Molecular insights and immune responses of big belly seahorse syndecan-2 (CD362): Involvement of ectodomain in regulating cell survival, proliferation, and wound healing

다국어 초록 (Multilingual Abstract)

Syndecan-2, also known as CD362, is a transmembrane heparan sulfate proteoglycan which regulates cell growth, proliferation, cell adhesion, wound healing, and recruits immune cells. In the present study, we performed bioinformatics, spatial and temporal expression analyses of Hippocampus abdominalis syndecan-2 (HaSDC-2). Additionally, functional assays were conducted. HaSDC-2 has five major domains; an extracellular heparan sulfate attachment domain, a co-receptor binding domain, a transmembrane domain, two conserved domains (C1 domain, C2 domain), and a variable (V) domain. The ectodomain contained a signal peptide and GAG attachment sites. In-silico analysis revealed that HaSDC-2 contained a 798 bp long ORF and protein sequence of 265 amino acid residues. Further analysis of the amino acid sequence predicted a 28.9 kDa molecular weight and a 4.13 theoretical isoelectric point. The spatial expression of HaSDC-2 was ubiquitous in all tested tissues. HaSDC-2 expression in the liver was upregulated 24 h post-injection in response to all stimuli. Further, HaSDC-2 expression in blood cells was upregulated at 12 and 72 h post-injection in response to all the stimuli. HaSDC-2 + pcDNA™3.1(+) transfected cells exhibited significant survival in response to cell stressors such as H2O2 and HED. The ectodomain of recombinant HaSDC-2 treated cells showed significant cell proliferation in a concentration-dependent manner. The scratch wound healing assay showed significant Δ gap closures with increasing concentrations of HaSDC-2. Collectively, these results indicated that syndecan-2 was involved in regulating immune responses and cell stress conditions.

목차 (Table of Contents)

• ABSTRACT
• 1. Introduction
• 2. Materials and methods
• 2.1. Identification of HaSDC-2 sequence and characterization
• 2.2. Rearing of fish, immune challenge and isolation of tissues
• 2.3. Total RNA extraction and cDNA synthesis
• 2.4. Spatial and temporal expression analysis
• 2.5. Preparation of plasmids, cell culture maintenance, and transfection
• 2.6. Cellular toxicity/viability assay and cell proliferation assay
• 2.7. Scratch wound healing assay
• 3. Results and discussion
• 3.1. Sequence characterization
• 3.2. Spatial and temporal expression analysis
• 3.3. HaSDC-2 overexpression in FHM cells leads to cell survival
• 3.4. Effect of recombinant HaSDC-2-ED on cell proliferation
• 3.5. Recombinant HaSDC-2-ED in wound healing
• 4. Conclusion
• Acknowledgement
• References