Plasmodium chitinases: revisiting a target of transmission-blockade against malaria

Abstract

Malaria is a life-threatening disease caused by one of the five species of Plasmodium, among which Plasmodium falciparum cause the deadliest form of the disease. Plasmodium species are dependent on a vertebrate host and a blood-sucking insect vector to complete their life cycle. Plasmodium chitinases belonging to the GH18 family are secreted inside the mosquito midgut, during the ookinete stage of the parasite. Chitinases mediate the penetration of parasite through the peritrophic membrane, facilitating access to the gut epithelial layer. In this review, we describe Plasmodium chitinases with special emphasis on chitinases from P. falciparum and P. vivax, the representative examples of the short and long forms of this protein. In addition to the chitinase domain, chitinases belonging to the long form contain a pro-domain and chitin-binding domain. Amino acid sequence alignment of long and short form chitinase domains reveals multiple positions containing variant residues. A subset of these positions was found to be conserved or invariant within long or short forms, indicating the role of these positions in attributing form-specific activity. The reported differences in affinities to allosamidin for P. vivax and P. falciparum were predicted to be due to different residues at two amino acid positions, resulting in altered interactions with the inhibitor. Understanding the role of these amino acids in Plasmodium chitinases will help us elucidate the mechanism of catalysis and the mode of inhibition, which will be the key for identification of potent inhibitors or antibodies demonstrating transmission-blocking activity.

Keywords: Anopheles; Plasmodium; allosamidin; chitinase; malaria; transmission-blocking.

FIGURE 1

A ribbon representation of the TIM barrel fold from the predicted model structures of Chitinases from (a) Plasmodium falciparum and (b) Plasmodium vivax prepared by PyMOL. TIM Barrel is an eight stranded α‐β fold with β sheets forming a barrel (black), surrounded by α helices. Plasmodium falciparum chitinase contains a single catalytic domain (depicted in green and black comprising of α helices and β sheets, respectively). In Plasmodium vivax, the pro‐domain (red) and Chitin binding domain (orange) flanks the catalytic domain. (c) depicts the domain architecture of Plasmodium chitinases. Short form (PfCHT1, PgCHT2, PrCHT1, PreCHT2) and Long forms (PvCHT1, PgCHT1, PbCHT1, PreCHT1) of Plasmodium chitinases are annotated at the catalytic domain and chitin binding domains as reported elsewhere. The signal sequence ranges from 1–20 (observed in the long form) to 1–31/32 residues (observed in PgCHT2, a representative of short form). Long form of chitinases harbor pro‐domain, catalytic domain, and chitin binding domain. Pro‐domain although not annotated in most cases, is found to be located at the N‐terminal end of the catalytic domain in PvCHT1 and PbCHT1. Represented chitinases are PgCHT1 and PgCHT2: Plasmodium gallinaceum chitinase, PfCHT1: Plasmodium falciparum chitinase, PvCHT1: Plasmodium vivax chitinase, PbCHT1: Plasmodium berghei chitinase, PrCHT1: Plasmodium reichenowei chitinase, PreCHT1 and PreCHT2: Plasmodium relictum chitinase

FIGURE 2

Sequence alignment of Plasmodium chitinases using T‐Coffee and shaded with Box shade method. The long [L] and short [S] forms are indicated in parenthesis. Substrate binding site and catalytic binding sites are labeled accordingly. The residues predicted to interact with allosamidin are boxed and highlighted using asterisk (*). The catalytic site contains a position (marked as +) in which either Tyr or Trp present in short or long forms, defines the pH optimum. Represented chitinases are PgCHT1 and PgCHT2: Plasmodium gallinaceum chitinase, PfCHT1: Plasmodium falciparum chitinase, PvCHT1: Plasmodium vivax chitinase, PbCHT1: Plasmodium berghei chitinase, PrCHT1: Plasmodium reichenowei chitinase, PreCHT1 and PreCHT2: Plasmodium relictum chitinases. Although, P. knowlesi, P. chabaudi and P. yoelii chitinases have not been included in the sequence alignment, the same trend with sequence conservation is observed at the indicated positions

FIGURE 3

Comparison of the consensus amino acid sequences of the long and short forms of Plasmodium chitinases. The individual consensus sequence for long and short forms were derived by sequence alignment of chitinases from four species each (the sequences of which are compared in Figure 2). At the positions displayed in the figure, differences in amino acids were observed. For example, position 283 contains Gly (nonpolar residue) in the short and Asp (negatively charged residue) in the long form. + indicates the predicted residue position involved in binding to allosamidin. Residue numbering is with respect to PvCHT1

FIGURE 4

Prediction on binding mode of allosamidin with PfCHT1. (a) Two‐dimensional structural representation of allosamidin (C₂₅H₄₂N₄O₁₄). (b) Representation of binding mode of allosamidin to Plasmodium falciparum chitinase showing conserved residues – Glu259 and Thr260 forming polar hydrogen bonding interactions with –OH of tetrahydro‐4H‐cyclopenta[d]oxazole. Unlike His472 in PvCHT1, Ser472 in PfCHT1 orients the amide moiety of allosamidin for optimal interaction. Lys410 (Pf) interacts with the terminal glucose ring in allosamidin whereas His472 and Val410 of Plasmodium vivax (Pv) chitinase fails to make polar interactions. Residue numbering is with respect to PvCHT1