Structure and Characterization of Eriphia verrucosa Hemocyanin

Abstract

Arthropod hemocyanins (Hcs) are a family of large extracellular oxygen-transporting proteins with high molecular mass and hexameric or multi-hexameric molecular assembly. This study reports for the first time the isolation and characterization of the structure of an arthropod hemocyanin from crab Eriphia verrucosa (EvH) living in the Black Sea. Its oligomeric quaternary structure is based on different arrangements of a basic 6 × 75 kDa hexameric unit, and four of them (EvH1, EvH2, EvH3, and EvH4) were identified using ion-exchange chromatography. Subunit 3 (EvH3) shows high similarity scores (75.0, 87.5, 91.7, and 75.0 %, respectively) by comparison of the N-terminal sequence of subunit 1 from Cancer pagurus of the North Sea (Cp1), subunits 3 and 6 of Cancer magister (Cm3 and Cm6), and subunit 2 of Carcinus aestuarii (CaSS2), respectively. Moreover, a partial cDNA sequence (1309 bp) of E. verrucosa hemocyanin encoding a protein of 435 amino acids was isolated. The deduced amino acid sequence shows a high degree of similarity with subunits 3, 4, 5, and 6 of C. magister (81-84 %). Most of the hemocyanins are glycosylated, and three putative O-linkage sites were identified in the partial amino acid sequence of EvH at positions 444-446, 478-480, and 547-549, respectively. The higher stability of native Hc in comparison to its subunit EvH4 as determined by circular dichroism (CD) could be explained with the formation of a stabilizing quaternary structure.

Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca)

“…In our preliminary study, we have identified a high identity (65%) between EvH and CaeSS2 of C. aestuarii (P 84293.1). These values are very close to the homology reported between the subunits of other hemocyanins and we have suggested that this fragment belongs to EvH4 or EvHc5 [54]. The isolated here Hc subunit of E. verrucosa showed high sequence identity (>60%) with hemocyanins from organisms belonging to the class Malacostraca: Decapoda, Amphipoda, and Isopoda.…”

“…Glycosylation is frequently observed in crustacean hemocyanins [27,[50][51][52][53]. The native hemocyanin isolated from E. verrucosa is glycosylated but the concentration of monosaccharides is very low [54]. To determine the position of potential glycosylated sites of EvHc5, a 3D-model was performed, which showed the glycosylated sites exposed on the surface of the subunit at the same place as in P. interruptus (Figure 5).…”

Identification and in silico characterization of hemocyanin γ-type subunit protein in Vibrio harveyi infected freshwater prawn, Macrobrachium rosenbergii

“…A 2 x 6-meric hemocyanin nearly identical to hemocyanin found in decapod crustaceans has been studied from springtail taxa (Collembola) (Schmidt et al, 2019). Further, 6 x 75 kDa hexameric unit oligomeric quaternary structure has been refined for hemocyanin from crab Eriphia verrucosa (Dolashki et al, 2015). As understood from the sequence-level features and domain analysis of Mr_HC_1, the hemocyanin_N is all alpha domains in all chains with beta-fold recognized in the hemocyanin_M and hemocyanin_C domains.…”

Jumping on the Edge—First Evidence for a 2 × 6-meric Hemocyanin in Springtails

“…Further investigations are necessary to determine the complete sequence of subunit ScuHc2 and to answer this question. For example, the complete amino acid sequence and the glycosylation of the hemocyanins from the malacostracean taxa Carcinus aestuarii and Eriphia verrucosa helped to understand the quaternary structure of the molecules [31,32]. These analyses could also help to gain a better understanding of the quaternary structure of hexapod hemocyanins.…”