Biosynthesis and intracellular transport of α‐glucosidase and cathepsin D in normal and mutant human fibroblasts

Abstract

In order to study the intracellular localization of the proteolytic processing steps in the maturation of α‐glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl‐l‐phenylalanine‐β‐naphthylamide (Gly‐Phe‐NH‐Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux‐de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219, 965–970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly‐Phe‐NH‐Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes α‐glucosidase and β‐hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20–30%. In contrast, treatment with Gly‐Phe‐NH‐Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of α‐glucosidase and cathepsin D in fibroblasts was studied by pulse‐labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly‐Phe‐NH‐Nap prior to immunoprecipitation, 70–80% of all proteolytically processed forms of metabolically labelled α‐glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of α‐glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose‐6‐phosphate‐specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, α‐glucosidase was detected only after 2–4 h. When a pulse‐chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of α‐glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled α‐glucosidase to the lysosomes. However, the early processing steps in which the 110‐kDa precursor is converted to the 95‐kDa intermediate form of the enzyme were delayed, a transient 105‐kDa form was observed and the conversion of the 95‐kDa intermediate form to the 76‐kDa mature form of the enzyme was completely inhibited. Two cell lines from patients with glycogenosis type II have been described in which newly synthesized α‐glucosidase is not phosphorylated [Reuser, A. J. J., Kroos, M., Oude Elferink, R. P. J. & Tager, J. M. (1985) J. Biol. Chem. 260, 8336–8341]; in these specific cell lines newly synthesized α‐glucosidase is not transported to the lysosomes but is rapidly degraded in a prelysosomal compartment. In a third glycogenosis type II cell line, in which phosphorylation of α‐glucosidase is normal yet no proteolytic processing occurs (loc. cit.), there is no transport of the enzyme to the lysosomes. Copyright © 1985, Wiley Blackwell. All rights reserved