Triple Helical Structure Of Collagen Research Articles

Insights into the Structural and Thermal Properties of Pepsin-Soluble Collagen from Daggertooth pike conger (Muraenesox cinereus)

Pepsin soluble collagen (PSC) is a type-1 collagen which is abundant in skin. The pepsin soluble collagen was successfully extracted from the skin of Daggertooth pike conger (Muraenesox cinereus) by using the conventional method of salt precipitation followed by dialysis. Pepsin-soluble collagen showed a yield of 10.12% on wet matter basis and 25.5% on dry matter basis. The triple helical structure of collagen was confirmed by Fourier Transform Infrared Spectroscopy (FTIR) and UV-Visible spectrophotometry. Isolated PSC was confirmed as type 1 collagen by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). FTIR spectra of PSC suggested that the secondary structure of the triple helical collagen was intact even after pepsin digestion. Pepsin-soluble collagen was soluble in acidic pH with maximum solubility at pH 4. The solubility was found to be decreased with an increase in the concentration of sodium chloride with a minimum solubility at 4%. The present PSC isolate from eel fish exhibited comparatively high thermal stability of 36 ͦ C. Morphological analysis by Scanning electron microscopy (SEM) revealed a porous structure and comparatively high thermal stability makes it a promising biomaterial.

Impact of SiO2 nanoparticles on the structure and property of type I collagen in three different forms

Silica-based nanoparticles have found application in the development of biocomposites involving reconstituted collagen in tissue engineering and wound healing, and leather modification, specifically targeting collagen fibers. However, a comprehensive investigation into the interaction between collagen-silica nanoparticles and different forms of collagen using biophysical methods remains unexplored. In this study, we examined the interaction between silica (SiO2) nanoparticles and collagen in its fiber, microfibril, and monomer forms through high-resolution scanning electron microscopy, circular dichroism, Fourier-transform infrared spectroscopy, fluorescence analysis, zeta potential measurements, and turbidity assays. Our results reveal that SiO2 nanoparticles exhibited a non-specific attraction towards collagen fibers without disrupting their structural integrity. Interestingly, SiO2 nanoparticles influenced the process of microfibrillation, resulting in heterogeneous fibril diameters while maintaining the natural D-periodicity. This finding is significant, as fibril size variations can impact the properties of collagen composites. Notably, the triple helical structure of collagen in its monomer form remained unaffected in the presence of SiO2 nanoparticles, indicating that the nanoparticles did not disrupt the electrostatic interactions that stabilize the triple helix. Additionally, the increased stability of SiO2 nanoparticles in the presence of collagen confirmed their interaction. These findings provide a promising avenue for the development of SiO2-based nanoparticles to enhance the stability of collagen fibers and control fiber sizes for biomaterial preparation. Moreover, this study advances the potential application of SiO2-based nanoparticles in leather tanning, an emerging field where nanoparticles can play a crucial role.

Impact of Ultrasonication on the Self-Assembly Behavior and Gel Properties of Bovine Bone Collagen I.

This study deliberated the effect of ultrasonic treatment on collagen self-assembly behavior and collagen fibril gel properties. Bovine bone collagen I which had undergone ultrasonic treatment with different power (0-400 W) and duration (0-60 min) was analyzed. SDS-PAGE and spectroscopic analysis revealed that ultrasonic treatment decreased collagen molecular order degree and the number of hydrogen bonds, stretching collagen telopeptide regions while maintaining the integrity of the collagen triple-helical structure. Ultrasonic treatment (p ≤ 200 W, t ≤ 15 min) dispersed the collagen aggregates more evenly, and accelerated collagen self-assembly rate with a decreased but more homogeneous fibril diameter (82.78 ± 16.47-115.52 ± 19.51 nm) and D-periodicity lengths (62.1 ± 2.9-66.5 ± 1.8 nm) than that of the untreated collagen (119.15 ± 27.89 nm; 66.5 ± 1.8 nm). Meanwhile, ultrasonic treatment (p ≤ 200 W, t ≤ 15 min) decreased the viscoelasticity index and gel strength, enhancing thermal stability and promoting specific surface area and porosity of collagen fibril gels than that of the untreated collagen fibril gel. These results testified that collagen self-assembly behavior and collagen fibril gel properties can be regulated by ultrasonic treatment through multi-hierarchical structural alteration. This study provided a new approach for controlling in vitro collagen fibrillogenesis process so as to manufacture novel desirable collagen-based biomaterials with propitious performances for further valorization.

