Isolation and partial characterisation of a novel lectin from Aegle marmelos fruit and its effect on adherence and invasion of Shigellae to HT29 cells

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 μg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.     

Purification and characterization of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases

We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.     

Information-theoretic dissection of pairwise contact potentials

Pairwise contact potentials have a long, successful history in protein structure prediction. They provide an easily-estimated representation of many attributes of protein structures, such as the hydrophobic effect. In order to improve on existing potentials, one should develop a clear understanding of precisely what information they convey. Here, using mutual information, we quantified the information in amino acid potentials, and the importance of hydropathy, charge, disulfide bonding, and burial. Sampling error in mutual information was controlled for by estimating how much information cannot be attributed to sampling bias. We found the information in amino acid contacts to be modest: 0.04 bits per contact. Of that, only 0.01 bits of information could not be attributed to hydropathy, charge, disulfide bonding, or burial.     

Anacardic acids and ferric ion chelation

6-Pentadeca(e)nylsalicylic acids isolated from the cashew Anacardium occidentale L. (Anacardiaceae), commonly known as anacardic acids, inhibited the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) competitively without prooxidant effects. Their parent compound, salicylic acid, did not have this inhibitory activity up to 800 pm, indicating that the pentadeca(e)nyl group is an essential element to elicit the activity. The inhibition is attributed to its ability to chelate iron in the enzyme. Thus, anacardic acids chelate iron in the active site of the enzyme and then the hydrophobic tail portion slowly begins to interact with the hydrophobic domain close to the active site. Formation of the anacardic acids-ferric ion complex was detected in the ratio of 2:1 as the base peak in the negative ion electrospray ionization mass spectrometry. Hence, anacardic acids inhibit both Eox and Ered forms.     

Amino acid sequence around the serine phosphorylated by casein kinase II in brain myosin heavy chain

Casein kinase II from bovine brain transfers about one mole of phosphate to a serine residue near the COOH terminus of the heavy chain of myosin isolated from bovine brain. We have purified and characterized a peptide that contains this phosphoserine. The peptide was generated by chymotryptic and thermolytic digestion and was isolated by gel filtration, Fe3+ affinity chromatography, and reverse-phase high pressure liquid chromatography. Its sequence, Leu-Glu-Leu-Ser(PO4)-Asp-Asp-Asp-Asp-Glu-Ser-Lys-Ala-Ser-(Xaa)-Ile-Asn-Glu-Thr- Gln-Pro-Pro-Gln, shows that the Ser(PO4) is in an acidic environment, as is typical for casein kinase II phosphorylation sites. The "hydrophobic repeat" typical of alpha-helical coiled-coils is absent, suggesting that the sequence is part of a non-helical "tail piece" of the heavy chain. A synthetic peptide corresponding to residues 1-9 is shown to be an effective substrate for casein kinase II.     

Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite Plasmodium falciparum

Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.     

Solubility of peptides in trichloroacetic acid (TCA) solutions. Hypothesis on the precipitation mechanism

The assessment of proteolysis levels is often achieved by global quantification of the peptides soluble at different TCA concentrations, but little information is available on the features of this precipitation mechanism. Peptic, tryptic and chymotryptic digests of alpha s1, beta, and kappa caseins have been prepared and fractionated by RP-HPLC and each isolated peptide was identified. Each digest was precipitated by adding TCA to different final concentrations (2, 4, 8, and 12%). The soluble fraction was analysed by RP-HPLC. Relationships have been searched between the properties of 75 peptides obtained in this way, and their solubilities in TCA. The best correlation was found with the peptide retention time in RP-HPLC, which can be regarded as the experimental measure of peptide hydrophobicity. We concluded that TCA, by interacting with peptides, induces an increase of the hydrophobicity of peptides which can lead to aggregation through hydrophobic interactions.     

Identification of an invasion regulatory domain within the core protein of syndecan-1

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions, and thus far, the biological activity of syndecans has been attributed largely to their heparan sulfate chains. We have previously demonstrated that cell surface syndecan-1 inhibits invasion of tumor cells into three-dimensional gels composed of type I collagen. Inhibition of invasion is dependent on the syndecan heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein was also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated, and each construct was transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (amino acids 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading, or targeting of syndecan-1 to specific cell surface domains. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion, thus explaining the observed loss of syndecan-1 in some highly invasive cancers.     

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.     

Brain plasmamembrane NA+:,K+-ATPase is inhibited by acetylated tubulin

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the -subunit of Na⁺,K⁺-ATPase. In the present work we show that the interaction of tubulin with Na⁺K⁺-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na⁺K⁺-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.     

Molecular Dynamics Simulations of Micelle Formation around Dimeric Glycophorin A Transmembrane Helices

Insertion and formation of membrane proteins involves the interaction of protein helices with one another in lipid environments. Researchers have studied glycophorin A (GpA) transmembrane helices embedded in sodium dodecyl sulfate (SDS) micelles to identify contacts significant for helix dimerization. However, a detailed picture of the conformation and dynamics of the GpA-SDS system cannot be obtained solely through experiment. Molecular dynamics simulations of SDS and a GpA dimer can provide an atomic-level picture of SDS aggregation and helix association. We report 2.5-ns simulations of GpA wild-type and mutants in a preformed micelle as well as a 32-ns simulation showing the formation of a complete micelle around wild-type GpA from an initially random placement of SDS molecules in an aqueous environment. In the latter case, an initial instability of GpA helices in water is reversed after the helices become surrounded by SDS. The properties of the spontaneously formed micelle surrounding the GpA are indistinguishable from those of the preformed micelle surrounding the GpA dimer.     

