Scorpion toxins from Buthus martensii Karsch all possess a predicted α-tight-turn

We have purified a new toxin (BmK 17[4]) from Asian scorption (Buthus martensii Karsch) venom that possesses a distinctive structural motif in its N-terminal (positions 8–12) that is similarly found in two other previously described α-like toxins. BmK 17[4] prolongs action potentials (APs) in frog nerve and was purified using gel filtration, ion exchange, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). BmK 17[4] significantly prolonged frog APs but it did not alter APs from an insect ventral nerve cord at similar doses. When applied to voltage-clamped frog muscle single fibers, BmK 17[4] prolonged fast inactivation. Because the polypeptide prolongs APs when both K⁺ and Ca²⁺ channels were blocked, BMK 17[4] acts to selectively alter Na⁺ channel inactivation. The N-terminal sequence of BmK 17[4] was found to be VRDAYIAKPENCVYXC—. The molar mass of BmK 17[4] was determined by LC/MS/MS to be 7097 Daltons. The N-terminal motif (KPENC), which introduces a reverse turn in residues 8–12, does not appear in previously characterized BmK α-toxins and may be characteristic of α-like toxins. Sequence similarity database searches were used to test whether the N-terminal sequences of α-like polypeptide toxins from B. martensii Karsch possess a distinctive structural motif in its 5-residue reverse turn (α-turn) that is conserved. Sequence similarities with putative polypetides encoded by cDNAs obtained from a cDNA library [Zhu, S. Y., Li, W. X., Zenq, X. C., et al. (2000) Nine novel precursors of Buthus martensii scorpiox alpha-toxin homologues. Toxicon 38, 1653–1661] from BmK venom glands showed that an active polypeptide toxin cleaved from the putative propolypeptide toxin BmK M9 is likely identical to BmK 17[4]. Sequence comparisons with toxins and putative toxins from B. martensii Karsch and other species revealed that a group of these toxins possess a common structural motif in their α-turn. A neighbor-joining phylogenetic analysis suggests that there are two phylogenetic sister groups of related BmK polypeptides; one possesses the KPENC motif and the other possesses a modifed version (KPHNC) of it.     

The collagenolytic action of MMP-1 is regulated by the interaction between the catalytic domain and the hinge region

The role of the hinge region in the unwinding and cleavage of type I collagen by interstitial collagenase (MMP-1) has been studied at 37 °C and pH 7.3. The collagenolytic processing by MMP-1 displays a very similar overall rate for both chains of collagen I, even though the affinity is higher for the α-1 chain and the cleavage rate is faster for the α-2 chain. MMP-1 binding to collagen I brings about a significant unwinding of the triple-helical arrangement only after the first cleavage step of the α-1 and α-2 chains. The proteolytic processing by wild-type MMP-1 on a synthetic substrate and collagen I has been compared with that observed for site-directed mutants obtained either by truncating the hinge region (∆255–272) or by individually replacing the conserved amino acids Val268, Gly272, and Lys277 of the hinge region with residues observed for the corresponding position in stromelysin-1 (MMP-3), a noncollagenolytic metalloproteinase. The ∆256–272 mutant has no collagenolytic activity, clearly demonstrating the crucial role of this region for the enzymatic processing of collagen I. However, among various mutants investigated, only Gly272Asp shows a dramatically reduced enzymatic activity both on the synthetic substrate and on collagen I. This effect, however, is clearly related to the substituting residue, since substitution of Ala or Asn for Gly272 does not have any effect on the kinetic properties of MMP-1. These data suggest that the substrate specificity of MMP-1 is dictated by the reciprocal structural relationships between the catalytic domain and the carboxy-terminal domain through the conformational arrangement of the hinge region.     

Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains,modules, and exosites

The function of ancillary domains and modules attached to the catalytic domain of mutidomain proteases, such as the matrix metalloproteinases (MMPs), are not well understood. The importance of discrete MMP substrate binding sites termed exosites on domains located outside the catalytic domain was first demonstrated for native collagenolysis. The essential role of hemopexin carboxyl-domain exosites in the cleavage of noncollagenous substrates such as chemokines has also been recently revealed. This article updates a previous review of the role of substrate recognition by MMP exosites in both preparing complex substrates, such as collagen, for cleavage and for tethering noncollagenous substrates to MMPs for more efficient proteolysis. Exosite domain interaction and movements—“molecular tectonics”—that are required for native collagen triple helicase activity are discussed. The potential role of collagen binding in regulating MMP-2 (gelatinase A) activation at the cell surface reveals unexpected consequences of substrate interactions that can lead to collagen cleavage and regulation of the activation and activity of downstream proteinases necessary to complete the collagenolytic cascade.     

