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PLoS One
2020 Jan 01;159:e0239044. doi: 10.1371/journal.pone.0239044.
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First echinoderm alpha-amylase from a tropical sea cucumber (Holothuria leucospilota): Molecular cloning, tissue distribution, cellular localization and functional production in a heterogenous E.coli system with codon optimization.
Wu X
,
Ruan Y
,
Chen T
,
Yu Z
,
Huo D
,
Li X
,
Wu F
,
Jiang X
,
Ren C
.
Abstract
Holothuria leucospilota (Echinodermata: Holothuroidea) is a widespread tropical sea cucumber with strong value for the ecological restoration of coral reefs. Therefore, some studies regarding the artificial breeding and cultivation of H. leucospilota have been undertaken recently. However, the biological functions of the digestive system of this species have not been elucidated. In this study, a cDNA coding for α-amylase, an indicator of digestive maturity in animals, was identified from H. leucospilota and designated Hl-Amy. The full-length cDNA of the Hl-Amy gene, which is 1734 bp in length with an open reading frame (ORF) of 1578 bp, encodes a 525 amino acid (a.a.) protein with a deduced molecular weight of 59.34 kDa. According to the CaZy database annotation, Hl-Amy belongs to the class of GH-H with the official nomenclature of α-amylase (EC 3.2.1.1) or 4-α-D-glucan glucanohydrolase. The Hl-Amy protein contains a signal peptide at the N-terminal followed by a functional amylase domain, which includes the catalytic activity site. The mRNA expression of Hl-Amy was abundantly exhibited in the intestine, followed by the transverse vessel with a low level, but was hardly detected in other selected tissues. During embryonic and larval development, Hl-Amy was constitutively expressed in all stages, and the highest expression level was observed in the blastula. By in situ hybridization (ISH), positive Hl-Amy signals were observed in different parts of the three different intestinal segments (foregut, midgut and hindgut). The Hl-Amy recombinant protein was generated in an E. coli system with codon optimization, which is necessary for Hl-Amy successfully expressed in this heterogenous system. The Hl-Amy recombinant protein was purified by immobilized metal ion affinity chromatography (IMAC), and its activity of starch hydrolysis was further detected. The optimal temperatures and pH for Hl-Amy recombinant protein were 55°C and 6.0, respectively, with an activity of 62.2 U/mg. In summary, this current study has filled a knowledge gap on the biological function and expression profiles of an essential digestive enzyme in sea cucumber, which may encourage future investigation toward rationalized diets for H. leucospilota in artificial cultivation, and optimized heterogenous prokaryotic systems for producing recombinant enzymes of marine origins.
Fig 1. Full-length cDNA sequence and structural analysis of Hl-Amy.A: Full-length cDNA sequence and deduced amino acid sequence of Hl-Amy. The translational start codon (atg) and stop codon (taa) are shown in red, and the polyadenylation signal (aataaa) in the 3’-UTR is underlined. The signal peptide, domain A, domain B and domain C are shown in the boxes with different colors, and the active site, Ca2+ binding site and catalytic site are indicated with different symbols. B: Structural domain of Hl-Amy predicted using the SMART, ScanProsite and BLAST programs. The signal peptide (SP), α-amylase domain (Aamy) and α-amylase C-terminal domain (Aamy_C) are boxed, and the active site, Ca2+ binding site and catalytic site are shown.
Fig 2. Amino acid sequence alignment, phylogenetic and 3D structure analysis of Hl-Amy.A: Amino acid sequence alignment of Hl-Amy and amylase in other deuterostome animal species. The conserved amino acid residues are boxed in dark gray and similar amino acid residues are labeled in light gray. The α-helices, β-sheets are underlined the conserved cysteine residues are indicated, and the active site, Ca2+ binding site and catalytic site are shown. B: Phylogenetic analysis of amylase in various species using the Neighbor-Joining method with a bootstrap value of 1000. The amylases of echinoderms are boxed, and our newly identified Hl-Amy is shown with a red arrow. C: Comparison of the 3D structures models for human (H. sapiens) and sea cucumber (H. leucospilota) amylases. The structural domain A, B and C are indicated.
Fig 3. Tissue distribution and embryonic and larval developmental expression of Hl-Amy mRNA.A: Expression profiles of Hl-Amy mRNA in different tissues of H. leucospilota, including intestine (In), transverse vessel (TV), esophagus (Ep), cuvierian tubules (CT), respiratory trees (RT), body wall (BW), muscle (Ms), polian vesicle (PV), coelomocytes (Co), rete mirabile (RM), ovary (Ov) and testis (Ts). B: Expression profiles of Hl-Amy mRNA in embryonic and larval development of H. leucospilota, including fertilized egg (FE), blastula (Bs), early gastrula (EG), late gastrula (LG), early auricularia (EA), mid auricularia (MA), late auricularia (LA), doliolaria (Dl), pentactula (Pt) and juvenile (Jv). The data presented are expressed as the mean± S.E with three biological replicates.
Fig 4. Localization of Hl-Amy positive cells in the sea cucumber intestine.Localization of Hl-Amy positive cells were detected in the H. leucospilota intestine. The intestine is divided into the foregut (FG), midgut (MG) and hindgut (HG). H/E stain is the section stained with hematoxylin and eosin. Negative control of hybridization was performed without a DIG-labeled DNA template. The substructures of the H. leucospilota intestine included submucosa (sub), serosa (ser), muscle (mus), mucosa (muc), lumen (lum) and brush borders (bb).
Fig 5. Expression and purification of recombination of Hl-Amy protein.A: Codon optimization of Hl-Amy mRNA. Original n. t. sequence are shown in green, while optimized n. t. sequence are shown in blue with the optimized codon were highlighted and without changing the A.A. sequence (the red letter). B: Time course of IPTG induction for recombinant Hl-Amy protein expression in BL21 E. coli with its original nucleotide sequence. C: Time course of IPTG induction for recombinant Hl-Amy protein expression in BL21 E. coli with its optimal nucleotide sequence. The cell lysates were collected from 0, 2, 4, 6, and 8 h after 1 mM IPTG induction or collected from 4 h after 0.1, 0.3, 0.5, 0.7, and 1 mM IPTG induction. D: Purification of Hl-Amy recombinant protein cell lysate with 0.5 mM IPTG induction. M: Protein marker, IF: insoluble fraction of the cell lysate, FT-1: flow-through sample after washing with binding buffer, FT-2: flow-through sample after 20 mM imidazole elution; FT-3, FT-4: flow-through sample after 50 mM imidazole elution; PP: Purified protein after 500 mM imidazole elution.
Fig 6. Activity and characterization of Hl-Amy recombinant protein.A: Standard curve of D-glucose measurement. The X axis represents the concentration of D-glucose (mg/mL) and the Y axis shows the absorbance of amylase Hl-Amy at 562 nm (A562). B: Effect of temperature on the activity of Hl-Amy. The X axis represents the value of temperature (°C) and the Y axis shows the relative activity (%) of the optimal temperature. C: Effect of pH on the activity of Hl-Amy. The X axis represents the value of pH and the Y axis shows the relative activity (%) of the optimal pH.
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