Limits...
Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

Lakhal-Naouar I, Jardim A, Strasser R, Luo S, Kozakai Y, Nakhasi HL, Duncan RC - PLoS Negl Trop Dis (2012)

Bottom Line: Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid).The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA.Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, MD, USA.

ABSTRACT

Background: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/principal findings: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/significance: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Show MeSH

Related in: MedlinePlus

ASS activity assay.(A) Sample of recombinant ASS proteins affinity purified from 108 cells of vector control (lane 1), V5His-LdASSWT (lane 2) and V5His-LdASSG128S (lane 3) over expressing parasites using Ni-NTA resin beads, analyzed on an SDS gel and visualized with Gel code blue staining. (B) ASS activity measured by release of Pi in the presence of aspartate and citrulline substrates, obtained with Leishmania recombinant ASS proteins purified from the over expressing cell lines, in the absence (black bars), or presence of 1 mM (grey bars) or 5 mM of inhibitor (interrupted line). (C) Activity determined for Mouse Liver Extract (MLE) as a positive control for the assay. (D) HPLC analysis: Fluorescence intensity of OPA labeled components of ASS enzyme reaction. Overlay of elution profiles showing resolution of ASA (blue line: reaction mix, green line: 1 nmoles of ASA mixed with the reaction mix, red line: pulled down protein from V5His-LdASSWT cell line and incubated in presence of the reaction mix, turquoise line: pulled down protein from V5His-LdASSG128S cell line incubated in presence of the reaction mix, orange line: pull down from vector control incubated in presence of the reaction mix. The star indicates the ASA peak. The graphs shown are representative of 3 replicates of the assay.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475689&req=5

pntd-0001849-g002: ASS activity assay.(A) Sample of recombinant ASS proteins affinity purified from 108 cells of vector control (lane 1), V5His-LdASSWT (lane 2) and V5His-LdASSG128S (lane 3) over expressing parasites using Ni-NTA resin beads, analyzed on an SDS gel and visualized with Gel code blue staining. (B) ASS activity measured by release of Pi in the presence of aspartate and citrulline substrates, obtained with Leishmania recombinant ASS proteins purified from the over expressing cell lines, in the absence (black bars), or presence of 1 mM (grey bars) or 5 mM of inhibitor (interrupted line). (C) Activity determined for Mouse Liver Extract (MLE) as a positive control for the assay. (D) HPLC analysis: Fluorescence intensity of OPA labeled components of ASS enzyme reaction. Overlay of elution profiles showing resolution of ASA (blue line: reaction mix, green line: 1 nmoles of ASA mixed with the reaction mix, red line: pulled down protein from V5His-LdASSWT cell line and incubated in presence of the reaction mix, turquoise line: pulled down protein from V5His-LdASSG128S cell line incubated in presence of the reaction mix, orange line: pull down from vector control incubated in presence of the reaction mix. The star indicates the ASA peak. The graphs shown are representative of 3 replicates of the assay.

Mentions: Preliminary studies showed that the endogenous enzymatic activity of ASS in L. donovani is very low; hence we were not able to detect it (data not shown). In order to demonstrate the activity of LdASS in the parasite, we used the two cell lines over expressing LdASS with an N-terminal tag epitope for the in vitro assay. Leishmania parasites (1×108) expressing V5His-LdASSWT or V5His-LdASSG128S (point mutant) were used as the starting material to affinity purify these proteins from the respective transfectants by metal affinity chromatography using Ni2+-NTA agarose. A protein stained SDS gel showed that a band of the same intensity corresponding to the exogenous ASS was pulled down from both transfectant cell lines (Fig. 2A, lanes 2 and 3). As expected, no visible protein band was purified from the cells transfected with the empty vector control (Fig. 2A, lane 1).


Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

Lakhal-Naouar I, Jardim A, Strasser R, Luo S, Kozakai Y, Nakhasi HL, Duncan RC - PLoS Negl Trop Dis (2012)

ASS activity assay.(A) Sample of recombinant ASS proteins affinity purified from 108 cells of vector control (lane 1), V5His-LdASSWT (lane 2) and V5His-LdASSG128S (lane 3) over expressing parasites using Ni-NTA resin beads, analyzed on an SDS gel and visualized with Gel code blue staining. (B) ASS activity measured by release of Pi in the presence of aspartate and citrulline substrates, obtained with Leishmania recombinant ASS proteins purified from the over expressing cell lines, in the absence (black bars), or presence of 1 mM (grey bars) or 5 mM of inhibitor (interrupted line). (C) Activity determined for Mouse Liver Extract (MLE) as a positive control for the assay. (D) HPLC analysis: Fluorescence intensity of OPA labeled components of ASS enzyme reaction. Overlay of elution profiles showing resolution of ASA (blue line: reaction mix, green line: 1 nmoles of ASA mixed with the reaction mix, red line: pulled down protein from V5His-LdASSWT cell line and incubated in presence of the reaction mix, turquoise line: pulled down protein from V5His-LdASSG128S cell line incubated in presence of the reaction mix, orange line: pull down from vector control incubated in presence of the reaction mix. The star indicates the ASA peak. The graphs shown are representative of 3 replicates of the assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475689&req=5

pntd-0001849-g002: ASS activity assay.(A) Sample of recombinant ASS proteins affinity purified from 108 cells of vector control (lane 1), V5His-LdASSWT (lane 2) and V5His-LdASSG128S (lane 3) over expressing parasites using Ni-NTA resin beads, analyzed on an SDS gel and visualized with Gel code blue staining. (B) ASS activity measured by release of Pi in the presence of aspartate and citrulline substrates, obtained with Leishmania recombinant ASS proteins purified from the over expressing cell lines, in the absence (black bars), or presence of 1 mM (grey bars) or 5 mM of inhibitor (interrupted line). (C) Activity determined for Mouse Liver Extract (MLE) as a positive control for the assay. (D) HPLC analysis: Fluorescence intensity of OPA labeled components of ASS enzyme reaction. Overlay of elution profiles showing resolution of ASA (blue line: reaction mix, green line: 1 nmoles of ASA mixed with the reaction mix, red line: pulled down protein from V5His-LdASSWT cell line and incubated in presence of the reaction mix, turquoise line: pulled down protein from V5His-LdASSG128S cell line incubated in presence of the reaction mix, orange line: pull down from vector control incubated in presence of the reaction mix. The star indicates the ASA peak. The graphs shown are representative of 3 replicates of the assay.
Mentions: Preliminary studies showed that the endogenous enzymatic activity of ASS in L. donovani is very low; hence we were not able to detect it (data not shown). In order to demonstrate the activity of LdASS in the parasite, we used the two cell lines over expressing LdASS with an N-terminal tag epitope for the in vitro assay. Leishmania parasites (1×108) expressing V5His-LdASSWT or V5His-LdASSG128S (point mutant) were used as the starting material to affinity purify these proteins from the respective transfectants by metal affinity chromatography using Ni2+-NTA agarose. A protein stained SDS gel showed that a band of the same intensity corresponding to the exogenous ASS was pulled down from both transfectant cell lines (Fig. 2A, lanes 2 and 3). As expected, no visible protein band was purified from the cells transfected with the empty vector control (Fig. 2A, lane 1).

Bottom Line: Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid).The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA.Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, MD, USA.

ABSTRACT

Background: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/principal findings: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/significance: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Show MeSH
Related in: MedlinePlus