A mutant α-amylase with only part of the catalytic domain and its structural implication

A truncated mutant α-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type α-amylases (Xa-WT) through random mutagenesis that contained 167 amino acid residues (approx 65% shorter than that of Xa-WT). Secondary structure prediction implied that Xa-S2, would be unable to form the whole (β/α)₈-barrel catalytic domain and did not have the three conserved catalytic residues of wild type α-amylase, but it still displays the starch-hydrolyzing activity. Xa-S2 was prepared, characterized and compared to the recombinant wild-type enzymes. The K _m for starch was 32 mg/ml; activity was optimal at pH 6.2 and 30°C. In contrast, the K _m for starch of Xa-WT was 8 mg/ml and optimal enzyme activity was at pH 6.0–6.2 and 45–50°C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of α-1,4-glucosidic bonds. T. Ke and X. D. Ma contributed equally to this work     

Cloning,expression, and pharmacological activity of BmK AS,an active peptide from scorpion <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch

BmK AS is a β long-chain scorpion peptide from the venom of Buthus martensii Karsch (BmK). It was efficiently expressed as a soluble and functional peptide in Escherichia coli, and purified by metal chelating chromatography. About 4.2 mg/l purified recombinant BmK AS could be obtained. The recombinant BmK AS maintained a similar analgesic activity to the natural one in both the mouse-twisting test and hot-plate procedure. It also exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. BmK AS is the first long-chain scorpion peptide reported to have antimicrobial activity, and is a valuable molecular scaffold for pharmacological research.     

Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs

We describe a simple method for real-time monitoring of matrix metalloproteinase-9 (MMP-9) collagenolytic activity for native triple helical collagen IV with a surface plasmon resonance (SPR) biosensor. The proteolytic activity of MMP-9 is measured as a decrease in the SPR signal resulting from the cleavage of collagen IV immobilized on the sensor surface. The kinetic parameters of full-length MMP-9 and its catalytic domain—catalytic constant (k_cat), association rate constant (k_a), and dissociation rate constant (k_d)—were estimated by the SPR method. The presence of sodium chloride and a nonionic detergent Brij-35 in a reaction solution led to the lower collagenolytic activity of MMP-9, whereas they suppressed the nonspecific interaction between MMP-9 and a cysteamine-modified chip. The comparison of kinetic parameters between MMP-9 and its catalytic domain revealed that the association constant of MMP-9 is much larger than that of the catalytic domain, suggesting that the interplay among hemopexin-like domain, fibronectin type II repeats motif, and linker region (O-glycosylated domain) plays an important role in recognizing collagen IV.     

Regulation of MMP-2 expression and activity by β-1,3-<Emphasis Type="Italic">N</Emphasis>-acetylglucosaminyltransferase-8 in AGS gastric cancer cells

β-1,3-N-acetylglucosaminyltransferase-8(β3Gn-T8) catalyzes the transfer of GlcNAc to the non-reducing terminus of the Galβ1-4GlcNAc of tetraantennary N-glycan in vitro. It has been reported to be involved in malignant tumors, but a comprehensive understanding of how the glycolsyltransferase correlates with the invasive potential of human gastric cancer is not currently available. Therefore, we investigated the ability and possible mechanism involved with β3Gn-T8 in modulating matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in AGS gastric cancer cells. Here, we found out that siRNA-mediated suppression of the β3Gn-T8 could directly reduce the MMP-2 expression and activity as observed in RT-PCR, western blot and gelatin zymography analysis. Meanwhile, TIMP-2 expression had been increased. Cell invasion assay using matrigel matrix-coated transwell inserts showed that the invasive property was greatly suppressed in β3Gn-T8 siRNA transfected cells. Furthermore, cells overexpressing β3Gn-T8 gene (when transfected with pEGFP-C1 plasmid) also expressed MMP-2 gene, but TIMP-2 expression had been inhibited. The invasive ability of these cells was also enhanced. Protein–protein interaction analysis using STRING database showed that β3Gn-T8 and MMP-2 may have related signal pathway. In summary, our results reveal a new mechanism by which β3Gn-T8 can regulate MMP-2 and TIMP-2. We suggest that β3Gn-T8 can be used as a novel therapeutic target for human gastric treatment.     

