Triple-helical Configuration Research Articles

Optimization and Physicochemical Characterization of Polysaccharide Purified from Sonneratia caseolaris Mangrove Leaves: a Potential Antioxidant and Antibiofilm Agent.

The Box-Behnken design was applied to determine the optimal parameters of the extraction condition by using the response surface methodology (RSM) from the leaves of Sonneratia caseolaris L. The result indicates the best-optimized conditions used for the extraction of polysaccharides at 84.02°C temperature, 3.12h time, and 27.31mL/g for the water-to-material ratio. The maximum experimental yield of 8.81 ± 0.09% was obtained which is in agreement with the predicted value of 8.79%. Thereafter, low molecular weight polysaccharide (SCLP) was separated after sequentially being purified through column chromatography with a relative molecular weight of 3.74kDa. The physicochemical properties were evaluated by characterization techniques such as FT-IR spectra, NMR spectrum, and SEM analysis. RP-HPLC analysis confirmed that SCLP was a heteropolysaccharide, majorly comprising rhamnose (28.25%), and xylose (27.17%) residues, followed by mannose (18.90%), and galactose (17.17%), respectively. Thermal analysis (TGA-DSC) results showed that SCLP is a highly thermostable polymer with a degradation temperature of 361.63°C. X-ray diffraction patterns and tertiary structure analyses indicate that SCLP had a semi-crystalline polymer having a triple-helical configuration. Moreover, SCLP displayed potential antibiofilm ability for all the tested pathogens while stronger activity against Klebsiella pneumoniae and Pseudomonas aeruginosa. In addition, SCLP has potential in vitro antioxidant activity on DPPH, ABTS radical, superoxide, and Fe2+ chelating. These findings indicate that the polysaccharide has potentially been used in functional food, cosmetics, and pharmacological industries.

Acid and Pepsin Soluble Collagens from Skin By-product of Red-bellied Pacu (Piaractus brachypomus): Extraction and Comparative Characterizations Towards Finding Substitute to Bovine and Porcine Collagen

ABSTRACT This investigation focused on the sustainable valorization of red-bellied pacu (RBP) skin towards finding an alternative to bovine and porcine collagen. Acid-soluble collagen (ASC, 45.8 ± 0.7%) and pepsin soluble collagen (PSC, 57.8 ± 1.9%) were successfully extracted from RBP skin and comparatively elucidated their characteristics and in-vitro fibril-forming potential. The extracted collagens were characterized as type I collagen and confirmed the well-preserved triple-helical configuration by sodium dodecyl sulphate polyacrylamide gel electrophoresis and Fourier transform-infrared spectroscopy analysis. Both ASC and PSC exhibited relatively better thermo-stability, and the denaturation temperatures were found to be 35.5–35.29 °C by differential scanning calorimetry analysis. Both collagens displayed higher (p ≤ 0.05) solubility at low NaCl concentrations. The extracted ASC and PSC showed the same degree of fibril-forming capacities and confirmed well-defined fibril morphologies. Overall, the results showed the possibilities of effectively exploring RBP skin as a potential alternative source for realistic and safe collagen for food, nutrition, pharmaceutical, and other applications.

Selective Small-Molecule Targeting of a Triple Helix Encoded by the Long Noncoding RNA, MALAT1.

Metastasis-associated lung adenocarcinoma transcript 1 ( Malat1/ MALAT1, mouse/human), a highly conserved long noncoding (lnc) RNA, has been linked with several physiological processes, including the alternative splicing, nuclear organization, and epigenetic modulation of gene expression. MALAT1 has also been implicated in metastasis and tumor proliferation in multiple cancer types. The 3' terminal stability element for nuclear expression (ENE) assumes a triple-helical configuration that promotes its nuclear accumulation and persistent function. Utilizing a novel small molecule microarray strategy, we identified multiple Malat1 ENE triplex-binding chemotypes, among which compounds 5 and 16 reduced Malat1 RNA levels and branching morphogenesis in a mammary tumor organoid model. Computational modeling and Förster resonance energy transfer experiments demonstrate distinct binding modes for each chemotype, conferring opposing structural changes to the triplex. Compound 5 modulates Malat1 downstream genes without affecting Neat1, a nuclear lncRNA encoded in the same chromosomal region as Malat1 with a structurally similar ENE triplex. Supporting this observation, the specificity of compound 5 for Malat1 over Neat1 and a virus-coded ENE was demonstrated by nuclear magnetic resonance spectroscopy. Small molecules specifically targeting the MALAT1 ENE triplex lay the foundation for new classes of anticancer therapeutics and molecular probes for the treatment and investigation of MALAT1-driven cancers.

