Synthesis of Hemolymph High-Density Lipoprotein β-Glucan Binding Protein by Penaeus vannamei Shrimp Hepatopancreas

It was recently shown that the shrimp high-density lipoprotein (HDL) and the β-1,3-glucan binding protein (BGBP) are identical, implying dual functions for the same protein: lipid transport and involvement in the defense system. Because this protein is present in plasma, and the hepatopancreas is a major lipid storage gland, we investigated the presence of the HDL/BGBP polypeptide and its messenger RNA in this tissue using a monospecific antibody against HDL/BGBP. Hepatopancreas crude protein extracts, as well as polypeptides produced by poly(A)⁺ RNA in vitro translation, were recognized by the anti-HDL/BGBP. Furthermore, a specific pattern was revealed in hepatopancreas thin sections by immunodetection. Strong recognition was seen in the epithelial cells of hepatopancreatic tubules, probably related to the secretion process of this protein. Received September 24, 1999; accepted April 5, 2000.     

1,3-beta-D glucan binding protein (BGBP) from the white shrimp,Penaeus vannamei,is also a heparin binding protein

Shrimp BGBP was purified as a 100 kDa glycoprotein by affinity chromatography using immobilised heparin. BGBP bound simple carbohydrates, glycosaminoglycans like heparin sulphate and glycoproteins, but it was unable to agglutinate erythrocytes. Using an ELISA-based microplate assay, it was shown that simple carbohydrates such as n-glucose and D-mannose are competitive inhibitors of heparin sulphate binding to BGBP. Based on these properties BGBP is considered as a new type of heparin binding protein.     

Multiple sugar binding sites in alpha-glucosidase

Twenty-five analogs of D-glucose were examined as reversible inhibitors of yeast alpha-glucosidase (EC 3.2.1.20). The K(i) values range from 0.38 mM for 6-deoxy-D-glucose (quinovose) to 1.0 M for D-lyxose at pH=6.3 (0.1 M NaCl, 25 degrees ). All the monosaccharides and the three disaccharides (maltose, isomaltose and alpha,alpha-trehalose) were found to be linear competitive inhibitors with respect to alpha-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K(i)=1.8(+/-0.1) mM], one by D-galactose [K(i)=164(+/-11) mM] and one by D-mannose [K(i)=120(+/-9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK(a1)=5.55(+/-0.15), pK(a2)=6.79(+/-0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme-product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Purification and Characterization of an Endo-(1,3)-beta-d-Glucanase from Trichoderma longibrachiatum

A laminarinase [endo-(1,3)-beta-d-glucanase] has been purified from Trichoderma longibrachiatum cultivated with d-glucose as the growth substrate. The enzyme was found to hydrolyze laminarin to oligosaccharides varying in size from glucose to pentaose and to lesser amounts of larger oligosaccharides. The enzyme was unable to cleave laminaribiose but hydrolyzed triose to laminaribiose and glucose. The enzyme cleaved laminaritetraose, yielding laminaritriose, laminaribiose, and glucose, and similarly cleaved laminaripentaose, yielding laminaritetraose, laminaritriose, laminaribiose, and glucose. The enzyme cleaved only glucans containing beta-1,3 linkages. The pH and temperature optima were 4.8 and 55 degrees C, respectively. Stability in the absence of a substrate was observed at temperatures up to 50 degrees C and at pH values between 4.9 and 9.3. The molecular mass was determined to be 70 kilodaltons by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, and the pI was 7.2. Enzyme activity was significantly inhibited in the presence of HgCl(2), MnCl(2), KMnO(4), and N-bromosuccinimide. The K(m) of the enzyme on laminarin was 0.0016%, and the V(max) on laminarin was 3,170 mumol of glucose equivalents per mg of the pure enzyme per min.     

Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens.

