Purification and characterization of guanine phosphoribosyltransferase from Giardia lamblia

Giardia lamblia, a flagellated parasitic protozoan and the causative agent of giardiasis, lacks de novo purine biosynthesis and exists on salvage of adenine and guanine by adenine phosphoribosyltransferase and guanine phosphoribosyltransferase. Guanine phosphoribosyltransferase from G. lamblia crude extracts has been purified to apparent homogeneity by Sephacryl S-200 gel filtration followed by C-8-GMP-agarose and 2',3'-GMP-agarose affinity chromatography, resulting in an overall recovery of 77% and a purification of 83,000-fold. The molecular weight of the native enzyme as estimated by gel filtration and isokinetic sucrose gradients was found to be 58,000-63,000, with a subunit molecular weight of approximately 29,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mono P chromatofocusing chromatography gives rise to a major activity peak eluting from the column at a pH of 6.75 and two minor activity peaks at pH of 5.3 and 5.2. Hypoxanthine and xanthine can be recognized by the enzyme as substrates but at Km values 20 times higher than that observed with guanine. G. lamblia guanine phosphoribosyltransferase is immunologically distinct from human hypoxanthine-guanine phosphoribosyltransferase and Escherichia coli xanthine-guanine phosphoribosyltransferase, and G. lamblia DNA fragments are incapable of hybridizing with mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase DNA or E. coli xanthine phosphoribosyltransferase DNA under relatively relaxed conditions. All evidence presented suggests that G. lamblia guanine phosphoribosyltransferase may be qualified as a potential target for antigiardiasis chemotherapy.     

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.     

Characterization of two DNA populations of Giardia lamblia

Total DNA was isolated from the parasitic protozoan Giardia lamblia and separated into two distinct populations of different densities by centrifugation through CsCl gradients containing Hoechst dye 33258. The two populations obtained were characterized by restriction enzyme analysis and nucleic acid hybridization. The less-dense population contains non-repetitive DNA and may encode mainly structural genes, such as those for alpha- and beta-tubulin. Digestion of the DNA with several restriction endonucleases showed that the denser band was composed of a 5.5 kb unit which contains the G. lamblia ribosomal RNA cistron in tandem repeated organization.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Purification of cysteine proteinases from adult Schistosoma mansoni

Proteolytic activity against hemoglobin and low molecular weight synthetic substrates has been previously found in homogenates and excretion/secretion products of adult Schistosoma mansoni worms. This activity is stimulated in the presence of thiol compounds and is maximally active at acidic pH. To characterize further this proteolytic activity, lyophilized adult worms were extracted, and proteinases were isolated and purified. From extracts prepared in 0.2 M citrate buffer, pH 4.9, two proteinase species were purified to homogeneity by centrifugation, gel filtration, dialysis, and chromatofocusing chromatography. The proteinases, designated SMw32 and SMw28, have apparent molecular weights (SDS-PAGE) of 31,700 +/- 1400 and 27,800 +/- 1700, respectively. Both are thiol-dependent, acidic endopeptidases that cleave hemoglobin and a synthetic substrate, CBZ-arg-arg-AFC. A statistical comparison of amino acid compositions reveals that the proteinases are highly related.     

Purification and Substrate Specificity of Two Cysteine Proteinases of Giardia lamblia

The proteinase activity present in homogenates of trophozoites of Giardia lamblia , active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of M_r= 95,000 and 35,000 ± 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (M_r= 95,000) and proteinase II (M_r= 35,000) were active against the β-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1–6, 8–18, and 20–30 of the insulin β-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.

After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases.     

Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.     

Molecular modeling and substrate specificity of discrete cruzipain-like and cathepsin L-like cysteine proteinases of the human blood fluke Schistosoma mansoni