Aggregation and self assembly of non-enzymatic glycation of collagen in the presence of amino guanidine and aspirin: An in vitro study

Non-enzymatic glycation of collagen has been used in modern biomaterials science. This paper deals with in vitro studies on the effects of amino guanidine (AG) and aspirin in the non-enzymatic glycation (NEG) of collagen using thermal, conformational, fluorescence, turbidity and powder XRD measurements. There is no significant change in the fluorescence emission spectra for different concentrations of AG treated NEG of collagen whereas the emission intensity decreases as the concentration of aspirin increases. Circular dichroism (CD) revealed the disappearance of the positive peak at 220 nm for glycated collagen in the presence of amino guanidine and aspirin suggesting the collapse of triple helical configuration. Nearly 15 °C decrease is observed in shrinkage temperature of glycated rat tail tendon (RTT) collagen fibres in the presence of aspirin. Powder XRD of glycated collagen nano-fibrils in the presence of amino guanidine reveals high crystalline nature and the enhancement of self assembly processes when compared to aspirin. To the best of our knowledge, this is the first report of powder XRD of the self assembly of collagen nano-fibrils without mineralization. Our experimental results suggest that in the non-enzymatic glycation of collagen both AG and aspirin play a pivotal role in the aggregation and self assembly processes. From the present study, it is possible to conclude that while AG significantly influences the self assembly processes, aspirin facilitates the aggregation processes.

Renaturation of triple helical polysaccharide lentinan in water-diluted dimethylsulfoxide solution

Triple helical lentinan, a β-(1→3)- d-glucan from Lentinus edodes, was denatured in dimethylsulfoxide (DMSO) into single random coils. The randomly coiled lentinan/DMSO solutions were diluted with pure water to w H (the weight fraction of water in the mixed solvent) of 95%, and their intrinsic viscosity [ η], weight-average molecular weight M w, radius of gyration R g, and hydrodynamic radius R h were investigated at 25 °C after over 5-day storage. The [ η] and M w values, especially the conformation parameter ρ (≡ R g/ R h), of the renatured lentinan were close to those of the originally extracted one, suggesting that random lentinan chains in DMSO were reassembled into triple helical structures. Moreover, the renatured lentinan in 95% water/5% DMSO solution exhibited a unique behavior of triple helical glucans that shear modulus G′ decreased sharply at temperature from 8.4 °C to 13.3 °C with increasing temperature, which was ascribed to the intramolecular conformation transition from ordered triple helical I to disordered triple helical II. The AFM images gave was suggested intuitively evidence that the renatured lentinan mainly existed as rod-like chains, supporting that formation of triple helical structure. The optimal lentinan concentration for triple helical configuration formation was estimated to be over 0.04%. The time dependence of R h and UV absorption of the water-diluted lentinan/DMSO solution with an indicator of azo dye of Congo red suggested that renaturation of triple helix was a very rapid process. Moreover, the blue-shift of UV–vis absorption spectra suggested that the dye molecules of Congo red were assembled into supramolecular structure in the hydrophobic cavity of the renatured triple helical lentinan. All the results showed that the triple helical structure formed once the randomly coiled lentinan/DMSO was diluted to the final water content of 95%.

Identification and Characterization of α-Spectrin Mutations Associated with Inherited Hemolytic Anemia.

Spectrin, the major structural component of the erythrocyte membrane skeleton, is composed of α and β chains that self-associate to form tetramers. These tetramers provide the structural integrity and flexibility critical for erythrocyte stability and shape. Mutations of α spectrin have been associated with hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP). The large size of the spectrin molecule has complicated its study. We developed a high-throughput capillary nucleotide sequencing strategy to identify mutations of the α-spectrin gene in a group of patients with spectrin-linked HS, HE, or HPP. We identified several variants including 8 nonsense, 4 splice junction, and 4 deletion/insertion mutations.(Mutations in >1 patient are counted only once.) We were interested in the identification of missense mutations, as we hypothesize that defects in α-spectrin occur in regions of structural and functional importance and their identification and characterization will provide important information about spectrin and the membrane skeleton. We identified 16 missense mutations in the region encoding the spectrin self-association site; 6 were proline substitutions and 2 were glycine substitutions, both predicted to disrupt the triple helical configuration of spectrin. Outside the self-association site, excluding 3 common protein polymorphisms, we identified 13 missense mutations; 3 were proline substitutions. To begin to study the functional significance of these mutations, we prepared 15 recombinant spectrin-GST fusion peptides containing residues 1–158 of α spectrin, the self-association contact site, representing wild type (WT) or 14 different missense mutations. After expression and purification, purity was assured by SDS-PAGE, absence of aggregation was verified by analytical HPLC gel filtration, and mass confirmed by MS analyses. Analyses by circular dichroism demonstrated that none of the missense mutations significantly modified secondary structure of the recombinant peptide. WT and mutant peptides exhibited a helical content of ∼65%. Ultracentrifugation studies verified that all peptides were monomeric at 4 and 30°C. Differential scanning calorimetry showed that the WT peptide was very stable with a single reversible 2-state transition with a Tm of 54.6°C. All mutations, except R34W, showed transitions similar to WT. R34W unfolded at a much lower Tm, 49.1°C, with a broader single peak transition. Analysis of spectrin tetramerization between α-spectrin peptides and a recombinant β-spectrin peptide (repeats 16, 17 and COOH-terminus) was performed using an analytical HPLC gel filtration assay. A wide range of binding affinities was observed: WT binding Kd=0.43μM at 23°C; group I: I24S, R28C, R28H, R28L, R28S, R45S, no binding; group II: I24T, R41W, L49F, much weaker binding than WT; group III: V31A, R45T, G46V, binding weaker than WT, and R34W and K48R, binding equal to WT. Quantitative thermodynamic analyses of spectrin tetramerization site formation between α and β spectrin peptides were assessed by isothermal calorimetry. These results were essentially comparable to the gel filtration data except the R34W mutant bound β-spectrin more avidly than WT. The identification and characterization of variants associated with HS, HE and HPP continues to extend our understanding and knowledge of both normal membrane biology and human disease pathogenesis.

