Three tetraspanins from Chinese shrimp, Fenneropenaeus chinensis, may play important roles in WSSV infection

Abstract

Three members of the tetraspanin/TM(4)SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM(4)SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene (FcCD9) was specifically expressed in the hepatopancreas. While for the CD63 gene (FcCD63), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63, the tetraspanin-3 gene (FcTetraspanin-3) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9, FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM(4)SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection.

Blocking the large extracellular loop (LEL) domain of FcTetraspanin-3 could inhibit the infection of white spot syndrome virus (WSSV) in Chinese shrimp, Fenneropenaeus chinensis

“…2), which illuminated the FcTetraspanin-3 might be important and constitutive. The transcript level of FcTetraspanin-3 was detected also in all the examined tissues, and high transcription was observed in intestine and ventral nerve cord [24]. However, the relation between mRNA and protein was not strictly linear.…”

“…Hepatopancreas is the digestive organ in crustaceans that performs functions similar to those of the liver and pancreas in vertebrates, it is crucial in the immune system in penaeid shrimp [45]. The human Tetraspanin-3 could express highly in liver [40], and the three tetraspanins found in shrimp, including FcTetraspanin-3, FcCD63 and FcCD9 were observed in hepatopancreas [24]. It can be concluded that hepatopancreas not only initiates the humoral immune response in shrimp, but also maintains highly specialized phagocytes and cells that function in the cellular immune response [46,47].…”

“…FcTetraspanin-3 (EF032649) contains a 720 bp open reading frame (ORF), which encodes 239 amino acids [24]. The position of LEL motif in FcTetraspanin-3 is from 104 to 205, containing 101 amino acids, and the molecular weight (MW) of LEL is about 11 kDa.…”

“…Restriction enzyme cutting site sequence of BamHⅠ and SalⅠ were introduced separately to the 5 0 end of LEL-f and LEL-r, and obtained LEL-F and LEL-R (shown in Table 1). PCR was performed using the previously cloned shrimp cDNA [24] and primers LEL-F and LEL-R, under the following conditions: one cycle of 94 C for 5 min; 35 cycles including denaturation at 94 C for 30 s, annealing at 58 C for 30 s and extension at 72 C for 30 s; followed by one cycle of 72 C for 10 min. Then the amplified fragment was ligated into pMD-19T vector (Invitrogen).…”

“…Our previous study cloned and identified the full length cDNA of one tetraspanin (FcTetraspanin-3) from Chinese shrimp Fenneropenaeus chinensis, and observed that it was markedly up-regulated in the live WSSV-challenged shrimp tissues [24]. In order to understand the function of FcTetraspanin-3 during the WSSV infection, the LEL motif sequence of FcTetraspanin-3 was cloned into pGEX-4T-1 vector and the recombinant peptide was expressed in Escherichia coli BL21 (DE3).…”