Ionic environment effects on collagen type II persistence length and assembly.

Collagen type II is a main structural protein in cartilage and forms fibrils. The radius of the fibrils ranges from 50nm to a few hundred nm, and previous theoretical studies point to electrostatics and collagen elasticity (measured as the persistence length, lp) as the main origin for the self-limiting size scales. In this study, we have investigated the collagen triple helical structure and fibril size scales in pH 2 solutions with varying NaCl concentrations from 10-4 to 100 mM, at which collagen is positively charged, and in pH 7.4 solutions, with varying ionic strengths from 100 to 250 mM, at which collagen is both positively and negatively charged. Using static and dynamic light scattering, the radius of gyration (Rg), hydrodynamic radius (Rh), and second virial coefficient (A2) of collagen triple helices are determined, and lp is calculated. With increasing ionic strength, triple helical lp decreases in pH 2 solutions and increases in pH 7.4 solutions. The value ranges from 60 to 100nm depending on the ionic environment, but at the salt concentration at which A2 is near zero, there are no net backbone interactions in solution, and the intrinsic collagen triple helix lp is determined to be 90-95nm. Electron microscopy is used to determine the diameter of fibrils assembled in pH 7.4 conditions, and we compare lp of the collagen triple helices and fibril diameter using recent theory on fibril assembly. By better understanding collagen lp and fibril assembly, we can further understand mechanisms of biomacromolecule self-assembly.

Predicting Collagen Triple Helix Stability through Additive Effects of Terminal Residues and Caps.

Collagen model peptides (CMPs) consisting of proline-(2S,4R)-hydroxyproline-glycine (POG) repeats have provided a breadth of knowledge of the triple helical structure of collagen, the most abundant protein in mammals. Predictive tools for triple helix stability have, however, lagged behind since the effect of CMPs with different frames ([POG]n , [OGP]n , or [GPO]n ) and capped or uncapped termini have so far been underestimated. Here, we elucidated the impact of the frame, terminal functional group and its charge on the stability of collagen triple helices. Combined experimental and theoretical studies with frame-shifted, capped and uncapped CMPs revealed that electrostatic interactions, strand preorganization, interstrand H-bonding, and steric repulsion at the termini contribute to triple helix stability. We show that these individual contributions are additive and allow for the prediction of the melting temperatures of CMP trimers.

Predicting Collagen Triple Helix Stability through Additive Effects of Terminal Residues and Caps

AbstractCollagen model peptides (CMPs) consisting of proline‐(2S,4R)‐hydroxyproline‐glycine (POG) repeats have provided a breadth of knowledge of the triple helical structure of collagen, the most abundant protein in mammals. Predictive tools for triple helix stability have, however, lagged behind since the effect of CMPs with different frames ([POG]n, [OGP]n, or [GPO]n) and capped or uncapped termini have so far been underestimated. Here, we elucidated the impact of the frame, terminal functional group and its charge on the stability of collagen triple helices. Combined experimental and theoretical studies with frame‐shifted, capped and uncapped CMPs revealed that electrostatic interactions, strand preorganization, interstrand H‐bonding, and steric repulsion at the termini contribute to triple helix stability. We show that these individual contributions are additive and allow for the prediction of the melting temperatures of CMP trimers.

