Title:Biophysical Evidence for the Amyloid Formation of a Recombinant Rab2
Isoform of Leishmania donovani
Volume: 31
Issue: 4
Author(s): Roshanara, Shivani A. Muthu, Gulafsha, Rati Tandon, Angamuthu Selvapandiyan and Basir Ahmad*
Affiliation:
- Protein Assembly Laboratory, Department of Medical Elementology and Toxicology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi 110062, India
Keywords:
Recombinant Rab2, protein unfolding, protein aggregation, protofibrils, circular dichroism, intrinsic fluorescence.
Abstract:
Background: The most fatal form of Visceral leishmaniasis or kala-azar is caused by
the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway
of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab
proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion
abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are
proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of
protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will
determine their physical impact on the disease pathogenesis and individual health.
Objective: This work investigates the acidic pH-induced unfolding and aggregation of a
recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes.
Methods: The acidic unfolding of rLdRab2 is characterised by intrinsic fluorescence and ANS
assay, while aggregation is determined by Thioflavin-T and 90⁰ light scattering assay. Circular
dichroism determined the secondary structure of monomers and aggregates. The aggregate
morphology was imaged by transmission electron microscopy.
Results: rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced
unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded
intermediate (PFI) state characterised by ~ 30% structural loss and exposed hydrophobic core was
found to accumulate. The PFI state slowly converted into well-developed protofibrils at high
protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was
also observed to be aggregation-prone at high protein concentrations. However, it formed
amorphous aggregation instead of fibrils.
Conclusion: To our knowledge, this is the first study to report in vitro amyloid-like behaviour of
Rab proteins in L donovani. This study provides a novel opportunity to understand the complete
biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.
