**3.2 Determined fine specificity of galectin-1, -3 and the two galectin-8 CRDs**

Although the general fold of all galectins is highly conserved, single galectins are characterised by specific binding interactions with single carbohydrate ligands. Differences in fine specificity have been analysed using different binding assays (as mentioned above). Moreover extensive theoretical evaluation of the putative interactions between single amino acids and functional groups of the bound glycan has been done by modelling and calculation. Some specific ligands with high affinity for the single galectin CRDs are mentioned in Table 1.

The recognition of galactose is common for all galectins but the interaction with the monosaccharide alone is very weak (Carlsson et al., 2007; Knibbs et al., 1993; Salameh et al., 2010). Disaccharides containing galactose β-glycosidic bound to GlcNAc, Glc or GalNAc are bound with significantly increased affinities. Different galectins thereby show high affinity to specific disaccharides. Galectin-3, galectin-1 and the C-terminal CRD of galectin-8 bind preferentially LacNAc units of type I and type II while the N-terminal CRD of galectin-8 shows highest affinity for lactose (Carlsson et al., 2007; Ideo et al., 2011; Salomonsson et al., 2010).

Extensions of the bound galactose moiety effect glycan binding in dependence on the galectin. Galectin-3 tolerates due to its enlarged binding pocket extensions at the galactose 3`-OH-group for example repetitive LacNAc (type II) –structures (poly-LacNAc), showing even higher affinities for repetitive LacNAc structures compared to single LacNAc units (Hirabayashi et al., 2002; Rapoport et al., 2008; Salomonsson et al., 2010). In contrast galectin-1 recognises single LacNAc units presented at the non-reducing terminus of glycans not showing preference for extended poly-LacNAc glycans (Leppänen et al., 2005). Most authors agree that galectin-1 is not able to bind internal galactose moieties in poly-LacNAc-glycans (GlcNAc-β3- **Gal**-β4-GlcNAc) (Leppänen et al., 2005; Stowell et al., 2004; Stowell et al., 2008a) but depending on the assay set-up some publications report affinity to this sugar unit (Di Virgilio et al., 1999; Zhou & Cummings, 1993). These different results prove the importance of evaluation of the test set-up and critical examination of the measured binding data.

Other extensions at the 3`-OH-group of galactose such as sulphate or neuraminic acid increase the affinity of galectin-3, galectin-1 and especially galectin-8 N-CRD to the core disaccharide (Carlsson et al., 2007; Sörme et al., 2002; Stowell et al., 2008a). In contrast the C-terminal galectin-8 domain fails to bind 3`-sulfated or 3`-sialylated galactose (Ideo et al., 2003).

Modification at the 6`-OH-group for example with neuraminic acid reduces binding of all four discussed galectin CRDs (Ideo et al., 2003; Stowell et al., 2008a). Therefore α6-sialylation is discussed as regulatory modification for galectin-mediated functions (Zhuo & Bellis, 2011). Galectin-3 and galectin-8 C-CRD show high affinity for blood-group antigens (Hirabayashi et al., 2002; Yamamoto et al., 2008)

calorimetry experiments are suitable for comparative studies of different glycans but do not lead to accurate calculation of affinity constants (Ahmad et al., 2004b). Another way to determine the direct interaction of soluble galectins and glycans is the use of hemagglutination assays, but those are limited to multivalent glycans or the inhibition of interactions between galectins and multivalent glycans or erythrocytes (Ahmad et al., 2004a;

Although the general fold of all galectins is highly conserved, single galectins are characterised by specific binding interactions with single carbohydrate ligands. Differences in fine specificity have been analysed using different binding assays (as mentioned above). Moreover extensive theoretical evaluation of the putative interactions between single amino acids and functional groups of the bound glycan has been done by modelling and calculation. Some specific ligands with high affinity for the single galectin CRDs are

The recognition of galactose is common for all galectins but the interaction with the monosaccharide alone is very weak (Carlsson et al., 2007; Knibbs et al., 1993; Salameh et al., 2010). Disaccharides containing galactose β-glycosidic bound to GlcNAc, Glc or GalNAc are bound with significantly increased affinities. Different galectins thereby show high affinity to specific disaccharides. Galectin-3, galectin-1 and the C-terminal CRD of galectin-8 bind preferentially LacNAc units of type I and type II while the N-terminal CRD of galectin-8 shows highest affinity for lactose (Carlsson et al., 2007; Ideo et al., 2011; Salomonsson et al.,

Extensions of the bound galactose moiety effect glycan binding in dependence on the galectin. Galectin-3 tolerates due to its enlarged binding pocket extensions at the galactose 3`-OH-group for example repetitive LacNAc (type II) –structures (poly-LacNAc), showing even higher affinities for repetitive LacNAc structures compared to single LacNAc units (Hirabayashi et al., 2002; Rapoport et al., 2008; Salomonsson et al., 2010). In contrast galectin-1 recognises single LacNAc units presented at the non-reducing terminus of glycans not showing preference for extended poly-LacNAc glycans (Leppänen et al., 2005). Most authors agree that galectin-1 is not able to bind internal galactose moieties in poly-LacNAc-glycans (GlcNAc-β3- **Gal**-β4-GlcNAc) (Leppänen et al., 2005; Stowell et al., 2004; Stowell et al., 2008a) but depending on the assay set-up some publications report affinity to this sugar unit (Di Virgilio et al., 1999; Zhou & Cummings, 1993). These different results prove the importance of

evaluation of the test set-up and critical examination of the measured binding data.

galectin-8 domain fails to bind 3`-sulfated or 3`-sialylated galactose (Ideo et al., 2003).