Adult Schistosoma mansoni blood flukes express two discrete cysteine proteinases, SmCL1 and SmCL2, both of which are related to the cathepsin L-like enzymes of the C1 family of peptidases. Our previous phylogenetic analysis indicated that SmCL1 is more closely related to cruzipain from the parasitic protozoa Trypanosoma cruzi than to human cathepsin L, whereas the converse situation applies with SmCL2. To characterize their catalytic subsites and substrate specificities, we have now developed three-dimensional (3D) homology models of SmCL1 and SmCL2 using the structure of cruzipain and cathepsin L. Eisenberg analysis of the 3D models revealed self-compatibility scores of 90.1 and 96.1 out of a possible score of 97.6 for SmCL1 and SmCL2, respectively, verifying the accuracy and utility of the models. Substrate preferences of recombinant SmCL1 and SmCL2 at positions P3, P2, and P1 conformed to the substrate specificity predicted by the models. In particular, SmCL1 and SmCL2 both exhibited high affinity (k(cat)/K(m)) for substrates with hydrophobic residues at P2 including Z-Leu-Arg-NHMec (773.4 and 548.5 mM(-1) s(-1), respectively), Boc-Val-Leu-Lys-NHMec (116.8 and 306.5 mM(-1) s(-1)), and Z-Phe-Arg-NHMec (38.9 and 113.4 mM(-1) s(-1)). SmCL1 exhibited only a low affinity for the cathepsin B diagnostic substrate Z-Arg-Arg-NHMec while SmCL2 failed to cleave this substrate. The substrate specificities of SmCL1 and SmCL2 were clearly differentiated with H-Leu-Val-Tyr-NHMec and Suc-Leu-Tyr-NHMec since SmCL1 cleaved both efficiently (k(cat)/K(m) values of 51.9 and 41.1 mM(-1) s(-1), respectively), whereas SmCL2 cleaved neither. The 3D models revealed that this difference in specificity was due to restrictions imposed on the S3 subsite of SmCL2 as a result of insertion of two amino acids vicinal to residue 60.     

Detection of Giardia lamblia by immunofluorescence.

High-titer immune sera to cysts of Giardia lamblia, produced in guinea pigs, were labeled with fluorescein isothiocyanate. The resulting conjugates were used to detect G. lamblia in stool specimens by fluorescence microscopy. The sera also reacted with cysts of Chilomastix mesnili, but the two organisms could be differentiated by their size.     

Electron microscopy of Giardia lamblia cysts.

The flagellated protozoan Giardia lamblia is a recognized public health problem. Intestinal infection can result in acute or chronic diarrhea with associated symptoms in humans. As part of a study to evaluate removal of G. lamblia cysts from drinking water by the processes of coagulation and dual-media filtration, we developed a methodology by using 5.0-microns-porosity membrane filters to evaluate the filtration efficiency. We found that recovery rates of G. lamblia cysts by membrane filtration varied depending upon the type and diameter of the membrane filter. Examination of membrane-filtered samples by scanning electron microscopy revealed flexible and flattened G. lamblia cysts on the filter surface. This feature may be responsible for the low recovery rates with certain filters and, moreover, may have implications in water treatment technology. Formation of the cyst wall is discussed. Electron micrographs of cysts apparently undergoing binary fission and cysts exhibiting a possible bacterial association are shown.     

Comparison of Giardia lamblia and Giardia muris cyst inactivation by ozone.

Inactivation of Giardia lamblia and Giardia muris cysts was compared by using an ozone demand-free 0.05 M phosphate buffer in bench-scale batch reactors at 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times of 2 and 5 min. The viability of the control and treated cysts was evaluated by using the C3H/HeN mouse and Mongolian gerbil models for G. muris and G. lamblia, respectively. The resistance of G. lamblia to ozone was not significantly different from that of G. muris under the study conditions, contrary to previously reported data that suggested G. lamblia was significantly more sensitive to ozone than G. muris was. The simple Ct value for 2 log unit inactivation of G. lamblia was 2.4 times higher than the Ct value recommended by the Surface Water Treatment Rule.     

Giardia lamblia: isolation and axenic cultivation.

Giardia lamblia trophozoites have been axenically cultured for more than a year. Initially, organisms were established in a complex liquid medium in the presence of the host's intestinal fungi; subcultures were made of these protozoa-fungus mixtures. G. lamblia trophozoites, free of yeast, were obtained by inoculating a protozoafungus culture in one arm of a U-tube, then later removing, from the other arm of the tube, Giardia trophozoites that had migrated across the base. Medium was changed at 2- or 3-day intervals; numerous subcultures were made. Tests for the possible presence of other organisms in these axenic cultures were negative. Trophozoite cultures remained viable, after freezing in the presence of glycerol, for 14 months. This is the first reported axenic culture of this common human intestinal parasite and pathogen; its study in pure culture is now possible.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain.     

Isoenzyme comparison of axenic Giardia lamblia strains.

We obtained isoenzyme patterns by polyacrylamide gradient gel electrophoresis (PGGE) of water-soluble protein fractions prepared from trophozoites of 11 axenic G. lamblia strains. The strains were isolated from animals and humans (both symptomatic and asymptomatic) from various geographic locations. Isoenzymes were also separated by isoelectric focusing. Of 12 enzymes attempted, eight exhibited well-defined and reproducible isoenzyme patterns by PGGE, based on which the strains were grouped into four zymodemes. Although the 11 strains were grouped into four zymodemes based on PGGE, no correlation between zymodeme and the known characteristics of the strains existed. Thus, a high degree of characteristic sharing appears to occur among genetically different G. lamblia strains.