Sialic acids substructural specificities for MAG-binding

Sialic acids occur on cell surfaces at exposed positions, mostly terminal components with different linkages to the glycans of glycoconjugates. A unique feature of sialic acids is their structural variability leading to more than 40 naturally occurring modifications. Therefore, sialic acids are thought to play important roles in cellular interactions.80,81

As discussed before, the sialic acids of gangliosides are highly essential for MAG-binding. To further investigate the contributions of substructural features of sialic acid for MAG-binding affinity, chemically synthesized and modified gangliosides were employed for extensive structure-function study.

Research in this field was carried out mostly by two groups: Schnaar et al.56,57,71,75,82

studied adhesion between natural and synthetic gangliosides adsorbed onto microwell plastic plates and full-length MAG expressed on the transiently transfected COS cell surface; Kelm et al.79,83 _{employed “heptan inhibition assay” which observed the}

binding between Fc-chimera containing N-terminal 3 or 5 domains of MAG (Fc- MAG1-3 or Fc-MAG1-5) and soluble natural and synthetic gangliosides in the presence

of human erythrocytes. Due to the different assays, some conflicting data were reported by these two groups. However, most results are consistent, or supplementary with each other, which made the picture of sialic acid substructural specificities for MAG binding more and more clear.

α2,3-linked terminal sialic acid  Sia(I)

When GD1a is modified at the carboxylic acid by esterification, amidation, or reduction, MAG-binding is abolished, strongly demonstrating that the anionic charge is crucial for protein binding (data not shown).71

Additionally, this carboxylic acid needs to be in axial position, since only α-anomer, the natural conformation in sialylated glycans support MAG-binding.83

It is in good agreement with the X-ray structure of the N-terminal immunoglobin-like domain of the sialoadhesin in complex with 2,3-sialyllactose, which shows the axial carboxylic acid of the terminal sialic acid directedly involved in binding by forming a salt bridge with Arg97.84

It should be noted that this Arg is highly conserved in the first Ig-domain of other members of siglec family, while Arg97 in sialoadhesin corresponds to Arg118 in MAG.35

The glycerol side chain of sialic acid was shown to be crucial for binding in Schnaar’s studies with modified GD1a. The binding affinity to MAG was abolished with truncated glycerol chain, 7/8-aldehydes, 7/8-alcohols, as did deoxy and/or methoxy derivatives of the 7, 8 or 9- position of Sia(I) of GM3 (data not shown).71,82

These results suggested that the intact glycerol chain is needed for binding and that eliminating or derivating any of the OHs in this part reduced binding. The role of 9- OH attracts the interest based on the X-ray of sialoadhesin cocrystallized with sialyllactose, which implicates that an additional contact with the protein at that place might be exist.84

structural information was obtained demonstrating the requirement of 9-OH for binding (table 3).83

Replacement of 9-OH with hydrogen (entry 3) or halogens (entries 4 to 6) did not support binding, while a 9-NH2-analogue (entry 8) showed a 3-

fold increase in binding, suggesting 9-OH functions as a hydrogen bond donor for one or more amino acids in the binding pocket. The introduction of a thiol group resulted in much lower affinity (entry 7), because SH forms weaker hydrogen bonds with the protein, and the steric constraints by the large sulfur atom may also be the reason. More recently, a systematic study based on C(9)-NH2 was carried out by differently

substituting the amine.85

Notably, acyl groups such as benzoyl, biphenylcarbonyl, naphthylcarbonyl (entries 9 to 11) increased the binding dramatically. Among them, methyl-α-9-N-benzamido-9-deoxy-Neu5Ac enhanced the binding as much as 700- fold compared with the reference (entry 1). This indicates indeed an additional hydrophobic contact with the Tyr 44, 46 residues of MAG.86

Table 3: Relative inhibitory potencies (rIPs) of modified methyl sialosides for MAG

Entry Compound rIP [Ref.]

