Molecular cloning and localization of a calpain-like protease from the abdominal muscle of Norway lobster Nephrops norvegicus.
Calpains are ubiquitous cysteine-proteases found in many, if not all, living organisms and their roles within these organisms are diverse, ranging from the mediation of cytoskeletal remodeling to the regulation of gene expression. In crustaceans calpains have so far been shown to be important mainly during moulting and growth. In the present study we report the expression of a calpain in the abdominal muscle of Norway lobster (Nephrops norvegicus) using degenerate primer, rapid amplification of cDNA ends (5'-3'-RACE), reverse transcriptase-PCR and RNA in situ hybridization approaches. The full-length mRNA sequence (2,774 bp) was found to include an open reading frame (bp 225-1,940) encoding a 572 amino acid polypeptide with a predicted mass of 65.9 kDa and a predicted pI of 5.17. The calpain was found to be an arthropod M-class calpain homologue to Homarus americanus Calpain M (Ha-CalpM) and has thus been termed Nephrops norvegicus calpain M (Nn-CalpM). When its expression pattern in abdominal muscle of adult intermoult Nephrops norvegicus was investigated an exclusive expression in a thin layer of connective tissue cells surrounding muscle fibres was found. This localization suggests a role in tenderizing connective tissue networks during growth and moulting.
Gornik
S G
SG
Division of Ecology and Evolutionary Biology (DEEB), Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK. sebastian.gornik@googlemail.com
Westrop
G D
GD
Coombs
G H
GH
Neil
D M
DM
D000005:Abdomen; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002154:Calpain / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004076:Digoxigenin / Q000378:metabolism; D005786:Gene Expression Regulation; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D008121:Nephropidae / Q000201:enzymology*; D009664:Norway; D010802:Phylogeny; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D018076:DNA, Complementary; D002154:Calpain; D004076:Digoxigenin