The term sialic acid describes a large group of 9-carbon monosaccharides located on the outermost position of glycoproteins and glycolipids of higher animals and in some bacteria. The group comprises 50 members, the most common one being
N-acetyl-neuraminic acid (Neu5Ac). The carbon 1 carries a carboxyl group that is responsible for the negative charge of the molecule. Hydroxylation of the N-acetyl group of Neu5Ac results in N-glycolyl-neuraminic acid (Neu5Gc). Another derivative of Neu5Ac is KDN (2-keto-3-deoxy-nonulosonic acid) which is obtained when the 5-amino group is re-placed by a hydroxyl group. Further possible modifications are acetylation, methylation or the addition of phoshates or sulphates. If the exact chemical structure is not speci-fied, the respective molecules are termed sialic acids, abbreviated Sia (A. Varki [1997]).
The addition of Sias to glycan chains is catalyzed by enzymes called sialyltransferases.
Those are localized in the Golgi as part of the complex glycosylation machinery. Each sialyltransferase transfers a sialic acid residue to a specific sugar in a certain orien-tation. Most common are the α2,3- and α2,6-linkages to an underlying galactose, N-acetylglucosamine or N-acetylgalactosamine molecule. Also possible is the attach-ment to another sialic acid in α2,8 linkage (Nicholls et al. [2008], Kleineidam et al.
[1997]).
The expression of sialic acids is tissue-dependent and developmentally regulated. In humans only Neu5Ac is expressed, Neu5Gc can only be found in certain tumors. In other species like pigs Neu5Gc is quite abundant. Due to their negative charge Sias repel each other, giving mucus its viscosity or preventing thrombocytes from clump-ing. They play an important role in cellular and molecular recognition. The immune system employs Sias for self/non-self recognition. In cancer sialylation patterns are often changed, it has been shown for example that the ST6Gal1 sialyltransferase is upregulated in carcinomas of the colon, cervix, breast and others (N. Varki & Varki [2007]). Also erythrocytes carry sialic acids and therefore participate to the glycosyla-tion pattern resulting in different blood types (Traving & Schauer [1998]). In the form of polysialic acids, Sias play an important role in neural development and plasticity (A. Varki & Schauer [2009]).
However, sialic acids are not only recognized by cells but also by different pathogens.
The invasion of red blood cells by Plasmodium falciparium, the causative agent of malaria, depends on the expression of certain Sias. The bacteriumHelicobacter pylori binds to gastric mucins and bacterial toxins like cholera toxin also recognize sialic acids (Traving & Schauer [1998]).
In 1941 George Hirst discovered that influenza viruses are able to agglutinate chicken red blood cells and later Gottschalk showed that this effect is prevented by removal of Sias (Nicholls et al. [2008]). In the next decades many efforts were undertaken to elu-cidate the receptor binding of influenza viruses. So it is known that influenza C viruses and some coronaviruses use 9-O-acetylated sialic acids as receptors (Herrler et al.
[1985], Schultze & Herrler [1992]).
General methods to investigate the distribution of sialic acids on cells and tissues is the histological or immunofluorescent staining with plant lectins. It is possible to discrim-inate between α2,3- and α2,6-linked sialic acids by using lectins derived from Sam-bucus nigra (SNA, α2,6-linkage) and Maackia amurensis (MAA, α2,3-linkage). Stud-ies using these lectins often led to different results because of the two isoforms of MAA, for example when addressing the question whether ciliated cells in the human lung expressed α2,3- or α2,6-linked sialic acids (Nicholls et al. [2008]). MAAII has a more restricted binding pattern and recognizes Sia-α2,3-Gal-β1,2(Siaα2,6)-GalNAc whereas MAAI also binds to other sialylated glycans (Sia-α2,3-Gal-β1,4-GlcNAc/Glc and even to non-sialic acid residues (Nicholls et al. [2007]). Furthermore lectins differ in their affinity to inner components of carbohydrate chains, which is also known to af-fect the binding of different HAs (A. Gambaryan et al. [2005]). Thus lectins might bind to sialoglycans that do not serve as receptors for influenza viruses and some glycans recognized by influenza viruses might not be detected by lectin staining.
