Atsuro Okuno

Ligand-Selective Potentiation of Rat Mineralocorticoid Receptor Activation Function 1 by a CBP-Containing Histone Acetyltransferase Complex

ABSTRACT The rat mineralocorticoid receptor (MR) has two activation functions in distinct regions of the A/B domain, designated activation function 1a (AF-1a; amino acids 1 to 169) and AF-1b (amino acids 451 to 600). Since the p160 family protein TIF2, a known component of the AF-2 coactivator complex, potentiates the transactivation function of AF-1b but not that of AF-1a, it is likely that some other, novel protein complex interacts with the AF-1a region. Therefore, we attempted to identify such coactivator complexes from HeLa nuclear extracts by biochemical purification using a glutathione S-transferase-MR AF-1a fusion protein. Purified AF-1a region-interacting proteins were found to contain RNA helicase A (RHA) and CBP. Further analysis showed that RHA interacted with the AF-1a region directly and then recruited a complex with histone acetyltransferase (HAT) activity that contained CBP. For full-length MR, aldosterone, but not hydrocortisone, was found to induce the binding of RHA/CBP complexes to the AF-1a region, as well as to allow the cooperative potentiation of MR transcriptional activity by RHA and CBP. In addition, a chromatin immunoprecipitation assay showed that aldosterone-bound MR, but not hydrocortisone-bound MR, recruited RHA/CBP complexes to native MR target gene promoters. Our results suggested that an altered conformation of the A/B region induced by aldosterone, but not hydrocortisone, might determine the accessibility of MR AF-1a to RHA/CBP complexes.

Molecular cloning and expression of an otolith matrix protein cDNA from the rainbow trout, Oncorhynchus mykiss

The fish otolith is a hard tissue consisting of calcium carbonate and organic matrices. The matrix proteins play important roles in otolith formation, but little is known about the nature of these proteins. In this study, matrix proteins were extracted from the otoliths of rainbow trout, Oncorhynchus mykiss, and chum salmon, Oncorhynchus keta. EDTA-soluble matrix proteins were separated by SDS-PAGE, revealing two major components in the otoliths of both species with apparent molecular masses of 55 and 43 kDa. N-terminal and some internal amino acid sequences of the 55-kDa otolith matrix protein were determined. A cDNA fragment encoding this protein of O. mykiss was amplified by reverse transcription PCR using two degenerate primers designed from the amino acid sequences. A cDNA encoding this protein was obtained by screening a saccular cDNA library using the amplified cDNA fragment as a probe. Nucleotide sequence analysis revealed that the cDNA clone has a sequence of 2.5 kb and the open reading frame encoding 344 amino acid residues. Northern blot analysis showed that mRNA of this protein is expressed specifically in the sacculus, and consistently during the day.

Molecular Cloning and Expression Analysis of cDNAs Encoding Androgenic Gland Hormone Precursors from Two Porcellionidae Species, Porcellio scaber and P. dilatatus

Abstract Male sexual characteristics in Crustacea are induced by androgenic gland hormone (AGH), which is produced by the male-specific androgenic gland. Recently, AGH in the terrestrial isopod Armadillidium vulgare was characterized and its cDNA cloned, the first example in which the structure of AGH was elucidated. We report here the molecular cloning of cDNAs encoding AGH precursors from two additional terrestrial isopods, Porcellio scaber and P. dilatatus. cDNA fragments encoding Porcellio scaber AGH (Pos-AGH) and P. dilatatus AGH (Pod-AGH) were amplified by RT-PCR using degenerate oligonucleotide primers designed based on the amino acid sequence of A. vulgare AGH (Arv-AGH). Subsequently, full length cDNAs were obtained by 5′- and 3′-RACE. Both AGH precursors consisted of a signal peptide, B chain, C peptide and A chain, and exhibited the same organization as that of Arv-AGH. The amino acid sequences of the A and B chains, which comprise mature AGH peptide, were highly conserved among the three species, while that of the C peptide showed only low sequence similarity. In Northern blot analysis, each cDNA fragment used as a probe specifically hybridized with a single band (0.75 kb) in mRNA extracted from whole male reproductive organs. In analysis of the tissue-specific gene expression of these two AGHs by RT-PCR, it was revealed that both AGH transcripts were detected only in cDNA synthesized using total RNA from the testis carrying the androgenic glands, but not in that from testis only, seminal vesicle, vas deferens, or hepatopancreas.

PURIFICATION AND PROPERTIES OF ANDROGENIC GLAND HORMONE FROM THE TERRESTRIAL ISOPOD ARMADILLIDIUM VULGARE

Abstract The androgenic gland hormone (AGH) is known to control sex differentiation in crustaceans. AGH was purified from isolated androgenic glands (AGs) of the male isopod Armadillidium vulgare by three steps of reverse-phase high performance liquid chromatography (RP-HPLC) and its chemical properties were examined. The purified AGH-active fraction showed masculinizing activity when 38 pg of this preparation was injected into a young female of the same species. Only 160 ng of the material was obtainable from 2000 animals at about an 11% rate of recovery. The elution of AGH activity by molecular sieve HPLC indicated that molecular weight of AGH was 11,000 ∼ 13,000. AGH was inactivated by treatment with trypsin or by reductive carboxymethylation. The AGH activity was not affected by heat treatment at 100°C for 3 min. These results indicated that AGH was a heat-stable protein with disulfide bond(s).

Preparation of an active recombinant peptide of crustacean androgenic gland hormone

In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone.

Immunological identification of crustacean androgenic gland hormone, a glycopeptide.

Androgenic gland hormone (AGH) is known to be responsible for sex differentiation in crustaceans. The amino acid sequence of AGH-active fraction purified from androgenic glands of the terrestrial isopod Armadillidium vulgare was determined by immunoprecipitation employing three types of antibodies raised against differing parts of the amino acid sequence deduced from the putative AGH cDNA sequence. As all antibodies adsorbed AGH activity, it was confirmed that the sequence examined was that of AGH. The affinity of AGH to certain lectins indicated that AGH possesses a carbohydrate moiety, which is in agreement with the observation that AGH possesses an N-glycosylation consensus sequence.