Genetic Variation in the Plasmodium falciparum Circumsporozoite Protein in India and Its Relevance to RTS,S Malaria Vaccine

RTS,S is the most advanced malaria vaccine candidate, currently under phase-III clinical trials in Africa. This Plasmodium falciparum vaccine contains part of the central repeat region and the complete C-terminal T cell epitope region (Th2R and Th3R) of the circumsporozoite protein (CSP). Since naturally occurring polymorphisms at the vaccine candidate loci are critical determinants of the protective efficacy of the vaccines, it is imperative to investigate these polymorphisms in field isolates. In this study we have investigated the genetic diversity at the central repeat, C-terminal T cell epitope (Th2R and Th3R) and N-terminal T cell epitope regions of the CSP, in P. falciparum isolates from Madhya Pradesh state of India. These isolates were collected through a 5-year prospective study aimed to develop a well-characterized field-site for the future evaluation of malaria vaccine in India. Our results revealed that the central repeat (63 haplotypes, n = 161) and C-terminal Th2R/Th3R epitope (24 haplotypes, n = 179) regions were highly polymorphic, whereas N-terminal non-repeat region was less polymorphic (5 haplotypes, n = 161) in this population. We did not find any evidence of the role of positive natural selection in maintaining the genetic diversity at the Th2R/Th3R regions of CSP. Comparative analysis of the Th2R/Th3R sequences from this study to the global isolates (n = 1160) retrieved from the GenBank database revealed two important points. First, the majority of the sequences (∼61%, n = 179) from this study were identical to the Dd2/Indochina type, which is also the predominant Th2R/Th3R haplotype in Asia (∼59%, n = 974). Second, the Th2R/Th3R sequences in Asia, South America and Africa are geographically distinct with little allele sharing between continents. In conclusion, this study provides an insight on the existing polymorphisms in the CSP in a parasite population from India that could potentially influence the efficacy of RTS,S vaccine in this region.     

In vitro evolved non-aggregating and thermostable lipase: structural and thermodynamic investigation

Rational and in vitro evolutionary approaches to improve either protein stability or aggregation resistance were successful, but empirical rules for simultaneous improvement of both stability and aggregation resistance under denaturing conditions are still to be ascertained. We have created a robust variant of a lipase from Bacillus subtilis named “6B” using multiple rounds of in vitro evolution. T_m and optimum activity temperature of 6B is 78 °C and 65 °C, respectively, which is ∼ 22 °C and 30 °C higher than that of wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis of remarkable thermostability and non-aggregating behavior of 6B was explored using X-ray crystallography, NMR and differential scanning calorimetry. Our structural investigations highlight the importance of tightening of mobile regions of the molecule such as loops and helix termini to attain higher thermostability. Accordingly, NMR studies suggest a very rigid structure of 6B lipase. Further investigation suggested that reduction/perturbation of the large hydrophobic patches present in the wild-type protein structure, decreased propensity of amino acid sequence for aggregation and absence of aggregation-prone intermediate during thermal unfolding of 6B can account for its resistance to aggregation. Overall, our study suggest that better anchoring of the loops with the rest of the protein molecule through mutations particularly on the sites that perturb/disturb the exposed hydrophobic patches can simultaneously increase protein stability and aggregation resistance.     

Stabilizing effect of polyols is sensitive to inherent stability of protein

In studies on polyol-mediated protein stabilization, the polyols are the preferred variable and less importance is given to the intrinsic properties of the protein used. We investigated the stabilizing effects of glycerol on three in vitro evolved lipase mutants with varying stabilities and also in a broad pH range of 3.3-12.1. Significant linear negative correlation between increment in stability due to glycerol and prior stability suggests that stabilizing effects of glycerol depend on the prior stability of the protein. Polar/nonpolar surface area and charge do not have a bearing on the stabilizing effects of glycerol.     

Engineering deamidation-susceptible asparagines leads to improved stability to thermal cycling in a lipase

At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75°C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90°C. MutC has retained more than 50% activity even after incubation at 100°C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.     

Cellular localization, oligomerization, and membrane association of the hereditary spastic paraplegia 3A (SPG3A) protein atlastin

Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.     

A lignan from Lonicera hypoleuca

A new lignan characterised as (-)-4-hydroxy-2,6-di-(4′-hydroxy-3′-methoxy)phenyl-3,7-dioxabicyclo-(3.3.0)octane along with n-10-nonacosanol, scopoletin, syringic acid, β-sitosterol and its glucoside, has been isolated from the aerial parts of Lonicera hypoleuca. The stereochemistry of the lignan has been established by its spectroscopic analysis and those of its derivatives, and by its conversion to (+)-pinoresinol. β-Sitosterol-β-D-glucoside displayed good spasmolytic activity.     

Bacteriostatic heterocycles from Euodia lunu-ankenda

A new constituent characterized as 8-acetyl-3,4-dihydroxy-5,7-dimethoxy-2,2-dimethylchroman has been isolated together with alloevodionol-7-methyl ether, 4-methoxy-1-methyl-2(1H)quinolinone, evolitrine, isoevodionol and its methyl ether from the aerial parts of Euodia lunu-ankenda. Its structure was confirmed by its transformation to alloevodionol-7-methyl ether. 4-Methoxy-1-methyl-2(1H)quinolinone and its isomer were synthesized by a modified procedure.     

8-hydroxydihydrochelerythrine and arnottianamide from roots of Toddalia asiatica

In addition to hexacosanoic acid, β-sitosterol and arnottianamide, 8-hydroxydihydrochelerythrine, a new benzophenanthridine alkaloid has been isolate     

Toddasin,a new dimeric coumarin from Toddalia asiatica

A new dimeric coumarin, named toddasin and possessing a cyclohexene ring with a vinylic side-chain interposed between the two coumarin moieties has been isolated from the roots of Toddalia asiatica. It has been characterized as (E)-8,8′-[1″,4″-dimethyl-3″ -cyclohexen-1″,2″-ylene vinylene]-bis-[5,7-dimethoxycoumarin] (1). The proposed structure is supported by the mass fragmentation of its dihydro derivative (2).     

Efficient synthesis of thymidine boranophosphoramidates conjugated with amino acids

An efficient synthesis of a thymidine boranophosphoramidate prodrug was accomplished using a phosphoramidite approach in high yield. This new class of compounds is designed to have improved antiviral and anticancer advantages conferred by combining the boranophosphate and normal nucleoside amino acid phosphoramidate. Compounds were characterized by MS and 31P NMR.