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction by decreasing the apparent binding affinity of heparin for thrombin

The present study has shown that calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction. The initial rate of thrombin (4.0 nM) inhibition by antithrombin III (200 nM) in the presence of heparin (2.5 ng/ml) decreased from 3.6 nM/min (in the absence of calcium) to 0.12 nM/min in the presence of 10 mM calcium. In the absence of heparin, the initial rate of thrombin inhibition by antithrombin III was not affected by calcium. The heparin-catalyzed antithrombin III/thrombin reaction is described by the general rate equation for a random-order, bireactant, enzyme-catalyzed reaction (M. J. Griffith (1982) J. Biol. Chem. 257, 13899-13902). As such, the reaction is saturable with respect to both thrombin and antithrombin III. The apparent kinetic parameters for the heparin-catalyzed antithrombin III/thrombin reaction were determined in the presence and absence of calcium. The apparent heparin/antithrombin III dissociation constant values were not measurably different in the presence of 0, 1.0, and 3.0 mM calcium. The apparent heparin/thrombin dissociation constant value increased from 7.0 nM, in the absence of calcium, to 10 and 30 nM in the presence of 1.0 and 3.0 mM calcium, respectively. The maximum reaction velocity, at saturation with respect to both proteins, was not affected by calcium. It is concluded that calcium binds to functional groups within the heparin molecule which are required for thrombin binding.     

Biophysical characterization of the non-fusogenic interaction between liposomes and the shrimp bifunctional lipoprotein-beta-glucan binding protein HDL/BGBP

Shrimp High Density Lipoprotein-beta-Glucan Binding Protein (HDL/BGBP) has been studied by its role in nutrition and innate defense. Although the mechanisms of lipid loading are still unknown, HDL-BGBP binds and aggregates phospholipids vesicles in vitro. To gain insights into the HDL-BGBP mechanism of interaction with membranes, we have used fluorescence spectroscopy and electron microscopy. Data show that HDL-BGBP does not induce membrane fusion, leakage nor lipid exchange, although microstructural changes are clearly observed. This work supports a model where protein aggregation leads to liposome clustering. Such interaction may be a critical factor for the activation of the shrimp blood cell in vivo.     

Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.     

Characterization of recombinant yeast exo-beta-1,3-glucanase (Exg 1p) expressed in Escherichia coli cells

Yeast exo-beta-1,3-glucanase gene (EXG1) was expressed in Escherichia coli and the recombinant enzyme (Exg1p) was characterized. The recombinant Exglp had an apparent molecular mass of 45 kDa by SDS-PAGE and the enzyme has a broad specificity for beta-1,3-linkages as well as beta-1,6-linkages, and also for other beta-glucosidic linked substrates, such as cellobiose and pNPG. Kinetic analyses indicate that the enzyme prefers small substrates such as laminaribiose, gentiobiose, and pNPG rather than polysaccharide substrates, such as laminaran or pustulan. With a high concentration of laminaribiose, the enzyme catalyzed transglucosidation forming laminarioligosaccharides. The enzyme was strongly inhibited with high concentrations of laminaran.     

Surface plasmon resonance characterization of calspermin-calmodulin binding kinetics

We cloned, expressed, and purified a chimeric fusion between a soluble green fluorescent protein (smGFP) and the calmodulin binding protein calspermin. We have shown that the fusion protein, labeled smGN, has a K(i) in the calmodulin-dependent cyclic nucleotide phosphodiesterase activity assay of 1.97 nM, i.e., 3800 times smaller than that of the commonly used calmodulin inhibitor W7. Association and dissociation rate constants (k(a) and k(d)) and the dissociation equilibrium constant (K(D)) of smGN for calmodulin were determined using surface plasmon resonance (SPR). The k(a)=1.24 x 10(6)M(-1)s(-1), the k(d)=5.49 x 10(-3)s(-1), and the K(D)=4.42 x 10(-9)M. We also found that the GFP moiety was important for successfully binding calspermin to the surface of the CM5 flow cell at a sufficiently high concentration for SPR, and that this procedure may be used for SPR analysis of other acidic polypeptides, whose pI< or =4. To determine whether smGN might also bind to other calmodulin-like proteins in a heterologous system, we purified proteins from a plant total cell extract or a plant total protein extract by affinity chromatography against smGN. The purified proteins were identified as calmodulins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry, indicating a high level of specificity. We conclude that the high affinity and specific binding between smGN and calmodulin make it an easily localized recombinant alternative to chemical calmodulin inhibitors.     

