Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues

Abstract

Full-length human protein arginine methyltransferase 7 (PRMT7) expressed as a fusion protein in Escherichia coli was initially found to generate only ω-N(G)-monomethylated arginine residues in small peptides, suggesting that it is a type III enzyme. A later study, however, characterized fusion proteins of PRMT7 expressed in bacterial and mammalian cells as a type II/type I enzyme, capable of producing symmetrically dimethylated arginine (type II activity) as well as small amounts of asymmetric dimethylarginine (type I activity). We have sought to clarify the enzymatic activity of human PRMT7. We analyzed the in vitro methylation products of a glutathione S-transferase (GST)-PRMT7 fusion protein with robust activity using a variety of arginine-containing synthetic peptides and protein substrates, including a GST fusion with the N-terminal domain of fibrillarin (GST-GAR), myelin basic protein, and recombinant human histones H2A, H2B, H3, and H4. Regardless of the methylation reaction conditions (incubation time, reaction volume, and substrate concentration), we found that PRMT7 only produces ω-N(G)-monomethylarginine with these substrates. In control experiments, we showed that mammalian GST-PRMT1 and Myc-PRMT5 were, unlike PRMT7, able to dimethylate both peptide P-SmD3 and SmB/D3 to give the expected asymmetric and symmetric products, respectively. These experiments show that PRMT7 is indeed a type III human methyltransferase capable of forming only ω-N(G)-monomethylarginine, not asymmetric ω-N(G),N(G)-dimethylarginine or symmetric ω-N(G),N(G')-dimethylarginine, under the conditions tested.

FIGURE 1.

Purification of human GST-PRMT7. SDS-12.6% polyacrylamide gel electrophoresis of GST-PRMT7 following purification as described under “Experimental Procedures.” GST-purified protein (3 μg) was analyzed in the left lane; molecular mass standards (Bio-Rad low range containing phosphorylase b, 97.4 kDa; serum albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; and trypsin inhibitor, 21.5 kDa) were analyzed in the right lane. Following electrophoresis, the gel was stained with Coomassie Blue and dried.

FIGURE 2.

GST-PRMT7 can monomethylate peptide R1 (GGFGGRGGFGG-amide). In vitro methylation reactions in a final reaction volume of 60 μl were carried out with GST-PRMT7 (6 μg; 0.95 μm) in the presence of 0.7 μm [³H]AdoMet with peptide substrate R1 (52 μg; 1.0 mm) as described under “Experimental Procedures.” After acid hydrolysis, methylated amino acid derivatives were analyzed by high-resolution cation-exchange chromatography as described under “Experimental Procedures.” Radioactivity (solid lines) from a 1- (■, panel A), 5- (♦, panel B), and 20-h (●, panel B) incubation is shown for complete reaction mixtures. Control incubations were also performed in the absence of peptide substrate (○) or PRMT7 enzyme (♢) for 1 (panel A) or 20 h (panel B). Radioactivity and ninhydrin color of the methylated arginine standards (100 μl analyzed; □, dashed lines) were determined as described under “Experimental Procedures.” Due to a tritium isotope effect, the ³H-methyl derivatives of ADMA, SDMA, and ω-MMA elute on high-resolution cation-exchange chromatography columns slightly earlier than the non-isotopically labeled standards (22, 26, 30, 31, 32). The inset in panel B shows a magnification of the radioactivity in the elution region of ADMA and SDMA.

FIGURE 3.

GST-PRMT7 fails to dimethylate peptide P-SmD3. In vitro methylation reactions were carried out for 20 h in a final volume of 60 μl with the substrate P-SmD3 (AGRGRGKAAILKAQVAARGRGRGMGRGN-amide) (8.4 μg; 50 μm) in the presence of 0.7 μm [³H]AdoMet and GST-PRMT7 (4 μg; 0.63 μm) (●, panel A) or GST-PRMT1 (4 μg; 0.98 μm) (●, panel B) as described under “Experimental Procedures.” Enzyme alone controls (▴, PRMT7, panel A, or ▴, PRMT1, panel B), and substrate alone controls (■, peptide P-SmD3) were carried out under the same conditions. Chromatography of the hydrolyzed reaction products was carried out as described under “Experimental Procedures” using 100 μl for ninhydrin analysis of the standards (□, dashed lines). Radioactivity is shown by solid lines. The inset in both panels shows a magnification of the radioactivity in the elution region of ADMA and SDMA.

FIGURE 4.

GST-PRMT7 fails to dimethylate protein complex SmB/D3. In vitro methylation reactions were carried out for 1 h in a final volume of 60 μl with the protein substrate SmB/D3 (7 μg; 5.8 μm) in the presence of GST-PRMT7 (15 μg; 2.4 μm) (●, panel A), GST-PRMT1 (2 μg; 0.49 μm) with 7.5 μg of SmB/D3 (5.4 μm) (●, panel B) or Myc-PRMT5 (4 μg; 0.81 μm) with 13 μg of SmB/D3 (10.8 μm) (●, panel C) as described under “Experimental Procedures.” Enzyme alone controls (▴) and substrate alone controls (■) were carried out under the same conditions for each enzyme. Chromatography of the hydrolyzed reaction products was carried out as described under “Experimental Procedures” using 100 μl for ninhydrin analysis of the standards (□, dashed lines). The insets show a magnification of the radioactivity in the elution region of ADMA and SDMA.

FIGURE 5.

GST-PRMT7 fails to form ADMA or SDMA with increased substrate concentrations. In vitro methylation reactions (60 μl) of GST-PRMT7 (2 μg; 0.32 μm) with peptide P-SmD3 (1 μg (■), or 5 μg (♦), or 50 μg (●) (6.0, 30, and 300 μm, respectively)) were carried out for 21 h in the presence of 0.7 μm [³H]AdoMet as described under ”Experimental Procedures” (panel A). In vitro methylation reactions of GST-PRMT7 (2 μg; 0.32 μm) with peptide P-SmD3 (8.3 μg; 50 μm) were performed for 21 h in the presence of either 0.7 μm [³H]AdoMet (♦) or 1.4 μm [³H]AdoMet (●) (panel B). Chromatography of the hydrolyzed reaction products was carried out as described under “Experimental Procedures” using 100 μl for ninhydrin analysis of the standards (□, dashed lines). The insets show a magnification of the radioactivity in the elution region of ADMA and SDMA.

FIGURE 6.

GST-PRMT7 can methylate histone H2B producing only ω-MMA. In vitro methylation reaction of GST-PRMT7 (15 μg; 2.4 μm) with histone H2B (10 μg; 12.1 μm) was carried out for 1 (■), 5 (♦), and 25 h (●) in a final volume of 60 μl. Chromatography of the hydrolyzed reaction products was carried out as described under “Experimental Procedures” using 50 μl for ninhydrin analysis of the standards (□, dashed lines). The inset shows a magnification of the radioactivity in the elution region of ADMA and SDMA.