D. Sellos

Adaptation of trypsin, chymotrypsin and α-amylase to casein level and protein source in Penaeus vannamei (Crustacea Decapoda)

Abstract Adaptation of digestive enzymes to dietary proteins was studied in Penaeus vannamei at the level of enzyme activity, synthesis measured by an immunoquantitative assay and polymorphism estimated for enzymes by electrophoresis and for mRNA by RT-PCR amplification. An initial experiment was conducted over 20 days with dietary casein increasing between 25 and 48%, starch and the other constituants remaining unchanged. A significant dose response effect was established between trypsin and casein ranging from 25 to 40% in the diet. No apparent change in the isoform pattern was recorded. Chymotrypsin varied neither at the level of specific activities nor at the level of polymorphism. Amylase activities and amounts decreased while casein increased. Two major isoforms were detected for 25% casein in the diet and only one for 40% casein. In order to establish the level of polymorphism of the expressed amylase mRNAs, a 378 bp fragment was obtained by RT-PCR on total RNA. In the case of the 25% casein diet, two different fragments, which corresponded to two proteins differing by an acidic charge, were amplified while only one was amplified in the case of the 40% casein diet. A comparison of casein with other sources of protein, gelatin, squid meal and fish protein soluble concentrate at two different concentrations in the diets (25 and 40%, respectively) confirmed the stimulation of trypsin by casein. No dose response was measured with other protein sources: moreover, an inhibition of trypsin activity was measured with gelatin in the diet. Highest activities of chymotrypsin were measured with casein and squid meal diets and lowest activities with gelatin and FPSC diets. Concerning amylase, the same variations were measured. Moreover, all the 40% protein diets inhibited the expression of one isoform.

Molecular cloning of hemocyanin cDNA from Penaeus vannamei (Crustacea, Decapoda): structure, evolution and physiological aspects

Hemocyanin is present as 2 subunits in the hemolymph of Penaeus vannamei. Isolated from a hepatopancreas cDNA library of this penaeid shrimp, the cDNA chain (2095 bp) corresponds to a full length hemocyanin messenger as determined by Northern hybridization, with a short 5′ untranslated region (17 bp), an open reading frame (1989 bp counting initiation and termination codons) coding for a signal peptide (13 residues) and a mature hemocyanin (648 amino acids), and a 3′ untranslated region (89 bp) followed by the polyadenylated track. It is the first time that the existence of a hydrophobic signal peptide is shown in arthropod hemocyanin. Two primary N‐terminal sequences are determined and a 3‐fold increase of mRNA content, measured in the hepatopancreas during the premoult stages, is reported. The low level of polymorphism shown by P. vannamei hemocyanin, along with its weak percentage identity with counterparts and its similarity with hemocyanin from Panulirus interruptus, suggests that this arthropod hemocyanin may be a primitive subunit that has evolved independently, following gene duplication.

Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda)

Two clones were isolated by screening a shrimp hepatopancreas cDNA library with a DNA fragment obtained by PCR amplification using two oligonucleotides based on the partial protein sequence of Penaeus vanamei chymotrypsin purified earlier. One of these clones, PVC 7 contains a complete cDNA coding for a serine protease. The deduced amino acid sequence shows the existence of a 270 residue‐long preproenzyme containing a highly hydrophobic signal peptide of 14 amino acids. This suggests the existence of a putative zymogen form of the enzyme containing a 30 amino acid‐long peptide which is cleaved to give a mature protein of 226 residues. A highly preferred codon usage is observed for this protein. The other obtained cDNA was found to encode the less predominant variant of the protein. Sequence alignments show that shrimp chymotrypsin is highly homologous with crab collagenase (77% homology taking into account the same amino acid at the same position, and 83% homology taking into account amino acids wall conserved function) and that it is more similar to mouse trypsin (41% homology of strictly conserved amino acids) than to hornet chymotrypsin (35% homology).

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

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.

Purification, biochemical characterization and N-terminal sequence of a serine-protease with chymotrypsic and collagenolytic activities in a tropical shrimp, penaeus vannamei (crustacea, decapoda)

1. Two chymotrypsin variants, with collagenolytic activities, were purified from the hepatopancreas of Penaeus vannamei using radioactive protein as the substrate. 2. These proteases are very close as far as amino acid composition, molecular weight, inhibitors studies and specificity against small synthetic substrates are concerned. 3. N-terminal amino acid sequences of both variants are identical and are very close to other known crustacean serine proteases.

CLONING AND SEQUENCING ANALYSIS OF THREE AMYLASE CDNAS IN THE SHRIMP PENAEUS VANNAMEI (CRUSTACEA DECAPODA) : EVOLUTIONARY ASPECTS

InPenaeus vannamei, α-amylase is the most important glucosidase and is present as at least two major isoenzymes which have been purified. In order to obtain information on their structure, a hepatopancreas cDNA library constructed in phage lambda-Zap II (Strategene) was screened using a synthetic oligonucleotide based on the amino acid sequence of a V8 staphylococcal protease peptide ofP. vannamei α-amylase. Three clones were selected: AMY SK 37 (EMBL sequence accession number: X 77318) is the most complete of the analyzed clones and was completely sequenced. It contains the complete cDNA sequence coding for one of the major isoenzymes of shrimp amylase. The deduced amino acid sequence shows the existence of a 511-residue-long pre-enzyme containing a highly hydrophobic signal peptide of 16 amino acids. Northern hybridization of total RNA with the amylase cDNA confirms the size of the messenger at around 1,600 bases. AMY SK 28, which contains the complete mature sequence of amylase, belonged to the same family characterized by a common 3′ terminus and presented four amino acid changes. Some other variants of this family were also partially sequenced. AMY SK 20 was found to encode a minor variant of the protein with a different 3′ terminus and 57 amino acid changes.Phylogenetic analysis established with the conserved amino acid regions of the (β/α) eight-barrel domain and with the total sequence ofP. vannamei showed close evolutionary relationships with mammals (59–63% identity) and with insect α-amylase (52–62% identity). The use of conserved sequences increased the level of similarity but it did not alter the ordering of the groupings. Location of the secondary structure elements confirmed the high level of sequence similarity of shrimp α-amylase with pig α-amylase.

