Strategies for humanizing glycosylation pathways and producing recombinant glycoproteins in microbial expression systems

Homogeneously glycosylated proteins are essential for analyzing the structure of N-glycans, studying their consequences inside cells, and developing therapeutic glycoproteins. However, the isolation of glycoproteins with homogeneous glycans from human is difficult since glycoforms slightly differ from each other with respect to molecular weight and charge. Microbial expression systems have numerous benefits in expression technology and have gained great attention, because they are more adaptable to the biotechnology industry. While selecting an expression host, the glycosylation pattern must be taken into account, because glycosylation strongly depends on cellular production system and selected production clone. This review discussed the technological developments in glycoengineering of microbial expression systems for humanizing the glycosylation profile and highlighted the expression potential of Leishmania expression system. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2752, 2019.     

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells – the influence of phosphoglycans

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.     

Isolation of Kinetoplast-Mitochondrial Complexes from Leishmania tarentolae*

SYNOPSIS. Kinetoplast-mitochondrial complexes were liberated from Leishmania tarentolae by passing hypotonically swollen cells in dilute Tris-EDTA through a needle at 100 1bs/in². The complexes formed an equilibrium band by flotation in Renografin gradients at a density of 1.22 g/ml. The band was monitored by several mitochondrial and kinetoplastic markers: [³H]DNA, succinate-cytochrome c reductase activity, [⁵⁰Fe]hemoproteins and optical density at 600 nm. Electron microscopy showed that the sole component of the 1.22 g/ml band was the kinetoplast-mitochondrial complex.     

Leishmania molecules that mediate intracellular pathogenesis

Parasites of the Leishmania genus are the causative agents of a complex disease called leishmaniasis. Many activities of infected cells including their responses to a range of stimuli are modulated by Leishmania parasites. This review will profile some of the parasite molecules that target host cell processes for which there has been recent progress.     

Leishmania donovani flagellum-specific epitopes mediating host-parasite interactions

Abstract Monoclonal antibodies were developed against flagellar components of promastigotes of Leishmania donovani . The monoclonal antibody produced by clone A₁₁ (mAb A₁₁) recognised epitopes in the polypeptides with molecular weights of 86, 66 and weakly 53 kDa. These epitopes were found to be distributed along the flagellum and at the anterior end of promastigotes. The mAb A₁₁ of IgG₁ isotype strongly agglutinated the promastigotes of L. donovani . The prior treatment of promastigotes of L. donovani with mAb A₁₁ resulted in a significant ( P < 0.001) reduction in the attachment of promastigotes to cultured mouse peritoneal macrophages of line J774G8. The affinity-purified epitopes identified by mAb A₁₁ were recognised by human sera of cases of visceral leishmaniasis. The present study suggest that flagellar-specific epitopes mediate host-parasite interactions and, therefore, the role of these epitopes in the disease process is speculated.     

Antileishmanial activity of fucosterol recovered from Lessonia vadosa Searles (Lessoniaceae) by SFE,PSE and CPC

Fucosterol, a triterpene derivative encountered in several alga species, provides a wide range of biological activities, such as protection against metabolic syndrome, or against UV-induced skin damage. We describe here the comparison of extraction by supercritical fluid (SFE) and pressurized solvent (PSE) of the brown alga Lessonia vadosa mainly abundant in the coastal water of Patagonia, followed by the isolation of fucosterol, using centrifugal partition chromatography (CPC), in association with HPLC–UV quantification. After collection, the seaweed was dried, ground and extracted either by PSE or by SFE under various conditions. The yield and the content in fucosterol of each extract were determined by HPLC–UV. Optimization of a biphasic solvent system and K_D calculation led to the isolation of pure fucosterol with high recovery rate. Extraction by SFE using CO₂ at 180 bar and 50 °C with 20 to 30 % of cellulose as modifier and CPC purification by cyclohexane/acetone/methanol/water 10/1/10/1 with lower layer as mobile phase led to the best results in terms of yield, purity, time and solvent consumption. Natural and semisynthetic steroid derivatives have been previously shown to be potential drug candidates against parasitic diseases including leishmaniasis. In this context fucosterol was evaluated and demonstrated noticeable antileishmanial activity (IC₅₀ < 10 μM) against intracellular amastigotes with limited or no cytotoxicity in host cell macrophages. These results make this compound a valuable starting scaffold for pharmacomodulation. Adaptation of the procedure by slight modifications of the extraction and/or isolation conditions could permit the exploitation of other alga species as raw material. Since SFE and CPC are available for pilot and batch production, this work may serve as a model for further scale-up and industrial development.