Identification of Insulin Domains Important for Binding to and Degradation by Endosomal Acidic Insulinase

• Published in: Endocrinology (Philadelphia) 2001-01, Vol.142 (1), p.276-289
• Main Authors: Authier, François, Danielsen, Gillian M, Kouach, Mostafa, Briand, Gilbert, Chauvet, Geneviève
• Format: Article
• Language: English
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.142.1.7916

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase (EAI), which hydrolyzes internalized insulin at a limited number of sites. Although the positions of these cleavages are partially known, the residues of insulin important in its binding to and proteolysis by EAI have not been defined. To this end, we have studied the degradation over time of native human insulin and three insulin-analog peptides using a soluble endosomal extract from rat liver parenchyma followed by purification of the products by HPLC and determination of their structure by mass spectrometry. We found variable rates of ligand processing, i.e. high ([AspB10]- and[ GluA13,GluB10]-insulin), moderate (insulin) and low (the H2-analog). On the basis of IC50 values, competition studies revealed that human and mutant insulins display nearly equivalent affinity for the EAI. Proteolysis of human and mutant insulins by EAI resulted in eight cleavages in the B-chain which occurred in the central region (GluB13-LeuB17) and at the C-terminus (ArgB22-ThrB27), the latter region comprising the initial cleavages at PheB24-PheB25 (major pathway) and PheB25-TyrB26 (minor pathway) bonds. Except for the [GluA13,GluB10]-insulin mutant, only one cleavage on the A-chain was observed at residues GlnA15-LeuA16. Analysis of the nine cleavage sites showed a preference for hydrophobic and aromatic amino acid residues on both the carboxyl and amino sides of a cleaved peptide bond. Using the B-chain alone as a substrate resulted in a 30-fold increase in affinity for EAI and a 6-fold increase in the rate of hydrolysis compared with native insulin. A similar role for the C-terminal region of the B-chain of insulin in the high-affinity recognition of EAI was supported by the use of the corresponding B22-B30 peptide, which displayed an increase in EAI affinity similar to the entire B-chain vs. wild-type insulin. Thus, we have identified a highly specific molecular interaction of insulin with EAI at the aromatic locus PheB24-PheB25-TyrB26. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized[ 125I]TyrA14-H2-analog, a protease-resistant insulin analog, undergoes a greater lysosomal transfer and lesser degradation than [125I]TyrA14-insulin, confirming that endosomal sorting is regulated directly or indirectly by endosomal proteolysis.