The aspartic proteinases form a gene family whose sequences and three- dimensional structures are strongly conserved. These monomeric proteases are initially translated as prepro- precursors into the lumen of the endoplasmic reticulum (ER), whence they are post-translationally modified and sorted. We have studied three members of the gene family which are expressed in man. Procathepsin D carries N-linked oligosaccharides that are modified to contain mannose 6-phosphate residues. Mannose 6-phosphate receptors in the trans Golgi and on the plasma membrane bind and deliver the proenzyme to an acidic prelysosomal compartment and then recycle. Procathepsin D is processed to the mature enzyme and active at low pH (~4.5). Pepsinogen is the abundantly secreted precursor to gastric pepsin and is generally not glycosylated. Propeptide cleavage and activity occur extracellularly at acid pH (>3). Unlike cathepsin D and pepsin, renin shows exquisite specificity towards its physiological substrate, angiotensinogen, and is active at neutral pH in the circulation. Its glycosylation is variable, and cleavage of the propeptide takes place near neutrality at a paired basic residue site. These three aspartic proteinases share certain common features. Procathepsin D and pepsinogen are processed and active at acid pH. Procathepsin D and prorenin usually carry N-linked glycosylation sites. Procathepsin D is targeted to the lysosome, while pepsinogen and prorenin are sorted into secretory vesicles. Sorting into regulated secretory granules occurs in cells which express this specialized pathway. We were interested in the practicality of expressing chimeras between the human proenzyme forms of cathepsin D, pepsin and renin. The studies were aimed at testing whether such chimeras would be successfully synthesized by transfected mammalian cells and whether they would be sorted through the ER-Golgi pathway and either secreted or targeted to lysosomes. Kornfeld and colleagues have pioneered the use of pepsinogen as a framework for the substitution of segments from procathepsin D.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)