted on these film surfaces, they recognized the structures of the PI molecular chains and self-assembled to form cyclic-bending Col fibrils. Especially, in PI films subjected to three cycles of layering and baking, hemicircular-shaped Col fibrils were observed to be regularly arrayed. Additionally, these regularly cyclic-bending fibrils were aligned in the uniaxial direction through a uniaxial rubbing treatment of the PI films. This successful research is significant both as a method for controlling the morphologies of Col fibrils and as a study that explores the biomedical implications of Col fibril cyclic-bending in the living body.

Heterogeneous Structure and Dynamics of Water in a Hydrated Collagen Microfibril.

Collagen type I is well-known for its outstanding mechanical properties which it inherits from its hierarchical structure. Collagen type I fibrils may be viewed as a heterogeneous material made of protein, macromolecules (such as glycosaminoglycans and proteoglycans) and water. Water content modulates the properties of these fibrils. Yet, the properties of water and the fine interactions of water with the protein constituent of these heterofibrils have only received limited attention. Here, we propose to model collagen type I fibrils as a hydrated structure made of tropocollagen molecules assembled in a microfibril crystal. We perform large-scale all-atom molecular dynamics simulations of the hydration of collagen fibrils beyond the onset of disassembly. We found that the structural and dynamic properties of water vary strongly with the level of hydration of the microfibril. More importantly, we found that the properties vary spatially within the 67 nm D-spacing periodic structure. Alteration of the structural and dynamical properties of the collagen microfibril occur first in the gap region. Overall, we identify that the change in the role of water molecules from glue to lubricant between tropocollagen molecules arises around 100% hydration while the microfibril begins to disassemble beyond 130% water content. Our findings are supported by a decrease in hydrogen bonding, recovery of bulk water properties and amorphization of the tropocollagen molecules packing. Our simulations reveal the structure and dynamics of hydrated collagen fibrils with unprecedented spatial resolution from physiological conditions to disassembly. Beyond the process of self-assembly and the emergence of mechanical properties of collagen type I fibrils, our results may also provide new insights into mineralization of collagen fibrils.

Measuring mechanical cues for modeling the stromal matrix in 3D cell cultures.

A breast-cancer tumor develops within a stroma, a tissue where a complex extracellular matrix surrounds cells, mediating the cancer progression through biomechanical and -chemical cues. Current materials partially mimic the stromal matrix in 3D cell cultures but methods for measuring the mechanical properties of the matrix at cell-relevant-length scales and stromal-stiffness levels are lacking. Here, to address this gap, we developed a characterization approach that employs probe-based microrheometry and Bayesian modeling to quantify length-scale-dependent mechanics and mechanical heterogeneity as in the stromal matrix. We examined the interpenetrating network (IPN) composed of alginate scaffolds (for adjusting mechanics) and type-1 collagen (a stromal-matrix constituent). We analyzed viscoelasticity: absolute-shear moduli (stiffness/elasticity) and phase angles (viscous and elastic characteristics). We determined the relationship between microrheometry and rheometry information. Microrheometry reveals lower stiffness at cell-relevant scales, compared to macroscale rheometry, with dependency on the length scale (10 to 100 µm). These data show increasing IPN stiffness with crosslinking until saturation (≃15 mM of Ca2+). Furthermore, we report that IPN stiffness can be adjusted by modulating collagen concentration and interconnectivity (by polymerization temperature). The IPNs are heterogeneous structurally (in SEM) and mechanically. Interestingly, increased alginate crosslinking changes IPN heterogeneity in stiffness but not in phase angle, until the saturation. In contrast, such changes are undetectable in alginate scaffolds. Our nonlinear viscoelasticity analysis at tumor-cell-exerted strains shows that only the softer IPNs stiffen with strain, like the stromal-collagen constituent. In summary, our approach can quantify the stromal-matrix-related viscoelasticity and is likely applicable to other materials in 3D culture.

A comparative analysis of stem cell differentiation on 2D and 3D substrates using Raman microspectroscopy.

