Cloning and expression of cathepsin L -like proteinases in the hepatopancreas of the shrimp Penaeus vannamei during the intermolt cycle

Abstract

Cysteine protease activities have been characterized with benzyloxycarbonyl-lysine p-nitrophenyl ester as a synthetic substrate and E64 as a specific inhibitor in the hepatopancreas of the shrimp Penaeus vannamei. An optimum pH of 5.1 has been measured. To characterize these cysteine proteases, a hepatopancreas cDNA library was screened by hybridization to a Norway lobster cysteine protease cDNA fragment. Two cDNAs encoding P. vannamei cysteine protease precursors have been cloned and sequenced. The encoded polypeptides have 326 and 322 amino acid residues, respectively, each consisting of partial signal sequences (15 and 10 residues), a pro-region (93 and 94 residues), and a mature enzyme polypeptide (218 residues). Cys25, His159 and Asn175 form the catalytic triad in the putative active site of the mature enzymes. Compared with invertebrate cysteine proteases (Homarus and Fasciola), each of the two shrimp enzymes shows 70 and 52% amino acid sequence identity, respectively; 63% identity is shown with rat cathepsin L. Northern hybridization analysis showed the same size for the different cysteine protease transcripts in hepatopancreas tissue (approximately 1.1 kb). During intermolt cycles, variations in cysteine protease activity were correlated with the variations in the levels of specific mRNA.

Cysteine proteinases substitute for serine proteinases in the midgut glands of Crangon crangon and Crangon allmani (Decapoda: Caridea)

“…Maximum inhibition appeared within the peaks of highest total proteinase activity. The appearance of cathepsin isoforms in crustaceans is in agreement with Laycock et al (1989Laycock et al ( , 1991 and Le Boulay et al (1996) who isolated cathepsin-L-like cysteine proteinases from lobsters and shrimps. A detailed characterization of North Sea shrimp cathepsins, however, was beyond the scope of this work but will be the subject of future studies.…”

“…In contrast, cathepsin-like cysteine proteinases show highest activities between pH 4 and 7. The maximum activity of cysteine proteinase from P. vannamei was at pH 5.1 (Le Boulay et al, 1996). Because of the low pH-optimum in both Crangon species, we agree with Degkwitz (1957) that the majority of proteolytic enzymes in C. crangon and C. allmani may consist of cathepsin-like proteinases rather than trypsin-like enzymes.…”

“…In crustaceans, however, knowledge about cysteine proteinases is still limited. Sequences and gene structures were analysed for some cysteine proteinases (Laycock et al, 1991;Le Boulay et al, 1995, 1996, 1998; however, their role in the process of digestion is still poorly understood.…”

Molecular characterization of cathepsin L cDNA and its expression during oogenesis and embryogenesis in the oriental river prawn Macrobrachium nipponense (Palaemonidae)

“…The hepatopancreas is responsible for the synthesis of many digestive enzymes in decapods (Gibson and Barker, 1979). Therefore, the predominant expression of CatL in the hepatopancreas of oriental river prawn suggests its involvement in food digestion, since the association of CatL with food digestion has been found in other decapods such as American lobster H. americanus (Laycock et al, 1992) and Norway lobster N. norvegicus (Le Boulay et al, 1996). The evidence at the cellular level also shows that the CatL is localized in large digestive vacuoles of the digestive B cell of hepatopancreas in M. ensis (Hu and Leung, 2004).…”

“…CatL cDNAs were also cloned in Norway lobster (Nephrops norvegicus) (Le Boulay et al, 1996), northern shrimp (Pandalus borealis) (Aoki et al, 2004) and brine shrimp (Artemia franciscana) (Liu and Warner, 2006). However, there is little information on CatL cDNA in freshwater prawn species.…”