Biochemical and Functional Characterization of the Three Zebrafish Transglutaminases 2
Abstract
:1. Introduction
2. Results
2.1. Evolutionary History of Zebrafish Transglutaminase 2
2.2. Gene Expression Analysis of Zebrafish Transglutaminase 2 Genes
2.3. Zebrafish TGs2 Proteins Sequence and Structure Analysis
2.4. Recombinant zTGs2 Proteins Have a Ca2+-Dependent Activity
2.5. The zTGs2 Proteins Have a Ca2+-Dependent Activity but Are Not Inhibited by GTP
2.6. Intracellular zTGs2 Proteins in HEK293 Cells Mediate Different Effects against Cell Death in a Stimulus-Dependent Manner
2.7. The zTGs2 Proteins Mediate Can Mediate RGD-Independent Cell Adhesion and Spreading
3. Discussion
4. Materials and Methods
4.1. Genetic Analysis of TG2 Orthologues in the Zebrafish Genome
4.2. Zebrafish Embryos Handling and Maintenance
4.3. Gene Expression Study of the Three Zebrafish TG2 Genes during the Embryonic Development
4.4. Zebrafish Embryos Lysates Transamidation Activity Assay
4.5. Fluorescent Cadaverine Incorporation in Live Zebrafish Embryos
4.6. Molecular Cloning of Zebrafish Transglutaminase 2 Sequence into Bacterial Expression Vector
4.7. Recombinant Protein Production and Purification
4.8. zTGs2 Transamidation Activity Curve and Inhibition Assays
4.9. RGD-Independent Cell Adhesion Assay
4.10. Actin Cytoskeletal Staining
4.11. Molecular Cloning of Zebrafish Transglutaminase 2 Sequence into Eukaryotic Expression Vector
4.12. Transient Cell Transfection and Selection of Stable TG2-Expressing HEK293 Cells
4.13. HEK293 Anti-SV5 Tag Western Blot
4.14. HEK293 Cell Lysates Transamidation Activity Assay
4.15. HEK293 Cells Flow Cytometry Analysis
4.16. Apoptosis Assays with Hydrogen Peroxide and Thapsigargin
4.17. Caspase3 Activity Assay
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
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Primer Name | Sequence 5′-3′ |
---|---|
zTG2a Forward | CCAAAGCAGTGGGTCGAGAT |
zTG2a Reverse | GTGCGTAAAACATCACCCGG |
zTG2b Forward | GATGGGTCGTTCCGATCTCC |
zTG2b Reverse | GCTGATCTTCTGGCCCACTT |
zTG2like Forward | CACCTGCAGCCCTGAAAAAC |
zTG2like Reverse | TCTGGTTGGGATGCCAAGAC |
Beta actin Forward | CGAGCTGTCTTCCCATCCA |
Beta actin Reverse | TCACCAACGTAGCTGTCTTTCTG |
Lsm12b Forward | AGTTGTCCCAAGCCTATGCAATCAG |
Lsm12b Reverse | CCACTCAGGAGGATAAAGACGAGTC |
Hsp70 Forward | TCAAGCGCAACACAACCATC |
Hsp70 Reverse | ATTTGCCCAGCAGGTTGTTG |
Primer Name | Sequnce 5′-3′ |
---|---|
zTG2a sense KpnI | AGATCTGGGTACCATGGAGAGAGTGGTGGAG |
zTG2a anti HindIII | TCCTCGAGAAGCTTTTATTCATAGATAACCAG |
zTG2b sense KpnI | AGATCTGGGTACCATGGCTCTGGACATCGGC |
zTG2b anti HindIII | TCCTCGAGAAGCTTTCATTTCCCGATGATGAC |
zTG2like sense KpnI | AGATCTGGGTACCATGGCCAGCTATAATGCC |
zTG2like anti HindIII | TCCTCGAGAAGCTTCTAAATTTCTGGGACAGC |
Primer Name | Sequence 5′-3′ |
---|---|
hTG2-HYGRO-Sense_NheI | GCTGGCTAGCTGCCACCATGGCCGAGGAGCTGGTC |
hTG2-HYGRO-anti_SV5 | GATTGGTTTGCCACTAGTGGCGGGGCCAATGATGAC |
zTG2a-HYGRO-Sense_Xba | AGCTGTCTAGATGCCACCATGGAGAGAGTGGTGG |
zTG2a-HYGRO-anti_SV5 | GATTGGTTTGCCACTAGTTTCATAGATAACCAGATTC |
zTG2b-HYGRO-Sense_NheI | GCTGGCTAGCTGCCACCATGGCTCTGGACATCGGC |
zTG2b-HYGRO-anti_SV5 | GATTGGTTTGCCACTAGTTTTCCCGATGATGACGTTC |
zTG2like-HYGRO-Sense_Xba | GCTGTCTAGATGCCACCATGGCCAGCTATAATGCC |
zTG2like-HYGRO-anti_SV5 | GATTGGTTTGCCACTAGTAATTTCTGGGACAGCATTTAC |
SV5_HindIII_anti | AGCTAAGCTTTTAAGTACTATCCAGGCCCAGCAG TGGGTTTGGGATTGGTTTGCCACTAGT |
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Lisetto, M.; Fattorini, M.; Lanza, A.; Gerdol, M.; Griffin, M.; Wang, Z.; Ferrara, F.; Sblattero, D. Biochemical and Functional Characterization of the Three Zebrafish Transglutaminases 2. Int. J. Mol. Sci. 2023, 24, 12041. https://doi.org/10.3390/ijms241512041
Lisetto M, Fattorini M, Lanza A, Gerdol M, Griffin M, Wang Z, Ferrara F, Sblattero D. Biochemical and Functional Characterization of the Three Zebrafish Transglutaminases 2. International Journal of Molecular Sciences. 2023; 24(15):12041. https://doi.org/10.3390/ijms241512041
Chicago/Turabian StyleLisetto, Manuel, Mariagiulia Fattorini, Andrea Lanza, Marco Gerdol, Martin Griffin, Zhuo Wang, Fortunato Ferrara, and Daniele Sblattero. 2023. "Biochemical and Functional Characterization of the Three Zebrafish Transglutaminases 2" International Journal of Molecular Sciences 24, no. 15: 12041. https://doi.org/10.3390/ijms241512041