Two Activin Type 2B Receptors from Sea Bream Function Similarly in vitro

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Myostatin (MSTN) and activin are crucial members of the transforming growth factor-β superfamily, signaling via activin type II receptors (ActRII and ActRIIB).
• Previous research identified two distinct fish ActRIIB genes, ActRIIB-1 and ActRIIB-2, with differing amino acid sequences.
• Soluble fish ActRIIB-1 (extracellular domain; ECD) demonstrated inhibition of MSTN activity in vitro.

Purpose of the Study:

• To investigate the functionality and inhibitory potential of the fish ActRIIB-2a isoform.
• To compare the inhibitory effects of fish ActRIIB-1 and ActRIIB-2a on mammalian MSTN and activin A.
• To determine if functional diversification (neofunctionalization) occurred between the two fish ActRIIB paralogs.

Main Methods:

• Production of soluble ActRIIB-2a extracellular domain (ECD) in the yeast Pichia pastoris.
• Confirmation of N-glycosylation in soluble ActRIIB-2a, similar to ActRIIB-1.
• Inhibition assays using CAGA-luciferase reporter gene in mammalian A204 cells to compare ActRIIB-1 and ActRIIB-2a efficacy against MSTN and activin A.

Main Results:

• Both affinity-purified soluble fish ActRIIB-1 and ActRIIB-2a are N-glycosylated.
• Both ActRIIB isoforms demonstrated comparable inhibitory potency against mammalian MSTN and activin A.
• Amino acid differences in the ECD between ActRIIB-1 and ActRIIB-2a do not affect their ability to bind and inhibit mammalian ligands.

Conclusions:

• Fish ActRIIB-1 and ActRIIB-2a paralogs, likely arising from gene duplication, have retained similar inhibitory functions.
• Both isoforms are equally effective in inhibiting mammalian MSTN and activin A in a mammalian cell system.
• Key residues for ligand binding appear conserved across both fish ActRIIB paralogs, ensuring recognition of mammalian MSTN and activin A.