1215 LYSOSOMAL EFFLUX OF FREE SIALIC ACID: IMPLICATIONS FOR SALLA DISEASE

Abstract

Salla disease is characterized biochemically by greatly increased levels of free sialic acid in lysosomes. In order to study the metabolism of free sialic acid (N-acetyl neuraminic acid, NANA) in lysosomes, highly purified lysosomes were prepared from rat liver by Percoll gradient centrifugation. These lysosomes were loaded with sialic acid by incubation in 250 mM 14C-NANA. Lysosomal loading was both temperature and time dependent with optimal conditions obtained at 37° C for 20 minutes. Lysosomes treated in this manner and then subjected to freeze/thawing lost both 90% of their sialic acid content and 90% of their β-hexosaminidase activity. The lysosomal sialic acid thus appeared to be soluble rather than protein bound. Lysosomes containing 5,500 pmol sialic acid/μmol/min β-hexosaminidase activity lost 204 pmol/min or 37% of their sialic acid content over 10 minutes at 25° C without evidence of lysosomal breakage. Loss of sialic acid slowed markedly after 10 minutes to 40 pmol/min. Addition of 2 mM Mgcl2/ATP to the lysosomes resulted in a 50% increase in the loss of sialic acid during a 10 minute incubation without change in lysosomal integrity as measured by β-hexosaminidase activity. These studies suggest that lysosomes may contain a transport system for the efflux of free sialic acid. Accumulation of free sialic acid in Salla disease would be consistent with defective lysosomal efflux of sialic acid.