Elucidation of Carbohydrate Molecular Interaction Mechanism of Recombinant and Native ArtinM David Gime ́ nez-Romero,* ,† Paulo R. Bueno,* ,‡ Naira C. Pesquero, ‡ Isidro S. Monzó , § Rosa Puchades, † and A ́ ngel Maquieira † † Institute of Molecular Recognition and Technological Development, Department of Chemistry, Universitat Polite ̀ cnica de Vale ̀ ncia, Camino de Vera s/n, 46022 Valencia, Spain ‡ Institute of Chemistry, Department of Physical Chemistry, Universidade Estadual Paulista (UNESP), Rua Prof. Francisco Degni 55, 14800-900 Araraquara, Sã o Paulo, Brazil § Department of Physical Chemistry, University of Valencia, C/Dr Moliner 50, 46100 Burjassot, Valencia, Spain * S Supporting Information ABSTRACT: The quartz crystal microbalance (QCM) technique has been applied for monitoring the biorecognition of ArtinM lectins at low horseradish peroxidase glycoprotein (HRP) concentrations, using a simple kinetic model based on Langmuir isotherm in previous work. 18 The latter approach was consistent with the data at dilute conditions but it fails to explain the small differences existing in the jArtinM and rArtinM due to ligand binding concentration limit. Here we extend this analysis to differentiate sugar-binding event of recombinant (rArtinM) and native (jArtinM) ArtinM lectins beyond dilute conditions. Equivalently, functionalized quartz crystal microbalance with dissipation monitoring (QCM-D) was used as real-time label-free technique but structural-dependent kinetic features of the interaction were detailed by using combined analysis of mass and dissipation factor variation. The stated kinetic model not only was able to predict the diluted conditions but also allowed to differentiate ArtinM avidities. For instance, it was found that rArtinM avidity is higher than jArtinM avidity whereas their conformational flexibility is lower. Additionally, it was possible to monitor the hydration shell of the binding complex with ArtinM lectins under dynamic conditions. Such information is key in understanding and differentiating protein binding avidity, biological functionality, and kinetics. ■ INTRODUCTION ArtinM, also known as KM+ or Artocarpin, a plant lectin from Artocarpus integrifolia 1 belonging to the jacalin-related lectin family, is a tetrameric nonglycosylated protein composed of identical 16 kDa protomers. The protein carbohydrate recognition domains (1 per protomer and 4 per whole protein) preferentially bind to D-mannose, recognizing N-linked glycans containing the trimannoside core Manα1-3[Manα1-6]Man. 2 Lectin-carbohydrate interactions mediate biological pro- cesses such as cell-cell recognition, host-pathogen inter- actions, cancer metastasis, and cell differentiation. Studies of avidity for carbohydrate together with investigation of carbohydrate structure of glycoproteins by the use of lectins are the main subject for the emerging field of lectinomics and lectin microarrays. 3 Elucidation of carbohydrate selectivity of human and animal lectins is of great importance for better understanding of many biological processes involving cell communication. ArtinM possesses relevant biological properties. It acts on neutrophils, inducing haptotactic migration and phenotypic and functional changes. 4 Furthermore, an amplification loop for in vivo ArtinM inflammatory activity is provided by induction of mast cell degranulation. 5 ArtinM stimulates macrophage and dendritic cells to release Interleukin 12, thereby establishing in vivo Type 1 helper T cells immunity and conferring protection against several intracellular pathogens. 6 However, evaluation of uses of ArtinM has been limited by the lectin paucity in the extract of Artocarpus heterophyllus seeds less than 0.5% of the total protein content. 7 Thus, the abundance of recombinant forms should ease the pharmaceutical application of the ArtinM lectin, as well as the study of its biological properties. The study of native and recombinant lectins compatibility is very important in the medical field, because plant lectins have a limited availability, which would be compensated by the use of recombinant lectins. Recombinant ArtinM (rArtinM) reproduces well the bio- logical properties of native ArtinM (jArtinM). As jArtinM is a tetramer and rArtinM a monomer, biological functions of both lectins are not identical. For example, jArtinM induces macrophages to produce high levels of pro-inflammatory Received: March 28, 2013 Revised: June 19, 2013 Published: June 25, 2013 Article pubs.acs.org/JPCB © 2013 American Chemical Society 8360 dx.doi.org/10.1021/jp403087p | J. Phys. Chem. B 2013, 117, 8360-8369