Alterations of the posttranslational processing of a lysosomal enzyme in C₆ glioma cells

Abstract

Cathepsin D was assessed in C6 glioma cells grown in medium with an intermediate- or low-percent composition of serum. The amount, form, and subcellular location of cathepsin D differed after treatment with cyanate or monensin in cells grown in a low-serum, growth-factor-supplemented medium. Immunoblotting showed that cathepsin D in the lysosomal fraction of the C6 cell line had a molecular weight (Mr) of 42 kD, whereas that in the microsomal fraction had Mr's of 42, 47, and 78 kD. After treatment for 1 to 16 hr with 4 mmol/L cyanate and subcellular fractionation, the molecular weight of lysosomal cathepsin D was the same in treated and untreated cells, but more enzyme was found in lysosomes of treated cells at 8 and 16 hr. In the microsomal fraction, the amounts of both the 42 and 47 kD forms were increased after 1 to 16 hr of treatment. When exposed to 20 mmol/L cyanate, C6 cells remained viable, but compared with untreated cells, they showed 25% less lysosomal cathepsin D, with increased amounts found in the microsomal fraction. The 78 kD protein detected by immunoblotting was present in both the lysosomal and microsomal fractions but was predominant in the latter. The apparent molecular weight of this protein was the same after cyanate but differed with monensin, where Mr's of 39, 42, and 73 kD were found. Monensin-treated cells had less lysosomal cathepsin D and relatively more microsomal enzyme. The differing molecular weights of cathepsin D from cyanate- and monensin-treated cells suggest that their inhibitions occur at different processing loci in distal elements of the Golgi stacks. The differences in the pI of cathepsin D and the number of its forms from cyanate- and monensin-treated cells are also consistent with interference in the late stages of glycoprotein maturation. In this paper we show that the amount, molecular form, and consequent intracellular location of cathepsin D in cells of the C6 line can be affected by agents that selectively disrupt stages in Golgi-related protein modification and transport.

Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin

“…Their identity and relationship to the other molecular species of cathepsin D are unknown. Similar to the primary astrocytes studied in this investigation, cathepsin D is present in C6 glioma cells (Snyder and Whitaker, 1988). Cathepsin D in C6 glioma cells appears in different sizes besides the dominant 42-kDa form and can be modified by cyanate and monensin presumably through an effect on the Golgi apparatus (Snyder and Whitaker, 1988).…”

“…In the synthesis of cathepsin D, the proform may have a molecular mass of 46-52 kDa (Hasilik and Neufeld, 1980;Westley and May, 1987). Higher molecular weight bands of immunoreactive cathepsin D of different sizes have been noted in human promonocytes (Stein et al, 1987), human skin fibroblasts (Hasilik and Neufeld, 1980), and C6 glioma cells (Snyder and Whitaker, 1988). Their identity and relationship to the other molecular species of cathepsin D are unknown.…”