Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 95, Num. 4, 2000, pp. 591-594

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 95(4) Jul./Aug. 2000, pp: 591-594

SHORT COMMUNICATION

Acidic Ribosomal Proteins and Histone H3 from Leishmania Present a High Rate of Divergence

Ysabel Montoya/⁺, Carlos Padilla, Maxy De Los Santos, Teresa Barreto,
Douglas Barker*, Carlos Carrillo**

Instituto Nacional de Salud, Capac Yupanqui 1400, Lima 11, Peru *University of Cambridge, UK **Universidad Peruana Cayetano Heredia, Honorio Delgado, Lima 31, Peru

⁺Corresponding author. Fax: +511-471.2529. E-mail: ymontoya@ins.sld.pe

Received 13 April 2000
Accepted 15 May 2000

Code Number: oc00094

Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms.

These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V.) peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V.) braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

Key words: Leishmania - acidic ribosomal proteins - histone H3

Acidic ribosomal proteins known as P (phospho) proteins are present in the ribosomes belonging to the eubacteria, archaebacteria and eukaryotes. These conserved proteins are placed in the large ribosomal sub-unit forming a highly flexible lateral projection referred as the ribosomal stalk (Wittman 1983, Lake 1985). The stalk is involved in the interaction between the translation factors and the ribosome during protein synthesis (Shimmin et al. 1989).

Acidic ribosomal P1 protein in trypano-somatids had not been reported in the Leishmania genus previous to this work. However, the P0 and P2 ribosomal proteins had been characterised in L. infantum (Soto et al. 1995a, b), L. chagasi (Skeiky et al. 1994) and L. donovani (Kunz et al. 1993). Moreover, Trypanosoma cruzi acidic ribosomal protein belong to a gene family found in multiple copies (TcP0, TcP1, TcP2a and TcP2b). The P proteins are important antigens that generate humoral response in leishmaniasis, Chagas disease and systemic lupus erythematosus (Elkon et al. 1986).

Despite of presenting histones, the trypa-nosomatids do not condense their chromatin during mitosis (Solari 1980). Likewise, trypano-somatids histone H3 presents an extremely divergent N-terminal domain (Galanti et al. 1998). Another important peculiarity is that trypano-somatids histone H3 lacks of the amino acids sequence KSTGGKA at the N-terminal end, which is present in the consensus sequence of higher eukaryotes (Wells 1986).

Three clones from L. (V.) peruviana (T26-U3) and L. (V.) braziliensis cDNA libraries (T166-U19 and T166-M49) were previously selected by their sero-reactivity with leishmaniasis patients (Montoya 1993). The DNA insert corresponding to each of them was amplified and sub-cloned in pUC18/EcoRI/BAP (Pharmacia) plasmid vector.

After sequence analysis, the T26-U3 clone was identified as a L. (V.) peruviana acidic ribosomal protein P1 being referred as LpP1. The clone T166-U19 was recognised as a L. (V.) braziliensis acidic ribosomal protein P2b and referred as LbP2b. Finally, the T166-M49 clone was identified as a L. (V.) braziliensis histone H3 being referred as LbH3.

The deduced amino acid sequence of the LpP1 had a divergence of 57.4% in comparison with T. cruzi P1 protein (TcP1). LpP2b showed 17.9% of divergence with the L. donovani ribosomal P2 protein (LdP2) and 41.7% of divergence with their homologous in T. cruzi (TcP2). Finally, LbH3 presented 17.7% and 31.8% of divergence in comparison with the sequences of L. infantum (LiH3) and T. cruzi (TcH3) respectively (Table).

Multiple alignment of sequences was performed using CLUSTALW 1.5 program and also for establishing the phylogenetic relationships among these proteins was used PHYLIP 3.5c program. For this purpose, eight eukaryotic P2 proteins (Fig. 1) and six histones H3 (Fig. 2) were included. The bootstrap values (bv) were obtained from a consensus tree based on 100 randomly generated trees.

The genetic distances were obtained by Kimura method showing a higher value between LpP1 and TcP1 (0.83902), in contrast with the value observed between LbP2b and TcP2 (0.39811), and also the value obtained between LbH3 and TcH3 (0.38273) (Table). Likewise, LbP2b was very related to L. donovani P2 protein whose phylogenetic distance was 0.18489 and their divergence rate was 17.9%. Therefore, our results show that these structural proteins present a high rate of divergence regarding to their homologous protein with T. cruzi.

Our results based on the analysis sequence of the L.(V.) braziliensis histone H3 and L. (V.) peruviana P2 proteins indicate that Leishmania is monophyletic. Trypanosomatids P2 clade was separated from the higher eukaryotes clade and sustained with a high bootstrap value (100%) (Fig. 1). In a similar way, trypanosomatids histone H3 clade was separated from the higher eukaryotes clade with a high bootstrap value (99%) (Fig. 2).

Histones H3 are structural proteins considered as one of the most conserved proteins. For example, the divergence rate reported previously between human and Saccharomyces histone H3 was just 9.6% (Table). However, in Leishmania a high rate of divergence at the N-terminal end and the separation of the trypanosomatids clade out of the higher eukaryotes clade is reported here (Fig. 2). This changes in the N-terminal end could contribute in the lost of chromatin condensation.

The divergence rate observed between P1 proteins (LpP1 and TcP1 57.4%) showed a value fluctuating in the range of the higher eukaryotes (44% to 64.1%) (Table). The low number of sequences reported to this protein did not allow establish the phylogenetic relationship among these proteins.

Further studies should be performed to relate the high divergence rate detected in these parasites with their functional implications during the translation.

ACKNOWLEDGEMENT

To Dr Jr Stevens for helping with the manuscript.

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Y Montoya received partial support from a re-entry grant provided by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. This work also received partial financial support from the STD-3 Programme of the European Community, contract TS3*CT92-0123.

Copyright 2000 Fundacao Oswaldo Cruz Fiocruz

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