The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells.
Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.
Laboratory of Animal Reproduction and Physiology, Faculty of Agriculture, Department of Applied Biological Chemistry, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan.
Siqin
Itaru
I
Minagawa
Mitsutoshi
M
Okuno
Kimihiko
K
Yamada
Yasushi
Y
Sugawara
Yoshio
Y
Nagura
Koh-Ichi
K
Hamano
Enoch Y
EY
Park
Hiroshi
H
Sasada
Tetsuya
T
Kohsaka
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D007985:Leydig Cells / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013737:Testis / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism
D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins