Comparative Characterization of Two cxcl8 Homologs in Oplegnathus fasciatus: Genomic, Transcriptional and Functional Analyses
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals, Reagents, Experimental Fish, and Immune Stimulants
2.2. Animal Rearing and Ethics
2.3. Determination of Complementary- and Genomic-DNA (cDNA and gDNA) Sequences of Ofcxcl8 Homologs
2.4. Molecular Profiling of Rock Bream cxcl8 Homologs by Computational Tools
2.5. Transcriptional Profiling of Rock Bream cxcl8 Homologs
2.5.1. Immune Challenge Experiments
2.5.2. Sampling for Spatial Tissue Distribution of Ofcxcl8 Homologs in Unchallenged Animals
2.5.3. Sampling for Temporal Transcriptional Analyses of Ofcxcl8 Homologs in Immune-Challenged Animals
2.5.4. In Vitro Concanavalin A (Con A) Stimulation of Peripheral Blood Leukocytes (PBLs)
2.5.5. Total RNA Extraction and cDNA Synthesis
2.5.6. Determination of Ofcxcl8 Transcript Levels by Quantitative Real-Time PCR (qPCR)
2.6. Functional Characterization of Rock Bream OfCXCL8 Recombinant Proteins
2.6.1. Construction of Recombinant Plasmids
2.6.2. Bacterial Expression of Recombinant OfCXCL8 (rOfCXCL8) Proteins
2.6.3. Purification and Evaluation of Recombinant OfCXCL8 (rOfCXCL8) Proteins
2.6.4. Cell Migration Assay
2.6.5. Cell Proliferation Assay
2.7. Statistical Analysis
3. Results
3.1. Molecular Profiling of Structural Features
3.1.1. Identification of Rock Bream cDNAs for cxcl8 Homologs and Their Features
3.1.2. Recovery of Rock Bream cxcl8 Genomic Sequences and Their Genomic Structures
3.1.3. Amino Acid Sequences of Rock Bream CXCL8 Proteins: Homology and Phylogeny
3.2. Profiling of Genomic Arrangement and Promoter Sequences
3.2.1. Genomic Comparison of Vertebrate Orthologs of cxcl8 (il-8) Gene
3.2.2. Putative Promoter Sequences of Rock Bream cxcl8 (il-8) Homologs
3.3. Profiling of Transcriptional Gene Expression of Rock Bream cxcl8 (il-8) Homologs
3.3.1. Tissue mRNA Expression of Ofcxcl8 Homologs in Unchallenged Animals
3.3.2. Detection of Ofcxcl8 mRNAs in Fish Injected with FLA-ST
3.3.3. Detection of Ofcxcl8 mRNAs in Fish Injected with LPS, Poly(I:C), and Pathogens
3.3.4. Detection of Ofcxcl8 mRNAs in Con A-stimulated PBLs
3.4. Functional Characterization of Rock Bream CXCL8 Homologs Using Recombinant Proteins
3.4.1. Bacterial Expression of Recombinant Proteins and Purification
3.4.2. Chemotactic Response of Leukocytes Towards Recombinant OfCXCL8 Proteins
3.4.3. Induction of Leukocyte Proliferation by Recombinant OfCXCL8 Proteins
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Taxonomy | Species | Accession No | Protein Name | OfCXCL8-L1 | Species | Accession No | Protein Name | OfCXCL8-L3 | ||
---|---|---|---|---|---|---|---|---|---|---|
I% | S% | I% | S% | |||||||
Fish | Oplegnathus fasciatus | AGR27882.1 | Interleukin-8 (OfCXCL8-L1) | 100.0 | 100.0 | Oplegnathus fasciatus | AGR27882.1 | Interleukin-8 (OfCXCL8-L1) | 30.60 | 53.30 |
Fish | Oplegnathus fasciatus | ADK35757.1 | Interleukin-8 | 98.0 | 99.0 | Oplegnathus fasciatus | ADK35757.1 | Interleukin-8 | 29.70 | 53.30 |
Fish | Oplegnathus fasciatus | AGR27883.1 | Interleukin-8 like (OfCXCL8-L3) | 30.6 | 53.3 | Oplegnathus fasciatus | AGR27883.1 | Interleukin-8 like (OfCXCL8-L3) | 100.00 | 100.00 |
Fish | Siniperca chuatsi | AFK65606.1 | Interleukin-8 | 88.9 | 94.9 | Lateolabrax japonicus | QFG40053.1 | Interleukin-8 | 91.40 | 96.20 |
