**4. Glycoproteins as binding partners of galectins**

Galectins can act intracellular or in the extracellular space, where they have different functions regulated by protein-protein or protein-glycan interactions. In the extracellular space they interact with different glycoproteins influencing cell adhesion, signalling and proliferation events. Thereby they interact with ECM-glycoproteins forming the extracellular matrix and with glycosylated transmembrane or membrane associated proteins on the cell surface (table 2). Following we present some selected binding partners of the three different galectins discussed so far.

### **4.1 ECM glycoproteins as binding partners of galectins**

Different extracellular matrix proteins contribute to structural and functional aspects of the extracellular space. Galectin-1 interacts strongly with different extracellular matrix proteins. It has affinity to several glycoproteins as with increasing affinity osteopontin, vitronectin, thrombospondin, cellular fibronectin and laminin (Moiseeva et al., 2003). Most of these interactions depend on the carbohydrate recognition domain and can be inhibited with soluble glycan ligands (Cooper, 1997; Moiseeva et al., 2003; Ozeki et al., 1995; Zhou & Cummings, 1993). Galectin-3 also shows high affinity for some ECM-glycoproteins (Dumic et al., 2006; Kuwabara & Liu, 1996; Massa et al., 1993; Matarrese et al., 2000; Ochieng et al., 1998b; Sato & Hughes, 1992). The best binding candidates fibronectin and laminin are heavily glycosylated (5-7% and at least 12-15% respectively), carrying mainly *N*-glycans (Paul & Hynes, 1984; Tanzer et al., 1993). *N*-glycans are among the main binding partners of galectin-1, -3 and –8 (Patnaik et al., 2006) (although galectin-8 also shows high affinity to some glycosphingolipids (Ideo et al., 2003; Yamamoto et al., 2008)). One third of laminin *N*glycans is composed of repetitive *"N*-acetyllactosamine" units (shown for mouse EHSlaminin) which are preferentially recognised by galectin-3 but also by galectin-1 and to less extent galectin-8 (Arumugham et al., 1986; Hirabayashi et al., 2002; Knibbs et al., 1989; Sato & Hughes, 1992; Zhou & Cummings, 1993). The other ECM-glycoproteins carry also *N*glycans but are less glycosylated (Bunkenborg et al., 2004; Chen et al., 2009; Liu et al., 2005). For example osteopontin from human bone shows only two *N*-glycans with binding sites which are partially blocked by α6-bound sialic acid (Ideo et al., 2003; Masuda et al., 2000; Stowell et al., 2008a). Another extracellular matrix protein interacting with galectin-1 and -3 is the Mac-2 binding protein or 90K antigen which influences adhesion processes (Sasaki et al., 1998; Tinari et al., 2001).

The different ECM-proteins which are bound by galectins can interact with other ECMglycoproteins and/or integrins (Adams, 2001; Janik et al., 2010; Kariya et al., 2008; Singh et al., 2010). These interactions can lead to regulatory effects, lattice formation and signalling cascades.

#### **4.2 Cell-surface glycoproteins as binding partners of galectins**

Beside these soluble ECM components also some membrane-bound proteins are recognised by galectins. One of these is the lysosome associated membrane glycoprotein 1 (LAMP-1) which is known to carry several *N*-glycans partly presenting poly-lactosamine glycans recognised by galectin-3 and -1 (Chen et al., 2009; Do et al., 1990; Dong & Hughes, 1997). LAMP-1 is also known as CD107a. Several other membrane proteins associated in the cluster of differentiation such as CD3, 4, 7, 8, 43 and 45 which are presented on T-cells are

Galectins can act intracellular or in the extracellular space, where they have different functions regulated by protein-protein or protein-glycan interactions. In the extracellular space they interact with different glycoproteins influencing cell adhesion, signalling and proliferation events. Thereby they interact with ECM-glycoproteins forming the extracellular matrix and with glycosylated transmembrane or membrane associated proteins on the cell surface (table 2). Following we present some selected binding partners of the

