Structures at the proteolytic processing region of cathepsin D.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Cathepsin D is a key aspartic protease involved in various cellular processes.
• Understanding its proteolytic processing is crucial for elucidating enzyme function and regulation.
• Previous studies have focused on specific species, lacking a comparative analysis of processing regions.

Purpose of the Study:

• To determine the amino acid sequences of cathepsin D proteolytic processing regions in bovine, porcine, and rat enzymes.
• To deduce the cleavage sites and understand the functional significance of proteolytic processing.
• To investigate the structural role of the processing region in procathepsin D conformation and activation.

Main Methods:

• Amino acid sequencing of peptides isolated from single-chain enzymes (bovine, rat).
• cDNA structure analysis (porcine).
• Sequence homology comparisons with known aspartic proteases (pepsinogen, penicillopepsin).
• Model building of processing region conformation using homology to porcine pepsinogen.

Main Results:

• Significant variations in the size and amino acid sequence of cathepsin D processing regions across species (rat: 6, cow: 9, pig: 9, human: 11 residues).
• Distinct proteolytic cleavage sites identified in bovine (two Ser-Ser bonds) and porcine (Asn-Ser, Gly-Val bonds) cathepsin D.
• Structural modeling revealed the processing region forms a beta-hairpin loop that interacts with the activation peptide and may stabilize procathepsin D conformation.
• The Cys residue in the processing region is positioned for disulfide linkage with Cys-27 of the light chain.

Conclusions:

• Proteolytic processing of single-chain cathepsin D likely does not involve a specific processing protease due to varied cleavage sites.
• The processing region's beta-hairpin structure plays a critical role in stabilizing procathepsin D.
• The processing region may be integral to the activation mechanism of cathepsin D.