Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp Macrobrachium carcinus José M. Viader-Salvadó1, José Alberto Aguilar Briseño1, Juan A. Gallegos-López1, José A. Fuentes-Garibay1, Carlos Alfonso Alvarez-González2 and Martha Guerrero-Olazarán1 1 Instituto de Biotecnología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Nuevo León, Mexico 2 Laboratorio de Acuicultura Tropical, División Académica de Ciencias Biológicas, Universidad Juárez Autónoma de Tabasco, Villahermosa, Tabasco, Mexico ABSTRACT Macrobrachium carcinus (Linnaeus, 1758) is a species of freshwater shrimp widely distributed from Florida southwards to southern Brazil, including southeast of Mexico. In the present work, we identified a putative trypsin-like protease cDNA fragment of 736 nucleotides from M. carcinus hepatopancreas tissue by the 3′RACE technique and compared the deduced amino acid sequence to other trypsin-related proteases to describe its structure and function relationship. The bioinformatics analyses showed that the deduced amino acid sequence likely corresponds to a trypsin-like protease closely related to brachyurins, which comprise a subset of serine proteases with collagenolytic activity found in crabs and other crustacea. The M. carcinus trypsin-like protease sequence showed a global sequence identity of 94% with an unpublished trypsin from Macrobrachium rosenbergii (GenBank accession no. AMQ98968), and only 57% with Penaeus vannamei trypsin (GenBank Submitted 23 December 2019 accession no. CAA60129). A detailed analysis of the amino acid sequence revealed 31 March 2020 Accepted specific differences with crustacean trypsins, such as the sequence motif at the Published 23 April 2020 beginning of the mature protein, activation mechanism of the corresponding Corresponding authors José M. Viader-Salvadó, zymogen, amino acid residues of the catalytic triad and residues responsible for [email protected] substrate specificity. Martha Guerrero-Olazarán, [email protected] Subjects Aquaculture, Fisheries and Fish Science, Biochemistry, Bioinformatics, Computational Academic editor Vladimir Uversky Biology Keywords Brachyurins, Macrobrachium carcinus, Serine proteases, Threonine proteases, Additional Information and Trypsin-like protease Declarations can be found on page 12 INTRODUCTION DOI 10.7717/peerj.9030 In crustacea, the organ in the digestive tract called the hepatopancreas or midgut gland is Copyright 2020 Viader-Salvadó et al. known to have high proteolytic activity and it is responsible for assimilating ingested Distributed under proteins. The proteolytic activity is mainly due to the presence of serine proteases, with Creative Commons CC-BY 4.0 trypsins being the main digestive endopeptidases (Muhlia-Almazán, Sánchez-Paz & García-Carreño, 2008; Linton et al., 2014; Perera et al., 2015). Trypsins (EC 3.4.21.4) are How to cite this article Viader-Salvadó JM, Aguilar Briseño JA, Gallegos-López JA, Fuentes-Garibay JA, Alvarez-González CA, Guerrero- Olazarán M. 2020. Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp Macrobrachium carcinus. PeerJ 8:e9030 DOI 10.7717/peerj.9030 serine proteases found in the digestive systems of many vertebrates and invertebrates that cleave peptides at the carboxyl side of Lys or Arg residues. Because trypsins can cause tissue damage due to autologous protein hydrolysis, they are synthesized as inactive precursors (proenzymes or zymogens) called trypsinogens. The importance of trypsins in protein digestion is given by its role in the activation of other digestive proenzymes, and trypsinogen itself. The conversion of trypsinogen to active trypsin is initiated by the specific cleavage of a small N-terminal peptide by an autoactivation process or in mammals, by the action of enterokinase. In the shrimp Penaeus vannamei, trypsin synthesis from trypsinogen has been proposed from sequence analysis (Klein et al., 1996), the demonstration of trypsinogen storage in the midgut gland has been proved (Sainz et al., 2004), and recently we demonstrated a rapid trypsinogen autoactivation process (Guerrero-Olazarán et al., 2019). Trypsin sequences from different species have been used as a model for studies of evolution. A continuous evolutionary divergence of trypsins from a common ancestor has been proposed (Rypniewski et al., 1994). Comparisons of activation peptide sequences have also been used to study the evolution and function of trypsinogen activation (Chen et al., 2003). Macrobrachium carcinus (Linnaeus, 1758) is a species of freshwater shrimp widely distributed from Florida southwards to southern Brazil, including southeast of Mexico. This species has great aquaculture potential due to its large size, high fertility in captivity