Overexpression of human α-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion

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Abstract

Human lysosomal α-galactosidase A (α-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3- 1000Mx, which was the highest enzyme overexpressor, produced intracellular α-Gal A levels of 20,900 U/mg (~100 μg of enzyme/107 cells) and secreted ~13,000 U (or 75 μg/107 cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 μm crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human α-Gal A antibodies. Pulse-chase studies revealed that ~65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the α-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6- phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted α-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal α-N- acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted α-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

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Ioannou, Y. A., Bishop, D. F., & Desnick, R. J. (1992). Overexpression of human α-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion. Journal of Cell Biology, 119(5), 1137–1150. https://doi.org/10.1083/jcb.119.5.1137

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