Molecular characterization and phylogenetic relationships of a protein with potential oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects?

Abstract

Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.

Cuticular Pro-phenoloxidase of the Silkworm, Bombyx mori

“…The transcription of the gene was actually shown to be up-regulated by ecdysone (41). Furthermore, the transcript of the gene encoding a protein that possesses two copper binding sites, as observed in proPO or hemocyanin, was detected in the hemocytes of early locust embryos (42). Nascent polypeptide of the locust hemocyanin-like protein has a putative signal sequence for secretion in the deduced sequence, although none of the cDNAs of proPOs that have been isolated contain such signal sequences.…”

A respiratory hemocyanin from an insect

“…Approximately 5 micrograms of purified poly(A) ϩ RNA extracted from 3-year-old female nymphs of Perla marginata were used for the construction of a directionally cloned cDNA expression Lambda ZAP library (Stratagene). Various degenerated oligonucleotide primers were designed according to conserved amino acid sequences of the crustacean Hcs (20) and the embryonic hemolymph protein of S. americana (16). These primers were tested in RT-PCR experiments with the total RNA from P. marginata nymphs, applying the One Step kit according to the manufacturer's instructions (Qiagen, Valencia, CA).…”

“…Hc-like larval storage proteins (hexamerins) are present in all insect taxa studied, but they lack functional copper-binding sites and thus do not transport O 2 (14,15). The six-histidine copper-binding sites are present in another Hc-like protein identified from the embryonic hemolymph of the grasshopper Schistocerca americana (16). However, the function of this protein is uncertain because neither O 2 binding nor its expression in later developmental stages could be demonstrated.…”

The analysis of large-scale gene expression correlated to the phase changes of the migratory locust

“…Among them, 71 unigenes showed differential or specific expression at a high confidence level (P Ͻ 0.01), and 40 of them yielded functional assignments based on GO databases. The most outstanding difference is the expression of a group of unigenes that are related to a superfamily of genes that include the larval serum protein complex and juvenile hormone-binding protein, hexamerins, prophenoloxidase, and hemocyanins (JHPH) (32)(33)(34). We refer to this superfamily of genes as the JHPH superfamily.…”