Two hexameric cyanoprotein subunits from an insect, Riptortus clavatus

Abstract

Hemolymph of a hemipteran insect, Riptortus clavatus, contains four distinct hexameric proteins (cyanoproteins) composed of two distinct subunits, CPA and CPβ, which show profiles of abundance depending on developmental stage, diapause status controlled by juvenile hormone, and sex. We have isolated cDNA clones encoding the two subunits and determined the complete sequences. The nucleotide sequences predict polypeptides of 693 and 691 residues for CPA and CPβ, respectively, including identical 16-residue signal peptides. The deduced amino acid sequences of both CPA and CPβ have significant similarity to other hemocyanin-related proteins, indicating that the cyanoproteins represent hexamerins. Phylogenetic analyses show that the cyanoprotein subunits can be grouped together with other hexamerins from exopterygote insects. Developmental Northern-blot analyses suggest that the expression of the cyanoproteins is regulated at the level of transcript abundance. In addition, the expression of the CPA subunit in female adults has been shown to be enhanced by juvenile hormone (JH) while the expression of the CPβ subunit is suppressed by the same hormone. To our knowledge, the CPA subunit of R. clavatus is the first case of a JH-enhanceable hexamerin whose sequence has been determined.Keywords : insect; hexamerin; juvenile hormone regulation; molecular phylogeny.The hexamerins are arthropod hexameric or multi-hexameric serum proteins composed of identical or distinct subunits with molecular masses of around 80 kDa. The hexamerin superfamily includes insect hemolymph proteins as well as hemocyanins of crustaceans, chelicerates and myriapods [1]. In contrast to the established function of hemocyanins as copper-binding oxygen carriers [2], the insect hexamerins perform several functions, such as amino acid reservoirs (storage proteins) for non-feeding developmental stages [3Ϫ7], as a major yolk protein [8,9], and as binding proteins for riboflavin [10,11] and juvenile hormone (JH) [12].Insect hexamerins so far sequenced have been classified into the following five groups primarily based on amino acid sequence alignment [13,14]: (a) lepidopteran methionine-rich hexamerins; (b) lepidopteran aromatic-amino-acid-rich arylphorins ; (c) lepidopteran JH-suppressible hexamerins; (d) dipteran arylphorins; (e) coleopteran and dictyopteran JH-suppressible arylphorins. Our present understanding of insect hexamerins, thus, is mainly from the studies on higher endopterygote insect orders. Recently, several research groups have reported the cDNA cloning of proenzymes of insect and crustacean pheCorrespondence to K. Miura, Department of Medical Zoology, School of Medicine, Mie University, 2-174 Edobashi, Tsu City, Japan 514Fax: ϩ81 59 231 5215. E-mail: k-miura@doc.medic.mie-u.ac.jp Abbreviations. CP, cyanoprotein; JH, juvenile hormone; JHA, juvenile hormone analog.Note. The nucleotide sequence data of CPA and CPβ reported in this paper appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the accession numbers D87272 an...

Characterization of the Drosophila Methoprene ‐tolerant gene product

“…Total RNA was isolated from S2 cells as described previously [50]. First-strand cDNA was synthesized by Superscript reverse transcriptase II (Invitrogen) with oligo dT primer, and used as a template for RT-PCR.…”

Isolation, Characterisation and Molecular Imaging of a High-Molecular-Weight Insect Biliprotein, a Member of the Hexameric Arylphorin Protein Family

“…They are present in eggs and hemolymph as complexes formed from two different subunits. 15,30 However, the proportion of aromatic amino acids present is only about 15%, significantly lower than that of typical arylphorins possibly indicating a different role of these cyanoproteins the expression of which is activated by juvenile hormone.…”

“…Examples of these are the arylphorins from dipterans, hexamerins binding juvenile hormone, riboflavin, or biliverdin, hexamerins the expression of which is repressed or enhanced by juvenile hormone, and hexamerins rich in glutamine. [12][13][14][15][16][17] In many cases, the expression of hexamerins is under developmental and tissue-specific control. Arylphorins, for example, are typically produced in the larval fat body, accumulate in the hemolymph before larval-pupal transformation and are thought to deliver amino acids required for the formation of the adult insect and of the sclerotized cuticle that consumes large quantities of aromatic amino acids.…”

A COUP-TF/Svp homolog is highly expressed during vitellogenesis in the mosquito Aedes aegypti

“…Total RNA was prepared from the fat body using the guanidine-isothiocyanate method as described previously (Miura et al 1998) with the modification that all isopropanol precipitation steps were done without low-temperature incubation to avoid co-precipitation of glycogen and salts. Poly(A + ) RNA was isolated utilizing the QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech Inc.).…”