Kwang-Poo Chang

Acid protease activity of a major surface membrane glycoprotein (gp63) from Leishmania mexicana promastigotes

A unique protease with activity optimal at pH 4.0 and trailing toward the alkaline pH spectrum was detected with intact glutaraldehyde-fixed promastigotes of Leishmania mexicana amazonensis, indicating surface localisation of the enzyme. That this surface protease may be a virulence factor is suggested by its apparent roles in multiple steps during leishmanial infections of macrophages. Indeed, its specific activity was 2-2.5 fold higher on virulent cells than on avirulent cells. Several lines of evidence indicate that this acid protease activity is expressed by the major surface glycoprotein (gp63) of L. m. amazonensis. Monoclonal antibody affinity purified gp63 degraded serum albumin, hemoglobin, complement C3, immunoglobulin G and purified rat liver lysosomal proteins in their native forms. The specific activity is about 20-fold higher at pH 4.0 than at pH 7.5 and is about four-fold higher at the body temperature of the mammalian host (37 degrees C) than at that of the insect host (27 degrees C). The protease activity is sodium dodecyl sulphate-sensitive. Among various protease inhibitors tested, only heavy metal ions (1 mM), 1,10-orthophenanthroline (1 mM) and bestatin (100 ng ml-1) significantly inhibited gp63 acid protease activity by up to 80%. N-linked oligosaccharides of gp63 appear to be important for the stability of this molecule, possibly by preventing its autodegradation. Purified gp63 effected limited proteolysis of human complement C3 molecules at the physiological serum pH of 7.5 in a manner, which supports the idea of its participation in complement-receptor mediated endocytosis of promastigotes by macrophages.

Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis.

The inability to synthesize heme, a well known metabolic defect of trypanosomatid protozoa, accounts for their growth requirement for heme compounds in vitro. We now extend this finding to a pathogen Leishmania mexicana amazonensis, especially the intracellular replicative stage of amastigotes in the macrophage. We measured the level of heme and its biosynthetic enzymes, aminolevulinate dehydratase and porphobilinogen deaminase in the parasites and in infected and non-infected macrophages of J774G8 line. Succinylacetone was used to inhibit heme biosynthesis. Leishmanias transform and grow only in medium containing either heme (usually supplied as hemin) or protoporphyrin IX (the latter is leishmanicidal at high concentrations). We detected 1.2, 8.5 and 25 pmol mg-1 protein of heme in amastigotes, promastigotes and macrophages, respectively. The activities of porphobilinogen deaminase and aminolevulinate dehydratase in macrophages were 70 and 2400 pmol h-1 mg-1 protein, respectively. Leishmania-infected macrophages gave the same results and leishmanias had negligible activities of these enzymes. Succinylacetone at 10(-9)-10(-3) M had no effect on leishmanias, but dose-dependently inhibited the activity of aminolevulinate dehydratase to a negligible level and lowered that of porphobilinogen deaminase in macrophages, resulting in a maximum of 66% reduction in intracellular heme. Amastigotes grew equally well in succinylacetone-treated and control untreated macrophages. The results suggest that L. m. amazonensis, incapable of heme biosynthesis, acquires heme exogenously from the culture medium, i.e., fetal bovine serum, independent of the heme synthesized by the macrophages.

Expression and size heterogeneity of a 63 kilodalton membrane glycoprotein during growth and transformation of Leishmania mexicana amazonensis

Our previous work by immunoprecipitation with a specific monoclonal antibody showed multiple, closely apposed electrophoretic bands of a major surface antigen specific to the promastigote stage of Leishmania mexicana amazonensis. Here, we analyzed the antigen during growth and transformation of this parasite with particular emphasis on the origin of the multiple bands. Immunobinding assays revealed the presence of the antigen throughout all phases of growth of cloned and uncloned promastigotes in various media for different number of generations. More antigen is expressed by promastigotes grown in Medium 199 plus fetal bovine serum than those in serum-supplemented Schneiders medium or a defined medium; however, this is clone-dependent. Purified monoclonal antibody coupled to Affi-Gel 10 gave a high capacity of antigen binding, resolving four electrophoretic bands of 60-66 kDa. A 63 kDa membrane protein, representing one of the four bands, became predominant after [35S]methionine label and chase. Pretreatment of promastigotes with 10 micrograms ml-1 tunicamycin reduces the antigen to a single band of 54 kDa. Treatment of the antigen bound to the affinity gel with endoglycosidase-H produces similar, but less complete effect. These results indicate glycosylation of this antigen with asparagine-linked oligosaccharides, which appears to account at least in part for its expression as multiple, closely apposed bands during biosynthesis. Binding of fluorescein isothiocyanate-labeled 6H12 monoclonal IgG or Fab to the promastigotes showed an even distribution of the antigen over the cell surface and its capping upon the addition of rabbit anti-mouse IgG. Additional hybridomas prepared against amastigotes yielded monoclonal antibodies which recognized surface antigens common to both stages of the parasite.