Studies on stabilization of collagen using Cr-doped polydopamine complex

The stabilization of collagen using different small molecules have been explored for the development of biomaterials. One of the most studied molecules in biomaterials research is polydopamine (PDA) due to its ability to bind to different substrates that ranges from metal surface to collagen. Similarly, in leather tanning, chromium has been an extensively used metal ion as it binds strongly with collagen and enhances its stability. However, as per regulatory authority, the presence of chromium in leather has been restricted to minimum level. Here, we studied the application of chromium doped polydopamine (Cr-PDA) complex as collagen stabilizing agent. The preparation and characterization of Cr-PDA were confirmed using FE-SEM, DLS and FT-IR techniques. Cr-PDA did not alter the triple helical structure of collagen as evidenced from the CD spectral data. Cr-PDA delays the fibrillation in collagen compared to collagen or PDA alone. Calorimetric data shows the enhanced stability of collagen when treated with Cr-PDA compared to collagen control but lesser than PDA alone. Viscometry studies have shown that Cr-PDA reduces the viscosity of collagen compared to PDA or collagen alone. Contact angle studies showed that PDA and Cr-PDA imparts more hydrophobicity to collagen compared to control. Tensile strength studies showed that addition of Cr-PDA or PDA increases the tensile strength of the collagen fiber. The present study on stabilization of collagen using Cr-PDA might be helpful in development of crosslinking agents with eco-friendly approach.

Calendula flower extract loaded collagen film exhibits superior wound healing potential: Preparation, evaluation, in-vitro &in-vivo wound healing study

The present study was aimed to prepare Calendula flower extract loaded collagen film as a safe and effective wound dressing. Collagen film was prepared by solvent casting method and characterized for physicochemical properties like water absorption capacity, total phenolic content, total flavonoid content, antioxidant activity, infrared spectra, biodegradation, moisture content, differential scanning calorimetry, and sterility. The triple helical structure of collagen in the formulation was confirmed by gel electrophoresis and atomic force microscopy study. The safety and efficacy of developed formulation were investigated by in-vitro cell culture experiments including MTT assay, scratch assay on 3T3 Swiss albino cell line, In-vitro hemolysis study and In-vivo wound healing animal study on Wistar rats. In-vitro cell cytotoxicity test showed that the developed film was non-cytotoxic. In-vivo animal study on Wistar rats showed rapid wound healing compared to non-treated and placebo-treated group. The rate of wound contraction was significantly (p < 0.001) high in CFE loaded collagen film treated wounds (92.18 ± 1.2%) when compared to the control group (56.25 ± 4.1%), placebo-treated group (62.5 ± 5.1%) and marketed product treated group (68.75 ± 6.1%) on day 21. The prepared biodegradable collagen film incorporated with calendula flower extract was safe, stable, and effective as a wound healing dressing.

Monitoring the Degradation of Collagen Hydrogels by Collagenase Clostridium histolyticum.

Collagen-based hydrogels are investigated extensively in tissue engineering for their tunable physiochemical properties, biocompatibility and biodegradability. However, the effect of the integrity of the collagen triple helical structure on biodegradability is yet to be studied. In this study, we monitored the degradation of intact collagen (C-coll) and hydrolyzed collagen (D-coll) hydrogels in collagenase Clostridium histolyticum to understand their degradation process. Our results show that when peptides are present on the surface of the fibrils of D-coll hydrogels, cleavage of amide bonds occur at a much higher rate. The fibrillar structure of D-coll hydrogel results in a more pronounced breakdown of the gel network and dissolution of collagen peptides. The results from this work will improve the understanding of enzymatic degradation and the resulting bioabsorption of collagen materials used in drug delivery systems and scaffolds.

N-Vanillylnonanamide, a natural product from capsicum oleoresin, as potential inhibitor of collagen fibrillation

Inhibition of collagen fibrillation by small molecules is of growing interest to develop therapeutics for the illnesses related to excess deposition of collagen. In this context, we have studied the inhibitory effect of N-Vanillylnonanamide (NVA), a natural product from capsicum oleoresin and an analog of capsaicin (a known inhibitor of collagen fibrillation), on collagen self-assembly that leads to fibrillation in vitro. Commercially, capsaicin was found to be expensive than NVA. Therefore, it would be an advantage economically if NVA could display a similar/better inhibitory activity compared to capsaicin. The conventional turbidity measurements indicate that NVA completely inhibits collagen fibrillation at body temperature (37 °C) and its inhibition were concentration-dependent. The inhibition efficiency was observed to reduce at room temperature (25 °C). NVA protects the triple helical structure of collagen while it increases the thermal stability of collagen compared to collagen alone. Fluorescence results suggest that NVA binds in both telopeptide and triple helical regions of collagen and thereby prevents collagen self-assembly. The present results thus indicate that NVA is a potential inhibitor and, economically, it could be a better choice as a therapeutic agent compared to capsaicin in evolving treatment for disorders associated with excessive collagen deposition.