Fish | Dicentrarchus labrax | CAM32186.1 | Interleukin-8 | 87.9 | 93.9 | Sparus aurata | XP_030285781.1 | Permeability factor 2 | 89.50 | 96.20 |
Fish | Latris lineata | ACQ99511.1 | Interleukin-8 | 86.9 | 94.9 | Lutjanus peru | ASK51661.1 | Interleukin-8 | 89.50 | 95.20 |
Fish | Pagrus major | ADK35756.1 | Interleukin-8 | 85.0 | 92.0 | Dicentrarchus labrax | AKC57337.1 | Interleukin-8 | 87.60 | 94.30 |
Fish | Anoplopoma fimbria | ACQ57874.1 | Interleukin-8 | 79.8 | 91.9 | Salarias fasciatus | XP_029943750.1 | Interleukin-8 | 81.90 | 89.50 |
Fish | Cyprinus carpio | ABE47600.1 | Interleukin-8 | 60.6 | 77.6 | Anoplopoma fimbria | ACQ58275.1 | Interleukin-8 | 78.10 | 83.80 |
Fish | Danio rerio | XP_001342606.2 | Permeability factor 2 IF X1 | 58.6 | 76.5 | Danio rerio | XP_001342606.2 | Permeability factor 2 IF X1 | 29.70 | 55.20 |
Fish | Oncorhynchus mykiss | AAO25640.1 | Interleukin-8 | 57.1 | 76.5 | Tetraodon nigroviridis | CAF90539.1 | Unnamed protein * | 68.60 | 81.90 |
Amphibia | Xenopus tropicalis | XP_002942577.1 | Interleukin-8 | 39.3 | 60.0 | Xenopus tropicalis | XP_002942577.1 | Interleukin-8 | 31.20 | 52.40 |
Amphibia | Xenopus laevis | AEB96252.1 | Interleukin-8 | 37.1 | 63.1 | Xenopus laevis | AEB96252.1 | Interleukin-8 | 33.90 | 55.20 |
Reptilia | Anolis carolinensis | XP_003230087.1 | Interleukin-8 | 41.2 | 65.0 | Anolis carolinensis | XP_003230087.1 | Interleukin-8 | 33.90 | 50.50 |
Aves | Gallus gallus | NP_990349.1 | Interleukin-8 | 41.5 | 63.5 | Gallus gallus | NP_990349.1 | Interleukin-8 | 30.30 | 53.30 |
Mammalia | Sus scrofa | NP_999032.1 | Interleukin-8 | 34.3 | 56.3 | Sus scrofa | NP_999032.1 | Interleukin-8 | 27.50 | 49.50 |
Mammalia | Homo sapiens | NP_000575.1 | Interleukin-8 IF 1 | 34.7 | 57.6 | Homo sapiens | NP_000575.1 | Interleukin-8 IF 1 | 25.20 | 56.20 |
Mammalia | Oryctolagus cuniculus | NP_001075762.1 | Interleukin-8 | 33.0 | 56.4 | Oryctolagus cuniculus | NP_001075762.1 | Interleukin-8 | 27.50 | 52.40 |
Gene Symbol 1 | Accession Number/Reference | Organism | Exon 1 3 | Intron 1 | Exon 2 | Intron 2 | Exon 3 4 | Intron 3 | Exon 4 4 | Group | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|
5′ UTR | CDSP | CDS | CDS | CDSX,Q | 3′ UTR | |||||||
cxcl8 | ENST00000307407 2 | Homo sapiens | 153 | 64 | 819 | 136 | 271 | 84 | 416 | 16 | 1252 | Mammals |
cxcl8 | ENSBTAT00000026275 2 | Bos taurus | 70 | 64 | 1573 | 136 | 273 | 84 | 439 | 22 | 1105 | Mammals |
cxcl8 | ENSSSCT00000009807 2 | Sus scrofa | 83 | 64 | 1018 | 136 | 288 | 84 | 427 | 28 | 1093 | Mammals |
cxcl8 | ENSOCUT00000030077 2 | Oryctolagus cuniculus | 265 | 64 | 516 | 136 | 269 | 84 | 448 | 22 | 1148 | Mammals |
il-8 | ENSGALT00000042745 2 | Gallus gallus | - | 61 | 774 | 136 | 642 | 84 | 568 | 31 | 2661 | Birds |
il-8 like | XM_005498533 | Columba livia | 63 | 61 | 773 | 136 | 700 | 84 | 1249 | 31 | 169 | Birds |
il-8like | ENSACAT00000011352 2 | Anolis carolinensis | - | 64 | 993 | 136 | 265 | 80 | 339 | 32 | - | Reptiles |
il-8 | XM_004911120 | Xenopus tropicalis | 81 | 64 | 263 | 124 | 676 | 88 | 1490 | 12 | 294 | Amphibians |
Ofcxcl8-L1 | This study | Oplegnathus fasciatus | 200 | 67 | 194 | 133 | 146 | 87 | 142 | 10 | 358 | Fish |
il-8 | Laing et al. (2002) | Oncorhynchus mykiss | - | 64 | 341 | 133 | 247 | 87 | 292 | 10 | - | Fish |