1 Neu5Ac-α-Me 1.00 83 2 Neu5Ac-α-Bn 9.80 83 3 9-deoxy-Neu5Ac-α-Me n.a. 83 4 9-Cl-Neu5Ac-α-Me n.a. 83 5 9-Br-Neu5Ac-α-Me n.a. 83 6 9-I-Neu5Ac-α-Me n.a. 83 7 9-thio-Neu5Ac-α-Me n.a. 83 8 9-NH2-Neu5Ac-α-Me 2.98 83 9 9-benzoyl-NH-Neu5Ac-α-Me 704 85 10 9-bipheyl-4-carbonyl-NH-Neu5Ac-α-Me 218 85 11 9-naphthyl-2-carbonyl-NH-Neu5Ac-α-Me 236 85 12 Neu5Propyl-α-Me 1.56 83 13 Neu5aminoAc-α-Me n.a. 83 14 Neu5ThioAc-α-Me 3.85 83 15 Neu5FAc-α-Me 16.94 83 16 Neu5ClAc-α-Me 7.00 83 17 Neu5F3Ac-α-Me 4.04 83

Concerning the modifications of the N-acetyl group at C5, R.L. Schnaar reported that GM3 bearing N-glycolyl neuraminic acid (NeuGc), which is rare in humans but common in rodents, did not support adhesion.82

Furthermore, GM4 derivatives bearing a 5-deaminated neuraminic acid (KDN-GM4) failed to support binding as well (data not shown).71

Both results clearly demonstrated that modifications at 5- position are not tolerated by the protein. Based on the X-ray structure of sialoadhesin cocrystallized with sialyllactose showing that a Trp residue in the binding site interacts specifically with the methyl group of the acetyl moiety, Kelm et al. investigated this position in much detail.83

Except the contradictory finding about the terminal KDN of a pentasaccharide, which bind to MAG 6.47 times stronger than its NeuAc analogue (data not shown), Kelm’s investigation confirmed the crucial role of N-acetyl group for recognition. In a study where propionyl (entry 12), amionacetyl (entry 13), thioacetyl (entry 14) or halogenated acetyl groups (entries 15-17) were compared with the acetyl residue, MAG showed significant enhancement of the binding affinity with halogenated acetyl groups. It should be noted that N-fluoroacetyl sialic acid derivative (entry 15) is bound about 17-fold better than the reference, while N-chloroacetyl and N-trifluroacetyl analogues increased affinities with 7- and 4-fold, respectively (entries 16 and 17). The enhanced affinity can be rationalized by halogen mediated additional contacts with the protein or by electronic effects on the amide, which results in a weaker hydrogen bond acceptor quality of the carbonyl oxygen and a significantly increased hydrogen bond donor quality of the amino group.83

α2,6-linked internal sialic acid  Sia (II)

The increased potency of Chol-1 gangliosides, like GQ1bα, to support MAG- mediated adhesion suggests a potential direct interaction between the α2,6-linked internal sialic acid and the protein. Extended explorations of the substructural specificity of Sia(II) to further define the determinant for MAG binding are reported in the literature.75

To test the role of the anionic charge, the entire Sia(II) moiety of GD1a was replaced with a sulfate group (table 2, entry 17) which had only a small influence on MAG

binding, indicating negative charge to be the key pharmacophore in the Sia(II) moiety.75

Notably, an analogue of GT1aα bearing two sulfate groups instead of two sialic acid moieties showed a 10-fold increase in binding affinity for MAG, compared with GT1aα (entries 9 and 18).75

Unlike for Sia(I), modifications of the exocyclic glycerol chain (GD1α bearing a 7-, 8- or 9-deoxy) supported MAG-mediated adhesion, suggesting that the OHs on this exocyclic glycerol chain are not strictly required for enhanced recognition.75

In summarize, numerous evidence showed that gangliosides are MAG ligands. Furthermore, they are both necessary and sufficient to support MAG-mediated neurite outgrowth inhibition. Structure-activity relationship (SAR) studies with natural gangliosides suggest to focus on the tetrasaccharide terminus Neu5Acα(2→3) Galβ(1→3)[Neu5Acα(2→6)]GalNAc for high binding affinity with MAG, where both sialic acids are essential for recognition, while the Galβ(1→3)GalNAc core seems to act predominantly as a linker (figure 8). Extended investigation of substructural specificity of Sia (I), Sia (II), mapped the epitopes important for binding: the intact Sia (I) moiety is necessary for binding, while modification at 5-, 9- positions can greatly enhanced the affinity; the anionic charge is the key point for the interaction of the Sia (II) moiety.

O HO O OH O HO O NHAc R O CO2H OH AcHN OH OH O OH HO O HO AcHN OH OH HO CO2H Carboxyl group

- probably interacts with Arg - via a salt bridge

- needs to be in axial position

Hydroxyl groups at C-4, C-8 and C-9

probably interact with MAG via hydrogen bonds