Calcium- and pH-dependent aggregation of carboxypeptidase E

Area of Science:

• Biochemistry
• Cell Biology
• Neuroendocrinology

Background:

• Carboxypeptidase E (CPE) is vital for peptide hormone and neurotransmitter biosynthesis.
• CPE exists in soluble (S-CPE) and membrane-associated (M1-CPE, M2-CPE) forms in neuroendocrine cells.
• Regulated sorting of proteins into secretory vesicles may involve Ca2+ and pH-induced aggregation.

Purpose of the Study:

• To investigate the aggregation properties of Carboxypeptidase E (CPE) under conditions mimicking the trans Golgi network.
• To determine the influence of pH and calcium on CPE aggregation and its different forms.
• To elucidate the role of CPE aggregation in its sorting into regulated secretory pathways.

Main Methods:

• Purification of CPE from bovine pituitary membranes.
• Assessment of CPE aggregation at varying concentrations, pH levels (5.0-6.0), and calcium concentrations (1-30 mM).
• Solubilization assays using Triton X-100 and pH changes to differentiate aggregation from membrane binding.

Main Results:

• CPE aggregation is concentration-dependent, occurring at pH 5.5 with higher concentrations (≥0.3 µg/µL).
• Aggregation is pH-dependent, with minimal aggregation above pH 6.0.
• The M2-CPE form exhibits greater aggregation tendency at pH 5.0-5.5. Calcium (1-30 mM) enhances M1- and M2-CPE aggregation at pH 6.0.
• M2-CPE aggregation does not fully explain its membrane association, as aggregates are solubilized under conditions where M2-CPE remains membrane-bound.

Conclusions:

• Decreasing pH and increasing Ca2+ in the trans Golgi network likely induce CPE aggregation.
• CPE aggregation is a key mechanism contributing to its sorting into regulated secretory vesicles.
• This aggregation mechanism is critical for the proper biosynthesis and secretion of peptide hormones and neurotransmitters.