Chemical synthesis and biological activity of rat INSL3

被引:26
作者
Smith, KJ
Wade, JD
Claasz, AA
Otvos, L
Temelcos, C
Kubota, Y
Hutson, JM
Tregear, GW
Bathgate, RA [1 ]
机构
[1] Univ Melbourne, Howard Florey Inst Expt Physiol & Med, Melbourne, Vic 3010, Australia
[2] Wistar Inst Anat & Biol, Philadelphia, PA 19104 USA
[3] Murdoch Childrens Res Inst, F Douglas Stephens Surg Res Lab, Parkville, Vic, Australia
关键词
insulin; 3; relaxin-like factor; testis descent; gubernaculum; solid phase peptide synthesis;
D O I
10.1002/psc.344
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The recently identified protein, insulin 3 (INSL3), has structural features that make it a bonafide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure With conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum. to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. Me purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3,3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the super-family, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high beta -turn and alpha -helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL-3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure. Copyright (C) 2001 European Peptide Society and John Wiley & Sons, Ltd.
引用
收藏
页码:495 / 501
页数:7
相关论文
共 19 条
[1]   Specific, high affinity relaxin-like factor receptors [J].
Büllesbach, EE ;
Schwabe, C .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (32) :22354-22358
[2]   A NOVEL LEYDIG-CELL CDNA-DERIVED PROTEIN IS A RELAXIN-LIKE FACTOR [J].
BULLESBACH, EE ;
SCHWABE, C .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (27) :16011-16015
[3]  
CHENG Y, 1973, BIOCHEM PHARMACOL, V22, P3099
[4]   Solid-phase synthesis of ovine Leydig cell insulin-like peptide - a putative ovine relaxin? [J].
Dawson, NF ;
Tan, YY ;
Macris, M ;
Otvos, L ;
Summers, RJ ;
Tregear, GW ;
Wade, JD .
JOURNAL OF PEPTIDE RESEARCH, 1999, 53 (05) :542-547
[5]   Hormonal control of gubernaculum development during testis descent:: Gubernaculum outgrowth in vitro requires both insulin-like factor and androgen [J].
Emmen, JMA ;
McLuskey, A ;
Adham, IM ;
Engel, W ;
Grootegoed, JA ;
Brinkmann, AO .
ENDOCRINOLOGY, 2000, 141 (12) :4720-4727
[6]   COMPUTED CIRCULAR DICHROISM SPECTRA FOR EVALUATION OF PROTEIN CONFORMATION [J].
GREENFIE.N ;
FASMAN, GD .
BIOCHEMISTRY, 1969, 8 (10) :4108-&
[7]  
Ivell R, 1997, Rev Reprod, V2, P133, DOI 10.1530/ror.0.0020133
[8]   Cryptorchidism in mice mutant for Insl3 [J].
Nef, S ;
Parada, LF .
NATURE GENETICS, 1999, 22 (03) :295-299
[9]   Relaxin binds to and elicits a response from cells of the human monocytic cell line, THP-1 [J].
Parsell, DA ;
Mak, JY ;
Amento, EP ;
Unemori, EN .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (44) :27936-27941
[10]  
Spiess AN, 1999, MOL REPROD DEV, V54, P319, DOI 10.1002/(SICI)1098-2795(199912)54:4&lt