Annexin-1 is abnormally expressed in fragile X syndrome: Two-dimensional electrophoresis study in lymphocytes

The search for targets of FMRP (the product of FMR1, the mutated gene in Fr agile X syndrome) has predominantly focused on identifying transcripts that are regulated by this RNA-binding protein. This study introduces the use o f two-dimensional gel electrophoresis (2D PAGE) as a novel approach for dem onstrating changes in protein synthesis secondary to FMRP deficit. By a sta ndardized 2D PAGE protocol, we studied leukocyte homogenates from 30 males with different patterns of FMR1 mutation and different levels of FMRP. Samp les from these subjects were compared to those of 12 normal control males a nd eight subjects with other mental retardation-associated conditions (i.e. , Rett and Down syndromes). We found an abnormal pattern of a major leukocy tic protein, identified by 2D PAGE datasets and immunoblotting as annexin-1 (Anx-1). Anx-1 appeared in subjects with Fragile X as multiple rather than 1-2 spots, at similar to 37 kd, in the pI 5-7 range. The presence and inte nsity of this Anx-1 pattern was relatively independent of Anx-1 levels and inversely related to total and high MW FMRP immunoreactivities. Based on th e 2D PAGE pattern, without obvious AM change, and on dephosphorylation assa ys, we concluded that Anx-1's abnormality represents an aberrant posttransl ational modification other than phosphorylation. Comparisons of our data wi th published cytoskeletal protein 2D profiles suggest that Anx-1 may be abn ormally acetylated and, consequently, incapable of establishing appropriate N-terminal protein-protein interactions. In addition to its peripheral ant iinflammatory function, Anx-1 mediates glucocorticoid inhibition of the hyp othalamopituitary-adrenal axis. As the latter seems to be disrupted in Frag ile X syndrome, the reported Anx-1 abnormality could be responsible for som e aspects of the Fragile X neurobehavioral phenotype. Our data also emphasi ze the feasibility of using 2D PAGE for disclosing molecular abnormalities in Fragile X and other genetic disorders. (C) 2001 Wiley-Liss, Inc.