Several studies in the last years investigated the presence of receptor type sialic acids on the epithelium of the human respiratory tract to see whether humans would be sus-ceptible for infection with avian influenza viruses. These reports (M. N. Matrosovich et
al. [2004], Shinya et al. [2006], van Riel et al. [2006]) all came to different conclusion with respect to the cells in the human respiratory tract that are targeted by human or avian viruses and with respect to the type of sialic acids that is expressed on those cells (for review see Nicholls et al. [2008] and Hong [2009]). Therefore, Nicholls et al. [2007]
suggest to use both MAA isoforms as they could show using glycan array technology that both MAAI and H5N1 bound to certain sialoglycans that might not be detected by MAAII.
Similar studies showed, that pigs express both α2,3- and α2,6-linked sialic acids and, therefore, may serve as a mixing vessel for genetic reassortment between avian viruses, which favor α2,3-linked Sias, and human IAV, that preferentially bindα2,6-linked ones.
After several reports of direct bird-to-human transmission and studies that demon-strated the presence of both linkage types in the human lung, this notion had to be revised (Horimoto & Kawaoka [2005]). Recent detailed studies of the porcine respi-ratory epithelium have shown that indeedα2,6-linked sialic acids are the predominant sialic acid species and that pigs in this respect resemble humans and not birds (Bate-man et al. [2010]). Other studies using lectin staining have shown, that the respiratory epithelia of chicken, turkey and quail express both sialic acid linkage types and there-fore may allow the adaptation to "human type" receptor Sias (Kimble et al. [2010], Kuchipudi et al. [2009], Imai et al. [2012]).
To investigate receptor binding properties of different influenza strains methods like solid phase binding assays or hemagglutination assays with erythrocytes from differ-ent species or resialylated red blood cells were used. Avian strains were shown to bind preferentially α2,3-linked sialic acids. Human viruses on the other hand preferentially boundα2,6-linked sialic acids. New methods to investigate receptor binding profiles of different subtypes are glycan arrays, where different sialoglycans are printed on glass slides and the respective viruses are bound. The general preferences of human and avian strains for α2,6- or α2,3 respectively has not been refuted by these studies but
they add more detail to assessment of HA binding as they take underlying glycans into account. Still, these arrays, can not show on what cells and tissues these sugars are expressed (Blixt et al. [2004], J. K. Taubenberger [2008], Liao et al. [2010]) and so this question remains. First attempts have been undertaken by analyzing primary porcine or human cells with mass spectrometry with respect to the glycans present (Chandrasekaran et al. [2008] and Bateman et al. [2010]).
Glaser et al. [2007] showed, that infection of mice lacking aα2,6 sialyltransferase (thus without abundance of α2,6-linked Sias) with human influenza viruses led to equally high titers as in wildtype mice. Also influenza viruses were still able to bind to desialy-lated MDCK cells (Stray et al. [2000]).
The topology of the glycan also appears to be important and might play a role in recep-tor binding. One is the narrow cone-like topology in which sialic acid and the following sugars span like a cone into the HA RBS. This topology is adopted byα2,3 linked sialic acids and short α2,6 glycans. The amino acids associated withα2,3 specificity (E190 and Q226) are involved in this conformation. The degree of branching after the first trisaccharide plays a crucial role in the second possible, umbrella like, topology. The amino acids involved in the binding to these glycans are not conserved among-human adapted H1 and H3 viruses. The broader umbrella-like topology is unique to longα2,6 terminated glycans (Chandrasekaran et al. [2008], Viswanathan et al. [2010]).
Future studies will continue to clarify the relationship between sialic acid linkage, gly-can structure and receptor specificity as well as the distribution of influenza receptors and will determine what the prerequisites for an avian HA to bind to human cells are.