(Hirabayashi et al., 2002; Yamamoto et al., 2008)

Other extensions at the 3`-OH-group of galactose such as sulphate or neuraminic acid increase the affinity of galectin-3, galectin-1 and especially galectin-8 N-CRD to the core disaccharide (Carlsson et al., 2007; Sörme et al., 2002; Stowell et al., 2008a). In contrast the C-terminal

Modification at the 6`-OH-group for example with neuraminic acid reduces binding of all four discussed galectin CRDs (Ideo et al., 2003; Stowell et al., 2008a). Therefore α6-sialylation is discussed as regulatory modification for galectin-mediated functions (Zhuo & Bellis, 2011). Galectin-3 and galectin-8 C-CRD show high affinity for blood-group antigens

**3.2 Determined fine specificity of galectin-1, -3 and the two galectin-8 CRDs** 

Ahmad et al., 2004b; Appukuttan, 2002; Giguere et al., 2008).

mentioned in Table 1.

2010).

Table 1. Preferred ligands of the single carbohydrate recognition domains of galectin-1, -3 and -8 following Rapoport et al. 2002. Symbols according to the consortium of functional glycomics (Brewer, 2004; Carlsson et al., 2007; Dell, 2002; Hirabayashi et al., 2002; Ideo et al., 2003; Ideo et al., 2011; Leppänen et al., 2005; Patnaik et al., 2006; Rabinovich & Toscano, 2009; Rapoport et al., 2008; Salomonsson et al., 2010; Stowell et al., 2004; Stowell et al., 2008a; Yamamoto et al., 2008)

Galectins: Structures, Binding Properties and Function in Cell Adhesion 13

also recognised by galectin-1, showing the function of galectin-1 in immune response and inflammation (Liu, 2005; Nishi et al., 2008; Pace et al., 1999; Rabinovich et al., 2002a;

Binding partner Gal Cell type Process Reference

Influences integrinlaminin interaction

Influences adhesion and migration

modulating integrin-ECM interaction

Likely influences cell adhesion and signalling processes

Influences endothelial cell motility and morphogenesis

Influences cell adhesion and survival by

(Gu et al., 1994)

(Moiseeva et al., 1999)

(Hadari et al.,

(Probstmeier et al., 1995)

(Wen et al., 2006)

2000)

Skeletal muscle

muscle cells

cells; Myoblasts

cells

3 Neural tissue

pericytes

Table 2. Examples of cell-surface-glycoproteins interacting with galectins

This does not constitute a comprehensive list of cell-bound galectin-binding-glycoproteins, but just intends to show some examples which might be interesting for tissue engineering.

Similarly galectin-3 binds to CD98 on macrophages, CD66 on neutrophils and the T-cell receptor also showing functions in immune response and inflammation (Demetriou et al., 2001; Dong & Hughes, 1997; Dumic et al., 2006; Hughes, 2001). Other cell surface markers involved in cell-adhesion processes such as CD44 are bound by galectin-8 in a glycandependent manner underlining the importance of galectin-8 as matricellular protein

All three galectins mentioned in this review are able to bind different integrin subunits. All bind to β1-integrins (Dumic et al., 2006; Furtak et al., 2001; Hughes, 2001; Sakaguchi et al., 2010; Zick et al., 2004). In this context galectin-3 binding to β1-integrins leads to an internalisation signal, regulating receptor amount on the cell surface and thereby influencing cell signalling aspects (Furtak et al., 2001). Other integrins such as αvβ3 integrin on endothelial cells or the αM subunit on macrophages are also bound by galectin-3 (Dong & Hughes, 1997; Markowska et al., 2010). Galectin-8 is known to have a major function in integrin-binding and integrin-mediated signalling (Zick et al., 2004). The binding of galectin-8 N-CRD to the β1-integrin-sunbunit is especially good as high affinity α2-3-

Integrin α1β1 1 Vascular smooth

Integrin α3β1 8 e.g. endothelial

NG2 proteoglycan 3 Microvascular

Immune and tumor cells are not included in the list.

involved in the regulation of cell-adhesion (Sebban et al., 2007).

Rabinovich et al., 2002b).

Integrin α7β1 1

Cell recognition molecule L1; Myelin associated glycoprotein (MAG) Neural cell

adhesion molecule

(NCAM)