Chondrogenesis is a complex cellular process that involves the transformation of mesenchymal stem cells (MSCs) into chondrocytes, the specialised cells that form cartilage. In recent years, three-dimensional (3D) culture systems have emerged as a promising approach to studying cell behaviour and development in a more physiologically relevant environment compared to traditional two-dimensional (2D) cell culture. The use of these systems provided insights into the molecular mechanisms that regulate chondrogenesis and has the potential to revolutionise the development of new therapies for cartilage repair and regeneration. This study demonstrates the successful application of Raman microspectroscopy (RMS) as a label-free, non-destructive, and sensitive method to monitor the chondrogenic differentiation of bone marrow-derived rat mesenchymal stem cells (rMSCs) in a collagen type I hydrogel, and explores the potential benefits of 3D hydrogels compared to conventional 2D cell culture environments. rMSCs were cultured on 3D substrates for 3 weeks and their differentiation was monitored by measuring the spectral signatures of their subcellular compartments. Additionally, the evolution of high-density micromass cultures was investigated to provide a comprehensive understanding of the process and complex interactions between cells and their surrounding extracellular matrix. For comparison, rMSCs were induced into chondrogenesis in identical medium conditions for 21 days in monolayer culture. Raman spectra showed that rMSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway at a significantly higher rate than the 2D culture cells. 3D cultures expressed stronger and more homogeneous chondrogenesis-associated peaks such as collagens, glycosaminoglycans (GAGs), and aggrecan while manifesting changes in proteins and lipidic content. These results suggest that 3D type I collagen hydrogel substrates are promising for in vitro chondrogenesis studies, and that RMS is a valuable tool for monitoring chondrogenesis in 3D environments.

Spectroscopic analysis of the sum-frequency response of the carbon-hydrogen stretching modes in collagen type I.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its Xijk2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon-hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942 cm-1 and a shoulder at 2917 cm-1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870 cm-1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030 cm-1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements.

Utilization of Lumpfish (Cyclopterus lumpus) Skin as a Source for Gelatine Extraction Using Acid Hydrolysis.

Lumpfish (Cyclopterus lumpus) is an underutilized marine resource that is currently only being exploited for roe. Lumpfish skin was pre-treated with alkali (0.1M NaOH) and acid (0.1M HCl) at a skin to chemical ratio of 1:10 for 24 h at 5 °C to remove non-collagenous proteins and minerals. The pre-treated skin was washed, and gelatine was extracted with 0.1M of acetic acid at three different ratios (1:5, 1:10, and 1:15), time (12,18, and 24 h), and temperature combinations (12, 28, and 24 °C). The highest total extraction yield (>40%) was obtained with combinations of extraction ratios of 1:15 and 1:10 with a longer time (24 h) and higher temperature (18-24 °C). The highest gelatine content was obtained with an extraction period of 24 h and ratio of 1:10 (>80%). SDS-PAGE analysis confirmed the presence of type-I collagen. A rheological evaluation indicated melting and gelling temperatures, gel strength, and viscosity properties comparable to existing cold-water gelatine sources.

Versatile Self-Assembly of Triblock Peptides into Stable Collagen Mimetic Heterotrimers.

The construction of peptides to mimic heterogeneous proteins such as type I collagen plays a pivotal role in deciphering their function and pathogenesis. However, progress in the field has been severely hampered by the lack of capability to create stable heterotrimers with desired functional sequences and without the effect of homotrimers. We have herein developed a set of triblock peptides that can assemble into collagen mimetic heterotrimers with desired amino acids and are free from the interference of homotrimers. The triblock peptides comprise a central collagen-like block and two oppositely charged N-/C-terminal blocks, which display inherent incompetency of homotrimer formation. The favorable electrostatic attraction between two paired triblock peptides with complementary terminal charged sequences promptly leads to stable heterotrimers with controlled chain composition. The independence of the collagen-like block from the two terminal blocks endows this system with the adaptability to incorporate desired amino acid sequences while maintaining the heterotrimer structure. The triblock peptides provide a versatile and robust tool to mimic the composition and function of heterotrimer collagen and may have great potential in the design of innovative peptides mimicking heterogeneous proteins.

Prothymosin Alpha: A Novel Contributor to Estradiol Receptor Alpha-Mediated CD8+ T-Cell Pathogenic Responses and Recognition of Type 1 Collagen in Rheumatic Heart Valve Disease.

BACKGROUND: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. METHODS: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcuspyogenes proteomes were used to identify mimic epitopes. RESULTS: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8+ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8+ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8+ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and Spyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cell activation in vitro. CONCLUSIONS: ProTα-dependent CD8+ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8+ T cells, suggesting mechanisms provoking autoimmunity.

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2.

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.

Poly(acrylic acid)-Assisted Intrafibrillar Mineralization of Type I Collagen: A Review.

