Influence on Accumulation Levels and Subcellular Localization of Prolamins by Fusion with the Functional Peptide in...

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Immunology

Background:

• Sjögren's syndrome is an autoimmune disease targeting exocrine glands.
• Developing oral vaccines using transgenic plants offers a promising alternative to traditional methods.
• Altered peptide ligands (APLs) are crucial for modulating autoimmune responses.

Purpose of the Study:

• To develop a rice seed-based oral vaccine for Sjögren's syndrome.
• To express N-terminal 1 (N1-APL7) altered peptide ligand fused with four types of rice prolamins.
• To investigate the accumulation, localization, and digestion of these fusion proteins in transgenic rice.

Main Methods:

• Constructing fusion proteins of N1-APL7 with 10, 13, 14, and 16 kDa rice prolamins.
• Expressing fusion proteins in rice under native prolamin promoters.
• Analyzing protein accumulation, subcellular localization (PB-Is and PB-IIs), and in vitro proteolysis using pepsin.
• Employing RNA interference (RNAi) to suppress endogenous seed storage proteins.

Main Results:

• 10kD:N1-APL7 and 14kD:N1-APL7 showed high accumulation (287 and 58 µg/grain).
• Co-expression and RNAi-mediated suppression did not significantly enhance fusion protein accumulation.
• 10kD:N1-APL7 exhibited faster in vitro digestion and was localized in PB-IIs (glutelin-rich), unlike 14kD:N1-APL7 in PB-Is.
• N1-APL7 significantly impacted prolamin accumulation and subcellular localization.

Conclusions:

• Rice seed-based expression of prolamin:N1-APL7 fusion proteins is feasible for oral vaccine development.
• Accumulation levels and subcellular localization of prolamins are modulated by the fused N1-APL7 peptide.
• Further research is needed to optimize expression and validate the immunogenicity of these constructs for Sjögren's syndrome treatment.