Looking for inhibitor: structural and functional analysis of novel bangle lectin PHL from Photorhabdus asymbiotica

Josef Houser1,2, Gita Jančaříková1,2, Pavel Dobeš3, Eva Fujdiarová2, Pavel Hyršl3, Magdolna Csávás4, Jitka Moravcová5, Michaela Wimmerová1,2

1CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

2National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic,

3Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

4Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1, Η-4032 Debrecen, Hungary

5Department of Chemistry of Natural Compounds, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague, Czech Republic

 

Photorhabdus asymbiotica is a gram-negative bioluminescent bacterium living in a symbiosis with Heterorhabditis nematodes forming a highly entomopathogenic complex. Unlike other Photorhabdus species, P. asymbiotica can act as an emerging human pathogen as well. In its genome, we identified a gene for a putative lectin, and examined the corresponding recombinant protein PHL from functional and structural point of view. It exhibited high affinity for fucosylated carbohydrates and lower affinity to several other mono- and oligosaccharides including saccharides from bacterial cell wall or human blood epitopes. It was further shown to interact with all types of red blood cells and insect haemocytes, inhibit the production of reactive oxygen species in human blood and inhibit antimicrobial activity both in human blood, serum and insect haemolymph [1].

We succeeded in determining structure of PHL in complex with several monosaccharides revealing its unusual properties. It was shown that fucose and galactose occupy different group of well-defined sites, making PHL the first confirmed case of barrel-shape lectin with two sets of sites displaying different specificity and arranged in two layers. As it further forms dimer, the maximal number of potential binding sites per biological unit is 28. This arrangement lead to forming a new type of lectin called bangle lectin [1].

As being promising target for treatment of P. asymbiotica related infections, we tested a wide range of various mono-, di- and oligovalent carbohydrate-based molecules. Their ability to bind PHL and subsequently inhibit its interactions with natural ligands was studied using heamagglutination, isothermal titration calorimetry and surface plasmon resonance [2]. For several of them, we also succeeded in preparing crystals of corresponding PHL complexes and analyzing their structures (Fig. 1). Data gained so far show the way for design and synthesis of potential therapeutics.

 

This work was supported by the Czech Science Foundation (project 18-18964S) and from European Regional Development Fund-Project „CIISB4HEALTH“ (No. CZ.02.1.01/0.0/0.0/16_013/0001776). CIISB research infrastructure project LM2015043 funded by MEYS CR is also gratefully acknowledged for the financial support of the measurements at the CF Biomolecular Interactions and Crystallization, CF X-ray Diffraction and Bio-SAXS and CF Proteomics at CEITEC (Brno, Czech Republic). We wish to thank the BESSY II (Berlin-Adlershof, Germany) and PETRA III (Hamburg, Germany) for access to their synchrotron data collection facilities and allocation of synchrotron radiation beam time.

 

[1] Jancarikova, G. et al (2017) PlosPathogen, doi:10.1371/journal.ppat.1006564

[2] Jancarikova, G. et al (2018) Chem Eur J, doi:10.1002/chem.201705853

 

Figure 1: PHL dimer in complex with one of the studied inhibitors (shown as ball and sticks). Individual monomers shown in white and black, respectively, and recognized binding sites highlighted in orange.