The mineralization of type I collagen is a biological process occurring in vertebrates by which some hard tissues such as bone and dentin are constructed. Due to the extensive clinical needs for bone defect repair and remineralization of mineral-depleted dentin, biomimetic mineralization of collagen is attracting more and more interests. Synthetic analogs of noncollagenous proteins are necessary for directing the in vitro mineralization. In this paper, the function and mechanism of poly(acrylic acid) (PAA) in regulating the mineralization, especially intrafibrillar mineralization (IM) of collagen are reviewed. As two mineralization patterns (extrafibrillar and intrafibrillar) co-exist in natural hard tissues, differences between them in terms of microstructure, biodegradation, cytocompatibility, osteoinduction in vitro, and performance in vivo are systematically compared. Then the roles of PAA in biomimetic collagen IM within one-analog and two-analog systems are discussed, respectively. Moreover, mineralization of some self-mineralizable collagen matrices is described. Due to the interactions between collagen and PAA play a crucial role in the processes of collagen mineralization, some reference researches are also provided involving the collagen/PAA interactions in some other fields. Finally, this review is ended with an outlook for future potential improvements based on the collection of existing bottlenecks in this field.

Swim Bladder of Farmed Totoaba macdonaldi: A Source of Value-Added Collagen.

Finding strategies to use the swim bladder of farmed totoaba (Totoaba macdonaldi) is of the utmost need to reduce waste. Fish swim bladders are rich in collagen; hence, extracting collagen is a promising alternative with benefits for aquaculture of totoaba and the environment. The elemental biochemical composition of totoaba swim bladders, including their proximate and amino acid compositions, was determined. Pepsin-soluble collagen (PSC) was used to extract collagen from swim bladders, and its characteristics were analyzed. Alcalase and papain were used for the preparation of collagen hydrolysates. Swim bladders contained 95% protein, 2.4% fat, and 0.8% ash (on a dry basis). The essential amino acid content was low, but the functional amino acid content was high. The PSC yield was high, at 68% (dry weight). The amino acid composition profile, electrophoretic pattern, and structural integrity analyses of the isolated collagen suggested it is a typical type-I collagen with high purity. The denaturalization temperature was 32.5 °C, probably attributable to the imino acid content (205 residues/1000 residues). Papain-hydrolysates (≤3 kDa) of this collagen exhibited higher radical scavenging activity than Alcalase-hydrolysates. The swim bladder from the farmed totoaba could be an ideal source to produce high-quality type I collagen and may be considered an alternative to conventional collagen sources or bioactive peptides.

Extraction and Characterization of Pepsin- and Acid-Soluble Collagen from the Swim Bladders of Megalonibea fusca.

There is a growing demand for the identification of alternative sources of collagen not derived from land-dwelling animals. The present study explored the use of pepsin- and acid-based extraction protocols to isolate collagen from the swim bladders of Megalonibea fusca. After extraction, these acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) samples respectively were subjected to spectral analyses and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization, revealing both to be comprised of type I collagen with a triple-helical structure. The imino acid content of these ASC and PSC samples was 195 and 199 residues per 1000 residues, respectively. Scanning electron microscopy demonstrated that samples of freeze-dried collagen exhibited a compact lamellar structure, while transmission electron microscopy and atomic force microscopy confirmed the ability of these collagens to undergo self-assembly into fibers. ASC samples exhibited a larger fiber diameter than the PSC samples. The solubility of both ASC and PSC was highest under acidic pH conditions. Neither ASC nor PSC caused any cytotoxicity when tested in vitro, which met one of the requirements for the biological evaluation of medical devices. Thus, collagen isolated from the swim bladders of Megalonibea fusca holds great promise as a potential alternative to mammalian collagen.

A Thermostable Type I Collagen from Swim Bladder of Silver Carp (Hypophthalmichthys molitrix).

As a major component of the extracellular matrix, collagen has been used as a biomaterial for many purposes including tissue engineering. Commercial collagen derived from mammals is associated with a risk of prion diseases and religious restrictions, while fish-derived collagen can avoid such issues. In addition, fish-derived collagen is widely available and low-cost; however, it often suffers from poor thermal stability, which limits its biomedical application. In this study, collagen with a high thermal stability was successfully extracted from the swim bladder of silver carp (Hypophthalmichthys molitrix) (SCC). The results demonstrated that it was a type I collagen with high purity and well-preserved triple-helix structure. Amino acid composition assay showed that the amounts of threonine, methionine, isoleucine and phenylalanine in the collagen of swim bladder of silver carp were higher than those of bovine pericardium. After adding salt solution, swim-bladder-derived collagen could form fine and dense collagen fibers. In particular, SCC exhibited a higher thermal denaturation temperature (40.08 °C) compared with collagens from the swim bladder of grass carp (Ctenopharyngodon idellus) (GCC, 34.40 °C), bovine pericardium (BPC, 34.47 °C) and mouse tail (MTC, 37.11 °C). Furthermore, SCC also showed DPPH radical scavenging ability and reducing power. These results indicate that SCC presents a promising alternative source of mammalian collagen for pharmaceutical and biomedical applications.

