J. de Jersey

Structure, function, and regulation of tartrate-resistant acid phosphatase

The tartrate-resistant acid phosphatases (TRAPs) are a class of metalloenzymes that catalyze the hydrolysis of various phosphate esters and anhydrides under acidic reaction conditions. Because the bound metal ions confer an intense color on these enzymes they are also known as purple acid phosphatases (PAPs). Resistance to inhibition by high concentrations of the competitive inhibitor L 1 tartrate distinguishes TRAP from acid phosphatases of lysosomal or prostatic origin present in many mammalian cells and tissues. TRAP enzymes have been isolated from many mammalian sources, including: bovine and rat spleen; the spleens of patients affected with hairy cell leukemia and Gaucher’s disease; human and rat bone; and human lungs and placenta. The TRAP purified from porcine allantoic fluid, which is also known as uteroferrin, was originally recognized as an abundant basic protein in uterine secretions induced by progesterone. The catalytic mechanism, structure, and properties of the iron center of porcine TRAP have been studied extensively by our group. Mammalian isolated TRAP enzymes all have similar physical properties, including a molecular weight of about 35 kDa, a basic isoelectric point (pI 7.6–9.5), and optimal enzyme activity at an acidic pH. The enzyme can be isolated as a single chain polypeptide, but a dimeric nicked form arises from posttranslational cleavage of the single chain enzyme. Cleavage occurs in an exposed loop that is conserved in all mammalian TRAP enzymes and leads to an increase in Vmax/kcat of the enzyme by an unknown mechanism. Several proteolytic enzymes are able to cleave the exposed loop, but only the cysteine proteinases papain and cathepsin B have been able to cause activation among several tested. Ljusberg et al. put forth the view that TRAP, like several other hydrolases, is synthesized as a relatively inactive proenzyme, and cleavage is the physiological mechanism of proenzyme activation in osteoclasts. Mammalian TRAP enzymes are glycoproteins and, like most lysosomal enzymes, possess the mannose-6-phosphate lysosomal targeting sequence, which must presumably be cleaved or modified to permit secretion. TRAP isolated from allantoic fluid of the pig showed a single, unphosphorylated, high-mannose-type oligosaccharide composed of five or six mannose residues and two N-acetylglucosamine residues. In contrast, recombinant porcine TRAP secreted by Chinese hamster ovary (CHO) cells possessed N-linked, high-mannose oligosaccharide chains that were phosphorylated and could not be dephosphorylated by alkaline phosphatase treatment in vitro. This suggests that the uteroferrin oligosaccharide phosphates were not exposed, perhaps as a result of blocking by an N-acetylglucosamine residue. The glycoprotein structure of human bone TRAP was analyzed by lectin binding and, in agreement with the prior analysis of native uteroferrin, contained only N-linked high-mannose carbohydrates, implying that the native secreted protein is normally dephosphorylated. Analysis of TRAP activity present in electrophoretically separated human serum revealed two isoforms, termed 5a and 5b, with each isoform having a different pH optimum (5a: pH 4.9; 5b: pH 5.5–6.0). The carbohydrate content of the isoforms also differed with only isoform 5a containing sialic acid. TRAP contains two iron atoms at its active site, and the intense purple color of the enzyme results from a tyrosinate Fe(III) charge transfer. Reduction of the active site binuclear center to a mixed valency Fe(III)-Fe(II) form is required for activation and this corresponds to a shift in color from purple to pink. Further reduction or the presence of iron chelators can lead to reversible inactivation and formation of a colorless form of the enzyme. The enzyme is also inhibited noncompetitively by incubation with vanadate or simply following more extended incubation at 37°C. The latter case at least, produced a “yellowish” form of the enzyme. TRAP may become irreversibly inactivated by oxidation in the presence of ascorbate. The recent availability of monoclonal antibodies against TRAP has permitted the identification of an inactivated “yellowish” form of the enzyme as the major form in the circulation.

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model.

