Excretion-Reuptake Route of β-Hexosaminidase in Normal and I-Cell Disease Cultured Fibroblasts

Abstract

A B S T R A C T It has been proposed that in cultured fibroblasts the final packaging of enzymes in lysosomes re(luires excretion followed by pinocytosis by neighboring cells via a carbohydrate-specific receptor mechanism. It has also been proposed that the abnormally high activity of lysosomal enzymiies in the medium of cultured fibroblasts from patients with I-cell disease (mucolipidosis II) results from an altered carbohydrate recognition residue on the enzymes which prevents reuptake into the cells. With 8-hexosaminidase as a marker, and competitive inhibition of uptake by 2 mM mannose-6-phosphate, we have determined that only 12% of the total (intra-and extracellular) B-hexosaminidase in normal fibroblasts is channeled through the excretion-reuptake route. After 9 d of exposure to mannose-6-phosphate, normal fibroblast cultures accumulated in the medium only a fraction of the enzyme excreted by I-cell disease fibroblasts in the same period. Furthermore, this minimal loss of enzyme to the medium did not result in a decrease of intracellular enzyme activity. Finally, if the defect in I-cell disease were only because of an impairment of a reuptake mechanism that involves only 12% of the total enzyme, then 88% of the newly synthesized enzyme should be retained by I-cell fibroblasts, resulting in intracellular activity three to nine times higher than that which is observed. These data are consistent with our previous proposal that excessive lysosomal enzyme activity in the medium of I-cell disease fibroblasts results from preferential exocytosis.

Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture.

“…The MPRI/IGF II receptor is an attractive candidate, as it may not only be involved in endocytosis for catalytic activity in a prelysosomal compartment, but also possibly trigger mechanisms normally activated by the binding of IGF II, resulting in cell proliferation (31,32). Involvement of the MPRc/IGF II receptor, however, seems unlikely in our case because M6P at a concentration that effectively prevents lysosomal enzyme binding (20) did not significantly inhibit Hex A effect (Fig. 5).…”

Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts.

“…Chloroquine an(l NH4Cl, which inhibit enzyme uptake noncompetitively (28), likewise cause increased extracellular accumulation of enzyme and decrease(l incorporation into lysosomes, although by a mechanism different from the I-cell mutation (10). On the other hand, at a concentration that comnpletely inhibits uptake of exogenous enzyme, mannose 6-phosphate does not decrease the intracellular level of endogenous enzymes and increases only slightly the extracellular level (10,(29)(30)(31) …”

Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.

“…22); the dialyzed compounds were incubated for 30 min at 4°C in a 6:1 (TTC/Hex A) molar ratio to obtain mixed disulfides, followed by further dialysis in saline. Recovery was -50% by enzyme activity against 4-methylumbelliferyl N-acetylglucosaminide (4MUG) (23) (1 unit = 1 nmol of 4MUG cleaved per hr at 37°C), 4MUG 6-sulfate (24), and GM2 ganglioside [GalNAcI31--4(NeuAca2--*3)Galp1-4Glc---Cer] (GM2) (25) (27) were detectable in 42% and 71% of the cells by 3 and 4 DIV, respectively, consistent with neuronal identity. These cultures were used at 2-3 DIV, when the number of processbearing neurons was highest.…”