Direct Radiation Effects on the Structure and Stability of Collagen and Other Proteins.

In this review, recent progress in understanding the direct effects of radiation on the structure and stability of collagen, the most abundant protein in the human body, and other proteins is surveyed. Special emphasis is placed on the triple-helical structure of collagen, as studied by means of collagen mimetic peptides. The emerging patterns are the dose dependence of radiation processes and their abundance, the crucial role of radicals in covalent-bond formation (crosslinking) or cleavage, and the influence of the radiation energy and nature. Future research should allow fundamental questions, such as charge transfer and fragmentation dynamics triggered by ionization, to be answered, as well as developing applications such as protein-based biomaterials, notably with properties controlled by irradiation.

Stability of collagen in ionic liquids: Ion specific Hofmeister series effect.

In protein-ionic liquids (ILs) interactions, anions play an important role. In this work, imidazolium-based ILs (IILs) with varying anions namely dicyanamide (DCA), hydrogen sulfate (HS), dimethyl phosphate (DP), acetate (A), sulfate (S) and dihydrogen phosphate (DHP) have been chosen with the aim of understanding the role of anions in bringing about the destabilization effect on collagen based on the kosmotropicity and chaotropicity of ions. Imidazolium-based ILs destabilized the triple helical structure of collagen, thereby proving as strong denaturants for collagen and this was confirmed by various spectroscopic techniques viz., CD, FT-IR, viscosity and impedance measurements. The solution studies were in accordance to the changes in the dimensional stability of RTT collagen fibres at the fibrillar level. Imidazolium cations with varied anions have exhibited destabilizing effect on collagen in order of ions in Hofmeister series; IDP < IDHP < IA < IDCA < IS < IHS. Presumably, these notable effect and changes were facilitated by electrostatic interactions between the anions and amine functional groups of collagen.

Methods for the Construction of Collagen-Based Triple-Helical Peptides Designed as Matrix Metalloproteinase Inhibitors.

The triple-helical structure of collagen has been accurately reproduced in numerous chemical and recombinant model systems. Triple-helical peptides have found application for dissecting collagen-stabilizing forces, isolating receptor and protein binding sites in collagen, evaluating collagen-mediated cell signaling activities, mechanistic examination of collagenolytic proteases, and developing novel biomaterials and drug delivery vehicles. Due to their inherent stability to general proteolysis, triple-helical peptides present an opportunity as in vivo inhibitory agents. The present chapter provides methods for the construction of collagen-based triple-helical peptides designed as matrix metalloproteinase inhibitors.

A frameshift variant in the COL5A1 gene in a cat with Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome (EDS) is a group of heritable connective tissue disorders caused by defective collagen synthesis or incorrect assembly of the collagen triple helical structure. EDS is characterised by joint hypermobility, skin hyperextensibility, abnormal scarring, poor wound healing and tissue friability. Human EDS may be caused by variants in several different genes including COL5A1, which encodes the collagen type V alpha 1 chain. For the present study we investigated a 1.5-year-old, spayed female, domestic shorthair cat with EDS. The affected cat showed multiple recurrent skin tears, hyperextensibility of the skin and joint abnormalities. We obtained whole genome sequencing data from the affected cat and searched for variants in candidate genes known to cause EDS. We detected a heterozygous single base-pair deletion in exon 43 of the COL5A1 gene, namely c.3420delG. The deletion was predicted to result in a frameshift and premature stop codon: p.(Leu1141SerfsTer134). Sanger sequencing confirmed that the variant was present in the affected cat and absent from 103 unaffected cats from different breeds. The variant was also absent from a Burmese cat with EDS. Based on knowledge about the functional impact of COL5A1 variants in other species, COL5A1:c.3420delG represents a compelling candidate causative variant for the observed EDS in the affected cat.

