Gastrin biosynthesis in canine G cells

Area of Science:

• Endocrinology
• Molecular Biology
• Cell Biology

Background:

• Gastrin, a key hormone, requires complex posttranslational modifications for its biological activity.
• Progastrin processing involves cleavage at basic amino acid sites and C-terminal amidation.
• The specific enzymes involved in progastrin processing in primary cells remained to be fully elucidated.

Purpose of the Study:

• To investigate the precise biochemical pathway of progastrin processing in canine antral G cells.
• To identify the prohormone convertase responsible for the initial cleavage of progastrin.
• To confirm the role of peptidyl-glycine alpha-amidating monooxygenase (PAM) in the final amidation step.

Main Methods:

• Utilized [(35)S]methionine-labeled pulse-chase biosynthetic experiments in primary canine antral G cells.
• Employed PC2 antisense oligonucleotide probes to assess the role of prohormone convertase 2 (PC2).
• Measured the accumulation and conversion of radiolabeled progastrin and its intermediates.

Main Results:

• Progastrin processing intermediates, including glycine-extended gastrin (G-Gly), were identified and their temporal appearance characterized.
• The conversion of G-Gly to amidated gastrin was significantly enhanced by ascorbic acid, a cofactor for PAM.
• Inhibition of PC2 expression using antisense probes led to progastrin accumulation and delayed amidated gastrin formation.

Conclusions:

• Progastrin is cleaved by prohormone convertase 2 (PC2) in antral G cells to generate a glycine-extended intermediate (G-Gly).
• This G-Gly intermediate is subsequently converted to COOH-terminally amidated gastrin through the enzymatic action of peptidyl-glycine alpha-amidating monooxygenase (PAM).
• This study confirms the sequential enzymatic steps in gastrin biosynthesis within a relevant cellular context.