TY - JOUR

T1 - Photorhabdus luminescens lectin A (PllA) - a new probe for detecting α-galactoside-terminating glycoconjugates

AU - Beshr, Ghamdan

AU - Sikandar, Asfandyar

AU - Jemiller, Eva-Maria

AU - Klymiuk, Nikolai

AU - Hauck, Dirk

AU - Wagner, Stefanie

AU - Wolf, Eckhard

AU - Koehnke, Jesko

AU - Titz, Alexander

N1 - Acknowledgments We are grateful to Prof. Rolf Müller (HIPS Saarbrücken) for providing the P. luminescens strain used in this study and to the Consortium for Functional Glycomics (Core H, Protein–Glycan Interaction Resource of the Consortium for Functional Glycomics and National Institutes of Health Supporting Grant R24 GM098791) for the glycan array analysis of PllA. We acknowledge use of the ESRF synchrotron (beamlines ID23-2 and ID30-B). GTKO pigs were produced with funding from Deutsche Forschungsgemeinschaft Grant TRR 127 “Biology of xenogeneic cell, tissue, and organ transplantation – from bench to bedside.”

PY - 2017/12/1

Y1 - 2017/12/1

N2 - Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for -galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting -galactosides. The -Gal epitope is present on wild-type pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA’s high specificity for -galactoside–containing ligands, and we show that PllA can be used to visualize the -Gal epitope on porcine tissues.

AB - Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for -galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting -galactosides. The -Gal epitope is present on wild-type pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA’s high specificity for -galactoside–containing ligands, and we show that PllA can be used to visualize the -Gal epitope on porcine tissues.

UR - http://www.scopus.com/inward/record.url?scp=85036468397&partnerID=8YFLogxK

U2 - 10.1074/jbc.m117.812792

DO - 10.1074/jbc.m117.812792

M3 - Article

VL - 292

SP - 19935

EP - 19951

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 48

ER -