Inhibiting effect of αs1-casein on Aβ1-40 fibrillogenesis

Background

α_s1-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α_s1-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α_s1-casein on the fibrillogenesis of 1-40 β-amyloid peptide, involved in Alzheimer's disease.

Methods

The aggregation kinetics of β-peptide in presence and absence of α_s1-casein was followed under shear at 37 °C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α_s1-casein-Aβ_1-40 interaction.

Results and discussions

α_s1-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α_s1-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism.

General significance

Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.     

Characterization of the reciprocal binding sites on human α-thrombin and factor XIII A-chain

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-a-thrombin interactions. -Thrombin activated Factor XIII more efficiently (Km = 0.83 ± 0.08 × 10^-7 M; V/K = 14.90 ± 3.20 × 10^-3 min^-1) than -thrombin (Km = 6.14 ± 1.26 × 10^-7 M; V/K = 3.30 ± 1.00 × 10^-3 min^-1) or -thrombin (Km = 6.25 ± 1.15 × 10^-7 M; V/K = 3.00 ± 0.80 × 10^-3 min^-1). Immobilized FPR--thrombin bound plasma Factor XIII (Kd = 0.17 ± 0.04 × 10^-7 M) > Factor XIIIa (Kd = 0.69 ± 0.18 × 10^-7 M) > liver transglutaminase (Kd = 4.73 ± 1.01 × 10^-7 M) > Factor XIII A-chain (Kd = 49.00 ± 9.40 × 10^-7 M). FPR--thrombin and -thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 × 10^-7 M and 13.6 × 10^-7 M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR--thrombin: IC₅₀ = 1.0 × 10^-7 M, 3.0 × 10^-6 M and 1.52 × 10^-4 M, respectively. Transglutaminase also inhibited Factor XIII binding to ×-thrombin (IC₅₀ = 2.0 × 10^-6 M). Thrombin-binding site was localized to G^-38-M^-731 fragment of Factor XIII A-chain, probably within homologous regions (N^-72-A^-493) of transglutaminase. R^-320-E^-579 of -thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in -thrombin were essential for binding but not catalytic H^-363 or residues R^-382-N^-394 and R^-443-G^-475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.     

Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived α_s2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the α_s2-casein f(183–207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of α_s2-casein f(193–203) and α_s2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid α_s2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.     

Identification of high-affinity binding sites for the hepta-β-glucoside elicitor in membranes of the model legumes Medicago truncatula and Lotus japonicus

 Previous studies have led to the identification and characterization of specific, high-affinity binding sites for a hepta-β-glucoside elicitor in soybean. A survey of plant species for elicitor-binding activity reveals that among the plants tested, the hepta-β-glucoside elicitor is only recognized by plants belonging to the legume family. We have characterized in detail the glucan elicitor-binding site in the model legume Medicago truncatula Gaertn., and partially characterized the site in Lotus japonicus. These sites have characteristics that are very similar to the one in soybean, with dissociation constants of 4.7 and 8.9 nM respectively. The elicitor-binding sites from both plants are stable during solubilization with non-ionic alkylglycoside detergents. However, differences are observed in the abundance of the binding sites and their selectivity towards structurally related analogues of the hepta-β-glucoside elicitor. Our results suggest that similar, but perhaps not identical, binding sites for the hepta-β-glucoside elicitor exist in diverse legumes, but not in plants outside of the legume family. Received: 15 December 1999 / Accepted: 28 February 2000     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex

Background

The milk protein α_S1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4). In this study, α_S1-casein was identified as binder of theTLR4 ecto domain.

Specificity of the α-tocopherol (Vitamin E) effect on lifespan and fecundity of bdelloid rotifers

Experiments were undertaken to determine molecular specificity of Vitamin E-effects on lifespan and fecundity in four bdelloid rotifers (Habrotrocha sp., Philodina sp., Pleuretra sp., and Rotaria sp.). Results indicate that lifespan and fecundity could be significantly increased by addition of any one of three tocopherol compounds (d--, -, and -tocopherol) to the rotifer medium. Life table functions were increased the most by the d--tocopherol form. Improvement of these life table functions was not achieved by substitution of tocopherol analogs or other antioxidants in the rotifer medium.     