Ecdysteroid levels during embryogenesis in the shrimp, Palaemon serratus (Crustacea decapoda): Quantitative and qualitative changes

During the embryogenesis of Palaemon serratus profound changes in ecdysteroid concentrations were found. Ecdysteroid concentrations increase at the appearance of the Y-organ and a slight decrease is observed just before hatching. Two peaks of ecdysteroid concentration occur between these two events if Palaemon is reared at 19 degrees, but only one broad maximum is observed after rearing at 11-12 degrees. The pattern of ecdysteroids changes during embryogenesis; the ecdysone level decreases, while the 20-OH-ecdysone concentration and the amount of high polarity products increase. During the embryonic stage C the ratio of 20-OH-ecdysone to ecdysone was shown by HPLC analysis to steadily increase. After enzymatic hydrolysis of high-polarity products the main RIA-positive material elutes from reversed-phase HPLC columns as 20-OH-ecdysone.

Tissue expression of two α-amylase genes in the Pacific oyster Crassostrea gigas. Effects of two different food rations

A semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to estimate tissue-specific expression of two amylase genes in five different tissues of the Pacific oyster Crassostrea gigas and the experimental effects of food level on the cellular, enzymatic and gene expression changes of the digestive gland. Results indicated that both genes were transcribed at different levels depending on tissue type. A strong preferential expression of both amylase genes was observed in the digestive gland leading to high amylase activities, in accordance with the digestive function of this organ. Traces of gene expression and activity were observed in other analysed tissues. These appeared higher in labial palps than in other non-digestive tissues. After 4 weeks of feeding two groups of oysters on either a high or low food level, cellular growth of the digestive gland (+56%) and increase in the total activity of amylase enzymes and in specific amylase activity (+50%) were observed under high food conditions compared to low food conditions. A significant increase (+18%) in the expression of gene A was observed in the digestive gland of oysters fed the high ration compared to that of oysters fed on the low food level whereas no changes in the expression of gene B were observed. Gene A was expressed at a higher level than gene B in all tissues and at all conditions. A role of the amylase gene A in digestive processes and changes in its expression according to food availability is demonstrated in this study. The functional role of the α-amylase B gene remains to be elucidated. Author keywords: α-Amylase; Digestive enzyme activity; Food regulation; Gene expression; Oyster

CATHEPSIN L GENE ORGANIZATION IN CRUSTACEANS

The gene structure of a cathepsin L of the shrimp Penaeus vannamei has been determined by the polymerase chain reaction. It comprises six exons of various lengths spanning a total of 1792bp. This architecture is homologous to that of rat cathepsin L, three conserved sites of intron position have been effectively identified, with the exception of the third intron break-point located immediately after the cysteinyl active site. In contrast, no similarity is observed with Drosophila or Plasmodium cathepsin L-like gene organizations. This gene expresses a major cathepsin L enzyme in the hepatopancreas. The last intron is polymorphic, suggesting the presence of at least three different genes.

Structure of amylase genes in populations of Pacific Cupped oyster ( Crassostrea gigas): tissue expression and allelic polymorphism.

Using the previously determined complementary DNA Sequence of Crassostrea gigas amylase (Y08370), we designed several oligonucleotide primers and used them with polymerase chain reaction (PCR) technology to characterize oyster amylase gene sequences. Two genes encoding 2 different amylases were characterized and sequenced. The 2 genes are similarly organized with 8 exons and 7 introns. Intron insertions are found at the same location in the 2 genes. Sizes and nucleotide sequences are different for the different introns inside each gene and different for the corresponding introns in the 2 genes. Comparing the 2 genes, around 10% of the nucleotides are different along the exons, and comparing the 2 deduced protein sequences, a mean value of 10.4% of amino acids are changed. Genes A and B encode mature proteins of, respectively, 500 and 499 amino acids, which present 94% similarity. A microsatellite (TC37) that constitutes the largest part of intron 4 of gene A has been used as a polymorphic marker. A method consisting of a PCR step followed by EcoRI digestion of the obtained fragments was used to observe polymorphism in these 2 genes. Six and 4 alleles for genes A and B, respectively, have been sequenced, leading to a maximum of 2.9% base change. The 2 genes are ubiquitously expressed in the different digestive tissues with quantitative differences. Gene A is strongly expressed in the digestive gland and at a lower level in stomach, while gene B is preferentially expressed in the labial palps. The microsatellite repeat was used in the analysis of 4 populations of Crassostrea gigas from the French Atlantic coast. A high level of polymorphism observed with 30 different alleles of gene A inside the populations should allow their characterization using the mean value of the microsatellite allelic distribution. These populations showed a low level of differentiation (Fst between 0 and 0.011); however, the population of Bonne Anse appeared to be distinguished from the other populations.