Different extracellular matrix proteins contribute to structural and functional aspects of the extracellular space. Galectin-1 interacts strongly with different extracellular matrix proteins. It has affinity to several glycoproteins as with increasing affinity osteopontin, vitronectin, thrombospondin, cellular fibronectin and laminin (Moiseeva et al., 2003). Most of these interactions depend on the carbohydrate recognition domain and can be inhibited with soluble glycan ligands (Cooper, 1997; Moiseeva et al., 2003; Ozeki et al., 1995; Zhou & Cummings, 1993). Galectin-3 also shows high affinity for some ECM-glycoproteins (Dumic et al., 2006; Kuwabara & Liu, 1996; Massa et al., 1993; Matarrese et al., 2000; Ochieng et al., 1998b; Sato & Hughes, 1992). The best binding candidates fibronectin and laminin are heavily glycosylated (5-7% and at least 12-15% respectively), carrying mainly *N*-glycans (Paul & Hynes, 1984; Tanzer et al., 1993). *N*-glycans are among the main binding partners of galectin-1, -3 and –8 (Patnaik et al., 2006) (although galectin-8 also shows high affinity to some glycosphingolipids (Ideo et al., 2003; Yamamoto et al., 2008)). One third of laminin *N*glycans is composed of repetitive *"N*-acetyllactosamine" units (shown for mouse EHSlaminin) which are preferentially recognised by galectin-3 but also by galectin-1 and to less extent galectin-8 (Arumugham et al., 1986; Hirabayashi et al., 2002; Knibbs et al., 1989; Sato & Hughes, 1992; Zhou & Cummings, 1993). The other ECM-glycoproteins carry also *N*glycans but are less glycosylated (Bunkenborg et al., 2004; Chen et al., 2009; Liu et al., 2005). For example osteopontin from human bone shows only two *N*-glycans with binding sites which are partially blocked by α6-bound sialic acid (Ideo et al., 2003; Masuda et al., 2000; Stowell et al., 2008a). Another extracellular matrix protein interacting with galectin-1 and -3 is the Mac-2 binding protein or 90K antigen which influences adhesion processes (Sasaki et

The different ECM-proteins which are bound by galectins can interact with other ECMglycoproteins and/or integrins (Adams, 2001; Janik et al., 2010; Kariya et al., 2008; Singh et al., 2010). These interactions can lead to regulatory effects, lattice formation and signalling

Beside these soluble ECM components also some membrane-bound proteins are recognised by galectins. One of these is the lysosome associated membrane glycoprotein 1 (LAMP-1) which is known to carry several *N*-glycans partly presenting poly-lactosamine glycans recognised by galectin-3 and -1 (Chen et al., 2009; Do et al., 1990; Dong & Hughes, 1997). LAMP-1 is also known as CD107a. Several other membrane proteins associated in the cluster of differentiation such as CD3, 4, 7, 8, 43 and 45 which are presented on T-cells are

**4.2 Cell-surface glycoproteins as binding partners of galectins** 

**4. Glycoproteins as binding partners of galectins** 

**4.1 ECM glycoproteins as binding partners of galectins** 

three different galectins discussed so far.

al., 1998; Tinari et al., 2001).

cascades.

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; Rabinovich et al., 2002b).


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. Immune and tumor cells are not included in the list.

