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J. Biol. Chem., Vol. 276, Issue 46, 43253-43261, November 16, 2001

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Expression of a Mutant Form of Leishmania donovani Centrin Reduces the Growth of the Parasite^*

Angamuthu Selvapandiyan, Robert Duncan, Alain Debrabant, Sylvie Bertholet^§, Gannavaram Sreenivas^¶, Narender S. Negi, Poonam Salotra^¶, and Hira L. Nakhasi^**

From the  Laboratory of Bacterial, Parasitic, and Unconventional Agents, Division of Emerging and Transfusion Transmitted Disease, and the ^§ Laboratory of Parasitic Biology and Biochemistry, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and the ^¶ Institute of Pathology (Indian Council of Medical Research) and  Safdarjung Hospital, New Delhi, 110 029 India

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca²⁺ binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca²⁺ binding site suggest that Ca²⁺ binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G₂/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF406767 and to GenPept Data Bank with accession number(s) AAL01153.

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