Crystal structure of banana lectin reveals a novel second sugar binding site

Banana lectin (Banlec) is a dimeric plant lectin from the jacalin-related lectin family. Banlec belongs to a subgroup of this family that binds to glucose/mannose, but is unique in recognizing internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. Here we present the crystal structures of Banlec alone and with laminaribiose (LAM) (Glcbeta1, 3Glc) and Xyl-beta1,3-Man-alpha-O-Methyl. The structure of Banlec has a beta-prism-I fold, similar to other family members, but differs from them in its mode of sugar binding. The reducing unit of the sugar is inserted into the binding site causing the second saccharide unit to be placed in the opposite orientation compared with the other ligand-bound structures of family members. More importantly, our structures reveal the presence of a second sugar binding site that has not been previously reported in the literature. The residues involved in the second site are common to other lectins in this family, potentially signaling a new group of mannose-specific jacalin-related lectins (mJRL) with two sugar binding sites.     

Isoindol-1,3-dione and isoindol-1-one derivatives with high binding affinity to beta-amyloid fibrils

Based on the structural features of Indoprofen and PIB, a series of isoindol-1,3-diones 1a-k and isoindol-1-ones 2a-l were designed and synthesized. These 23 compounds were evaluated by competitive binding assay against aggregated Abeta42 fibrils using [(125)I]TZDM. All the isoindolone derivatives showed very good binding affinities with K(i) values in the subnanomolar range (0.42-0.94 nM). Among them, isoindol-1,3-diones 1i and 1k and isoindol-1-ones 2c and 2i exhibited excellent binding affinities (K(i)=0.42-0.44 and 0.46-0.49 nM) than those of Indoprofen (K(i)=0.52 nM) and PIB (K(i)=0.70 nM). These results suggest that isoindolones could be served as a scaffold for potential AD diagnostic probes to monitor Abeta fibrils.     

Synthesis and evaluation of in vitro bioactivity for vesicular acetylcholine transporter inhibitors containing two carbonyl groups

To identify selective high-affinity ligands for the vesicular acetylcholine transporter (VAChT), we have incorporated a carbonyl group into the structures of trozamicol and prezamicol scaffolds, and also converted the secondary amines of the piperidines of trozamicols and prezamicols into amides. Of 18 new racemic compounds, 4 compounds displayed high affinity for VAChT (K(i)=10-20 nM) and greater than 300-fold selectivity for VAChT over σ(1) and σ(2) receptors, namely (4-(4-fluorobenzoyl)-4'-hydroxy-[1,3'-bipiperidin]-1'-yl)(3-methylthiophen-2-yl)methanone oxalate (9g) (K(i-VAChT)=11.4 nM, VAChT/σ(1)=1063, VAChT/σ(2)=370), (1'-benzoyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10c) (K(i-VAChT)=15.4 nM, VAChT/σ(1)=374, VAChT/σ(2)=315), (4'-hydroxy-1'-(thiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10e) (K(i-VAChT)=19.0 nM, VAChT/σ(1)=1787, VAChT/σ(2)=335), and (4'-hydroxy-1'-(3-methylthiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10g) (K(i-VAChT)=10.2 nM, VAChT/σ(1)=1500, VAChT/σ(2)=2030). These four compounds can be radiosynthesized with C-11 or F-18 to validate their possibilities of serving as PET probes for quantifying the levels of VAChT in vivo.     

Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors

Phenylalanine ammonia-lyase (PAL) catalyzes the beta-elimination of ammonia from L-phenylalanine to trans-cinnamic acid. A study of inhibition of PAL by phenol, ortho-cresol, and meta-cresol gave mixed inhibition; para-cresol is not an inhibitor. The calculated values of K(i) and alphaK(i) are phenol, K(i)=2.1+/-0.5 mM and alphaK(i)=3.45+/-0.95 mM; ortho-cresol, K(i)=0.8+/-0.2 mM and alphaK(i)=3.4+/-0.2 mM; meta-cresol, K(i)=2.85+/-0.15 mM and alphaK(i)=18.5+/-1.5 mM. The synergistic inhibition of the same inhibitors with glycine showed a lack of inhibition with the para-cresol/glycine pair, while mixed inhibition was observed with the ortho-cresol/glycine pair (K(i)=0.038+/-0.008 mM, alphaK(i)=0.13+/-0.04 mM) and phenol/glycine pair (K(i)=0.014+/-0.003 mM, alphaK(i)=0.058+/-0.01 M). The meta-cresol/glycine pair gave competitive inhibition (K(i)=0.36+/-0.076 mM). The strong synergistic inhibition observed implies that the inhibitors bind at the active site: in fact, the inhibitors used imitate the structure of the substrate. The order of synergistic inhibition is the same for the sites related to K(i) and alphaK(i). These results are in agreement with the inhibitors entering two active sites located in two different subunits.     

