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Cloning and expression of cathepsinl-like proteinases in the hepatopancreas of the shrimpPenaeus vannamei during the intermolt cycle

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Abstract

Cysteine protease activities have been characterized with benzyloxycarbonyl-lysinep-nitrophenyl ester as a synthetic substrate and E64 as a specific inhibitor in the hepatopancreas of the shrimpPenaeus 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 encodingP. 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 andFasciola), 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.

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Abbreviations

Cbz-Lys-ONp :

benzyloxycarbonyllysinep-nitrophenyl ester

cDNA :

complementary DNA

EMBL :

accession n: X85127

FCP :

Fasciola hepatica cysteine protease

LCP :

lobster cysteine protease

NCE :

Norway lobster cysteine proteases from stomach

NCP :

Norway lobster cysteine proteases from nervous system

PAP :

papain

PCP 1 :

Penaeus vannamei cysteine protease 1

PCP 2 :

Penaeus vannamei cysteine protease 2

RCB :

rat cathepsin B

RCL :

rat cathepsin L

RCS :

rat cathepsin S

SDS :

sodium dodecyl suflate

SSC :

standard sodium citrate

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Authors and Affiliations

  1. Laboratoire de Biologie marine URM IFREMER-Collège de France, BP 225, F-29182, Concarneau Cedex, France

    C. Le Boulay, A. Van Wormhoudt & D. Sellos

Authors
  1. C. Le Boulay
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  2. A. Van Wormhoudt
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  3. D. Sellos
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Communicated by G. Heldmaier

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Le Boulay, C., Van Wormhoudt, A. & Sellos, D. Cloning and expression of cathepsinl-like proteinases in the hepatopancreas of the shrimpPenaeus vannamei during the intermolt cycle. J Comp Physiol B 166, 310–318 (1996). https://doi.org/10.1007/BF02439917

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  • DOI: https://doi.org/10.1007/BF02439917

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