Evaluation of -Acetylmannosamine Administration to Restore Sialylation in -Deficient Human Embryonal Kidney Cells.

Background: A key mechanism in the neuromuscular disease GNE myopathy (GNEM) is believed to be that point mutations in the gene impair sialic acid synthesis - maybe due to UDP--acetylglucosamine 2-epimerase/-acetylmannosamine kinase (GNE) activity restrictions - and resulting in muscle tissue loss. -acetylmannosamine (ManNAc) is the first product of the bifunctional GNE enzyme and can therefore be regarded as a precursor of sialic acids. This study investigates whether this is also a suitable substance for restoring the sialic acid content in -deficient cells.

Methods: A HEK-293 -knockout cell line was generated using CRISPR-Cas9 and analyzed for its ability to synthesize sialic acids. The cells were then supplemented with ManNAc to compensate for possible GNE inactivity and thereby restore sialic acid synthesis. Sialic acid levels were monitored by immunoblot and high performance liquid chromatography (HPLC).

Results: The HEK-293 -knockout cells showed almost no polysialylation signal (immunoblot) and a reduced overall (-71%) -acetylneuraminic acid (Neu5Ac) level (HPLC) relative to total protein and normalized to wild type level. Supplementation of -deficient HEK-293 cells with 2 mM ManNAc can restore polysialylation and free intracellular sialic acid levels to wild type levels. The addition of 1 mM ManNAc is sufficient to restore the membrane-bound sialic acid level.

Conclusions: Although the mechanism behind this needs further investigation and although it remains unclear why adding ManNAc to deficient cells is sufficient to elevate polysialylation back to wild type levels - since this substance is also converted by the GNE, all of this might yet prove helpful in the development of an appropriate therapy for GNEM.