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom ofPseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant,Ki ≈ 10−11 mol/l), while both of the latter bound less tightly (Ki ≈ 10−9 mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan,Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

Zomepirac acyl glucuronide covalently modifies tubulin in vitro and in vivo and inhibits its assembly in an in vitro system

Drugs possessing a carboxylate functional group usually form acyl glucuronides as major metabolites. These electrophilic metabolites can undergo several spontaneous reactions, including covalent adduct formation with proteins. The present study examined whether covalent adducts were formed with microtubular protein (MTP, 85%, alpha/beta-tubulin) and whether this influenced its ability to assemble into microtubules. Bovine brain microtubular protein (MTP) was purified by assembly-disassembly cycles and incubated with the nonsteroidal anti-inflammatory drug (NSAID) zomepirac (ZP), its acyl glucuronide (ZAG) and rearrangement isomers (iso-ZAG) at various concentrations for 2 h at room temperature and pH 7.5. Assembly was monitored by change in turbidity (increase in absorbance at 340 nm). Both ZAG and iso-ZAG caused dose-dependent inhibition of assembly (50% inhibition at about 1 mM), while ZP caused modest inhibition (< 50% inhibition at 4 mM). In a slightly different system, incubation of performed microtubules with 4 mM ZAG caused about 35% inhibition of reassembly ability, while modification of MTP under similar conditions resulted in about 85% reduction of assembly ability. Immunoblotting with a ZP antiserum showed that ZAG and iso-ZAG covalently modified MTP in a dose-dependent manner, while ZP itself caused no modification. Tubulin and many minor proteins comprising MTP were modified. ZP-modified tubulin was shown to be present in the cytosol of livers from rats dosed twice daily for 3 days with ZP at 50 mg/kg, using a sandwich ELISA with ZP and tubulin antisera. Whether any perturbation of microtubule assembly occurs in vivo as a result of this in vivo modification is currently under investigation.

Inflammatory Cells, Microglia and MHC Class II Antigen-Positive Cells in the Spinal Cord of Lewis Rats with Acute and Chronic Relapsing Experimental Autoimmune Encephalomyelitis

Chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord and adjuvants and treatment with low dose cyclosporin A (CsA). Acute EAE was induced by the same method without CsA treatment. Immunocytochemistry and flow cytometry were used to assess inflammatory cells and MHC class II (Ia) antigen expression in the central nervous system of these rats. The inflammatory infiltrate was composed mainly of CD4+ T cells and macrophages, and alpha beta T cells constituted about 65% of the CD2+ T cells. After recovery from acute EAE and during the first remission of CR-EAE, the number of T cells was significantly less than in the preceding episodes. The number of T cells was higher in the second episode of CR-EAE than in the first remission. Throughout the course of CR-EAE, the majority of the CD2+ T cells were CD45RC-. The ratio of IL-2R+ cells to CD2+ cells ranged from 10.5 to 24.0%. The ratio of CD4+ T cells to B cells was lower in the later episodes of CR-EAE than in the first episode. Ia antigen was expressed on infiltrating round cells at all stages of CR-EAE and on microglial cells (identified by dendritic morphology) with increasing intensity throughout the course of CR-EAE. With flow cytometry, the number of Ia+ cells obtained from the spinal cord rose throughout the course of CR-EAE. The number of FSClowOX1low cells, which we consider represent microglia, also increased during the course of CR-EAE.

Common evolution of waprin and kunitz-like toxin families in Australian venomous snakes

Abstract.The venoms of Australian snakes contain a myriad of pharmacologically active toxin components. This study describes the identification and comparative analysis of two distinct toxin families, the kunitztype serine protease inhibitors and waprins, and demonstrates a previously unknown evolutionary link between the two. Multiple cDNA and full-length gene isoforms were cloned and shown to be composed of three exons separated by two introns. A high degree of identity was observed solely within the first exon which coded for the propeptide sequence and its cleavage site, and indicates that each toxin family has arisen from a gene duplication event followed by diversification only within the portion of the gene coding for the functional toxin. It is proposed that while the mechanism of toxin secretion is highly conserved, diversification of mature toxin sequences allows for the existence of multiple protein isoforms in the venom to adapt to variations within the prey environment.

Expression of CD45RC and Ia antigen in the spinal cord in acute experimental allergic encephalomyelitis: an immunocytochemical and flow cytometric study.