Kinetic study of the cytotoxic effect of α-sarcin,a ribosome inactivating protein fromAspergillus giganteus,on tumour cell lines: protein biosynthesis inhibition and cell binding

-Sarcin is a ribosome inactivating protein produced by the mouldAspergillus giganteus. The effect of this protein on eight different tumour cell lines has been studied in the absence of any agent affecting membrane permeability. The protein is cytotoxic for all the tumour cell lines considered. -Sarcin modifies the cell proliferation pattern by inhibiting the protein biosynthesis of the cultured cells. No membrane damage produced by -sarcin has been observed by measuring lactic dehydrogenase leakage. Alteration on the cell mitochondrial activity has not been detected upon treatment with -sarcin. Differences on the extent of the protein binding to the cells have been observed by flow cytometric measurements. The kinetic analysis of the protein biosynthesis inhibition produced by -sarcin reveals an -sarcin concentration-dependent lag phase followed by a first order decrease of the protein synthesis rate. This parameter is dependent on the external -sarcin concentration. A saturable component for the action of -sarcin is also deduced from these experiments. Results are discussed in terms of the protein passage across the cell membrane as the potential rate-limiting step for the action of -sarcin.     

Enzymatic synthesis of unique sialyloligosaccharides using marine bacterial α-(2→3)- and α-(2→6)-sialyltransferases

We investigated the acceptor substrate specificities of marine bacterial α-(2→3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 and α-(2→6)-sialyltransferase cloned from Photobacterium damselae JT0160 using several saccharides as acceptor substrates. After purifying the enzymatic reaction products, we confirmed their structure by NMR spectroscopy. The α-(2→3)-sialyltransferase transferred N-acetylneuraminic acid (Neu5Ac) from cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) to the β-anomeric hydroxyl groups of mannose (Man) and α-Manp-(1→6)-Manp, and α-(2→6)-sialyltransferase transferred N-acetylneuraminic acid to the 6-OH groups of the non-reducing end galactose residues in β-Galp-(1→3)-GlcpNAc and β-Galp-(1→6)-GlcpNAc.     

The effect of sulphide on cytochromeaa3 Isosteric and allosteric shifts of the reduced α-peak

1. Sulphide, like cyanide, is a slow-binding inhibitor of cytochromeaa₃ with a high affinity (K_d < 0.1 μM).2. Unlike cyanide binding, the binding of sulphide is apparently independent of the redox state of components of the oxidase other than cytochromea₃and shows no anomalous kinetics during complex formation.3. Sulphide binding to cytochrome a₃³⁺ is accompanied by a blue-shift in the α-peak of the reduced enzyme (a²⁺ a₃³⁺H₂S), similar to but smaller than that induced by azide.4. The reduced sulphide-inhibited system shows a much higher Soret peak at 445 nm than the corresponding cyanide and azide complexes, suggesting that partial electron transfer from sulphide to haem may occur in the complex. No evidence was obtained for the formation of any sulfhaem derivatives of cytochromea₃.5. The influence of energization on the spectrum of mitochondrial cytochrome oxidase, and the effects of calcium on the α-peak of isolated cytochromeaa₃ (Wikstro¨m, M. K. F. (1974) Ann. N. Y. Acad. Sci. 227, 146–158) are distinct from the action of the cytochromea₃ligands.6. A classification of peak shifts in the α-region in terms of isosteric and allosteric ligands is proposed.     

The effect of charge reversal mutations in the α-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs,lysophospholipids and bile acids

Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of -helix II. The -helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on -helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the -helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group.     

Identification and partial characterization of α2-macroglobulin from the tuatara (Sphenodon punctatus)

α₂-Macroglobulin (α₂-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α₂-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α₂-M remains fairly constant. The subunits of tuatara α₂-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α₂-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α₂-M subunits are present in plasma of tuatara.     

The Primary Structure of Water Buffalo αs1- and β-Casein: Identification of Phosphorylation Sites and Characterization of a Novel β-Casein Variant

The primary structure of water buffalo _s1-casein and of -casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a -elimination/thiol derivatization. Water buffalo _s1-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine _s1-casein C variant, the water buffalo _s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine A₂-casein variant, the two water buffalo -casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo -casein variants seem to be homologous to bovine A₂-casein.     

