Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis.
The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.
Bulau
Patrick
P
Institute for Molecular Physiology and Developmental Biology, University of Bonn, Endenicher Allee 11-13, D-53115 Bonn, Germany. patrick.bulau@innere.med.uni-giessen.de
Okuno
Atsuro
A
Thome
Elke
E
Schmitz
Tina
T
Peter-Katalinic
Jasna
J
Keller
Rainer
R
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017952:Ganglia, Invertebrate / Q000737:chemistry / Q000378:metabolism; D007447:Invertebrate Hormones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D018978:Molting / Q000235:genetics*; D036361:Peptide Hormones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007447:Invertebrate Hormones; D036361:Peptide Hormones; D011498:Protein Precursors