Cloning and functional characterization of a complex endo-β-1,3-glucanase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp.

A β-1,3-glucanase gene, encoding a protein of 1,793 amino acids, was cloned from a strain of Paenibacillus sp. in this study. This large protein, designated as LamA, consists of many putative functional units, which include, from N to C terminus, a leader peptide, three repeats of the S-layer homologous module, a catalytic module of glycoside hydrolase family 16, four repeats of the carbohydrate-binding module of family CBM_4_9, and an analogue of coagulation factor Fa5/8C. Several truncated proteins, composed of the catalytic module with various organizations of the appended modules, were successfully expressed and characterized in this study. Data indicated that the catalytic module specifically hydrolyze β-1,3- and β-1,3–1,4-glucans. Also, laminaritriose was the major product upon endolytic hydrolysis of laminarin. The CBM repeats and Fa5/8C analogue substantially enhanced the hydrolyzing activity of the catalytic module, particularly toward insoluble complex substrates, suggesting their modulating functions in the enzymatic activity of LamA. Carbohydrate-binding assay confirmed the binding capabilities of the CBM repeats and Fa5/8C analogue to β-1,3-, β-1,3–1,4-, and even β-1,4-glucans. These appended modules also enhanced the inhibition effect of the catalytic module on the growth of Candida albicans and Rhizoctonia solani.     

Crystal structure determination of a neutral neurotoxin BmK M4 fromButhus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series. It is medially toxic and belongs to group III cc-toxins. The purified sample was crystallized in rhombic space group P6 Using an X-ray diffraction technique, the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution. The model was refined. The final crystallographic R factor was 0.142 and the free R factor was 0.173. The root mean square deviation is 0.001 5 nm for the bond length and 1.753° for the bond angles. 64 water molecules were added to the asymmetric unit. The refined structure showed an unusual non-prolyl cis peptide bond at residue 10. The structure was compared with group II a-toxin BmK M8 (an acidic, weak toxin). The potential structural implications of the cis peptide bond were discussed.     

Molecular characterization of xynX, a gene encoding a multidomain xylanase with a thermostabilizing domain from Clostridium thermocellum

A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined. The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa). Sequence analysis of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain. A deletion mutant of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability. In terms of half-life at 70 °C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme, XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain. The optimum temperature of XynX-C was about 5–10 °C lower than that of XynX-TC. Received: 12 January 2000 / Received revision: 24 April 2000 / Accepted: 1 May 2000     

Direct observation of multiple protonation states in recombinant human purple acid phosphatase

To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5–6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pH<pK _a,1), an active form (pK _a,1<pH<pK _a,2), and an inactive high pH form (pH>pK _a,2). The pK _a,1 values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme–phosphate complex (which should mimic the enzyme–substrate complex), the enzyme–fluoride complex, and the enzyme–fluoride–phosphate complex (which should mimic the ternary enzyme–substrate–hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.Electronic Supplementary Material Supplementary material is available for this article at     

Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N′-dimethylethylenediamine)2 2+ complex

Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C–H bonds, Stack and coworkers recently reported a synthetic μ-η²:η²-peroxodicopper(II)(DBED)₂ complex (DBED is N,N′-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-μ-oxo-dicopper(III)-phenolate complex, with the O–O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O–O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the μ-η²:η²-peroxodicopper(II)(DBED)₂ complex by N,N′-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2:1 Cu(I)O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The interface between catalytic and hemopexin domains in matrix metalloproteinase-1 conceals a collagen binding exosite

Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. The two components interact with 1:1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe(301), Val(319), and Asp(338) in collagen binding. Intriguingly, Phe(301) is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity.     

Cloning,co-expression with an amidating enzyme,and activity of the scorpion toxin BmK ITa1 cDNA in insect cells

BmK ITa1 is an insect-specific neurotoxin from the Chinese scorpion Buthus martensi Karsch (Bmk). We succeeded in obtaining biologically active recombinant BmK ITa1 protein by simultaneous expression in insect cells of BmK ITa1 cDNA with an amidating enzyme expressed by the rat peptidylglycine α-amidating monooxygenase (PAM) gene. We investigated the insecticidal efficacy of recombinant BmK ITa1/W (without coexpression of PAM), and of BmK ITa1/A (with coexpression of PAM) in 5th instar Bombyx mori, by injecting these recombinant toxins into larvae. The lethal time for 50% of larvae (LT₅₀) was 9 h for BmK ITa1/A and 17 h for BmK ITa1/W. At 19 h after injection all of the larvae exposed to BmK ITa1/A had been killed, whereas only half of the larvae exposed to BmK ITa1/W had been killed. These results show that the simultaneous expression of an amidating enzyme can result in apparently higher insecticidal activity of BmK ITa1.     

