Molecular cloning, expression analysis, and functional characterization of Intelectin-1 from Chinese giant salamanders (Andrias davidianus).

Abstract

Background: Intelectin (ITLN) plays a pivotal role in innate immunity, inflammatory responses, and tumor progression. However, its physiological roles in caudate amphibians, such as Andrias davidianus, remain elusive.

Objective: A proto-type of intelectin (AdITLN1) from A. davidianus was characterized, following which an AdITLN1 homology model was generated to analyze its expression profiles and functional characterizations.

Methods: The intelectin1 gene (AdITLN1) was cloned, and its evolutionary relationship with ITLN1 from other species was explored. Additionally, a homology model was generated using Molecular Operating Environment (MOE) software, and the active binding pocket of AdITLN1 was identified. Infections with Aeromonas hydrophila were conducted to analyze changes in ITLN1 transcript levels in liver tissue. Finally, recombinant A. davidianus ITLN1 protein (rAdITLN1) was synthesized using prokaryotic expression, and its bacterial agglutination activity and impact on macrophage phagocytosis were examined.

Results: Multiple sequence alignment and phylogenetic analysis indicated that AdITLN1 shared the closest evolutionary relationship with amphibians, with its structure being similar to that of human intelectin1 and Xenopus Embryonic Epidermal Lectin. Moreover, AdITLN1 mRNA was expressed in a wide range of tissues and was significantly up-regulated post-A. hydrophila infection. Meanwhile, the AdITLN1 protein was successfully expressed and purified in Escherichia coli (E. coli) BL21 (DE3). The recombinant AdITLN1 (rAdITLN1) displayed strong agglutination activity against different Gram-negative and Gram-positive bacteria. Lastly, The phagocytosis of rAdITLN1-treated E. coli by macrophages was significantly enhanced.

Conclusion: The results of the present study demonstrated that AdITLN1 was a multifunctional immune protein with potent immunomodulatory activity. This study offers valuable insights into disease control in giant salamanders and the conservation of natural resources.