Evaluation of posttranslational processing of proopiomelanocortin in the banded houndshark pituitary by combined cDNA cloning and mass spectrometry.
Proopiomelanocortin (POMC) is cleaved into small peptides, such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormones (MSHs), and beta-endorphin (beta-END), by tissue-specific posttranslational processing in the corticotrophs of the pars distalis (PD) and melanotrophs of the neurointermediate lobe (NIL) of the pituitary. We examined the posttranslational processing of POMC in the pituitary of the banded houndshark Triakis scyllium by molecular cloning and subsequent mass spectrometric identification of the POMC-derived peptides in the pituitary extracts. One-fifth of the randomly selected clones from a Triakis pituitary cDNA library contained a cDNA encoding for POMC. Triakis prePOMC contained 4 MSHs and a single beta-END, as has been observed in case of other cartilaginous fish POMCs. These predicted hormonal segments were flanked by basic amino acid residues, which are the cleavage sites for the processing enzymes, i.e., protein convertases. Mass spectrometry was performed using PD (including most parts of the rostral and proximal PD) and NIL extracts to detect mass values corresponding to the POMC-derived peptides. Consequently, ACTH, beta-END, and the joining peptide (JP) were detected in the PD extract, while MSHs, processed beta-END, and some other POMC-derived peptides were identified in the NIL extract; however, neither acetylated alpha-MSH nor acetylated beta-END was detected in the latter. These tissue-specific POMC processing patterns are similar to those of the other vertebrate pituitaries; however, the absence of acetylated peptides suggests the lack of an acetylation system in the melanotrophs in the NIL of the Triakis pituitary.
Takahashi
Akiyoshi
A
School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan. akiyoshi@kitasato-u.ac.jp
Kobayashi
Yuki
Y
Moriyama
Shunsuke
S
Hyodo
Susumu
S
D000107:Acetylation; D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009074:Melanocyte-Stimulating Hormones / Q000378:metabolism; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D012754:Sharks / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D001615:beta-Endorphin / Q000378:metabolism
D018076:DNA, Complementary; D029941:Fish Proteins; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D009074:Melanocyte-Stimulating Hormones