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Front Endocrinol (Lausanne)
2022 Jan 01;13:891714. doi: 10.3389/fendo.2022.891714.
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Teneurin and TCAP Phylogeny and Physiology: Molecular Analysis, Immune Activity, and Transcriptomic Analysis of the Stress Response in the Sydney Rock Oyster (Saccostrea glomerata) Hemocytes.
Abramov T
,
Suwansa-Ard S
,
da Silva PM
,
Wang T
,
Dove M
,
O'Connor W
,
Parker L
,
Lovejoy DA
,
Cummins SF
,
Elizur A
.
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Teneurin C-terminal associated peptide (TCAP) is an ancient bioactive peptide that is highly conserved in metazoans. TCAP administration reduces cellular and behavioral stress in vertebrate and urochordate models. There is little information for invertebrates regarding the existence or function of a TCAP. This study used the Sydney rock oyster (SRO) as a molluscan model to characterize an invertebrate TCAP, from molecular gene analysis to its physiological effects associated with hemocyte phagocytosis. We report a single teneurin gene (and 4 teneurin splice variants), which encodes a precursor with TCAP that shares a vertebrate-like motif, and is similar to that of other molluscan classes (gastropod, cephalopod), arthropods and echinoderms. TCAP was identified in all SRO tissues using western blotting at 1-2 different molecular weights (~22 kDa and ~37kDa), supporting precursor cleavage variation. In SRO hemolymph, TCAP was spatially localized to the cytosol of hemocytes, and with particularly high density immunoreactivity in granules. Based on 'pull-down' assays, the SRO TCAP binds to GAPDH, suggesting that TCAP may protect cells from apoptosis under oxidative stress. Compared to sham injection, the intramuscular administration of TCAP (5 pmol) into oysters modulated their immune system by significantly reducing hemocyte phagocytosis under stress conditions (low salinity and high temperature). TCAP administration also significantly reduced hemocyte reactive oxygen species production at ambient conditions and after 48 h stress, compared to sham injection. Transcriptomic hemocyte analysis of stressed oysters administered with TCAP demonstrated significant changes in expression of genes associated with key metabolic, protective and immune functions. In summary, this study established a role for TCAP in oysters through modulation of physiological and molecular functions associated with energy conservation, stress and cellular defense.
Figure 1. Teneurin predicted amino acid sequence and gene expression in SRO. (A)
S. glomerata teneurin splice variants; blue shading represented regions that were present in two out of the four isoforms. Purple shading indicates 100% sequence similarity. TMD- a single transmembrane domain. The amino acid sequence of the synthesized sroTCAP peptide (42 aa) used in this study is shown as a grey bar. Solid red boxes depict predicted cleavage sites (leading to 60 aa TCAP), and the dotted red box shows the conserved PELSD motif. (B) Heatmap of teneurin expression in different SRO tissues, normalized with Z-score analysis.
Figure 2. Teneurin/TCAP sequence analysis. (A) Phylogenetic tree of various invertebrates and vertebrates showing relationship based on teneurins, analyzed using Maximum Likelihood, 1000 bootstrap.(B) Homology alignment of TCAP sequences from invertebrates and vertebrates using MUSCLE, highlighting amino acid residues where >50% homology is observed. The red box shows the conserved motif among invertebrates and vertebrates. The orange asterisk shows the conserved cysteine residues among bivalvia and chepholopoda. (C) Conserved TCAP residues in various species show species specificity. Underlined residues- SRO and bivalve specific, red residues- newly identified in molluscs, echinoderms and arthropods. The size of residues reflects the relative level of conservation between the species investigated. The larger the size, the higher frequency a specific residue appears in the dataset and vice versa. (D) 3D model alignment of predicted sroTCAP with human TCAP-2, showing a top and side view. Blue ribbon- sroTCAP, red ribbon- human TCAP-2. N and C depict the N- and C- terminus, respectively.
