Biochemical and folding defects in a RAG1 variant associated with Omenn syndrome.

[omenn syndrome]

The RAG 1 and RAG 2 proteins are required to assemble mature Ag receptor genes in developing lymphocytes . Hypomorphic mutations in the gene encoding RAG 1 are associated with Omenn syndrome , a primary immunodeficiency . We explored the biochemical defects resulting from a mutation identified in an Omenn syndrome patient which generates an amino acid substitution in the RAG 1 RING finger /ubiquitin ligase domain (C 3 25 Y in murine RAG 1 ) as well as an adjacent substitution (P 326 G ). RAG 1 C 3 25 Y demonstrated a 50 -fold reduction in recombination activity in cultured pro-B cells despite the fact that its expression and localization to the nucleus were similar to the wild-type protein . The C 3 25 Y substitution severely abrogated ubiquitin ligase activity of the purified RAG 1 RING finger domain , and the tertiary structure of the domain was altered . The P 326 G substitution also abrogated ubiquitin ligase activity but had a less severe effect on protein folding . RAG 1 P 326 G also demonstrated a recombination impairment that was most pronounced when RAG 1 levels were limiting . Thus , a correctly folded RAG 1 RING finger domain is required for normal V (D )J recombination , and RAG 1 ubiquitin ligase activity can contribute when the protein is present at relatively low levels .