Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities

Christoph Geisler 0 Donald L Jarvis 0 0 Department of Molecular Biology, University of Wyoming , Laramie, WY 82071 , USA The Author 2011. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: Introduction Many researchers use MAL (Maackia amurensis leukoagglutinin) and MAH (M. amurensis hemagglutinin), the seed lectins from M. amurensis, as glycoanalytical tools to probe biological targets for 2-3-linked sialic acids. The carbohydratebinding specificities of these lectins have been carefully defined in several independent studies (Table I). However, investigators using M. amurensis lectins (MALs) for glycoanalysis often cite these specificities incorrectly (Table II). One reason for this discrepancy is that the M. amurensis seed lectins have been given a variety of different, but highly similar, names (Table I). This makes it particularly difficult to extract the correct binding specificities of these two distinct lectins (termed MAL and MAH herein) from the literature. Another reason for this discrepancy is that the technical specifications provided by some commercial vendors cite binding specificities that differ from those defined in direct scientific studies (Tables I and II). Thus, researchers who rely on vendors specifications can be misinformed. The confusion regarding the binding specificities of MAL and MAH and its impact on the accurate use of M. amurensis seed lectins for glycoanalysis was previously noted in two publications (Nicholls et al. 2007; Varki and Varki 2007). However, no communication has focused specifically on this topic. Having experienced serious confusion in this area, we thought it would be useful to review the binding properties of MAL and MAH and illustrate their utility as glycoanalytical tools in a controlled lectin-blotting assay. Literature review The presence of hemagglutinating activity in M. amurensis seed extracts was first described in the early 1960s (Boyd 1To whom correspondence should be addressed: Tel: +1-307-766-4282; Fax: +1-307-766-5098; e-mail: et al. 1961). Hemagglutinating proteins from M. amurensis seeds were subsequently isolated as a first fraction with relatively more potent hemagglutinating activity, which was called MAH, and a second fraction with relatively more potent lymphocyte mitogenic activity, which was called MAM (M. amurensis mitogen; Kawaguchi et al. 1974b). Initial studies suggested that MAH bound preferentially to sialylated O-linked glycans, whereas MAM bound preferentially to sialylated N-linked glycans (Kawaguchi et al. 1974a, 1974b; Kawaguchi and Osawa 1976). Later, MAM was found to bind strongly to leukocytes and re-designated MAL (Wang and Cummings 1987), which is the term we will use henceforth. Subsequent studies showed that MAL bound glycans with sialic acids in 2-3-, but not 2-6-linkages to galactose (Wang and Cummings 1988). This result was confirmed and extended by several investigators who observed that MAL bound most preferably to terminal Sia2-3Gal1-4Glc(NAc) in N-linked glycans (Knibbs et al. 1991; Kaku et al. 1993; Johansson et al. 1999; Imberty et al. 2000; Yamamoto et al. 2005; Nicholls et al. 2007). Nicholls et al. (2007) also recently found that MAL can bind to the unsialylated glycan, SO4-3-Gal1-4Glc(NAc). MAL and MAH have very similar amino acid sequences (86% identity) and probably have similar secondary and tertiary structures, as well (Yamamoto et al. 1994, 1997; Imberty et al. 2000). Thus, it is not surprising that their binding preferences are somewhat similar. Since its initial characterization, MAH was found to bind preferentially to an O-linked, disialylated tetrasaccharide with the structure Sia2-3Gal1-3 (Neu5Ac2-6)GalNAc, in which the 2-6-linked Neu5Ac is not required for binding (Konami et al. 1994; Imberty et al. 2000; Brinkman-Van der Linden et al. 2002; Maenuma et al. 2008, 2009). Like MAL, MAH also can bind unsialylated structures, such as glycans containing SO4-3-Gal1-3GalNAc (Bai et al. 2001; Maenuma et al. 2008). A defined glycan array also has been used to examine the binding specificities of MAL and MAH, and the results have been posted on the public website of the Consortium for Functional Glycomics (http://www.functionalglycomics.org/). Generally, the glycan array results confirmed the results of other direct studies and identified some new binding substrates. For example, they showed that MAL can bind the polysialylated structure Neu5Ac2-8Neu5Ac2-8Neu5Ac2- 3Gal1- 4Glc, indicating that this lectin tolerates substitution at C8 of Preferred binding substrates Sia2-3Gal1-4GlcNAc (Refs 18) MAL, MAM, MAL-I, MAA-1, MAA Weak binding substrates Haptenic inhibitors Neu5Ac2-3Gal1-4Glc (Refs 3, 4, 6) N-acetylneuraminic acid (Ref. 9) MAH, MAL-II, MAA-2, MAA Sia2-3Gal1-3(Sia2-6)GalNAc (Refs 1, 1014) SO4-3-Gal1-3(Sia2-6)GalNAc (Refs 12, 14, 15) None clearly identified N-acetylneuraminic acid (Ref. 9) Murine laminin (used for purification) (bovine fetuin) Human glycophorin A (also known as PSA-1) (bovine fetuin) References: 1, Imberty et al. (2000); 2, Johansson et al. (1999); 3, Kaku et al. (1993); 4, Knibbs et al. (1991); 5, Nicholls et al. (2007); 6, Wang and Cummings (1988); 7, Yamamoto et al. (2005); 8, CFG glycan array for MAL; 9, Kawaguchi et al. (1974b); 10, Brinkman-Van der Linden et al. (2002); 11, Konami et al. (1994); 12, Maenuma et al. (2008); 13, Maenuma et al. (2009); 14, CFG glycan array for MAH; 15, Bai et al. (2001). Some researchers investigated binding specificity using glycans with Glc instead of GlcNAc on the reducing end. MAL also recognizes the structure (Sia2-8)nSia2-3Gal1-4GlcNAc, but not polysialic acid (Sia2-8)n per se. Sia2-3Gal1-4Glc. In addition, the array results showed that MAH can bind to all structures containing SO4-3-Gal, underscoring the ability of this lectin to bind sulfated glycans. Table I summarizes the results of direct studies on the binding specificities of MAL and MAH, including the glycan array studies, lists haptenic inhibitors of lectin binding and includes suggested controls for lectin blotting experiments. As mentioned in the Introduction, the use of several different, but similar names for the M. amurensis seed lectins has contributed to the confusion surrounding their carbohydrate-binding specificities. For example, MAL is sometimes called MAL-I (e. g. Bai et al. 2001), MAA (M. amurensis agglutinin, e.g. Ohyama et al. 2004) or MAA-1 (e.g. Nicholls et al. 2007), and MAH is sometimes called MAL-II (e.g. Hennet et al. 1998), MAA (e.g. Nefkens et al. 2007) or MAA-2 (e.g. Nicholls et al. 2007). Furthermore, the terms MAA (indicating M. amurensis agglutinin) and MAL (indicating M. amurensis lectin) have been used to designate either the individual lectins or mixtures of both, often with no distinction. In fact, some vendors (e.g. EY Laboratories Inc., San Mateo, CA; Roche Diagnostics, Indianapolis, IN; Sigma-Aldrich Corporation, St Louis, MO) provide undefined mixt (...truncated)