We performed immunocytochemical studies to analyze the inflammatory infiltrate and major histocompatibility complex class II (Ia) antigen expression in the spinal cord of Lewis rats with acute experimental allergic encephalomyelitis (EAE) induced by inoculation with myelin basic protein and adjuvants. Using antibodies to lymphocyte markers and other monoclonal antibodies we found that during clinical episodes the inflammatory infiltrate was chiefly composed of T lymphocytes and macrophages. The majority of cells in the inflammatory infiltrate were stained by the W3/25 antibody to CD4 and a proportion was stained by OX22 which labels the high molecular weight form of the leucocyte common antigen (CD45RC). CD8+ T cells were sparse and B cells were not detected. There was minimal staining with the OX39 antibody to the interleukin-2 receptor. Presumptive microglia, identified by their dendritic morphology, expressed Ia antigen during the clinical episodes and after recovery. The prominence of Ia antigen expression after recovery could indicate that this Ia expression was associated with downregulation of the encephalitogenic immune response. We also performed flow cytometry studies on cells extracted from the spinal cord of rats before and during attacks of EAE. With flow cytometry, we found that in established disease a mean of 83(SD, 23)% of CD2+ cells were CD4+, and a mean of 27(SD, 12)% of CD2+ cells were CD45RC+. In rats sampled on the first day of signs, a mean of 43(SD, 22)% of CD2+ cells were CD45RC+. In the cells extracted from the spinal cord of rats with established disease a mean of 47(SD, 32)% of macrophages were CD45RC+. Our study has combined an immunocytochemical assessment of tissue sections with quantitative flow cytometry assessment of cells extracted from the spinal cord of rats with acute EAE. We have shown that the majority of T lymphocytes in the spinal cord are CD45RC-. We have also found prominent Ia expression on dendritic cells in acute EAE and after clinical recovery.

Isolation and characterization of a thermostable dextranase

A thermostable dextranase has been isolated from an anaerobic thermophilic bacterium, Rt364, collected from a New Zealand thermal spring. The enzyme was purified by ammonium sulfate precipitation and successive ion exchange, hydrophobic interaction, and size exclusion chromatographies. The enzyme exhibited an apparent molecular weight of ∼140 kDa, a temperature optimum of 80°C, and a pH optimum of ∼5.5. The enzyme was extremely stable. No activity was lost over 12 h at 75°C. It is more thermostable than the dextranase from Chaetomium gracile, the most thermostable dextranase previously characterized; however, the Rt364 dextranase has a much lower specific activity, 10 U mg−1, compared to 2,750 U mg−1 for the fungal enzyme at their respective temperature optima. The enzyme from Rt364 hydrolyzes dextran, starch, amylose, and amylopectin with approximately the same catalytic efficiencies but does not hydrolyze pullulan. It has therefore been designated an amylodextranase which is analogous to the recently characterized amylopullulanase.

A novel thermostable dextranase from a Thermoanaerobacter species cultured from the geothermal waters of the Great Artesian Basin of Australia

A Gram-negative sporulating thermophilic anaerobe, designated AB11Ad, was isolated from the heated waters of the Great Artesian Basin of Australia. It grew on a variety of carbohydrates including glucose, starch, and dextran and produced a thermostable and thermoactive extracellular endo-dextranase. The enzyme was produced more actively under pH controlled continuous culture conditions than under batch conditions. Ammonium sulfate precipitated crude dextranase exhibited a temperature optimum of 70 degrees C and a pH optimum between 5 and 6. The half life was approximately 6.5 h at 75 degrees C and 2 h at 80 degrees C at pH 5.0 and in the absence of added dextran. 16S rRNA sequence analysis indicated that isolate AB11Ad was a member of the genus Thermoanaerobacter.

Distinct activities of novel neurotoxins from Australian venomous snakes for nicotinic acetylcholine receptors

Abstract.Envenomation from Australian elapid snakes results in a myriad of neurological effects due to post-synaptic neurotoxins that bind and inhibit nicotinic acetylcholine receptors (nAChRs) of neurons and muscle fibres. However, despite the significant physiological effects of these toxins, they have remained largely undercharacterised at the molecular level. This study describes the identification and comparative analysis of multiple neurotoxin isoforms from ten Australian snakes, including functional characterisation of two of these isoforms, Os SNTX-1 from Oxyuranus scutellatus and the more potent Pt LNTX-1 from Pseudonaja textilis. Electrophysiological recordings from adrenal chromaffin cells demonstrate that both neurotoxins act as competitive antagonists of nAChRs in a concentration-dependent manner. Their effects upon spontaneous and nerveevoked membrane responses at the amphibian neuromuscular junction provide further evidence that both toxins bind muscle nAChRs in an irreversible manner. This study represents one of the most comprehensive descriptions to date of the sequences and activity of individual Australian elapid neurotoxins.