The effect of substrate modification on porcine pancreatic α-amylase subsite binding: Hydrolysis of substrates containing 2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose

Modified α-d-(1 → 4)-glucans containing a small proportion of ¹⁴C-labeled 2-deoxy-d-glucose or 2-amino-2-deoxy-d-glucose were examined as substrates for porcine pancreatic α-amylase (PPA). Cyclomaltoheptaose containing single 2-deoxy-d-glucose residues, synthesized by incubation of 2-deoxyglucosylglycogen with cyclomaltodextrin glucanotransferase in the presence of Triton X-100, was hydrolyzed by PPA to produce 2-deoxy-d-glucose; two isomers of 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxy-d-glucose may be productively bound at all five subsites of the PPA active site. Reaction kinetics and the distribution of products formed suggest, however, that productive binding of the modified residue does not occur readily at the point of catalytic attack (subsite 3) and that the preferred position of hydrolysis of modified substrates may be different from that of unmodified substrates. Results of PPA hydrolysis of glycogen containing [¹⁴C]-2-amino-2-deoxy-d-glucose showed that a modified trisaccharide and a modified disaccharide were the smallest substituted products formed. Analysis of these products indicated that they did not contain modified residues at their reducing ends. Formation of the observed 2-amino-2-deoxy-maltooligosaccharides is consistent with a scheme where productive binding of 2-amino-2-deoxy-d-glucose is allowed at subsites 1, 2, 4, and 5, but not at subsite 3, the subsite at which hydrolysis occurs.     

Effects of poly(ethylene glycol) and salt on the binding of α-amylase from the fermentation broth of Bacillus amyloliquefaciens by Cu2+-β-CD affinity adsorbent

α-Amylase from Bacillus amyloliquefaciens was purified by the immobilized metal ion affinity adsorbent, β-CD_cl-IDA-Cu²⁺. The adsorbent was prepared by reacting the cross-linked β-cyclodextrin (β-CD) with the ligand, iminodiacetic acid (IDA). The copper ion was further linked to the adsorbent. Poly(ethylene glycol) (PEG) was added to the fermentation broth to improve the adsorption efficiency of the adsorbent toward α-amylase. The effort was to provide hydrophobic interactions with the impurities which might interfere with the adsorption of α-amylase. It also provided a polymer shielding effect to prevent non-specific interactions. With the addition of PEG, the adsorption efficiency could be increased to 98%. Imidazole containing a phosphate buffer and NaCl was used to elute the bound α-amylase. By consecutive adsorption/desorption steps, up to 81% of the α-amylase activity could be recovered. Regarding the reutilization of the affinity adsorbents, α-amylase could be adsorbed and desorbed six times consecutively without a significant loss of α-amylase activity.     

Glycosylation of the N-terminal potential N- glycosylation sites in the human α1,3- fucosyltransferase V and -VI (hFucTV and -VI)

Human 1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and hFucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced by glutamine. Western blotting analyses demonstrated that both sites in hFucTV were glycosylated, whereas in hFucTVI only one of the sites (Asn91) was glycosylated. All the single mutants and the hFucTVI N46Q/N91Q double mutant exhibited enzyme activities that did not differ considerably from the wt activities. However, the enzyme activity of the hFucTV N60Q/N105Q double mutant was reduced to approximately 40% of the wt activity. In addition, castanospermine treatment diminished the enzyme activity and hence trimming of the N-linked glycans are required for expression of full enzyme activity of both hFucTV and -VI. The present study demonstrates that both of the N-terminal potential N-glycosylation sites in hFucTV and one of the sites in hFucTVI are glycosylated. Individually, their glycosylation does not contribute considerably to expression of enzyme activity. However, elimination of both sites in hFucTV reduces the enzyme activity.     

The effect of some ions on the activity of β-galactosidase and the inhibitory effect of tris (hydroxymethyl) aminomethane

The stimulating effect of phosphate and the inhibitory effect of tris-HCl on the activity of β-galactosidase inEscherichia coli was studied. The phosphate anion antagonizes the inhibitory effect of chloride. Since a similar effect is displayed by sulphate and arsenate no specific “stimulating” effect of phosphate can take place. The tris cation has also an inhibitory effect which is antagonized by univalent cations (K⁺). The resulting β-galactosidase activity reflects the antagonisms between cations and anions present in the reaction medium.