Limited Proteolysis of the /&Hexosaminidase Precursor in a Cell-free System*

Pulse-chase experiments had shown that @-hexosaminidase was synthesized in cultured human fibroblasts in precursor form and that during maturation of the enzyme the a-chains were converted from Mr = 67,000 to 54,000 and the /&chains from M, = 63,000 to 29,000 plus smaller fragments, probably through an intermediate form of M, = 52,000 (Hasilik, A., and Neufeld, E. F. (1980) J. Biol. Chem. 255,4937-4945). The lphexosaminidase precursor which is present in m’induced secretions has now been used as substrate to study these conversions in a cell-free system. A concentrate of such secretions, labeled biosynthetically, was incubated with cell fractions or purified proteinases; after the reaction, the &hexosaminidase was immunoprecipitated and its constituent polypeptides were denatured, reduced, and analyzed by polyacrylamide gel electrophoresis and fluorography. A 3,000-12,000 X g fraction of fibroblasts catalyzed the conversion of the a-chain of the precursor @-hexosaminidase from M, = 67,000 to 56,000 and of the 8chain from M, = 63,000 to 53,000. These products were similar to the mature a-chain and the intermediate form of the @-chain. The reaction occurred at acid pH, was stimulated by dithiothreitol, and was inhibited by iodoacetamide, N-ethylmaleimide, leupeptin, chymostatin, and antipain. Phenylmethylsulfonyl fluoride gave partial inhibition whereas EDTA, pepstatin A, and aprotinin had no effect. These properties indicate that the reaction was catalyzed by a lysosomal thiol proteinase; however, the enzyme was probably distinct from cathepsin B, as purified cathepsin B itself had no effect on the &hexosaminidase precursor. A similar reaction was also catalyzed by trypsin, chymotrypsin, and some related proteinases. Very high levels of trypsin released in addition polypeptides of M, = 30,000 and lower molecular weight, which resembled the products of intracellular &chain maturation. Formation of the 56,000 and 53,000 products by the lysosomal fraction, trypsin, and chymotrypsin proceeded equally well if the radioactive &hexosaminidase precursor was used as substrate in the form of an immunoprecipitate; the remainder of the precursor molecule appeared to be degraded. The limited proteolysis of the &hexosaminidase precursor did not affect its catalytic activity toward a synthetic substrate or its potential for uptake mediated by the mannose 6-phosphate recognition system.