Research paper Differential contributions of recognition factors of two plant lectins e Amaranthus caudatus lectin and Arachis hypogea agglutinin, reacting with Thomsen-Friedenreich disaccharide (Galb1e3GalNAca1eSer/Thr) Albert M. Wu a, * , June H. Wu b , Zhangung Yang a , Tanuja Singh a , Irwin J. Goldstein c , Nathan Sharon d a Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, Chang-Gung University Kwei-san, Tao-yuan, 333, Taiwan b Department of Microbiology and Immunology, Chang-Gung University Kwei-san, Tao-yuan, 333, Taiwan c Department of Biological Chemistry, University of Michigan, Medical School, Ann Arbor, MI 48109-0606, USA d Department of Biological Chemistry, Weizmann Institute of Science, IL-76100 Rehovoth, Israel Received 20 May 2008; accepted 25 August 2008 Available online 3 September 2008 Abstract Previous reports on the carbohydrate specificities of Amaranthus caudatus lectin (ACL) and peanut agglutinin (PNA, Arachis hypogea) indicated that they share the same specificity for the Thomsen-Friedenreich (T a , Galb1e3GalNAca1eSer/Thr) glycotope, but differ in monosaccharide binding e GalNAc [ Gal (inactive) for ACL; Gal [ GalNAc (weak) with respect to PNA. However, knowledge of the recognition factors of these lectins was based on studies with a small number monosaccharides and T -related oligosaccharides. In this study, a wider range of interacting factors of ACL and PNA toward known mammalian structural units, natural polyvalent glycotopes and glycans were examined by enzyme-linked lecti- nosorbent and inhibition assays. The results indicate that the main recognition factors of ACL, GalNAc was the only monosaccharide recognized by ACL as such, its polyvalent forms (poly GalNAca1eSer/Thr, Tn in asialo OSM) were not recognized much better. Human blood group precursor disaccharides Galb1e3/4GlcNAcb (I b /II b ) were weak ligands, while their clusters (multiantennary II b ) and polyvalent forms were active. The major recognition factors of PNAwere a combination of a or b anomers of T disaccharide and their polyvalent complexes. Although I b /II b were weak haptens, their polyvalent forms played a significant role in binding. From the 50% molar inhibition profile, the shape of the ACL combining site appears to be a cavity type and most complementary to a disaccharide of Galb1e3GalNAc (T), while the PNA binding domain is proposed to be Galb1e3GalNAca or b1- as the major combining site with an adjoining subsite (partial cavity type) for a disaccharide, and most complementary to the linear tetrasaccharide, Galb1e3GalNAcb1e4Galb1e4Glc (T b 1-4L, asialo GM 1 sequence). These results should help us understand the differential contributions of polyvalent ligands, glycotopes and subtopes for the interaction with these lectins to binding, and make them useful tools to study glycosciences, glycomarkers and their biological functions. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Carbohydrate specificities; Glycoprotein binding; Plant lectins; Polyvalency 1. Introduction Two plant lectins from Amaranthus caudatus (ACL) [1] and peanut (Arachis hypoge, PNA) [2] bind the same glycotope of Thomsen-Friedenreich antigen (T 1 antigen) with high affinity, Abbreviations: ACL, Amaranthus caudatus lectin; PNA, peanut (Arachis hypogea) agglutinin; ELLSA, enzyme-linked lectinosorbent assay; gp, glycoprotein; OSM, ovine salivary mucin; BSM, bovine salivary mucin; PSM, porcine salivary mucin; HSM, hamster submaxillary gp; T, Thomsen-Frie- denreich antigen, Galb1e3GalNAc. * Corresponding author. Tel.: þ886 3 211 8966; fax: þ886 3 211 8456. E-mail address: amwu@mail.cgu.edu.tw (A.M. Wu). 1 Bold letters are the mammalian carbohydrate structural units. 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.08.001 Available online at www.sciencedirect.com Biochimie 90 (2008) 1769e1780 www.elsevier.com/locate/biochi