Aspartic Cathepsin D Endopeptidase Contributes to Extracellular Digestion in Clawed Lobsters Homarus americanus and Homarus gammarus

Abstract

Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding.

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

“…We determined that cathepsin D1 expression occurs in the midgut tissue but not in muscle or gonads, while cathepsin D2 appears to be stably expressed at low levels in all three. In this proteomic study, we identified 9 peptides from cathepsin D1 but no peptides from cathepsin D2 which supports our previous hypothesis that cathepsin D2 functions in the endo-lysosomal pathway and not in gastric digestion of food (Rojo et al 2010a). Three peptides corresponding to a S28 family serine peptidase were identified in the gastric juice (GenBank: EX487603.1).…”

“…This enzyme has been isolated previously, and the pH and temperature for optimum activity has been determined (Rojo et al 2013). This enzyme can readily degrade hemoglobin (Rojo et al 2010a;Rojo et al 2013) and its substrate specificity profile is similar to cathepsin D isolated from the gut European hard tick, Ixodes ricinus (Sojka et al 2012). Both of these enzymes cleave between a pair of hydrophobic residues, particularly when phenylalanine is on the amino-terminal side of the scissile bond.…”

“…For fluorescent in-gel activity assays, the gel was washed in 2.5% Triton X-100 at room temperature for 30 min, followed by incubation in the dark for 20 min with 30 µM Z-FR-AMC in buffer containing 100 mM sodium acetate pH 4.7, 2 mM EDTA, 2 mM DTT and 0.05% Triton X-100. In-gel hemoglobinase assays were performed as described previously (Rojo et al 2010a). …”

“…Since then, proteins with amylase (Wojtowicz and Brockerhoff 1972), chitinase and chitobiase specificity (Lynn 1990) have been isolated and partially characterized; however the proteins responsible for these activities have not been determined. Two peptidases capable of degrading casein and hemoglobin have also been isolated from H. americanus gastric juice (Laycock et al 1989;Laycock et al 1991;Rojo et al 2010a;Rojo et al 2010b;Rojo et al 2013). N-terminal sequencing of these proteins identified them as cathepsin L1, a cysteine peptidase (GenBank X63567) and cathepsin D1, an aspartic acid peptidase (GenBank EU687261), respectively.…”

Is digestive cathepsin D the rule in decapod crustaceans?

“…Additionally, the aspartic endopeptidase, cathepsin D, was found in the gastric fluid of the clawed lobsters Homarus gammarus and H. americanus and was identified as a fully operational extracellular digestive enzyme at the acid pH of gastric fluid (Navarrete del Rojo et al, 2010a). The presence of cathepsin D in the gastric fluid seems unusual as this enzyme is known and well characterized as typical lysosomal and, thus, intracellular endopeptidase.…”