Defective lysosomal egress of free sialic acid (N-acetylneuraminic acid) in fibroblasts of patients with infantile free sialic acid storage disease.
Egress of free NeuAc from normal lysosome-rich granular fractions was assessed at NeuAc concentrations of up to 221 pmol/hexosaminidase unit, achieved by exposure of growing fibroblasts to 40-125 nM N-acetylmannosamine for up to 7 days. The normal velocity of NeuAc egress increased with NeuAc loading and with temperature, exhibiting a Q10 of 2.4, characteristic of carrier-mediated transport. Fibroblasts cultured from five patients with infantile free sialic acid storage disease (ISSD) contained approximately 139 nmol of free NeuAc/mg of whole cell protein, or 100 times the normal level. Differential centrifugation, as well as density gradient analysis using 25% Percoll, showed that the stored NeuAc cosedimented with the lysosomal enzyme beta-hexosaminidase. The velocity of appearance of free NeuAc outside ISSD granular fractions was negligible, even at initial loading levels of up to 3500 pmol/hexosaminidase unit. The lack of egress from ISSD granular fractions was found for both endogenous and N-acetylmannosamine-derived NeuAc. Fibroblasts from ISSD parents did not accumulate excess free NeuAc and did not display a velocity of NeuAc egress significantly different from normal. The defect in ISSD, like that in Salla disease, appears to be an impairment of carrier-mediated transport of free NeuAc across the lysosomal membrane. Clinical and biochemical differences between Salla disease and ISSD may reflect differences in the amount of residual NeuAc transport capacity.
Tietze
F
F
Laboratory of Molecular and Cell Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Seppala
R
R
Renlund
M
M
Hopwood
J J
JJ
Harper
G S
GS
Thomas
G H
GH
Gahl
W A
WA
D002239:Carbohydrate Metabolism, Inborn Errors / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism; D006580:Genetic Carrier Screening; D006801:Humans; D007223:Infant; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D019158:N-Acetylneuraminic Acid; D012016:Reference Values; D012794:Sialic Acids / Q000378:metabolism*
D012794:Sialic Acids; D019158:N-Acetylneuraminic Acid