METABOLIC PROCESSING OF GANGLIOSIDES BY NORMAL AND SALLA HUMAN FIBROBLASTS IN CULTURE - A STUDY PERFORMED BY ADMINISTERING RADIOACTIVE G(M3) GANGLIOSIDE

Cultured fibroblasts from normal subjects and from subjects affected b y Salla disease, characterized by the lack or misfunction of the membr ane carrier responsible for the egress of sialic acid from lysosomes, were fed with ganglioside G(M3) labeled at the sialic acid acetyl grou p, [Neu5Ac-H-3]G(M3), or at C-3 of sphingosine (Sph), [Sph-H-3]G(M3), or at C-1 of stearoyl chain, [stearoyl-C-14]G(M3). After a 15-h pulse the total amount of cell-bound G(M3) corresponded to about 2% of the e ndogenous ganglioside content. Cells were then subjected to a 72-h cha se, and the radioactive products from both ganglioside catabolism and salvage processes of catabolic fragments were measured. These data ind icated that about 50% of the cell-bound ganglioside underwent metaboli c processing, suggesting a ganglioside half-life of 2-3 days. [Neu5Ac- H-3] formed from [Neu5Ac-H-3]G(M3) degradation was mostly re-cycled fo r the biosynthesis of gangliosides and sialoglycoproteins, only a mino r part being degraded to [H-3]water, which constituted only 1.6% of to tal metabolite linked radioactivity. [Sph-H-3] from the [Sph-H-3]G(M3) degradation was partly re-cycled for the biosynthesis of gangliosides , neutral glycosphingolipids and sphingomyelin, and partly (about 20% of the total metabolite linked radioactivity) degraded to [H-3]water. In Salla fibroblasts metabolic processing of [Neu5Ac-H-3]G(M3) produce d large amounts of free [H-3]Neu5Ac, and a reduced incorporation of ra dioactivity into glycoconjugates (as compared to normal cells). Howeve r, the accumulation of free Neu5Ac was not accompanied by an increase of tritiated water. LacCer and Cer from [stearoyl-C-14]G(M3) catabolis m were found to accumulate in Salla fibroblasts, an indication that th e enzymes of glycosphingolipid metabolism were affected by the impairm ent of Neu5Ac egress from lysosomes. Particularly relevant was the acc umulation of ceramide which was hardly detectable in control cells.