Molecular cloning and characterization of Fc-TSP from the Chinese shrimp Fennerpenaeus chinensis.

Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that modulate cell behavior in homeostasis and during development, wound-healing, immune response and tumor growth of adult tissues in vertebrates. In invertebrates these proteins are a major component of cortical rods in mature oocytes. A fragment of a thrombospondin-like gene was generated by screening a subtractive cDNA library constructed from the hemocytes of Chinese shrimp, Fennerpenaeus chinensis. The full length F. chinensis cDNA of thrombospondin was cloned by 3'- and 5'-rapid amplification of cDNA ends (3'- and 5'-RACE). The complete cDNA sequence, named Fc-TSP, is 2886 bp and the open reading frame of the cDNA encodes a 938-residue protein that contains three ChtBD2 domains, an EGF domain, a TSP-3 domain and a common TSP-C (CTD) domain. The protein shares a high sequence identity with the mj-TSPa (46.3%), mj-TSPb (46.9%) and mj-TSPc (51.9%) of Marsupenaeus japonicus. The expression and distribution of Fc-TSP in both challenged and unchallenged shrimps were studied by Northern blot, RT-PCR and in situ hybridization. Northern blot analysis showed that the Fc-TSP transcripts were detected in the hemocytes, heart, intestine, stomach and ovary of both challenged and unchallenged shrimps, but the signal was much stronger in the challenged tissues. A strong hybridization signal was detected only in challenged hepatopancreas, with no signal in the unchallenged tissue. The RT-PCR showed that the Fc-TSP was detected in both challenged and unchallenged tissues including the hemocytes, heart, hepatopancreas, stomach, gills, intestine, spermary and ovary. Except for the ovary and spermary, the signal of challenged tissues was relatively stronger than that of unchallenged ones, especially in hepatopancreas. These results suggest that the thrombospondin was upregulated in the hemocytes, heart, intestine and stomach of challenged shrimp, and induced in the hepatopancreas of challenged shrimps. Therefore, Fc-TSP may be involved in the defense responses of the shrimp.