Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes.

In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO 4 ) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A ؊ and lpg5B ؊ mutants produced PGs, an lpg5A ؊ /5B ؊ double mutant was completely deficient. PG synthesis was restored in the lpg5A ؊ /5B ؊ mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A ؊ mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B ؊ mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis in Leishmania major

Capul

Barron

Dobson

et al. 2007

“…All of the samples were assayed in triplicate, and these assays were repeated on multiple samples. One unit of enzyme activity (19) reflects the hydrolysis of 1 nmol of pnitrophenyl phosphate (U.S. Biochemical Corp., Cleveland, OH) to pnitrophenol min Ϫ1 mg Ϫ1 of detergent-solubilized parasite surface membrane protein at 42°C. The percentage of activity immunoprecipitated was determined using the following formula: (activity bound/[activity bound ϩ activity unbound]) ϫ 100.…”

Section: Preparation Of Parasite Surface Membrane-enriched Fractions-mentioning

confidence: 99%

Molecular Dissection of the Functional Domains of a Unique, Tartrate-resistant, Surface Membrane Acid Phosphatase in the Primitive Human Pathogen Leishmania donovani

Shakarian¹,

Joshi²,

Ghedin³

et al. 2002

The primitive trypanosomatid pathogen of humans, Leishmania donovani, constitutively expresses a unique externally oriented, tartrate-resistant, acid phosphatase on its surface membrane. This is of interest because these organisms are obligate intracellular protozoan parasites that reside and multiply within the hydrolytic milieu of mammalian macrophage phago-lysosomes. Here we report the identification of the gene encoding this novel L. donovani enzyme. In addition, we characterized its structure, demonstrated its constitutive expression in both parasite developmental forms, and determined the cell surface membrane localization of its translated protein product. Further, we used a variety of green fluorescent protein chimeric constructs as reporters in a homologous leishmanial expression system to dissect the functional domains of this unique, tartrate-resistant, surface membrane enzyme.

“…Culture supernatants were harvested and further centrifuged at 10,000 ϫ g for 15 min at 4°C to eliminate remaining cellular debris. Cleared culture supernatants were assayed for acid phosphatase activity using paranitrophenyl phosphate (Sigma) as a substrate as described previously (18). Acid phosphatase enzymatic activity is expressed as a nanomole of the substrate hydrolyzed per minute per 10 7 cells (nmol/min/10 7 cells).…”

Section: Discussionmentioning

confidence: 99%

An Atypical Protein Disulfide Isomerase from the Protozoan Parasite Leishmania Containing a Single Thioredoxin-like Domain

Alejandro¹,

Noiva

Lee³

et al. 2003

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.