and resistance to handling and stress conditions. Furthermore, it has a short larval period, is omnivorous and its meat is of good quality and widely accepted. Another freshwater crustacean of interest to aquaculture, especially in Europe, is the crayfish Astacus leptodactylus that resembles a small lobster. Nevertheless, little is known about the digestive physiology of these species. Rapid amplification of cDNA ends (RACE) is a widely used technique for obtaining a cDNA copy of a specific RNA transcript from a cell. In the 3′RACE version of this technique, cDNAs are synthesized in a reverse transcription reaction using an oligo-dT- adaptor primer directed to the natural polyA tail of eukaryotic mRNAs. In the second step, specific cDNA is amplified by a polymerase chain reaction (PCR) using a sense gene-specific primer and an anti-sense primer that is complementary to the adaptor sequence of the primer used in the first step (Frohman, Dush & Martin, 1988). In this work, the cDNA sequence of a putative trypsin-like protease from M. carcinus hepatopancreas tissue was identified by the 3′RACE technique and the deduced amino acid sequence was compared to other trypsin-related proteases to describe structure and function relationship of the enzyme. Our findings contribute to the understanding of the digestive physiology of this species and the molecular mechanism of crustacean trypsins. MATERIALS AND METHODS Specimens, plasmids, medium composition, chemicals and enzymes Macrobrachium carcinus specimens were from the Grijalva River, Centla, Tabasco, Mexico (latitude 1814′11.9″ N, longitude 9239′49.4″). All oligonucleotides were purchased from Viader-Salvadó et al. (2020), PeerJ, DOI 10.7717/peerj.9030 2/15 Integrated (DNA Technologies, Inc., Coralville, IA, USA). Escherichia coli DH5a, pGEM-T easy vector, RQ1 RNase-free DNase, SV Total RNA Isolation System, and GoTaq DNA polymerase were purchased from Promega (Madison, WI, USA). Luria-Bertani (LB) agar plates (1% tryptone, 0.5% yeast extract, 1% NaCl, 15 g/L agar, pH 7.0) with 100 µg/mL ampicillin was used for E. coli transformants selection. PfuUltra II Fusion HotStart DNA polymerase and AccuScript High-Fidelity Reverse Transcriptase were from Agilent Technologies (Santa Clara, CA, USA). RNAlater was from Life Technologies (Gaithersburg, MD, USA). All chemicals were of analytical grade and purchased from Sigma–Aldrich Co. (St. Louis, MO, USA) or from Productos Químicos Monterrey (Monterrey, Nuevo León, Mexico). 3′RACE assay Total RNA from M. carcinus hepatopancreas tissue was isolated using SV Total RNA Isolation System, treated with RQ1 RNase-free DNase and used to synthesize cDNA by reverse transcription using 14.7 µM of T17AP primer (5′-GACTCGAGTCGACATC ′ GAT17-3 )(Frohman, Dush & Martin, 1988) and 2 µl of AccuScript High-Fidelity Reverse Transcriptase in a final reaction volume of 20 µl, according to the manufacturer’s recommendations. The cDNA was amplified by PCR using the RACEAP primer (5′-GAC TCGAGTCGACATCG-3′)(Frohman, Dush & Martin, 1988) and the consensus primer Pig1 (5′-CACTTCTGCGGCGCCTCCAT-3′) designed from a highly-conserved region of crustacean trypsin nucleotide sequences. The PCR was performed in a PCR Multigene Mini thermal cycler (Labnet International Inc., Edison, NJ, USA) in a 25 µl reaction volume containing 0.5 µM of each primer, 0.24 mM dNTP’s each, 1× buffer, 1.25 U PfuUltra II Fusion HotStart DNA polymerase, and 2 µl of primary cDNA, with a 40-cycle amplification program under the following conditions: 95 C for 20 s, 60 C for 20 s and 72 C for 30 s, with a first denaturation step at 95 C for 1 min and a final extension step at 72 C for 3 min. Cloning and sequencing The amplified cDNAs were adenylated and cloned into the pGEM-T easy vector according to the manufacturer’s instructions and using E. coli DH5a as the host strain. Transformants were selected for their ability to grow on LB-agar plates with 100 µg/mL ampicillin at 37 C. Colonies were then randomly selected and plasmids were isolated by alkaline lysis extraction. The presence of M. carcinus cDNAs in the plasmids was confirmed by PCR using the Pig1 and RACEAP primers, as described above for cDNA amplification but using GoTaq DNA polymerase. Positive plasmids, as determined by PCR analysis from the different colonies of E. coli, were sequenced using T7 and SP6 universal primers and an ABI Prism 310 sequencer (Applied Biosystems, Foster City, CA, USA) at the Molecular Biology Unit, Institute of Cellular Physiology, Universidad Nacional Autónoma de México. The sequences obtained with the T7 and SP6 primers were aligned using the Contig Assembly Program (CAP) module from the BioEdit 7.2.6 program (Hall, 1999). The consensus