Roles of free GPIs in amastigotes of Leishmania

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.

N-Glycosylation as a biochemical basis for virulence in Leishmania mexicana amazonensis

Promastigotes of Leishmania mexicana amazonensis grown in vitro under different conditions showed variable degrees of virulence, as determined quantitatively by the size of the lesions and the number of intracellular parasites produced in mice and in cultured macrophages, respectively. Promastigotes newly transformed from amastigotes gave the highest degree of virulence, which decreased progressively with periods of their continuous in vitro cultivation. This loss of virulence was prevented by making virulent wildtype promastigotes resistant to tunicamycin, an inhibitor of N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of protein glycosylation. In the wildtype cells, the progressive loss of virulence during a period of two years was marked by a gradual decrease in the activity of the glycosyltransferase, incorporation of 2-D-[3H]mannose and the expression of a surface glycoprotein (gp63). Virulent and avirulent wildtype cells differed in these activities by 3-4 fold. In contrast, during equivalent periods of in vitro cultivation, tunicamycin-resistant cells were found to consistently maintain the biochemical phenotypes of the virulent wildtype, except for a disproportional elevation of the glycosyltransferase activity. Thus, only a portion of the over-produced enzyme is relevant to leishmanial virulence in the drug-resistant variants. The bulk of its activity in these cells serves to overcome the inhibitory effect of tunicamycin responsible for their resistance to this drug, as shown previously. A role of N-glycosylation in leishmanial virulence is now indicated by studying cells of this phenotype selected by two independent methods. It is inferred that leishmanial virulence may be regulated by the level of the glycosyltransferases for N-glycosylation of proteins, e.g. gp63 in relation to their expression as virulent determinants.

Identification by extrachromosomal amplification and overexpression of ζ-crystallin/NADPH-oxidoreductase homologue constitutively expressed in Leishmania spp.

A gene which overexpresses a 36-kDa protein (p36) in tunicamycin-resistant Leishmania was mapped by transfection and overexpression to the upstream region of the drug maker in the extrachromosomal amplicon. Complete sequencing of this region revealed a single open reading frame of about 1 kb. Authenticity of the cloned gene is verified by immunologic specificity of its recombinant products and sequence identity with a p36 peptide. The gene shares an overall sequence similarity of about 50% with members of the eukaryote alcohol dehydrogenase family at the amino acid level, including essentially all 13 evolutionarily conserved residues and a nucleotide-binding domain. The binding ligands for both structurally and catalytically important zinc atoms are absent, similar to the zeta-crystallin/NADPH:quinone oxidoreductase gene. Consistent with hydrophilicity of its primary sequence and the presence of a nucleotide binding site, p36 is a soluble molecule non-sedimentable at 105,000 x g and binds Blue Sepharose, elutable only with NADPH. The p36 gene is expressed constitutively in both stages of the wild-type and is conserved among all Leishmania species examined, suggestive of its functional significance different from evolutionarily related homologues.

Tunicamycin-resistant variants from five species of Leishmania contain amplified DNA in extrachromosomal circles of different sizes with a transcriptionally active homologous region

Twelve independent variants were selected from five species of Leishmania for resistance to tunicamycin by exposure of cultured promastigotes to increasing concentrations of this antibiotic, an inhibitor of the microsomal N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of N-glycosylation. All variants obtained from all species, as found previously with Leishmania amazonensis, contain amplified chromosomal DNA exclusively as extrachromosomal circles. These circular amplicons hybridize with amplified DNAs cloned previously from tunicamycin-resistant Leishmania amazonensis, but not with those from Leishmania resistant to other drugs. The amplicons from tunicamycin-resistant cells vary with different species in size from 30 to 70 kb, but all share a homologous region of 20 kb. Multiple independent transcripts are overexpressed from this region. Elevation of the microsomal glycosyltransferase activity is demonstrated in these variants from representative species. The results thus provide further evidence that this enzyme is overexpressed due to amplification of the gene in these cells. The consistent observation of this event in all cases studied also suggests that this is the predominant, if not the only mechanism of tunicamycin resistance in Leishmania.