Figure 3. Western blot immuno-detection of TCAP in SRO tissues. Western blot using (A) 30 μg of total soluble protein from each tissue, and (B) 30 ng of 60 and 42 amino acid (aa) synthetic sroTCAP.
Figure 4. Localization of TCAP in SRO hemocytes. Left and middle: TCAP detected in the cytosol of hemocytes and in granule-like bodies (white arrows) showing 25 and 50 µm scale bar respectively. Right: No florescence was observed when pre-immune serum was used, scale bar 25 µm. Green signal: ir-TCAP. Blue signal: DAPI stained nuclei.
Figure 5. Phagocytosis activity of SRO hemocytes exposed to ambient and stress conditions injected with sroTCAP or FSSW. (A) Hemocyte phagocytosis activity following 3 h sroTCAP treatments (5, 10 and 20 pmol), compared to FSSW control (0 pmol). Oysters used were in ambient conditions of 34 ppt and 22°C (non-stressed). Lowercase letters indicate significant differences between treatments (ANOVA, p < 0.05). (B) Hemocyte phagocytosis activity at different time points (3, 12 and 24 h) post-stress (30°C, 15 ppt) following IM administration of 5 pmol sroTCAP or FSSW (control) treatment. Ambient control (non-stressed 34 ppt and 22°C) shows hemocyte phagocytosis activity 24 h post 5 pmol sroTCAP or FSSW IM administration. Y-axis shows the arbitrary fluorescence units, which reflects the phagocytosis activity; higher fluorescence demonstrates higher activity and vice versa (*P < 0.001).
Figure 6. ROS production in SRO hemocytes. Oysters were held in either ambient conditions (35 ppt and 22 ± 1°C) or stress conditions (15 ppt and 30 ± 1°C) for 12 – 48 h, and treated with either sroTCAP (5 pmol) or FSSW (negative control). Y-axis shows the arbitrary units of fluorescence which reflects the ROS activity, higher fluorescence demonstrates higher ROS and vice-versa (*P < 0.05, **P < 0.01).
Figure 7. Hemocyte gene expression in response to TCAP/FSSW and ambient/stress treatments (n=4) in different pair-wise comparisons (n=5). Venn diagrams show the total number of expressed genes between different comparisons, including unique and shared expression. Experimental groups show significant DEG of each pair-wise comparison. Blue: upregulated DEG, red: Down-regulated DEG. FDR ≤ 0.05, log fold change ≥ 1 and ≤ -1.
Figure 8. Changes in hemocyte gene expression in response to TCAP/FSSW and ambient/stress treatments. Plots showing the over-represented biological processes in the upregulated and downregulated DEG list in response to TCAP/FSSW and stress/ambient (transcriptome comparison). Each row represents a GO term category, and each column represents the treatment replicates. The most representative and significant biological processes are shown. The colour depicts the significance of the enrichment (-log10 (FDR-corrected P-values)). The X mark in the cells means that the GO term is not a representative term in the corresponding DEG list in a comparison. Asterisks represent enriched GO terms of interest.
Figure 9. Heatmap showing DGE of genes of interest in several relevant categories, colour coded within gene function. The colour of cells represents the log2 fold change (fc) expression value. Dendrograms of columns and rows show clustering based on average correlation. AS- Ambient condition and FSSW injected, AT- Ambient condition and TCAP injected, SS- Stressed and FSSW injected, ST- Stressed and TCAP injected. Numbers represent the biological replicates of each group (1–3).
Figure 10. Proposed model of TCAP and GAPDH interaction, leading to increased cell survival during stress. (A) Under oxidative conditions, GAPDH undergoes NO-S-nitrosylation, where it can then bind to E3 ubiquitin ligase. This protein complex transits to the nucleus, degrading nuclear proteins, resulting in apoptosis [modified from Hara et al. (51)]. (B) A proposed TCAP interaction with GAPDH leading to the inhibition of GAPDH binding with E3 ubiquitin ligase, thus preventing its downstream apoptotic actions. The inhibition of this pathway supports increased cell survival under stress. Created with BioRender.com.
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