New members of the brachyurins family in lobster include a trypsin‐like enzyme with amino acid substitutions in the substrate‐binding pocket

Abstract

Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed.

Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways

“…GU338026), PaTry2 (GU338028), PaTry3 (GU338029) and PaTry4 (GU338030). Specific primers (Biomers.net, Ulm, Germany) used for lobster trypsins were successfully employed before (Perera et al, 2010a) and are shown in Table2. The efficiency of target amplification for each primer set was optimized in the previous study by using 4pmoll -1 of primers and 60°C of annealing/extension (Perera et al, 2010a); thus, these conditions were employed here.…”

“…Specific primers (Biomers.net, Ulm, Germany) used for lobster trypsins were successfully employed before (Perera et al, 2010a) and are shown in Table2. The efficiency of target amplification for each primer set was optimized in the previous study by using 4pmoll -1 of primers and 60°C of annealing/extension (Perera et al, 2010a); thus, these conditions were employed here. By means of calibration curves (10-fold dilutions, corresponding to cDNA in reactions from 20ng to 0.2pg) all primer pairs were checked to produce similar efficiencies, except PaTry4.…”

“…cDNAs were synthesized using the qScript TM cDNA Synthesis Kit (Quanta BioSciences, Gaithersburg, MD, USA) and then used as templates for RT-qPCR on a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) using PerfeCTa TM SYBR ® Green Fast-Mix TM (Quanta BioSciences) in white wells twin.tec real-time PCR plates 96 (Eppendorf). Four previously isolated lobster trypsin cDNAs (Perera et al, 2010a) were studied: PaTry1a (GenBank accession no. GU338026), PaTry2 (GU338028), PaTry3 (GU338029) and PaTry4 (GU338030).…”

“…Four full-length lobster trypsin cDNAs were amplified as detailed before (Perera et al, 2010a) and cloned into plasmids using the TOPO TA Cloning ® Kit (Invitrogen Ltd, Paisley, UK). Plasmids were extracted from Transformed One Shot ® TOP10 competent Escherichia coli cells using the GenElute TM Five-Minute Plasmid Miniprep Kit (Sigma-Aldrich, St Louis, MO, USA).…”

“…In Panulirus argus, trypsin is a polymorphic enzyme (Perera et al, 2008a) and trypsin cDNA showing different expression rates has been cloned (Perera et al, 2010a). Additionally, trypsin isoforms in lobster appear to differ in catalytic properties or specificity as differences in digestion efficiency have been found among three distinct trypsin phenotypes (Perera et al, 2010b).…”