Molecular cloning and characterization of three novel Hemocyanins from Chinese mitten crab, Eriocheir sinensis

• Three novel hemocyanins were firstly identified from E. sinensis.
• EsHcs contain three tandem hemocyanin domains: hemocyanin N, M or tyrosinase, and C domain.
• EsHcs were mainly expressed in hepatopancreas and were also detected in hemocyticus.
• EsHcs showed significant increase after LPS, PGN, S. aureus, V. parahaemolyticus and A. hydrophila challenge.

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustaceans. However, little is known about the hemocyanin from the Chinese mitten crab, Eriocheir sinensis. In this study, three forms of hemocyanins designated as EsHc1, EsHc2 and EsHc3 were cloned from E. sinensis by using expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The open reading frames (ORFs) of EsHc1, EsHc2 and EsHc3 genes were 2182, 2580 and 2220 bp encoding proteins with 678, 662 and 691 amino acids, respectively, and all contain three tandem hemocyanin domains: 1) hemocyanin N, 2) hemocyanin M or tyrosinase, and 3) hemocyanin C domain. BLASTP and phylogenetic tree analysis showed that EsHc1 was clustered together with cryptocyanin 1 from Portunus pelagicus (PpCc1) and cryptocyanin from Metacarcinus magister (MmCc), EsHc2 and EsHc3 with hemocyanin from Pacifastacus leniusculus (PlHc) and hemocyanin subunit 1 from M. magister (MmHc1) were gathered into one clade. EsHc1 was mainly expressed in hepatopancreas and hemocytes with a lower level of expression in nerves, eyestalk, muscles, intestine, gills and heart; whereas EsHc2 and EsHc3 were mainly expressed in hepatopancreas and were also detected in hemocytes. Quantitative real-time RT-PCR analysis showed that EsHcs mRNA transcription in hepatopancreas was significantly expressed at various time points after infection with Lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, Vibrio parahaemolyticus and Aeromonas hydrophila. In summary, there is evidence that the three isoforms of hemocyanin genes participate in the innate immune response against bacteria infecting the Chinese mitten crab.