Shift in nucleotide conformational equilibrium contributes to increased rate of catalysis of GpAp versus GpA in barnase

The microbial ribonuclease barnase exhibits low catalytic activity toward GpN dinucleotides, where G is guanosine, p is phosphate and N represents any nucleoside. When a phosphate is added to the 3'-end, as in GpNp, substrate affinity is enhanced by one order of magnitude, and the catalytic rate by two. In order to gain insight into this phenomenon, we analyzed the nucleotide conformations and protein-nucleotide interactions of 4 ns molecular dynamics (MD) trajectories of complexes of barnase with guanylyl(3'-5') adenosine (GpA) and guanylyl(3'-5') adenosine 3'-monophosphate (GpAp), respectively, in the presence of solvent and counter ions. We found that, in a majority of the bound GpA conformations, the guanine base was firmly bound to the recognition site. The phosphate and adenosine moieties pointed into the solvent, and interactions with key catalytic residues were absent. In contrast, the bound GpAp adopted conformations in which all of the nucleotide portions remained tightly bound to the enzyme and interactions with key catalytic residues were maintained. These observations indicate that, for GpA, a significant proportion of the bound nucleotide adopts non-productive conformations and that adding the terminal phosphate as in GpAp shifts the equilibrium of the bound conformations towards structures capable of undergoing catalysis. Incorporating this property into the kinetic equations yields an increase in both the apparent rate constant (kcat) and the apparent dissociation constant (K(M)) for GpAp versus GpA. The increase in K(M), caused by the presence of additional non-productive binding modes for GpA, should however be counterbalanced by the propensity of free GpA to adopt folded conformations in solution, which are unable to bind the enzyme and by the tighter binding of GpAp (Giraldo J, Wodak SJ, Van Belle D. Conformational analysis of GpA and GpAp in aqueous solution by molecular dynamics and statistical methods. J Mol Biol 1998; 283:863-882). Addition of the terminal phosphate is shown to significantly influence the collective motion of the enzyme in a manner that fosters interactions with key catalytic residues, representing a further likely contribution to the catalytic rate enhancement.     

Amino acid sequence of 2 neurotoxins from the scorpion Buthus eupeus venom

Three polypeptides, M10, M14 and M9, toxic to mammals were isolated from the venom of the Central Asian scorpion Buthus eupeus. All the toxins were shown to be homogeneous according to disc-electrophoresis and N-terminal group analyses. The toxin M9 was digested with trypsin, Staphylococcus aureus proteinase and cleaved with BNPS-skatole. The toxin M14 was subjected to tryptic and chymotryptic hydrolyses. The complete amino acid sequences of the toxins M9 and M14 were established and it was shown that each of them consists of 66 amino acid residues with four intramolecular disulfide bonds.     

Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.     

Phosphorylation of the 49-kDa putative subunit of the chick cerebellar kainate receptor and its regulation by kainatergic ligands

The traffic of ions through the kainic acid (KA) receptor/channels present on chick cerebellar glia is modulated by intracellular events likely to involve phosphorylation reactions. The protein carrying the KA binding sites has been isolated from chick cerebellum and shown to be composed of a 49-kDa polypeptide. Its primary structure, established via cDNA cloning, shows the presence of two putative phosphorylation sites. We report here that this 49-kDa polypeptide is a substrate of the cAMP-dependent protein kinase which catalyzes the incorporation of up to 2 mol of phosphate/mol of KA binding site. KA prevents this phosphorylation reaction in a concentration range similar to that needed to activate the KA receptor/channels but higher by 3 orders of magnitude than that needed to saturate the KA binding sites. Kainatergic ligands produce similar effects to those of KA, but 1 mM N-methyl-D-aspartic acid and 1 mM quisqualic acid have no effect. However, 0.01 mM quisqualic acid prevents the inhibitory action of KA. These results raise the possibility that the phosphorylation of KA receptor/channels in their cellular environment is negatively regulated by KA.     

Polar residue tagging of transmembrane peptides

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.     

The protective effect of simvastatin against low dose streptozotocin induced type 1 diabetes in mice is independent of inhibition of HMG-CoA reductase

Besides a cholesterol-lowering effect, simvastatin possesses anti-inflammatory properties attributed to inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and/or direct binding to, and inhibition of, the integrin lymphocyte function associated antigen-1 (LFA-1). We have shown that simvastatin protects against multiple low dose streptozotocin (MLDS) induced type 1 diabetes in mice. Presently, we examined if this effect could be abolished by co-administration of mevalonic acid, thus determining if the protective effect is dependent or independent of inhibition of HMG-CoA reductase. Mevalonic acid did not affect the protective effect of simvastatin against MLDS diabetes. Moreover, spleens from these mice did not show any signs of toxic side-effects, thus excluding the possibility that the protective effect is secondary to a general inflammatory response. We suggest that simvastatin’s protective effect mainly is independent of HMG-CoA reductase inhibition. This implies that inhibition of LFA-1 activation is important for the protective effect exerted by simvastatin.     

Strong inhibition of peptide amyloid formation by a fatty acid

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer’s and Parkinson’s disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68–78 of the protein α-synuclein involved in Parkinson’s disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.     

Influence of the C-terminus of the glycophorin A transmembrane fragment on the dimerization process

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization.     

Primary structure of streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5.

The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.