Investigation on the behavior of collagen self-assembly in vitro via adding sodium silicate

Silicon, a trace element found in human body, plays a critical role in the process of collagen self-assembly. In this study, the intermolecular interaction and fibrillogenesis process were investigated to understand the effects of various concentrations of sodium silicate (SS) on collagen self-assembly in vitro. Fourier transform infrared spectroscopy analysis indicated that the triple helical structure of collagen was not significantly affected by SS. Hydrophobic interactions and particle sizes of collagen aggregates, which were measured using pyrene fluorescence and dynamic light scanning, enhanced via adding 2 mM SS whereas decreased with further increasing concentrations (4–8 mM). Kinetic analysis revealed that an increase in hydrophobic interactions boosted collagen self-assembly in the presence of 2 mM SS. The inhibition of self-assembly with the addition of 4–8 mM SS, as illustrated by a reduction in the fibrillogenesis rate and turbidity, was potentially attributed to weak hydrophobic interactions and strong electrostatic repulsion. The observation of microscopy demonstrated that the fibrils exhibited the characteristic D-periodicity at 2 mM SS. The inhibitory effect of 4 mM SS was slight and the fibrils still formed, while the microstructure was consisted of clustered collagen aggregates as SS ≥ 6 mM owing to serious inhibition on collagen self-assembly.

The Effects of Local and Systemic Administration of Proline on Wound Healing in Rats

ABSTRACTPurpose: Wound healing consists of a sequence of complex molecular and cellular events. Collagen is composed mainly of proline and hydroxyproline. Proline and hydroxyproline constitute 1/3 of the amino acids in collagen, which makes up approximately 30% of the proteins within the body. The hydroxylation of proline found in collagen determines the stability of the triple helical structure of collagen. In this study, we examined the effects of local and systemic administration of proline on wound healing. Materials and Methods: 24 female Sprague-Dawley rats were used in the study and divided into three groups. Group 1: The defect created in the backs of the subjects was left to secondary healing. Group 2: 200 µl proline per day was administered topically for 30 days on the defect in the backs of the subjects. Group 3: 200 µl per day was administered intraperitoneally for 30 days on the defect in the backs of the subjects. Results: On day 21, there was a statistically significant difference between the groups in terms of the mean re-epithelialization score. On days 7 and 14, there was a statistically significant difference between the groups in terms of the mean granulation score. On days 7, 14, and 21, there was a statistically significant difference between the groups in terms of the mean collagen accumulation score. On day 30, there was a statistically significant difference between Groups 1 and 3 in terms of the mean E-mode score on mechanical tensile test. Conclusion: Our study confirmed that proline has positive effects on wound healing. However, it revealed that systemic administration of proline is more effective than local administration of proline.

Choline-Based Amino Acid ILs-Collagen Interaction: Enunciating Its Role in Stabilization/Destabilization Phenomena.

Given the potential of productive interaction between choline-based amino acid ionic liquids (CAAILs) and collagen, we investigated the role of four CAAILs, viz., choline serinate, threoninate, lysinate, and phenylalaninate, and the changes mediated by them in the structure of collagen at different hierarchical orderings, that is, at molecular and fibrillar levels. The rheological, dielectric behavior and the secondary structural changes signify the alteration in the triple helical structure of collagen at higher concentrations of CAAILs. A marginal swelling and slight decrease in the thermal stability of rat tail tendon collagen fibers were observed for choline serinate and threoninate, albeit distortions in banding patterns were noticed for choline lysinate and phenylalaninate, suggesting chaotropicity of the ions at the fibrillar level. This signifies the changes in the hydrogen-bonding environment of collagen with increasing concentrations of CAAILs, which could be due to competitive hydrogen bonding between the carbonyl group of amino acid ionic liquids and the hydroxyl groups of collagen.

