In planta engineering of polysialylated glycoproteins using salmonid polysialyltransferases

Polysialic acid (polySia) is a glycan polymer composed of a linear chain of sialic acid residues detected on a small set of proteins in mammalian cells. This negatively charged glycan modulates various cellular functions and has potential biomedical applications. However, the targeted synthesis of polySia remains challenging due to biosynthetic and structural complexity. In this study, we investigated three polysialyltransferases from the salmonid Coregonus maraena (Cma-ST8Sia) for their application in glycoengineering. For this, the corresponding genes Cmast8Sia2-R1, Cmast8Sia2-R2, and Cmast8Sia4 were transiently expressed in Nicotiana benthamiana, and confocal laser scanning microscopy of GFP-fused Cma-ST8Sia exhibited trans-Golgi localization. Co-expression of Cma-ST8Sia with genes from the mammalian sialic acid biosynthesis pathway resulted in autopolysialylation of CmaST8SiaII-R1 and CmaST8SiaIV, but not of CmaST8SiaII-R2. Furthermore, co-expression with glycoproteins demonstrated polysialylation of recombinant proteins with distinct efficiencies and chain lengths. Inactive EndoN-coupled resin was used to enrich polysialylated proteins, and by the application of HPLC/DMB labeling, a degree of polymerization up to 45 was determined. Finally, a microglia inhibition assay demonstrated the biological activity of the engineered polySia. Collectively, these findings advance the capacity to engineer high-quality polySia in an eco-friendly, sustainable system, facilitating in-depth studies and the rational design of complex glycans.