Biosynthesis and Functions of KDNylated glycoproteins

All living cells are decorated with glycans that are involved in a myriad of essential cellular functions. In mammals, glycoproteins and glycolipids are commonly terminated with the sialic acid Neu5Ac, a negatively charged nine carbon sugar. In contrast to Neu5Ac, the biological functions and immunogenic profiles of a deaminated sialic acid variant called KDN are unknown. The unique features of KDN are (i) resistance to sialidase activity, (ii) termination of polysialic acid chain length, (iii) potential natural decoy against microorganisms that utilize glycoconjugates carrying the host Neu5Ac and, (iv) presence as "oncodevelopmental" antigen marker in various types of cancer. The unique features of KDN and lack of knowledge on its functions contributes to the importance of proposed research. The overall aims of the proposal are to generate KDNylated glycan structures and elucidate the structural and functional impact on selected human glycoproteins. We hypothesize that the coordinated expression of five non-plant enzymes from different species results in the formation of recombinant glycoproteins with KDNylated N-glycans that may have novel biological functions. Addressing the biological function of KDN-containing glycoproteins requires the engineering of the KDN biosynthetic pathway in a suitable host system. Transient expression in plants is a well-established approach for glycoengineering and recombinant protein production and plants are well suited because they lack the interfering Neu5Ac sialic acid biosynthesis pathway. The biosynthetic pathway of KDN will be introduced by transient expression method via agroinfiltration. To optimize expression and activity, a series of expression constructs that carry different combinations of promoter-terminator and subcellular localization signals will be generated and used to produce free KDN, CMP-KDN and KDN-containing N-glycans. The resulting products will be analysed by HPLC and mass spectrometry. In vitro binding assays will be performed to investigate a role of KDN on IgG1 N-glycans in Fcγ-receptor interaction and in vivo experiments will be conducted to investigate the pharmacokinetic profile of KDNylated glycoproteins. In mammalian cells, the biosynthetic pathways of CMP-Neu5Ac and CMP-KDN overlap at different levels. Hence, the presence of a native sialic acid pathway prevents the formation of homogenous KDNylated glycoproteins. Plants produce complex N-glycans but lack a sialic acid pathway. The heterologous expression of the KDN pathway will allow to produce for the first time KDNylated glycoproteins at high homogeneity to study their functional and biochemical characteristics.