Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Genetics and Genomics

Background:

• Carboxypeptidase D (CPD) is a newly identified 180-kD enzyme exhibiting carboxypeptidase E-like activity.
• CPD is hypothesized to function within the secretory pathway, potentially aiding peptide hormone processing, especially in the absence of functional Carboxypeptidase E (CPE).

Purpose of the Study:

• To characterize the structure, homology, gene mapping, and tissue distribution of Carboxypeptidase D (CPD).
• To elucidate the potential role of CPD in peptide processing within the secretory pathway.

Main Methods:

• Sequence analysis of cDNA clones encoding rat CPD.
• Homology comparison with duck homolog gp180.
• Gene mapping of the mouse Cpd gene.
• Northern blot analysis for CPD mRNA expression.
• In situ hybridization for CPD mRNA localization.

Main Results:

• Rat CPD cDNA reveals a protein with a signal peptide, three carboxypeptidase-like domains, a transmembrane domain, and a cytoplasmic tail.
• Conserved active site residues are present in the first two domains, but not the third, suggesting it may not be enzymatically active.
• High homology (75% amino acid identity) was observed between rat CPD and duck gp180 across domains.
• The mouse Cpd gene is located on chromosome 11.
• CPD mRNA (approx. 8 and 4 kb) is widely expressed in rat tissues, with highest abundance in hippocampus, spinal cord, heart atrium, colon, testis, and ovaries.
• In situ hybridization confirmed broad CPD mRNA distribution in rat brain and other tissues, notably high in hippocampus, olfactory bulb, and intermediate pituitary.

Conclusions:

• Carboxypeptidase D possesses structural features consistent with a transmembrane protein involved in the secretory pathway.
• The conserved domains and broad tissue expression suggest a significant role for CPD in the post-translational processing of numerous peptides and proteins.