Structural Analysis of Two Types of Tumor Necrosis Factor-? from Orange-spotted Grouper (Epinephelus coioides)

  • 游 敏

Student thesis: Doctoral Thesis

Abstract

Tumor necrosis factor-? (TNF-?) is known as a pleiotropic cytokine in the innate immune system However few studies focused on the investigation of TNF-? in teleost In previous study two types of TNF-? in orange-spotted groupers (Epinephelus coioides) have been identified named grouper TNF-?1 (gTNF-?1) and grouper TNF-?2 (gTNF-?2) respectively Both of proteins shared 35 6% amino acid sequence identity In the phylogenetic analysis for teleosts they were classified into two clusters of TNF-? from teleost Furthermore the amino acid sequence identity among gTNF-?1 gTNF-?2 in orange-spotted grouper and TNF-? in mammalian was about 26% However more evidence would be needed to reveal the difference on the functional and structural differences between the TNF-? proteins of these species In this study recombinant grouper TNF-?1 (rgTNF-?1) and rgTNF-?2 were expressed in Escherichia coli and purified by affinity chromatography Size exclusion chromatography analysis showed that the molecular size of rgTNF-?1 and rgTNF-?2 are closed to trimer size repectively The crystal structure of rgTNF-?2 was determined at 4 0 ? resolution by using the X-ray crystallography The structure also showed that rgTNF-?2 is a ringlike trimer conformation Additonally small-angle X-ray scattering analysis consistantly revealed that rgTNF-?2 formed a trimer with a cylindrical shape in the aqueous solution Comparing the structures of rgTNF-?2 with human TNF-? and mouse TNF-? respectively the protein structures were very similar among these proteins These results may suggest that TNF-? have a great structural homology between marine and terrestrial animals Moreover rgTNF-?2 was used as a template to mimic the putative protein structure of rgTNF-?1 Meanwhile it was found some highly conserved amino acids in difference species were located in jelly-roll motif It might mean that the trimeric structure of TNF-? in different species may maintain by the same amino acids
Date of Award2019
Original languageEnglish
SupervisorShih-Ming Lin (Supervisor)

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