Ingrid 't Hart

32 Chapter 2 2 Microarray binding studies TheSiglecsarea familyof transmembranecell surfacereceptorsexpressedonhemopoie�c cells that can bind specific sialic acid containing glycoconjugates. 21 Such binding events result in inhibitory signals that dampen innate and adap�ve immune responses. Siglec-7 is predominantly expressed on natural killer (NK) cells, and its engagement with specific sialoglycans on target cells results in inhibi�on of NK cellular toxicity. Over-expression of Siglec-7 ligands on cancer cells is a proposed mechanism of immune escape, and reversal of such interac�ons may lead to a new class of checkpoint inhibitors. 22 In vitro binding studies have indicated that Siglec-7 has a preference for glycoconjugates bearing a Neu5Acα(2,8)Neu5Ac mo�f such as present in b-series of gangliosides including GD3, GD2, GT1b and GQ1b. 23 It has been shown that the expressing of GD3 on a target cells leads to suppression of NK mediated cytoly�c ac�vity in a Siglec-7 dependent manner. 24 There are also indica�ons that glycans bearing an internal branching α(2,6)-linked sialic acid at GalNAc or GlcNAc, such as present in LSTb, disialyl Lewisa and DSGb5, can also be recognized by Siglec-7. 23, 25 A glycan microarray was created by piezoelectric non-contact prin�ng of compounds 4b , 5b and 6b and GM3 ( 17 ), GM2 ( 18 ), GM1a ( 19 ), GD3 ( 20 ) and GT1b ( 21 ) 13b,26 on N-hydroxysuccinimide (NHS)-ac�vated glass slides as replicates of 6. To validate proper prin�ng, the microarray was examined for binding of the bio�nylated lec�ns Maackia amurensis leukagglu�nin (MAL-II), soybean agglu�nin (SBA) and wheat germ agglu�nin (WGA). These lec�ns were preincubated with Streptavidin-AlexaFluor635 and binding of immobilized oligosaccharides was established by measuring fluorescence intensity using a microarray scanner. As an�cipated, MAL-II, which is known to recognize Neu5Ac(α2,3) Gal(β1,4)GlcNAc/Glc, did bind GM3 ( 17 ) and GD3 ( 20 ) having such an oligosaccharide fragment (Fig. 2). SBA recognized compounds 4b and 18 , which contain a terminal Gal and GalNAc residue, respec�vely that are known to be ligands for this lec�n. WGA, which binds the GlcNAc moiety but also some forms of sialic acid, bound compounds 5b , 6b and 21 , indica�ng it has a preference for Neu5Ac(α2,3)Gal(β1,3)GalNAc containing oligosaccharides. Next, the array was incubated with bio�n-conjugated ganglioside GM1 polyclonal an�body, and as an�cipated only binding to GM1 was detected. Interes�ngly, a similar binding experiment with recombinant human Siglec-7 comp showed binding to GD3 ( 20 ) and GT1b ( 21 ), however no recogni�on of DSGb5 ( 6b ) was observed. These results indicate that Siglec-7 has a strong preference for α2,8-Neu5Ac-α2,3-Neu5Ac containing oligosaccharides, and has low or no affinity for DSGb5, which has a branching α2,6- and a terminal α2,3-sialoside. 27 The previously proposed interac�on of DSGb5 with Siglec-7 was based on the observa�on that cells that express DSGb5 bind to Siglec-7 transfected COS-7 cells. Furthermore, knockdown of ST6GalNAc6 of renal cancer cells resulted in a reduced expression of DSGb5 and a substan�al lower binding of a Siglec- 7-Fc fusion protein. 25a,28 ST6GalNAc6 has been implicated in the biosynthesis of various other gangliosides including GM1b, GT1b and GD1a, and it is likely that knockdown of ST6GalNAc6 results in a lower expression of these gangliosides, which may affect Siglec-7 binding. 29 Furthermore, this transferase is also involved in the biosynthesis of disiayl Lewis a , which is also a proposed ligand for Siglec-7. 30