ORIGINAL PAPER Kinetics and Thermodynamics of Glycans and Glycoproteins Binding to Holothuria scabra Lectin: A Fluorescence and Surface Plasmon Resonance Spectroscopic Study Nagaraj M. Gowda & Sushama M. Gaikwad & M. Islam Khan Received: 27 January 2013 / Accepted: 22 May 2013 # Springer Science+Business Media New York 2013 Abstract Holothuria scabra produces a monomeric lectin (HSL) of 182 kDa. HSL showed strong antibacterial activity and induced bacterial agglutination under in vitro condi- tions, indicating its role in animals’ innate immune responses. Very few lectins have been reported from echinoderms and none of these lectins have been explored in detail for their sugar-binding kinetics. Affinity, kinetics and thermodynamic analysis of glycans and glycoproteins binding to HSL were studied by fluorescence and surface plasmon resonance spectroscopy. Lectin binds with higher affinity to O-linked than N-linked asialo glycans, and the affinities were relatively higher than that for sialated glycans and glycoproteins. T-antigen α-methyl glycoside was the most potent ligand having the highest affinity (Ka 8.32 ×10 7 M -1 ). Thermodynamic and kinetic analysis indicated that the binding of galactosyl Tn-antigen and asialo glycans is accompanied by an enthalpic contribution in addition to higher association rate coupled by low activation energy for the association process. Presence of sialic acid or protein matrix inhibits binding. Higher affinity of HSL for O-glycans than N-glycans had biological implications; since HSL specifically recognizes bacteria, which have mucin or O-glycan cognate on their cell surfaces and play a major role in animal innate immunity. Since, HSL had higher affinity to T-antigen, makes it a useful tool for cancer diagnostic purpose. Keywords Holothuria scabra . Lectin . Thermodynamic properties . Kinetic analysis . Surface plasmon resonance spectroscopy Abbreviations HSL Holothuria scabra lectin Galactosyl Tn-antigen (Gal β1-3 GalNAc α-1-O-L-Ser) Thomsen-Friedenreich antigen T-antigen α-methyl glycoside Gal β1-3 GalNAc α-1-O- Me T-antigen β–methyl glycoside Gal β1-3 GalNAc β-1-O- Me SPR Surface plasmon reso- nance spectroscopy Introduction Marine invertebrates rely solely on innate immunity, which includes both humoral and cellular responses, as they lack an adaptive immune system. Various methods employed to counteract infectious agents include, hemolymph coagulation, melanization, cell agglutination, encapsulation, nodule for- mation and phagocytosis [1, 2]. The microbial load in natural marine habitat can number up to 10 6 bacteria and 10 9 virus . mL -1 of seawater [3]. It is therefore imperative that animals develop a robust innate immune system for survival. Lectins are proteins with specificity for simple sugar, a sequence of sugars or their glycosidic linkages. Such ligands are known to occur on the surface and the capsule of bacteria. In invertebrates, lectins have been suggested to participate in innate immune response (humoral defense reaction) by inducing bacterial agglutination or by acting as opsonins to enhance phagocytosis by coelomycetes [2, 4]. Cell lysates and cell-free plasma of several invertebrates also expressed antibacterial activity, although the activity of the latter may possible be due to small antimicrobial proteins [5]. These This manuscript is dedicated to Dr. M. Islam Khan who expired during draft preparation. N. M. Gowda (*) Department of Anesthesiology and Critical Care Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA e-mail: nagaraj.mgowda@yahoo.com N. M. Gowda : S. M. Gaikwad : M. I. Khan Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India J Fluoresc DOI 10.1007/s10895-013-1244-4