Physicochemical characterization of the recombinant lectin scytovirin and microbicidal activity of the SD1 domain produced in rice against HIV-1

Victoria Armario-Najera,Julià Blanco,Jordana Muñoz-Basagoiti,Shilpa R Shenoy,Paul Christou,Teresa Capell,Amaya Blanco-Perera,Silvia Marfil,Daniel Perez-Zsolt,Barry R O’Keefe,Nuria Izquierdo-Useros,Yi Sun

doi:10.1007/s00299-022-02834-5

Abstract

Key messageRice-produced SD1 retains its physicochemical properties and provides efficient pre-exposure HIV-1 prophylaxis against infection in vitro.Scytovirin (SVN) is an HIV-neutralizing lectin that features two structural domains (SD1 and SD2) that bind to HIV-1 envelope glycoproteins. We expressed SD1 in rice seeds as a potential large-scale production platform and confirmed that rice-derived SD1 binds the HIV-1 envelope glycoprotein gp120 in vitro. We analyzed the thermodynamic properties of SD1 compared to full-size SVN (produced in E. coli) by isothermal titration and differential scanning calorimetry to characterize the specific interactions between SVN/SD1 and gp120 as well as to high-mannose oligosaccharides. SVN bound with moderate affinity (Kd = 1.5 µM) to recombinant gp120, with 2.5-fold weaker affinity to nonamannoside (Kd of 3.9 µM), and with tenfold weaker affinity to tetramannoside (13.8 µM). The melting temperature (Tm) of full-size SVN was 59.1 °C and the enthalpy of unfolding (ΔHunf) was 16.4 kcal/mol, but the Tm fell when SVN bound to nonamannoside (56.5 °C) and twice as much energy was required for unfolding (ΔHunf = 33.5 kcal/mol). Interestingly, binding to tetramannoside destabilized the structure of SD1 (ΔTm ~ 11.5 °C) and doubled the enthalpy of unfolding, suggesting a dimerization event. The similar melting phenomenon shared by SVN and SD1 in the presence of oligomannose confirmed their conserved oligosaccharide-binding mechanisms. SD1 expressed in transgenic rice was able to neutralize HIV-1 in vitro. SD1 expressed in rice, therefore, is suitable as a microbicide component.

Highlights

Lectins are carbohydrate-binding proteins that bind reversibly to specific glycan structures (Mitchell et al 2017; Singh et al 2017)
The presence of correctly assembled SD1 protein in the endosperm was confirmed by ELISA and the yield was determined by calculating the concentrations from titration curves based on positive controls spiked with known amounts of SD1 produced in E. coli and non-transformed rice endosperm as a negative control
Crude endosperm extracts from this transgenic line were tested for their ability to bind human immunodeficiency virus (HIV) gp120IIIB in vitro, using SD1 produced in E. coli as positive control and wild-type rice endosperm extracts as a negative control

Summary

Introduction

Lectins are carbohydrate-binding proteins that bind reversibly to specific glycan structures (Mitchell et al 2017; Singh et al 2017). (Mori et al 2005), both of which bind oligomannose structures on gp120 and can neutralize the virus before target cell uptake (Mori et al 2002) Both lectins have been investigated as topical microbicides to prevent HIV-1 transmission (Mori et al 2005; O’Keefe et al 2009; Moulaei et al 2010; Vamvaka et al 2016b, 2016c) and GRFT has recently entered human clinical trials (ClinicalTrials.gov identifier: NCT02875119 and NCT04032717). Another microbicide candidate is scytovirin (SVN) from the cyanobacterium Scytonema varium (Bokesch et al 2003). Native SVN is active against both laboratory strains and primary isolates of HIV-1, the latter providing a more faithful representation of the inoculum that humans encounter during HIV-1 transmission, with EC50 values ranging from 0.3 to 22 nM (Bokesch et al 2003)

Methods

Results

Conclusion