Functional and structural analysis of five mutations identified in methylmalonic aciduria cblB type

Area of Science:

• Biochemistry
• Genetics
• Molecular Biology

Background:

• ATP:cob(I)alamin adenosyltransferase (ATR) is crucial for synthesizing the adenosylcobalamin cofactor.
• Mutations in the MMAB gene, encoding ATR, lead to cblB type methylmalonic aciduria.
• Understanding the molecular basis of these mutations is key for therapeutic development.

Purpose of the Study:

• To functionally analyze five cblB mutations in the MMAB gene.
• To elucidate the molecular mechanisms underlying ATR dysfunction caused by these mutations.
• To provide a basis for developing targeted therapies for cblB methylmalonic aciduria.

Main Methods:

• Transcriptional profiling and minigene analysis to assess splicing defects.
• Expression and purification of wild-type and mutant ATR proteins in prokaryotic and eukaryotic systems.
• Enzymatic activity assays, stability studies, and oligomeric state analysis of mutant proteins.

Main Results:

• Three mutations (c.584G>A, c.349-1G>C, c.290G>A) were found to affect mRNA splicing.
• The p.I96T mutant showed reduced specific activity and altered stability and oligomeric state.
• The p.R191W mutant exhibited significant instability and potential misfolding, with a pronounced impact on protein recovery.

Conclusions:

• cblB mutations in MMAB can impair ATR function through splicing defects or by affecting protein stability and structure.
• Mutant proteins p.I96T and p.R191W display reduced stability and potential misfolding, contributing to disease pathology.
• These findings support the potential for pharmacological interventions to enhance residual ATR activity in patients with cblB methylmalonic aciduria.