Cathepsin D is membrane-associated in macrophage endosomes

Area of Science:

• Cell Biology
• Biochemistry
• Immunology

Background:

• Acid protease activity was previously identified in endosomes of rabbit alveolar macrophages.
• The specific identity and localization mechanism of this endosomal protease were not fully understood.

Purpose of the Study:

• To identify the endosomal protease in rabbit alveolar macrophages.
• To elucidate the targeting mechanism of cathepsin D to endosomes.
• To investigate the biosynthetic pathway and membrane association of cathepsin D.

Main Methods:

• Immunoprecipitation using polyclonal antibodies against rabbit cathepsin D.
• NH2-terminal sequencing of the endosomal protease.
• Analysis of membrane association of cathepsin D using Percoll density gradients and sequential washes.
• Solubilization studies at different pH and with detergents.
• Radioiodination of cathepsin D within endosomes and lysosomes.
• Biosynthetic studies of cathepsin D precursor processing.

Main Results:

• The endosomal protease was identified as cathepsin D.
• Cathepsin D was found to be membrane-associated with both light and heavy density vesicles from Percoll gradients.
• Membrane-associated cathepsin D could not be solubilized under non-denaturing conditions but was solubilized by Triton X-100.
• Radioiodination confirmed the presence of membrane-associated cathepsin D in endosomes.
• Biosynthetic studies revealed that cathepsin D is synthesized as an inactive membrane-associated precursor, which is processed to an active membrane-associated form, and subsequently to the active soluble form.

Conclusions:

• Cathepsin D is present in the endosomes of macrophages.
• Cathepsin D is transported to endosomes in a membrane-associated form.
• The membrane-associated form of cathepsin D serves as a biosynthetic precursor for the soluble form found in endosomes and lysosomes.