Background/Purpose

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with a diverse array of clinical manifestations that is characterized by the production of antibodies to components of the cell nucleus. Neurogranin(Ng), a calmodulin binding protein, was thought to be a neural-specific factor involved in learning and memory, while, its ectopic expression was found in peripheral blood cells, like T lymphocytes, B lymphocytes, etc. And its role in SLE has not been reported yet. Our aim was to compare the expression and structure of Ng in peripheral blood and central nervous system (CNS), and to explore its role in SLE.

Methods

Thirty-four patients with SLE and 22 healthy controls were enrolled. Splicing variants of Ng gene in peripheral blood mononuclear cells(PBMCs) were screened with  reverse transcription polymerase chain reaction (RT-PCR) combined with T/A clone, DNA sequencing , and reconfirmed using the primer-specific PCR . Expression of Ng splicing variants mRNA was detected by qRT-PCR. All subjects gave written informed consent prior to enrollment in the study, which was approved by the ethics committee of our hospital.

Results

Three splicing variants of Ng gene were cloned from SLE PBMCs, and two of which, named as Ng-mu1 and Ng-mu2 respectively, were not been reported yet.  Compared to wild type of Ng (Ng-WT), Ng-mu1 containing of a 55 bp intron retention insertion with 256-392 base sites deleted, Ng-mu2 with second exon partially deleted and termination codon early emerged. The IQ motifs of Ng-mu1 which is the binding site of calmodulin were impaired (Fig1). The ratio of Ng-WT to Ng-mu1 in healthy control PBMCs was lower than human brain total RNA. Further analysis showed the up-regulation of Ng-WT mRNA and the elevated ratio of Ng-WT to Ng-mu1 in SLE PBMCs, and the expression of Ng-WT was positively correlated with SLE disease activity index (SLEDAI) (Fig2).

Conclusion

The novel splicing variants of Ng and their ectopic expression in PBMCs may contribute to the pathogenesis of SLE through mediating Ca2+ signal transduction.

Fig1. Three splicing variants of Ng gene. A: Comparison of IQ motif of Ng-WT, Ng-mu1 and Ng-mu2. B: Schematic diagram of forming of Ng-WT, Ng-mu1 and Ng-mu2. C: Agarose gels with the analysis of the cDNA of Ng gene fragment with primers F2 and R2 as in fig1B. D: Agarose gels with the analysis of the cDNA of Ng with specific primers F2 and R2′ as in fig1B.

Fig2. Relative expression of Ng-WT mRNA and Ng-mu1 mRNA in SLE PBMCs. A: Expression of Ng-WT mRNA. B: Expression of Ng-mu1 mRNA. C: The ratio of Ng-WT to Ng-mu1. D: Expression of Ng-WT was positively correlated with SLEDAI.