Complete Hemocyanin Subunit Sequences of the Hunting SpiderCupiennius salei

Abstract

Hemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 ؋ 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many "modern" entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 ؋ 6-mer hemocyanins. Here we report the complete primary structure of the 2 ؋ 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrixassisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 ؋ 6-mer half-molecule most likely is composed of subunits 1-6, with subunit 1 linking the two hexamers via a disulfide bridge located in a C-terminal extension. The C. salei hemocyanin subunits all belong to the arachnid g-type, whereas the other six types (a-f) have been lost in evolution. The reconstruction of a complex hemocyanin from a single g-type subunit, which commenced about 190 million years ago and was completed about 90 million years ago, might be explained by physiological and behavioral changes that occurred during the evolution of the entelegyne spiders.

Cross-bracing uncalibrated nodes in molecular dating improves congruence of fossil and molecular age estimates

“…Contingency of subunit loss on physiology is supported by the observation that many derived spider species have lost most hemocyanin subunits and/or undergone duplications of remaining paralogs (e.g., the g paralogs of Cupiennius salei; absence of hemocyanin in Dysdera sp. [30,31]) Accordingly, biochemical assays have not identified hemocyanins in Pycnogonida, Solifugae, or Acariformes ( [26,27,60]). We note that hemocyanins are also not observed in the genomes of the mite Tetranychus urticae (Acariformes) or the tick Ixodes scapularis (Parasitiformes).…”

“…Most arachnids (terrestrial chelicerates) bear an archetypal 4 × 6 (24-mer) hemocyanin, whereas horseshoe crab hemocyanin consists of an 8 × 6 (48-mer) configuration [29]. Some variation in the 4 × 6 hemocyanin macromolecule of arachnids occurs in entelegyne spiders, wherein certain lineages have lost multiple subunits [26,27,30]. Hemocyanins do not occur in various terrestrial chelicerate orders that lack book lungs (e.g., Acariformes [mites]) or in sea spiders (Pycnogonida), although a recent study reported the presence of a single hemocyanin ortholog in the EST library of the pycnogonid Endeis spinosa [31].…”

Similar enzyme activation and catalysis in hemocyanins and tyrosinases

“…The differences in quaternary structures and consequences for the evolution of these proteins were discussed recently (van Holde et al, 2001). The basic hexameric arthropod hemocyanin consists of heterogeneous subunits with a M R of about 72 kDa, which have a common fold consisting of three domains (Ballweber et al, 2002). The "four α-helix bundle", which carries the active site with the two copper atoms, is located in the second domain ( Fig.…”

Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes

“…Arthropod hemocyanins, greatly different from those of molluscs in sequence, tertiary structure, and quaternary organization (reviewed in ref. 22), are built up in vivo as oligomers of hexamers, with each hexamer composed of heterogeneous subunits with a M r of Ϸ72 kDa (27). Arthropod hemocyanins are homologous to PPOs and storage hexamerins in insect hemolymph (26).…”