Are Mannan-binding Lectine Serin Protease-2 and Alpha-1-microglobulin and Bukinin Precursor the Potential Biomarkers of Manic Episode? A Study via Urinary Proetomic Analysis

Abstract

Objective: Investigating the molecular basis of bipolar disorder (BD) is crucial in terms of developing effective treatment strategies as well as objective laboratory-based diagnostic tools for the disease.

Methods: We examined the urine samples of BD patients both in manic episode and after remission and compared their urinary protein profiles with the controls. Twelve patients and twelve controls (C group) included to the study. Urinary samples of patients were first collected during manic episode (M group) and then after remission (R group). Two-dimensional gel electrophoresis (2-DE) coupled to MALDI-TOF/TOF massspectrometry approach and Western blot analysis were used.

Results: Alpha-1-microglobulin and bukinin precursor (AMBP), Mannan-binding lectine serin protease-2 (MASP-2), and Ig gamma-1-chain displayed significant increases in their abundance in the urine protein pool of M group in comparison to the C and R groups. Alpha-1B glycoprotein and prostaglandin-H2 D-isomerase (PGD2) levels were significantly higher in the urine protein pool of the M and R groups in comparison to the C group. Annexin A1 was downregulated significantly in the urine protein pool of the M group in comparison to the C group.

Conclusion: Intensities of MASP-2 and AMBP proteins discriminated manic episode from remission period and healthy controls indicating that these proteins may be candidate biomarkers for manic episode. The decrease in Annexin A1 and increase in Ig gamma-1 chain levels appeared to be associated with "Manic Episode" while the increase in PGD2 and alpha-1B glycoprotein levels appeared to be associated with "Bipolar Disorder".

Keywords: Alpha-1-microglobulin and bukinin precursor.; Biomarkers; Bipolar disorder; Manic state; Mannan-binding lectine serin protease-2; Proteomics.

Fig. 1

(A) A representative SDS-PAGE image of urine proteins with corresponding histograms. Twenty µg protein from each group was loaded to the gel, run at 180 V for 45 minutes before fixed and stained with colloidal Coomassie blue. (B) Representative two-dimensional gel electrophoresis (2-DE) images of the gels and identified protein spots. 2-DE electrophoresis was carried out with nonlinear 11 cm immobilized strips (pH 3−10) for the first dimension separation and 12% SDS-PAGE gels for the second dimension separation. Staining of the gels were performed in Colloidal Coomassie Blue. (C) The spots labeled with standard spot numbers were excised and identified. Protein identification was performed by peptide mass finger printing via MASCOT (Matrix Science, Boston, MA, USA). Proteins of interest that displayed changes in their urine levels were α-1-microglobulin and bukinin precursor (AMBP; SSP#21189), Mannan-binding lectine serin protease-2 (MASP-2; SSP#40069), alpha-1B- glycoprotein (SSP#3805), prostaglandin-H2 D-isomerase (PGD2; SSP#4108), Annexin A1 (SSP#8213) and Ig gamma-1-chain (SSP#8215). C, control group; M, manic episode group; R, remitted group.

Fig. 2

Close-up images of the Mannan-binding lectine serin protease-2 (MASP-2), α-1-microglobulin and bukinin precursor (AMBP), pro-staglandin-H2 D-isomerase (PGD2), α-1B-glycoprotein, Annexin A1 and Ig gamma-1-chain protein spots on the gels and the bar graphs (represented as mean ± standard deviation) showing the measured corresponding spot intensities. The protein spots were labeled with circles. Relative quantifi-cation of the spot intensities was performed using PD Quest Advanced software (Bio-Rad, Hercules, CA, USA). C, control group; M, manic episode group; R, remitted group; OD, optical density; NS, not significant. An asterisk (*) represents a significant difference between the groups (*p < 0.05; **p < 0.01, ***p < 0.001).

Fig. 3

(A) Representative Western blot images of Mannan-binding lectine serin protease-2 (MASP-2) and α-1-microglobulin and bukinin precursor (AMBP) proteins. (B) The bar graphs for the band intensities belonging to MASP-2 and AMBP (Graph bars represent mean ± standard deviation from three independent experiments). (C, D) Ponceau S and Coomassie Staining images of the membranes and gels, respectively, that were used in western blot analysis. Western blots for MASP-2 and AMBP were performed by loading 40 mg and 4 mg total protein per well and separating on 14% SDS-PAGE gels, respectively. C, control group; M, manic episode group; R, remitted group. An asterisk (*) represents a significant difference between the groups (*p < 0.05; **p < 0.01, ***p < 0.001).