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J. Biol. Chem., Vol. 280, Issue 7, 5884-5891, February 18, 2005

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Hemoglobin Receptor in Leishmania Is a Hexokinase Located in the Flagellar Pocket^*

Ganga Krishnamurthy, Rajagopal Vikram, Sudha B. Singh, Nitin Patel, Shruti Agarwal, Gauranga Mukhopadhyay¶, Sandip K. Basu, and Amitabha Mukhopadhyay||

From the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India and ^¶Center for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-C) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of ¹²⁵I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-C antibody and Ld-HbR-C, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis.

Received for publication, October 19, 2004 , and in revised form, November 30, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY659996.

^* This work was supported by a grant from the Department of Biotechnology, Government of India to the National Institute of Immunology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^|| To whom correspondence should be addressed. Tel.: 91-11-26703536 or 91-11-26703596; Fax: 91-11-26717104; E-mail: amitabha{at}nii.res.in.

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