il-8 | ENSTRUT00000016751 2/NC_042301 | Takifugu rubripes | 137 | 64 | 107 | 133 | 93 | 87 | 106 | 12 | 365 | Fish |
il-8 | Wang et al. (2013) | Ctenopharyngodon idella | - | 64 | 143 | 133 | 123 | 87 | 125 | 13 | - | Fish |
il-8 precursor | EU007442/ Seppola et al. (2008) | Gadus morhua | 122 | 73 | 109 | 133 | 151 | 87 | 202 | 13 | 169 | Fish |
il-8 | JQ407041/ Li et al. (2013) | Larimichthys crocea | - | 52 | 168 | 133 | 149 | 87 | 682 | 13 | - | Fish |
il-8 | KP202400/ Mu et al. (2015) | Larimichthys crocea | 64 | 137 | 133 | 129 | 87 | 124 | 16 | - | Fish | |
cxca | AJ421443/ Huising et al. (2003) | Cyprinus carpio | 81 | 58 | 117 | 133 | 126 | 88 | 148 | 19 | 247 | Fish |
cxcl8b.1 | XM_003198892/NM_001327985 | Danio rerio | 74 | 70 | 79 | 130 | 2660 | 69 | 2476 | 88 | 303 | Fish |
il-8 | Chen et al. (2005) | Ictalurus punctatus | - | 67 | 87 | 136 | 153 | 69 | 164 | 73 | - | Fish |
il-8 like | ENSTNIT000000059822 | Tetraodon nigroviridis | - | 52 | 427 | 136 | 140 | 130 | - | - | - | Fish |
il-8 like x2 | NW_004531887/XM_004570701 | Maylandia zebra | 46 | 52 | 1090 | 136 | 1252 | 121 | - | - | 413 | Fish |
Ofcxcl8-L3 | This study | Oplegnathus fasciatus | 59 | 52 | 2202 | 136 | 2209 | 130 | - | - | 625 | Fish |
Characteristics | Ofcxcl8-L1 | Ofcxcl8-L3 | |
---|---|---|---|
Complimentary DNA | GenBank accession No. | KC522966 | KC522965 |
Length of cDNA | 855 | 1002 | |
5′ UTR | 200 | 59 | |
CDS (bp) | 297 | 318 | |
3′ UTR | 358 | 625 | |
mRNA instability motif | 3 | 2 | |
Polyadenylation signal | 831AATAAA836 | 986AATAAA991 | |
Genome | Length of gDNA (bp) | 1337 | 5413 |
Number of exons | 4 | 3 | |
Number of introns | 3 | 2 | |
Protein | Peptide (aa) | 98 | 105 |
Molecular mass (Da) | 10,833 | 11,571.6 | |
Theoretical pI | 8.78 | 8.95 | |
Signal peptide | 1–23 | 1–18 | |
SCY domain | 32-93 | 23-88 | |
ELR-like motif | 32EMH34 | 27NSH29 | |
CXC motif | 35CRC37 | 30CRC32 | |
Invariant cysteines | 35, 37, 61, 78 | 30, 32, 57, 73 | |
Highest homology | Mandarin fish IL-8 | Japanese sea bass IL-8 | |
5′-flanking region | Transcription factor binding sites | NF-κB/ NF-κB1, C/EBPα, C/EBPβ (NF-IL6), Jun:Fos (AP1) and POU2F1 (Oct-1) | |
Transcripts | Dominant mRNA expression (qPCR) | gills, intestine, and PBCs | spleen and gills |
Functional characteristics | Chemotaxis index (100 ng/µL) Cell proliferation (OD450) | ~130 (significant) Significant at 10 and 100 ng/µL | ~19 (not significant) Not significant |
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Umasuthan, N.; Bathige, S.; Thulasitha, W.S.; Oh, M.; Lee, J. Comparative Characterization of Two cxcl8 Homologs in Oplegnathus fasciatus: Genomic, Transcriptional and Functional Analyses. Biomolecules 2020, 10, 1382. https://doi.org/10.3390/biom10101382
Umasuthan N, Bathige S, Thulasitha WS, Oh M, Lee J. Comparative Characterization of Two cxcl8 Homologs in Oplegnathus fasciatus: Genomic, Transcriptional and Functional Analyses. Biomolecules. 2020; 10(10):1382. https://doi.org/10.3390/biom10101382
Chicago/Turabian StyleUmasuthan, Navaneethaiyer, SDNK Bathige, William Shanthakumar Thulasitha, Minyoung Oh, and Jehee Lee. 2020. "Comparative Characterization of Two cxcl8 Homologs in Oplegnathus fasciatus: Genomic, Transcriptional and Functional Analyses" Biomolecules 10, no. 10: 1382. https://doi.org/10.3390/biom10101382