title: Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars.
abstract: RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B-C-A monomeric structure with full biological activity.
authors: Theravalues Research Institute, Theravalues, Osaka 567-0085, Japan.
authors: Itaru
authors: I
authors: Minagawa
authors: Masafumi
authors: M
authors: Fukuda
authors: Hisako
authors: H
authors: Ishige
authors: Hiroshi
authors: H
authors: Kohriki
authors: Masatoshi
authors: M
authors: Shibata
authors: Enoch Y
authors: EY
authors: Park
authors: Tatsuo
authors: T
authors: Kawarasaki
authors: Tetsuya
authors: T
authors: Kohsaka
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007985:Leydig Cells / Q000378:metabolism*; D008297:Male; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000502:physiology*
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled