Proteolytic processing of the beta-subunit of the lysosomal enzyme, beta-hexosaminidase, in normal human fibroblasts.
We have characterized the proteolytic processing of the beta-subunit of beta-hexosaminidase by identifying the amino termini of the various forms synthesized in cell-free translation and in cultured human fibroblasts. The procedures used had been developed for similar studies of the alpha-subunit (Little, L. E., Lau, M. M. H., Quon, D. V. K., Fowler, A. V., and Neufeld, E. F. (1988) J. Biol. Chem. 263, 4288-4292). Radioactive amino acids were incorporated biosynthetically into the different forms of the beta-subunit, which were isolated by immunoprecipitation, gel electrophoresis, and electroelution, and analyzed by automated Edman degradation. Translation by reticulocyte lysate in the presence of canine pancreas microsomes gave a product with alanine 43 at the amino terminus. The lysate could initiate translation at methionine 1 or methionine 13, depending on the SP6 mRNA provided. The product of signal peptidase action, the precursor form of the beta-subunit with amino-terminal alanine 43, was found in NH4+-induced secretions of cultured fibroblasts; intracellularly, this form was trimmed of two additional amino acids. The mature form was found to consist of three polypeptides joined by disulfide bonds; the amino termini were found to be valine 48, threonine 122, and lysine 315. Thus, in contrast to the alpha-subunit, the mature form of the beta-subunit of beta-hexosaminidase is derived from the precursor by internal proteolytic nicking rather than by removal of a large amino-terminal peptide segment.
Quon
D V
DV
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024.
Proia
R L
RL
Fowler
A V
AV
Bleibaum
J
J
Neufeld
E F
EF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D004285:Dogs; D004586:Electrophoresis; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D007163:Immunosorbent Techniques; D008247:Lysosomes / Q000201:enzymology*; D046911:Macromolecular Substances; D008861:Microsomes / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000648:ultrastructure; D010447:Peptide Hydrolases / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000096:biosynthesis / Q000378:metabolism*
D000596:Amino Acids; D046911:Macromolecular Substances; D011498:Protein Precursors; D001619:beta-N-Acetylhexosaminidases; D010447:Peptide Hydrolases