Abstract
The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.
Highlights
Proteins entering the endocytic pathway encounter an increasingly hydrolytic environment imposed by a progressive decrease in pH and an increase in protease concentrations
These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the PheB24-PheB25 bond, generating the inactive A1–21-B1–24 insulin intermediate
The events leading to the endosomal proteolysis of internalized insulin by endosomal acidic insulinase (EAI) involve initial binding of the C-terminal region of the B chain of insulin to EAI followed by two initial proteolytic cleavages at PheB24-PheB25 and PheB25-TyrB26 peptide bonds [11]
Summary
Generation of degradation products from unlabeled HI, we elucidated the entire structure of nine early insulin intermediates generated within hepatic endosomes, all of which contained the A14 tyrosine residue in their sequence and displayed a molecular mass higher than 3125 Da [11]. The trichloroacetic acid solubility of radioactive products, which greatly underestimates actual degradation, reflects additional and/or further processing of transient insulin intermediates and their late transformation into short end products and/or free amino acids by endosomal proteases that are possibly distinct from EAI. These concerns were addressed in this study by using native HI as the substrate and RP HPLC to improve the specificity of the degradation assay and identify the nature of EAI. These improvements in the methodology used to evaluate the proteolytic activity have allowed us to identify the role of the aspartic acid protease cathepsin D in the endosomal processing of insulin
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