Modular Autonomy, Ligand Specificity, and Functional Cooperativity of the Three In-tandem Fibronectin Type II Repeats from Human Matrix Metalloproteinase 2

Matrix metalloproteinase 2 (MMP-2) contains three fibronectin type II (col) modules that contribute to its collagen specificity. We observed that the CD spectra of the separate col modules account for the CD and temperature profiles of the in-tandem col-123 construct. Thus, to the extent of not significantly perturbing the secondary structure and thermal stability characteristics of the neighboring units, the domains within col-123 do not interact. Via NMR, we investigated ligand binding properties of the three repeats within col-123: col-123/1 (the col-1 domain within col-123), col-123/2, and col-123/3. Interactions of col-123 with the collagen mimic peptide (Pro-Pro-Gly)6 (PPG6) and propeptide segment PIIKFPGDVA (p33-42) were studied. While col-123/1 and col-123/2 bound PPG6, they interacted more weakly with p33-42. In contrast, col-123/3 exhibited a higher affinity for p33-42 than for PPG6. Thus, despite their structural homology, the col repeats of MMP-2 differ in substrate specificity. Furthermore the binding affinities toward the three in-tandem col repeats were close to those determined for the individual isolated domains or for col-12/1, indicating that vis-a-vis these ligands each module interacts essentially as an autonomous unit. Interestingly the domains within col-123 exhibited enhanced affinities for Hel3, a construct that contains ((Gly-Pro-Pro)12)3 in triple helical configuration. Nevertheless the affinities were significantly higher for col-123/1 and col-123/2 relative to col-123/3 in line with their behaviors toward PPG6. This hints at a cooperative participation toward Hel3, which is a closer mimic of collagen, a hypothesis that is supported by the detected lower affinities of col-12/1, col-12/2, col-2, col-23/2, col-3, and col-23/3 for Hel3.

Rat Sertoli-spermatogenic cell co-cultures established on collagen-glycosaminoglycan crosslinked copolymers

The role of permeable substrates on the proliferation and differentiation of rat spermatogenic cells in co-culture with Sertoli cells was evaluated. Co-cultures were prepared on substrate discs consisting of a thin, stable analog of extracellular matrix attached to a polyester mesh to facilitate handling. Substrate discs were used alone or mounted within a polysulfone reusable holder. Substrates included collagen type I alone or crosslinked with the glycosaminoglycans (GAG) chondroitin-6-sulfate (8%) or heparin (5%). Chemical crosslinking immobilizes collagen and GAG components, preserves the native triple-helical configuration of collagen type I, and renders the copolymer more resistant to degradation by cellular enzymes. Cell attachment and growth properties of Sertoli and spermatogenic cells on these substrates were compared with another permeable substrate (HATF, a mixture of cellulose esters) uncoated or coated with Matrigel (extracellular matrix material derived from EHS tumors) and with the more conventional nonpermeable glass or plastic substrates. We have found that Sertoli-spermatogenic cell co-cultures prepared from pubertal rats readily attach to collagen or collagen-GAG substrate discs as well as to HATF. However, the optical transparency of collagen or collagen-GAG, as compared to the opaque HATF substrate, facilitates monitoring cell attachment and growth by phase contrast microscopy. Substrate discs processed for light and electron microscopy using standard procedures demonstrate the organization of a structurally polarized epithelial layer with basally located Sertoli cells and spermatogenic cells associated by lateral and apical Sertoli cell surfaces. The permeable nature of the collagen-GAG substrate, its optical transparency, and the formation of an electrical-resistant, polarized Sertoli-spermatogenic cell epithelial layer opens new in vitro experimental possibilities for testing agents that may favor or disrupt the spermatogenic process.

