Purification and identification of native forms of goldfish neuromedin U from brain and gut

• Published in: Biochemical and biophysical research communications Vol. 517; no. 3; pp. 433 - 438
• Main Authors: Maruyama, Keisuke, Kaiya, Hiroyuki, Miyazato, Mikiya, Murakami, Noboru, Nakahara, Keiko, Matsuda, Kouhei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.09.2019
• Subjects: Amino Acid Sequence; Animals; Anorexigenic action; Biological Transport; Brain - metabolism; Calcium - metabolism; Chickens; Eating - physiology; Female; Fish Proteins - genetics; Fish Proteins - isolation & purification; Fish Proteins - metabolism; Fish Proteins - pharmacology; Gastrointestinal Tract - metabolism; Gene Expression; Goldfish; Goldfish - genetics; Goldfish - metabolism; HEK293 Cells; Humans; Male; Native form; Neuromedin U; Neuropeptides - genetics; Neuropeptides - isolation & purification; Neuropeptides - metabolism; Neuropeptides - pharmacology; Protein Isoforms - genetics; Protein Isoforms - isolation & purification; Protein Isoforms - metabolism; Protein Isoforms - pharmacology; Purification; Rats; Receptors, Neurotransmitter - genetics; Receptors, Neurotransmitter - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Transgenes; Anorexigenic action; Neuromedin U; Purification; Goldfish; Native form
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2019.07.108

Neuromedin U (NMU) plays important roles in energy homeostasis in rodents and birds. Previously, our group has isolated four cDNAs encoding precursor proteins of NMU from the goldfish brain and gut, and it was assumed that these transcripts are produced by alternative splicing. We have also demonstrated that intracerebroventricular (ICV) injection of putative goldfish NMU inhibits food intake. However, as native goldfish NMU has not yet been identified, we attempted to purify it from goldfish brain and gut extracts. To assess NMU activity in fractions at each purification step, we measured changes in the intracellular concentrations of Ca2+ using HEK293 cells expressing goldfish NMU-R1 or -R2. We isolated a 25-amino-acid peptide (NMU-25) from the brain and gut and found that its primary structure is similar to that of mammalian NMU. Another 21-amino-acid peptide (NMU-21) was purified from the brain, but not from the gut. Furthermore, a 9-amino-acid peptide (NMU-9) identical to the C-terminus of NMU-21 and -25 was also isolated from the brain and gut. Treatment with synthetic NMU-9, -21 and -25 dose-dependently increased the intracellular Ca2+ concentration in mammalian cells expressing goldfish NMU-R1 and -R2. We also examined the effect of ICV-administered synthetic goldfish NMUs on goldfish food intake. NMU-25 inhibited food intake to the same degree as NMU-21. However, the inhibitory effect of NMU-9 was slightly weaker than those of NMU-21 and -25. These results indicate that several molecular forms of NMU exist in the goldfish brain and gut, and that all of them play physiological roles via NMU-R1 and NMU-R2. •We isolated three native forms of NMU from goldfish brain and gut.•NMU-9 is expected to be produced by post-translational processing of longer forms.•The three NMUs evoked an increase in [Ca2+]i in gfNMU-R1- and -R2-expressing cells.•Injections of gfNMU-9 and -25 as well as gfNMU-21 suppressed food intake.