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

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-

Galectins: Structures, Binding Properties and Function in Cell Adhesion 15

**5.2 Selected examples of cell adhesion and motility regulated by galectins-1, -3 and -8**  We here present only few examples of galectin function in cell adhesion and motility processes. The list is by far not complete. Other review articles focus more detailed on cell

Galectin-1 is an important factor for the adhesion and proliferation of neural stem cells and neural progenitor cells. The adhesion is mediated by the carbohydrate recognition domain and interaction of this binding domain with integrin β1 subunit (Sakaguchi et al., 2006; Sakaguchi et al., 2010). Moreover galectin-1 can reduce the motility of immune cells which might explain parts of its anti-inflammatory effects (Elola et al., 2007; Liu, 2005; Rabinovich

One important function of galectin-3 is associated with angiogenesis (Nangia-Makker et al., 2000a; Nangia-Makker et al., 2000b). Galectin-3 increases for example angiogenesis by forming integrin αvβ3 lattices on the cell-surface leading to FAK regulated downstream signalling. Galectin-3 mediated angiogenesis depends on the growth factors VEGF and bFGF (Markowska et al., 2010). Another interesting function of galectin-3 is the chemotattraction of monocytes via a G-protein coupled receptor pathway and the role in eosinophil rolling to sites of inflammation (Rao et al., 2007; Sano et al., 2000). Most of those functions can only be performed by full length galectin-3 showing the importance of glycan binding and oligomerisation of the protein (Markowska et al., 2010; Sano et al., 2000). Different other biological activities are also depending on both N- and C-terminal domain (Nieminen et al., 2005; Ochieng et al., 1998a; Sano et al., 2000; Sato et al., 2002; Yamaoka et al., 1995). This proves the possibility of regulating galectin-3 function by protease cleavage

Galectin-8 has been assigned to matricellular proteins which are able to promote cell adhesion. CHO-cells on galectin-8 show similar binding kinetics as on fibronectin but differ in their formation of cytoskeleton (Boura-Halfon et al., 2003). Moreover the binding to galectin-8 triggers specific signalling cascades as Ras, MAPK and Erk pathway (Levy et al., 2003). A physiological function in human might be the modulation of neutrophil function. Galectin-8 promotes neutrophil adhesion by binding αM integrin and promatrix metalloproteinase-9. Moreover superoxide production which is essentiell for neutrophil function is triggered by galectin-8 C-terminal CRD (Nishi et al., 2003). Another galectin-8 function might be the promotion of angiogenesis as it was also shown for galectin-3. Galectin-8 increases tube formation *in vitro* and angiogenesis *in vivo* in dependence of its specific carbohydrate affinity at physiological concentrations. This regulatory function is at

As discussed in chapter 5.1 galectins can act pro- and antiadhesive which *in vivo* seems to be mainly regulated by concentration and oligomerisation status of the galectins. In the context of biomaterial research it is also of huge importance if the galectins are immobilised or soluble presented. Immobilised galectins act mainly proadhesive as they crosslink the surface they are immobilised on with glycosylated structures on the cell-membrane. Soluble galectins can either facilitate or reduce adhesion for example to functionalised surfaces as discussed for the *in vivo* situation in chapter 5.1 depending on concentration, oligomerisation and cell type (respectively receptor availability on this cell type) (Elola et al.,

adhesion events mediated by galectins (Elola et al., 2007; Hughes, 2001).

et al., 2002a; Rabinovich et al., 2002b).

as mentioned in chapter 2.3.3.

least partially depending on CD 166 (Delgado et al., 2011).

**6. Galectins in biomaterial research** 

2007).

sialylated ligands are presented on this subunit (Diskin et al., 2009). Beside the β1-sunbunit galectin-8 N-CRD also binds α5 and some other integrin-subunits, but literature does not give a clear picture about the exact integrin binding partners. For example *N*-glycans on the α4-subunit are once mentioned as main binding partner while other authors do not report binding to this subunit. Similar discrepancies were noticed for other subunits (Cárcamo et al., 2006; Diskin et al., 2009; Hadari et al., 2000; Nishi et al., 2003; Yamamoto et al., 2008). This might be explained by tissue- or cell-specific glycosylation patterns of the single subunits. In contrast to most interactions which are performed by the N-terminal galectin-8 CRD binding to the αM-subunit is performed by the C-terminal CRD (Nishi et al., 2003).
