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Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

Vudriko P, Masatani T, Cao S, Terkawi MA, Kamyingkird K, Mousa AA, Adjou Moumouni PF, Nishikawa Y, Xuan X - Drug Target Insights (2014)

Bottom Line: The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite.The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively.The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan. ; Department of Veterinary Pharmacy, Clinics and Comparative Medicine, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda.

ABSTRACT
Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.

No MeSH data available.


Related in: MedlinePlus

(A) Cytoplasmic localization of BmLDH in merozoite; (B) Nucleus localization of BmLDH; (1) Immunoflourescent staining of B. microti with polyclonal BmLDH and anti-mouse alexa-488; (2) B. microti merozoite nuclei stained with PI; and (3) merged image; scale bar is 5 and 2.5 μm for A and B, respectively.
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f4-dti-8-2014-031: (A) Cytoplasmic localization of BmLDH in merozoite; (B) Nucleus localization of BmLDH; (1) Immunoflourescent staining of B. microti with polyclonal BmLDH and anti-mouse alexa-488; (2) B. microti merozoite nuclei stained with PI; and (3) merged image; scale bar is 5 and 2.5 μm for A and B, respectively.

Mentions: The polyclonal antibody raised against recombinant BmLDH was able to detect the native BmLDH in infected RBCs by indirect IFAT. This was shown by specific green fluorescence observed in the cytoplasm and nucleus of B. microti merozoites (Fig. 4). The location of the green fluorescence in both the merozoite cytoplasm and nucleus is consistent with the finding from the cNLS mapper, which showed that BmLDH has both cytoplasmic and nucleus localization signal sequence from amino acid number 241–269 (EIIKLKGYTSWAIGLSVGDLSCSLIKNLR).


Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

Vudriko P, Masatani T, Cao S, Terkawi MA, Kamyingkird K, Mousa AA, Adjou Moumouni PF, Nishikawa Y, Xuan X - Drug Target Insights (2014)

(A) Cytoplasmic localization of BmLDH in merozoite; (B) Nucleus localization of BmLDH; (1) Immunoflourescent staining of B. microti with polyclonal BmLDH and anti-mouse alexa-488; (2) B. microti merozoite nuclei stained with PI; and (3) merged image; scale bar is 5 and 2.5 μm for A and B, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125376&req=5

f4-dti-8-2014-031: (A) Cytoplasmic localization of BmLDH in merozoite; (B) Nucleus localization of BmLDH; (1) Immunoflourescent staining of B. microti with polyclonal BmLDH and anti-mouse alexa-488; (2) B. microti merozoite nuclei stained with PI; and (3) merged image; scale bar is 5 and 2.5 μm for A and B, respectively.
Mentions: The polyclonal antibody raised against recombinant BmLDH was able to detect the native BmLDH in infected RBCs by indirect IFAT. This was shown by specific green fluorescence observed in the cytoplasm and nucleus of B. microti merozoites (Fig. 4). The location of the green fluorescence in both the merozoite cytoplasm and nucleus is consistent with the finding from the cNLS mapper, which showed that BmLDH has both cytoplasmic and nucleus localization signal sequence from amino acid number 241–269 (EIIKLKGYTSWAIGLSVGDLSCSLIKNLR).

Bottom Line: The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite.The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively.The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan. ; Department of Veterinary Pharmacy, Clinics and Comparative Medicine, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda.

ABSTRACT
Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.

No MeSH data available.


Related in: MedlinePlus