Human carboxypeptidase E. Isolation and characterization of the cDNA, sequence conservation, expression and...

Area of Science:

• Molecular Biology
• Neuroscience
• Protein Biochemistry

Background:

• Carboxypeptidase E (CPE) is crucial for neuropeptide processing and is subject to intracellular modification.
• Understanding CPE's structure and processing is key to deciphering its role in cellular functions.

Purpose of the Study:

• To isolate and characterize human Carboxypeptidase E (CPE) cDNA.
• To compare human CPE with rat CPE and other human carboxypeptidases (CPM, CPN).
• To investigate the processing, membrane association, and structural features of human CPE.

Main Methods:

• Isolation and sequencing of human CPE cDNA.
• Sequence comparison analysis between human and rat CPE, and human CPM/CPN.
• Antibody generation against human CPE fusion protein for immunological detection.
• In vitro translation of human CPE mRNA and analysis of protein processing using microsomal membranes.

Main Results:

• Human and rat CPE cDNAs exhibit high sequence similarity (79%), with 96% identity in predicted amino acid sequences.
• Immunological detection revealed two native CPE forms (approx. 50 kDa membrane-bound and soluble) in brain preparations.
• In vitro translation produced a 54 kDa precursor that was co-translationally processed into membrane-associated, glycosylated forms (56 kDa and 52 kDa), consistent with a peripheral membrane protein.

Conclusions:

• Human and rat CPE are highly conserved, suggesting conserved function.
• CPE exists as distinct membrane-associated and soluble forms, processed intracellularly.
• Structural differences in non-coding regions may dictate the differential cellular localization of carboxypeptidases.