Platelet-reactive sites in collagens type I and type III. Evidence for separate adhesion and aggregatory sites

The adhesion of human and rabbit platelets to collagens and collagen-derived fragments immobilized on plastic was investigated. Adhesion appeared to be independent of collagen conformation, since similar attachment occurred to collagen (type I) in monomeric form, as fibres or in denatured state. The adhesion of human platelets was stimulated to a variable degree by Mg2+, but rabbit platelet adhesion showed little if any dependence on this cation. Collagens type I, III, V and VI were all able to support adhesion, although that to collagen type V (native) was lower than that to the other collagens. Adhesion to a series of peptides derived from collagens I and III was measured. Attachment did not require the presence of peptides in triple-helical configuration. The extent of adhesion ranged from relatively high, as good as to the intact parent collagen molecule, to little if any adhesive activity beyond the non-specific (background) level. The existence of very different degrees of activity suggests that platelet adhesion is associated with specific structural sites in the collagen molecule. Adhesion in many instances was essentially in accord with the known platelet-aggregatory activity of individual peptides. However, two peptides, alpha 1(I)CB3 and alpha 1(III)CB1,8,10,2, exhibited good adhesive activity although possessing little if any aggregatory activity. Of particular interest, despite its near-total lack of aggregatory activity, adhesion to peptide alpha 1(I)CB3 was as good as that to the structurally homologous peptide alpha 1(III)CB4, in which is located a highly reactive aggregatory site. This implies that platelet adhesion to collagen may involve sites in the collagen molecule distinct from those more directly associated with aggregation.

The Platelet Reactivity of Collagen Type I: Evidence for Multiple Platelet-Reactive Sites in the Type I Collagen Molecule

In this study, the ability of peptides, obtained by fragmentation of the collagen type I molecule, to induce platelet aggregation has been examined. In order to satisfy requirements for tertiary and quaternary structure, peptides were first renatured (where necessary) to restore triple-helical configuration and then polymerised. Fragmentation with mammalian collagenase indicated the presence of platelet-reactive sites in both the N-terminal three-quarter and C-terminal one quarter fragment of the collagen molecule. Cleavage with cyanogen bromide indicated the presence in the constituent alpha 1(I)-chain of at least four platelet-reactive sites. Our results suggest a relatively wide distribution of platelet-binding sites situated throughout the length of the collagen (type I) molecule, each probably of relatively low affinity and low structural specificity, at least in terms of amino acid sequence, and probably of a similar nature to those that might be expected to exist in any collagen-like species.

Monoclonal antibodies to human type IV collagen: useful reagents to demonstrate the heterotrimeric nature of the molecule.

Monoclonal antibodies (mAbs) have been prepared against type IV collagen isolated from human kidney. Two mAbs, designated CIV 22 and CIV 16, were extensively characterized. CIV 22 reacted only with native type IV collagen, whereas CIV 16 also bound to fragments derived from the alpha 1(IV) chain after reduction and alkylation of the molecule. Therefore, CIV 22 recognizes a conformational epitope on the triple helical type IV collagen, whereas CIV 16 binds to a sequential determinant in the carboxyl-terminal half of the alpha 1(IV) chain. By immunofluorescence, typical basement membrane structures were stained with both mAbs on frozen sections of different human organs. The mAbs were used to investigate the chain composition of type IV collagen. Radiolabeled type IV collagen bound to CIV 22, proving its triple helical configuration. These native probes, containing both the alpha 1(IV) and the alpha 2(IV) chains, also bound to CIV 16. Since CIV 16 does not react with the isolated alpha 2(IV) chain, both chains must be arranged in a single triple helical molecule (heterotrimer).

Human procollagen: I. An anionic tropocollagen precursor from skin fibroblasts in culture

Tropocollagen is derived from an extracellular precursor, procollagen. Conversion to tropocollagen is accomplished by one or more tissue pioteases dependent in vitro on the presence of serum in the culture medium. Twenty-four hour cultures in which serum has been excluded yield an apparently undegraded precursor, procollagen I. The latter is approximately twice the size of tropocollagen, possesses an acidic pl in contrast to the alkaline pl of tropocollagen, and shares secondary structural characteristics common to tropocollagen. Procollagen I exhibits a sharp thermal transition point at 39° with a Δ T of 2° indicating that the collagenous portion of the molecule is in the triple helical configuration prior to proteolytic excision from the parent molecule. The amino acid composition is remarkable when compared to tropocollagen in the large quantity of acidic residues, decreased glycine and imino acids, and the presence of cystcine. Three models of procollagen I structure are presented and discussed relative to the available experimental evidence.