Characterization of heparin binding of human extracellular superoxide dismutase

The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K(d), of 0.12 microM, which is 700 and 10-20 times lower than the K(d) values for the synthetic peptide and the fusion proteins, respectively. However, when an alpha-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K(d) decreased to 0.64 microM. The differences in the K(d) values were mainly governed by differences in the association rate constants (k(ass)). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s(-)(1)), and a very high association rate constant (8 x 10(5) M(-)(1) s(-)(1)), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an alpha-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.     

Isolation and partial characterization of the lipoprotein families A and A-I from high-density lipoproteins of human serum

Procedures for the isolation of two lipoprotein fractions from plasma high-density lipoproteins (HDL), characterized by apolipoprotein A-I and apolipoprotein A-I together with apolipoprotein A-II, have been elaborated. Apolipoprotein A-I was identified as the protein moiety of one of these fractions (lipoprotein A-I) with polyacrylamide gel electrophoresis (at basic and acidic pH, as well as in the presence of sodium dodecyl sulphate), immuno-double-diffusion, and amino acid analysis. Apolipoproteins A-I and A-II were identified as the protein moiety of the other fraction (lipoprotein A) with polyacrylamide gel electrophoresis (basic and acidic pH) and immuno-double-diffusion. Lipoprotein A-I consisted of spherical particles with a diameter similar to that of HDL as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 8.7 nm from gel chromatography. Lipoprotein A-I migrated in the HDL position on crossed immunoelectrophoresis. On iso-electric focusing lipoprotein A-I appeared as multiple bands in the pH range 5.05-5.55. Lipoprotein A-I had the density of an HDL-2 fraction (rho: 1.063-1.105). Lipoprotein A consisted of spherical particles with a diameter similar to that of HDL, as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 7.9 nm from gel chromatography. The molar ratio between the A-I and A-II polypeptides was estimated to 1.3:1 with electroimmunoassay and calculations from the amino acid compositions. Lipoprotein A migrated in the position of HDL on crossed immuno-electrophoresis. On iso-electric focusing lipoprotein A appeared as one major and two minor bands in the pH range 5.10-5.30. Lipoprotein A had the hydrated density of an HDL-2 fraction.     

Identification of the catalytic subunit of acetohydroxyacid synthase in Haemophilus influenzae and its potent inhibitors

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate- (ThDP)- and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids (BCAAs) leucine, isoleucine, and valine. The gene from Haemophilus influenzae that encodes the AHAS catalytic subunit was cloned, overexpressed in Escherichia coli BL21(DE3), and purified to homogeneity. The purified H. influenzae AHAS catalytic subunit (Hin-AHAS) appeared as a single band on SDS-PAGE gel, with a molecular mass of approximately 63 kDa. The enzyme catalyzes the condensation of two molecules of pyruvate to form acetolactate, with a K(m) of 9.2mM and the specific activity of 1.5 micromol/min/mg. The cofactor activation constant (K(c)=13.5 microM) and the dissociation constant (K(d)=3.3 microM) of ThDP were also determined by enzymatic assay and tryptophan fluorescence quenching studies, respectively. We screened a chemical library to discover new inhibitors of the Hin AHAS catalytic subunit. Through which, AVS-2087 (IC(50)=0.53 microM), KSW30191 (IC(50)=1.42 microM), and KHG20612 (IC(50)=4.91 microM) displayed potent inhibition as compare to sulfometuron methyl (IC(50)=276.31 microM).