Heterologous expression,purification and characterization of the influenza A virus <Emphasis Type="Italic">M2e</Emphasis> gene fused to <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> HSP70<Subscript>359–610</Subscript> in prokaryotic system as a fusion protein

One of the concerns about influenza A vaccine based on M2e protein is their limited potency; hence, optimal approaches to enhance immunogenicity of M2e protein immunization remain to be established. It seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (HSP70_359–610), we can render it very immunogenic. According to previous reports, this study was designed to produce a novel influenza A virus recombinant fusion protein consisted of M2e, a potent immunogenic protein from influenza A virus, fused to C-terminal domain of mycobacterium tuberculosis HSP70, HSP70_359–610, as a carrier and adjuvant. We fused the genes of M2e and HSP70 _359–610 then inserted in pQE-60, prokaryotic expression vector. This recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M-15. The recombinant fusion protein was purified by Ni–NTA affinity chromatography under denaturing conditions, followed by urea gradient dialysis. The purified fusion protein was analyzed on SDS–PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial penta-His HRP conjugate antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on the infected MDCK cells surface by immunofluorescence and cell-ELISA assay using rabbit’s immunized antiserum. This observation suggest that the expressed fusion protein is useful as a universal recombinant vaccine for overcoming highly mutational influenza virus, but more immunological study in animal lab remains to be evaluated.     

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

The active site of the catalytic domain of stromelysin-1 (matrix metalloproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the environmentally sensitive fluorescent reporter molecule bisANS. Wavelength-dependent acrylamide quenching identified three distinct emitting tryptophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Significant changes in the tryptophan fluorescence polarization occur upon binding by any of the three hydroxamate inhibitors Batimastat, CAS108383-58-0, and Celltech CT1418, all of which bind in the P2′-P3′ region of the active site. In contrast, the inhibitor CGS27023A, which is t hought to bind in the P1-P1′ region, does not induce any change in tryptophan fluorescence polarization. The use of the fluorescent probe bisANS revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysin with a dissociation constant ofK _i=22 μM. In addition to this binding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40±0.17 μM. The auxiliary site is open, hydrophobic, and near the fluorescing tryptophans. The binding of bisANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.     

Expression,purification and characterization of ricin vectors used for exogenous antigen delivery into the MHC class I presentation pathway

Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smithet al. J Immunol 2002; 169:99–107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression inE. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterizedin vitro, via anN-glycosidase assay, andin vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass. Published: February 17, 2003     

Structural basis for matrix metalloproteinase-2 (MMP-2)-selective inhibitory action of β-amyloid precursor protein-derived inhibitor

Unlike other synthetic or physiological inhibitors for matrix metalloproteinases (MMPs), the β-amyloid precursor protein-derived inhibitory peptide (APP-IP) having an ISYGNDALMP sequence has a high selectivity toward MMP-2. Our previous study identified amino acid residues of MMP-2 essential for its selective inhibition by APP-IP and demonstrated that the N to C direction of the decapeptide inhibitor relative to the substrate-binding cleft of MMP-2 is opposite that of substrate. However, detailed interactions between the two molecules remained to be clarified. Here, we determined the crystal structure of the catalytic domain of MMP-2 in complex with APP-IP. We found that APP-IP in the complex is indeed embedded into the substrate-binding cleft of the catalytic domain in the N to C direction opposite that of substrate. With the crystal structure, it was first clarified that the aromatic side chain of Tyr(3) of the inhibitor is accommodated into the S1' pocket of the protease, and the carboxylate group of Asp(6) of APP-IP coordinates bidentately to the catalytic zinc of the enzyme. The Ala(7) to Pro(10) and Tyr(3) to Ile(1) strands of the inhibitor extend into the nonprime and the prime sides of the cleft, respectively. Therefore, the decapeptide inhibitor has long range contact with the substrate-binding cleft of the protease. This mode of interaction is probably essential for the high MMP-2 selectivity of the inhibitor because MMPs share a common architecture in the vicinity of the catalytic center, but whole structures of their substrate-binding clefts have sufficient variety for the inhibitor to distinguish MMP-2 from other MMPs.