Glutathione-S-transferases from chloroquine-resistant and -sensitive strains of Plasmodium falciparum: what are their differences?

Glutathione-S-transferases (GSTs) from chloroquine-resistant (CQR, K1) and -sensitive (CQS, T9/94) strains of Plasmodiumfalciparum were studied. The enzymes from both strains exhibited the optimal pH for enzyme catalysis, at pH 7.5, and were stable at temperatures below 60 degrees C. They also showed the highest specific activities toward CDNB and moderate activities to ethacrynic acid (40% of the activity to CDNB) but little or no activity for other substrates. Km and Vmax values for CDNB and GSH, calculated by Lineweaver-Burk plot from both CQR- and CQS-GSTs, were not statistically different (p<0.05). However, the GSTs activity from CQR appeared to be significantly higher than that from CQS. Therefore, we proposed that GSTs from both malarial strains are identical in their functional domain but different in level of gene expression. Furthermore, protein sequence alignment between P. falciparum GST and GSTs from other organisms suggested that the malarial enzyme is closely similar to other GSTs in Sigma, Alpha, Mu and Pi subclasses, probably most to the Alpha group. Characterization of the purified malarial GST in detail would reveal more precise classification and better understanding of its role in malarial detoxification.

Identification of a new mutation (Gly420Ser), distal to the active site, that leads to factor XIII deficiency.

The molecular defects of the factor XIII A subunit gene were studied in a patient with factor XIII deficiency. Mutation analysis was performed on amplified DNA from each exon of this gene by single-strand conformation polymorphism (SSCP) and DNA sequencing techniques. A substitution of guanine by adenine at nucleotide 1258 in exon 10 of the coagulation factor XIII A subunit gene has been identified in the patient. The mutation results in the replacement of Gly420 by Ser in the core domain of the enzyme. Restriction enzyme analysis of amplified exon 10 DNA confirmed that the patient was homozygous for this mutation. A family study revealed that the mutation was inherited from both parents, who were first cousins. The potential effects of the mutation were predicted by molecular modeling of the amino acid substitution within the coordinates of the crystal structure. The substitution occurred within the core domain of the enzyme at a residue completely conserved among all known members of the transglutaminase family. The model of the mutant protein suggests that although the substitution of Gly420 by Ser causes only minor readjustment of the residues and does not appear to be particularly deleterious in terms of structure, the mutation is, however, likely to decrease the molecule's ability to undergo the conformational change that is thought to be required for full transglutaminase activity. Our data strongly support the previously published information about the functional significance of the residues surrounding, but not forming, the catalytic pocket in the A subunit of factor XIII.

Identification and characterization of two missense mutations causing factor XIIIA deficiency.

In this study, two amino acid substitutions, Arg260His and Val414Phe, have been identified in the factor XIIIA subunits of factor XIII deficient patients of Syrian and Indian descent, respectively. To confirm the deleterious effects of these substitutions, both variant sequences have been engineered into cDNA clones and the mutant enzymes expressed in yeast. Determination of the transglutaminase activity and immuno detection of the mutant enzymes together with mRNA hybridization revealed that the mutations dramatically reduce both the catalytic activity and the level of enzyme expressed in yeast. The mutations Arg260His and Val414Phe occur within the 'core' domain of the enzyme. Computer modelling of the mutant enzymes reveals that the substitution of the Arg260 by His results in the loss of a conserved electrostatic interaction whereas the effect of the Val414Phe substitution is a consequence of the large increase in side-chain volume. Although both mutations do not effect the active site directly, they are predicted to reduce the stability of the enzyme. The effects of these two amino acid substitutions on enzyme expression and three-dimensional structure strongly confirm that residues which are located outside of the active site can have a significant effect on protein stability and function.

The Val34Leu polymorphism in the A subunit of coagulation factor XIII contributes to the large normal range in activity and demonstrates that the activation peptide plays a role in catalytic activity.

There is a wide normal range of coagulation factor XIII activity that has never been adequately explained. A polymorphism substituting leucine for valine at position 34 in the activation peptide of the A subunit of factor XIII has recently been discovered in nondeficient individuals, and the present studies indicate that the leucine substitution results in a significant increase in transglutaminase activity. The frequency of the Leu34 allele in the Australian Caucasian population is 0.27, which is high enough to suggest that the inheritance of either the Val34 or Leu34 alleles may contribute to the wide normal range of activity. Although there has been structural evidence indicating that the activation peptide does not dissociate from the enzyme after thrombin cleavage, the discovery of elevated activity resulting from the Leu34 substitution is the first direct evidence that the activation peptide plays a continuing role in the function of factor XIII.

