Molecular cloning and sequencing of two cDNAs encoding cathepsin L-related cysteine proteinases in the nervous system and in the stomach of the Norway lobster (Nephrops norvegicus)

Boulay, C. Le; Wormhoudt, Alain Van; Sellos, Daniel

doi:10.1016/0305-0491(95)00001-o

Cited by 28 publications

(8 citation statements)

References 11 publications

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“…The ATG start codon was found 41 bp from the 5¢ end of the cDNA. The short 5¢ untranslated sequence is a common feature of numerous invertebrate enzyme mRNA sequences [22][23][24][25][26]. In addition to the nucleotides encoding the N-terminus of the mature protein, those coding for a hydrophobic signal peptide of 16 residues were determined [27].…”

Section: R E S U L T S a N D Discussion Cdna Sequencementioning

confidence: 99%

Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) β‐mannanase gene

Sellos

Janson

2002

European Journal of Biochemistry

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Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a b-mannanase (mannan endo-1,4-b-mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several b-mannanases in glycoside hydrolase family 5. The b-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The b-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae a-factor signal sequence. The b-mannanase was produced in a 14-L fermenter with an expression level of 900 mgAEL. The expression level is strongly affected by the induction temperature. A two-step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure b-mannanase. However, due to post-translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulis b-mannanase was close to that of the wild-type enzyme. Also pH and temperature optima were the same as for the native protein.In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel b-mannanase. This is the first time a mollusc b-mannanase has been characterized at the DNA level.

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Section: R E S U L T S a N D Discussion Cdna Sequencementioning

confidence: 99%

Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) β‐mannanase gene

Sellos

Janson

2002

European Journal of Biochemistry

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“…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.…”

Section: The Presence Of Cysteine Proteinasesmentioning

confidence: 99%

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

Teschke

Saborowski

2005

Journal of Experimental Marine Biology and Ecology

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The utilization of dietary proteins in crustaceans is facilitated by a set of peptide hydrolases which are often dominated by btrypsin-likeQ serine proteinases. As expected, the North Sea shrimps Crangon crangon and Crangon allmani showed in their midgut glands high proteolytic activities. However, the majority of animals lacked trypsin and chymotrypsin. Conversely, a minority of about 10% of the animals had elevated trypsin activities. The appearance of trypsin was neither related to the mode of feeding nor to the nutritive state of the animals. When present, trypsin was expressed in both species as a single isoform of apparently 20 kDa. The lack of serine proteinases was also confirmed by inhibitor assays. AEBSF, a serine proteinase inhibitor, slightly reduced total proteinase activity by less than 10%. In contrast E 64, a cysteine proteinase inhibitor, caused a reduction of more than 70% of total proteinase activity, indicating that a substantial share of proteolytic activity is caused by cysteine proteinases. Cathepsin L-like proteinases were identified as major cysteine proteinases.A comparison with the eucarid crustaceans Pandalus montagui, Pagurus bernhardus, Cancer pagurus and Euphausia superba showed a similar high level of total proteinase activity in all species. Trypsin, however, varied significantly between species showing lowest activities in Caridea and the highest activity in E. superba. E 64 suppressed total proteinase activity by more than 70% in Crangon species but not in C. pagurus and E. superba. In contrast, the serine proteinase inhibitor AEBSF had only little effect in Caridea but was most effective in P. bernhardus, C. pagurus and E. superba. The results may indicate different traits of food utilization strategies in some eucarid crustaceans. Caridea may express predominantly cysteine proteinase, while in Anomura, Brachyura and Euphausiacea, serine proteinases may prevail. D

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“…Cathepsin L-like proteinases have also been identified as major gut enzymes in some crustaceans (Laycock et a/., 1989(Laycock et a/., , 1991Le Boulay et a/., 1995), and these enzymes may therefore have evolved a digestive function early in the arthropod lineage. However, CPl was also found to be expressed in other tissues such as garland cells, salivary glands, and 'macrophages' (Matsumoto et a/., 1995), indicating that this enzyme may also play other roles.…”

Section: Introductionmentioning

confidence: 98%

Cysteine proteinase 1 (CP1), a cathepsin L‐like enzyme expressed in the Drosophila melanogaster haemocyte cell line mbn‐2

Tryselius

Hultmark

1997

Insect Molecular Biology

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We have isolated cDNA clones encoding the full-length Drosophila melanogaster cysteine proteinase 1 (CP1). The clones were isolated from the Drosophila melanogaster haemocytic mbn-2 cell line, where the gene is relatively strongly expressed, giving a transcript of 1.6 kb in size. We present the sequence encoding the full-length protein, and deduced the genomic organization of the gene by comparison to previously published genomic partial sequence data. Immunofluorescence shows that CP1 is localized in small granules, probably lysosomes, in mbn-2 cells. The data presented suggest a role for cysteine proteinase in immune functions in insects. It is likely to participate in the degradation of internalized material in phagocytic cells.

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Molecular cloning and sequencing of two cDNAs encoding cathepsin L-related cysteine proteinases in the nervous system and in the stomach of the Norway lobster (Nephrops norvegicus)

Cited by 28 publications

References 11 publications

Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) β‐mannanase gene

Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) β‐mannanase gene

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

Cysteine proteinase 1 (CP1), a cathepsin L‐like enzyme expressed in the Drosophila melanogaster haemocyte cell line mbn‐2

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