Spatiotemporal regulation of PEDF signaling by type I collagen remodeling.

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.

Chain alignment of collagen I deciphered using computationally designed heterotrimers.

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.

Molecular investigations of the prenucleation mechanism of bone-like apatite assisted by type I collagen nanofibrils: insights into intrafibrillar mineralization.

Bone is a typical inorganic-organic composite material with a multilevel hierarchical organization. In the lowest level of bone tissue, inorganic minerals, which are mainly composed of hydroxyapatite, are mineralized within the type I collagen fibril scaffold. Understanding the crystal prenucleation mechanism and growth of the inorganic phase is particularly important in the design and development of materials with biomimetic nanostructures. In this study, we built an all-atom human type I collagen fibrillar model with a 67 nm overlap/gap D-periodicity. Arginine residues were shown to serve as the dominant cross-linker to stabilize the fibril scaffold. Subsequently, the prenucleation mechanism of collagen intrafibrillar mineralization was investigated using a molecular dynamics approach. Considering the physiological pH of the human body (i.e., ∼7.4), HPO42- was initially used to simulate the protonation state of the phosphate ions. Due to the spatially constrained effects resulting from the overlap/gap structure of the collagen fibrils, calcium phosphate clusters formed mainly inside the hole zone but with different spatial distributions along the long axis direction; this indicated that the nucleation of calcium phosphate may be highly site-selective. Furthermore, the model containing both HPO42- and PO43- in the solution phase formed significantly larger clusters without any change in the nucleation sites. This phenomenon suggests that the existence of PO43- is beneficial for the mineralization process, and so the conversion of HPO42- to PO43- was considered a critical step during mineralization. Finally, we summarize the nucleation mechanism for collagen intrafibrillar mineralization, which could contribute to the fabrication of mineralized collagen biomimetic materials.

Valorization of Fish Waste: Isolation and Characterization of Acid- and Pepsin-Soluble Collagen from the Scales of Mediterranean Fish and Fabrication of Collagen-Based Nanofibrous Scaffolds.

In search of alternative and sustainable sources of collagenous materials for biomedical applications, the scales of five Mediterranean fish species-fished in high tonnage in the Mediterranean region since they represent popular choices for the local diet-as well as those of the Atlantic salmon for comparison purposes, were comparatively studied for their acid- and pepsin-soluble collagen content. Fish scales that currently represent a discarded biomass of no value could be efficiently exploited for the production of a high added-value biomaterial. The isolated collagenous materials, which showed the typical electrophoretic patterns of type I collagen, were morphologically and physicochemically characterized. Using scanning electron microscopy the fibrous morphology of the isolated collagens was confirmed, while the hydroxyproline content, in conjunction with infrared spectroscopy and X-ray diffraction studies verified the characteristic for collagen amino acid profile and its secondary structure. The acid- and pepsin-soluble collagens isolated from the fish scales were blended with the bioactive sulfated marine polysaccharide ulvan and polyethylene oxide and electrospun to afford nanofibrous scaffolds that could find applications in the biomedical sector.

Physicochemical, Structural and Antioxidant Properties of Collagens from the Swim Bladder of Four Fish Species.

This study aimed to isolate and characterize pepsin-solubilized collagen (PSC) from marine and freshwater fish swim bladders. The physicochemical properties, protein pattern, amino acid composition, structure, thermal denaturation temperature, and antioxidant activity of PSC from four different swim bladder sources were investigated and compared. The results demonstrated that the four types of collagen extracted were all type I collagen. The yield of PSC extracted from grass carp (GCSB-PSC), bighead carp (BCSB-PSC), grouper (GSB-PSC), and monkfish swim bladders (MSB-PSC) were 38.98, 27.97, 18.16, and 10.35%, respectively. Compared to the other three PSCs, BCSB-PSC has the highest thermal denaturation temperature (38.60 °C). Based on FTIR spectroscopy and circular dichroism (CD) analysis, the extracted PSCs retained the triple helix and secondary structure well. Antioxidant studies showed that in the swim bladders of four species the swim bladder PSC could scavenge DPPH and ABTS radicals. Overall, swim bladders from marine and freshwater fish can be utilized as raw materials for collagen extraction, and the extracted collagen has potential commercial applications.