A novel Asn344 deletion in the core domain of coagulation factor XIII A subunit: its effects on protein structure and function.

In this study a previously undescribed 3 bp deletion, AAT1030-1032, in the factor XIII A subunit gene, has been detected in a Thai patient. The inframe deletion results in the translation of a factor XIII A subunit that lacks Asn344. This is the first inframe deletion to be identified in the factor XIII A subunit gene because six previously reported deletions have all caused frameshifts. The deletion has been introduced into a factor XIII A subunit cDNA and the deleted polypeptide expressed in yeast. The mRNA encoding the mutant enzyme appears to have normal stability but the translated protein is subject to premature degradation. In addition, the mutated enzyme exhibited very little transglutaminase activity compared with the wild-type enzyme. Structural modeling of the deleted enzyme suggests that the absence of Asn344 would have a potent impact on the catalytic activity by reorienting the residues associated with the catalytic center. Thus, the Asn344 deletion strongly confirms the significance of the residues surrounding the catalytic center of the factor XIII A subunit.

Application of HUMF13A01 (AAAG)n STR polymorphism to the genetic diagnosis of coagulation factor XIII deficiency.

Deficiency of the A subunit of coagulation factor XIII causes a severe bleeding disorder requiring life long replacement therapy. The mutations causing A subunit deficiency appear to be very heterogeneous, and it is impractical to identify each mutation before genetic counselling or prenatal diagnosis can be attempted. In this study we have shown that a highly polymorphic short tandem repeat element, HUMF13A01 (AAAG)n that occurs in the 5' flanking sequence of the factor XIII A subunit gene, can be used to follow the segregation of deficiency causing mutations. We studied 6 families with factor XIII A subunit deficiency from 5 different ethnic groups. All parents were heterozygous for the repetitive element and therefore all the families were informative. The linked polymorphism was used to carry out the first prenatal diagnosis of factor XIII A subunit deficiency. The analysis of this polymorphism by the polymerase chain reaction is rapid, reliable, requires little DNA and is ideal for the genetic analysis of factor XIII A subunit deficiency.

New mutations causing the premature termination of translation in the A subunit gene of coagulation factor XIII.

The amplification of factor XIII A subunit gene exons and heteroduplex analysis has been used to identify two new mutations that cause severe factor XIII deficiency. One mutation in a family of French origin results from a 4 bp deletion and leads to a premature termination of translation. The other mutation occurred in a Turkish family and results from a C-->T transition that inserts a premature translation stop signal at codon 400. Both mutations alter restriction enzyme sites and can be readily detected in amplified exon DNA for genetic counselling or prenatal diagnosis. The new mutations reflect the extensive molecular heterogeneity of factor XIII deficiency.

Molecular examination of GH gene deletion in familial growth hormone deficiency.

The human growth hormone gene (GH gene) from nine members of a family with familial growth hormone deficiency was examined. The patients were diagnosed as having growth hormone deficiency clinically and by response to hormonal treatment. PCR amplification was carried out using total DNA extracted from leukocytes. The flanking regions of the GH gene which are highly homologous were amplified by one pair of primers. PCR products of 1900 bp and 1919 bp were obtained. By using the combination of restriction enzymes BgII, HaeII and SmaI to digest these PCR products, the various sizes of GH gene deletion can be detected. None of the possible deletions was found in these patients and their relatives by either PCR or Southern blot analysis.

Random mating of natural Plasmodium populations demonstrated from individual oocysts.

DNA amplified from individual Plasmodium vivax oocysts, produced by feeding mosquitoes directly on naturally infected humans in Thailand, was used to study cross-mating of 2 polymorphs of the circumsporozoite (CS) gene, VK 210 and VK 247. Alleles were detected in matched blood parasites, sporozoites, and individual oocysts with oligoprobes specific to characteristic repeat units. Oocysts developing from 3 cases in which mixed alleles were present in the blood parasites had genotype frequencies, including hybrids, consistent with the Hardy-Weinberg equilibrium. There was apparently no barrier to hybridization of the 2 alleles nor a bias, as has been found in some laboratory experiments, favoring hybrid formation. These are the first measurements of cross-mating frequencies directly from natural Plasmodium infections and the first observations of genetic hybridization in P. vivax.