RANTES/CCL5 Induces Collagen Degradation by Activating MMP-1 and MMP-13 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) participates in rheumatoid arthritis (RA) pathogenesis by facilitating leukocyte infiltration, however, its other pathological functions are not fully defined in RA. In the present study, we evaluated the effect of RANTES/CCL5 on tissue degrading enzymes matrix metalloproteinase-1 (MMP-1) and MMP-13 expression and its contribution to the progressive joint damage by RA synovial fibroblasts (RASFs). Our results showed that RANTES/CCL5 dose dependently induced MMP-1 and MMP-13 expression in monolayers and three-dimensional (3D) micromass of human RASFs, which correlated with an increase in collagenase activity. This activation by RANTES/CCL5 was observed in RASF, but not in osteoarthritis SFs (OASFs). Evaluation of the signaling events showed that RANTES/CCL5 selectively activated PKCδ, JNK, and ERK proteins to induce MMP expression in human RASFs. Pretreatment with a functional antagonist (Met-RANTES) or heparinase III [an enzyme that selectively digests heparan sulfate proteoglycans (HSPGs)] completely abrogated RANTES/CCL5-induced MMP-1 and MMP-13 expression. Interestingly, the inhibition of RANTES/CCL5 using small-interfering RNA approach reduced the ability of interleukin-1β (IL-1β) to induce MMP-1 and MMP-13 expression, asserting its mediatory role in tissue remodeling. In the inhibitor study, only the selective inhibition of HSPGs or PKCδ, ERK, and JNK markedly inhibited RANTES/CCL5-induced MMP-1 and MMP-13 production. Circular dichroism spectroscopy results demonstrated the degradation of collagen triple-helical structure upon exposure to the conditioned media from RANTES/CCL5 stimulated RASFs, which was reverted by a broad-spectrum MMP inhibitor (GM6001). These findings suggest that RANTES/CCL5 not only upregulates MMP-1 and MMP-13 expression by partly utilizing HSPGs and/or PKCδ-JNK/ERK pathways but also mediates IL-1β-induced MMP-1 and MMP-13 expression.

Isolation and Characterization of Biocompatible Collagen from the Skin of Puffer Fish (Lagocephalus inermis)

In this present study, acid soluble collagen and pepsin soluble collagen from the skin of marine puffer fish Lagocephalus inermis was successfully isolated and characterized. The collagens extracted from the fish skin showed the high yields of both acid solubilized collagen (ASC) (43.1%) and Pepsin soluble collagen (PSC) (56.6%) on the dry weight basis, respectively. Based on electrophoretic patterns, both ASC and PSC consisted of two α- chains (α1 and α2) and were characterized as type I collagen. Amino acid analysis of both the collagen contained the imino acid of 190 and 198 residues/1000 residues, respectively. The ultraviolet absorption spectrum of collagen showed maximum absorption at 230 nm. Fourier transforms infrared spectra of both ASC, PSC was almost similar, and the acidic or enzymatic extraction had no effect on the triple helical structure of collagen. The denaturation temperature (Td) of the collagen was found to be 31.9 °C for ASC and 32.6 °C for PSC, respectively. Both ASC and PSC had highest solubility at acidic pH. Scanning electron microscopy images revealed the porous structure of collagens and both the collagens showed 100% biocompatible on NIH3T3 cell lines. These characteristic features are essential for wound dressing applications. From this study, a useful product recovery was achieved from the underutilized puffer fish, which may serve as an alternative source for mammalian collagen, as well as the management of natural wastes or ecological problems.

Disintegration of collagen fibrils by Glucono-δ-lactone: An implied lead for disintegration of fibrosis

Excess accumulation of collagen (fibrosis) undergoes self-aggregation, which leads to fibrillar collagen, on the extracellular matrix is the hallmark of a number of diseases such as keloids, hypertrophic scars, and systemic scleroderma. Direct inhibition or disintegration of collagen fibrils by small molecules offer a therapeutic approach to prevent or treat the diseases related to fibrosis. Herein, the anti-fibrotic property of Glucono-δ-lactone (GdL), known as acidifier, on the fibrillation and its disintegration of collagen was investigated. As collagen fibrillation is pH dependent, the pH modulation property of GdL is attractive to inhibit self-association of collagen. Optical density and microscopic data indicate that GdL elicits concentration-dependent fibril inhibition and also disintegrates pre-formed collagen fibrils. The simultaneous pH analysis showed that the modulation(lowering) of pH by GdL is the primary cause for its anti-fibrotic activity. The intact triple helical structure of collagen upon treatment of GdL suggests that collagen fibril disintegration can be achieved without affecting the native structure of collagen which is essential for any anti-fibrotic agents. Saturation transfer difference (STD) NMR result reveals that GdL is in proximity to collagen. The present results thus suggest that GdL provides a lead to design novel anti-fibrotic agents for the pathologies related to collagen deposition.