Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities (2011)

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 α2-3-linked sialic acids. The carbohydrate-binding 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 ​andII).II). Thus, researchers who rely on vendor's 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. 



Table I.

Maackia amurensis seed lectin-binding specificities defined by direct studies





Table II.

Wide variety of M. amurensis seed lectin-binding specificities cited in glycoanalytical studies or by commercial vendors




Literature review
The presence of hemagglutinating activity in M. amurensis seed extracts was first described in the early 1960s (Boyd 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 α2-3-, but not α2-6-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 Siaα2-3Galβ1-4Glc(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-Galβ1-4Glc(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 Siaα2-3Galβ1-3(Neu5Acα2-6)GalNAc, in which the α2-6-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-Galβ1-3GalNAc (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 Neu5Acα2-8Neu5Acα2-8Neu5Acα2- 3Galβ1- 4Glc, indicating that this lectin tolerates substitution at C8 of Siaα2-3Galβ1-4Glc. In addition, the array results showed that MAH can bind to all structures containing SO4-3-Galβ, 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 mixtures of M. amurensis seed lectins that can contain unequal amounts of MAL and MAH (Nicholls et al. 2007; Varki and Varki 2007). The binding specificities of these mixtures encompass those of both lectins, but can be more similar to that of either one of the individual lectins (Nicholls et al. 2007). Table II summarizes some incorrectly cited binding specificities for MAL and MAH from the scientific and commercial literature that have contributed to the confusion surrounding the use of these lectins for glycoanalysis.