Glycan arrays have been developed with the goal of analyzing biologically relevant protein-carbohydrate interactions. Most glycan arrays were constructed using synthetic glycans, but a move to creating naturally derived glycans is underway. Synthetic glycan arrays showcase only a fraction of the over 7000 predicted human glycans and cannot account for those that are undiscovered. Glycan arrays can be used in the study of influenza viral receptor preferences against several ligands including hemagglutinin protein(HA) and whole influenza virus. Current assays do not represent the host’s natural glycan profile. Studies performed on human lung and bronchial tissues have identified Sialα2,3- and Sialα2,6 terminated glycans with long poly LacNAc extensions (repeats of Gal α1,4GlcNAc disaccharides) (Walther, Karamanska, Chan et al. 2013). The study also noted that synthesized glycan arrays did not match the glycan profile of human tissues. Similarly studies on ferrets and CHO cells have also identified sialylated poly LacNAc glycans (Jia, Barclay, Roberts, et al. 2014; North, Huang, Sundaram, et al. 2009). Glycan array studies show synthetic glycans similar to poly LacNAcs have a strong binding affinity to HA (Smith and Cummings, 2014). In this study a natural glycan array will be produced using CHO cell glycans which serve as a model for human respiratory airways. Glycans will be derivatized using a bi-functional tag, 2-amino-N-(2-aminoethyl)-benzamide.  The glycans will be separated using a two-dimensional HPLC approach using NH2 first dimension, and porous graphite second (PGC) dimension HPLC based methods. Glycans will be quantitated using fluorescence signal of the AEAB tag. The second dimension PGC separation will be performed in line with an ion trap mass spectrometry to determine structure with quantitation based on fluorescence detection of aminated glycans. The resulting data will be processed with the aid of MSALAD, an in-house developed glycomics software used for analyses of glycan arrays. Our results of CHO K1 cell glycan extraction, permethylation and MALDI MS analysis have identified several sialylated poly LacNAc glycans which serve as a good model for human respiratory airways. These include NeuAc1Fuc1Hex7HexNAc6, NeuAc2Fuc1Hex7HexNAc6, and NeuAc1Fuc1Hex8HexNAc7 among others. Further work is being performed to derivatize the glycans and conjugate them to slides before probing the array with fluorescently labelled lectins, and influenza viruses.

Naturally derived glycan array from CHO cells to study influenza viral infection

256th National Meeting (2018)

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