Protein Arginine Methylation Patterns in Plasma Small Extracellular Vesicles Are Altered in Patients with Early-Stage Pancreatic Ductal Adenocarcinoma

Abstract

Small extracellular vesicles (sEVs) contain lipids, proteins and nucleic acids, which often resemble their cells of origin. Therefore, plasma sEVs are considered valuable resources for cancer biomarker development. However, previous efforts have been largely focused on the level of proteins and miRNAs in plasma sEVs, and the post-translational modifications of sEV proteins, such as arginine methylation, have not been explored. Protein arginine methylation, a relatively stable post-translational modification, is a newly described molecular feature of PDAC. The present study examined arginine methylation patterns in plasma sEVs derived from patients with early-stage PDAC (n = 23) and matched controls. By utilizing the arginine methylation-specific antibodies for western blotting, we found that protein arginine methylation patterns in plasma sEVs are altered in patients with early-stage PDAC. Specifically, we observed a reduction in the level of symmetric dimethyl arginine (SDMA) in plasma sEV proteins derived from patients with early- and late-stage PDAC. Importantly, immunoprecipitation followed by proteomics analysis identified a number of arginine-methylated proteins exclusively present in plasma sEVs derived from patients with early-stage PDAC. These results indicate that arginine methylation patterns in plasma sEVs are potential indicators of PDAC, a new concept meriting further investigation.

Keywords: arginine methylation; biomarkers; pancreatic ductal adenocarcinoma; small extracellular vesicles; symmetric dimethylarginine.

Figure 1

Validation of the arginine methylation antibodies and the sEVs isolated from human plasma. (A,B) Western blot detection of SDMA and MMA in PANC-1, MIA PaCa-2 and HPNE cell lysates. (C,D) Western blot detection of SDMA and MMA in PANC-1, MIA Paca-2 and HPNE cell derived sEV lysates. (E) Western blot detection of exosome markers (Flotillin-1, CD63 and CD9) and the negative marker, Calnexin, in human plasma sEVs. (F) Particle size determination of plasma sEVs using nanoparticle tracking analysis (NTA).

Figure 2

Arginine methylation patterns in plasma sEVs derived from healthy subjects and patients with PDAC and colon cancer. Western blot analysis of individual plasma sEV lysates was performed using antibodies against MMA, SDMA and ADMA. Representative gels are shown. (A) MMA detection: (left), Coomassie blue staining of the gel as loading control; (right), detection of MMA. (B) SDMA detection: (left), Coomassie blue staining of the gel as loading control; (right), detection of SDMA. (C) ADMA detection: (left), Coomassie blue staining of the gel as loading control; (right), detection of ADMA. (D) Quantification of the MMA band I and band II. (E) Quantification of SDMA band I and band II. (F) Quantification of the ADMA band I and band II. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons (n = 16).

Figure 3

Arginine methylation patterns in plasma sEVs derived from healthy subjects and patients with early- and late-stage PDAC. Western blot of individual plasma sEV lysates was performed using antibodies against MMA and SDMA. Representative gels are shown. (A) MMA and SDMA detection in plasma sEVs derived from patients with early- and late-stage PDAC: (left), Coomassie blue staining of the gel as loading control; (middle), detection of MMA; (right), detection of SDMA. (B) Quantification of MMA bands I and II. (C) Quantification of SDMA bands I and II. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons (n = 23 for early-stage PDAC and 22 for healthy subjects, n = 10 for late-stage PDAC).

Figure 4

Arginine methylation patterns in plasma sEVs derived from healthy subjects and patients with early-stage PDAC and chronic pancreatitis (CP). Western blot of plasma sEV lysates was performed using antibodies against MMA and SDMA. Representative gels are shown. (A) MMA and SDMA detection: left, Coomassie blue staining of the gel as loading control; middle, detection of MMA; right, detection of SDMA. (B) Quantification of MMA band I and II. (C) Quantification of SDMA band I and II. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons (n = 23 for early-stage PDAC and 22 for healthy subjects, and n = 8 for CP).

Figure 5

Identification of plasma sEV proteins corresponding to SDMA band I and band II. (A) The proteomics data obtained from digestion of gel portions corresponding to SDMA band I, and analyzed using Sequest: (left), Sequest score of the detected proteins; (right), abundance of the proteins. (B) The proteomics data obtained from the digestion of gel portions corresponding to SDMA band II, and analyzed using Sequest: (left), Sequest score of the detected proteins; (right), abundance of the proteins. (C) Proteins identified by Sequest analysis of the proteomics results.

Figure 6

Validation of arginine methylation of Complement C3 and Alpha-2-macroglobulin. (A) Arginine methylation of Complement C3 and (B) Alpha-2-macroglobulin predicted by PRmePRed. (C,D) Western blot detection of Complement C3 and Alpha-2-macroglobulin in the immunoprecipitated fraction of plasma sEV lysates ((C), SDMA; (D) MMA).

Figure 7

Proteomics analysis of the SDMA immunoprecipitated plasma sEV proteins from patients with early-stage PDAC and matched healthy subjects. Plasma sEV lysates (n = 18/group, pooled samples) from early-stage PDAC and healthy controls were immunoprecipitated with the SDMA antibody followed by proteomics analysis. (A) Western blot validation of immunoprecipitation by the SDMA antibody using PANC-1 cell lysates. (B) Venn-diagram showing numbers of arginine-methylated proteins that are shared or exclusively present in plasma sEV lysates from patients with early-stage PDAC (total 69 proteins) and healthy subjects (total 61 proteins). (C) Selected proteins that are exclusively arginine-methylated and closely associated with human cancer.