Generation of Msr overexpressing and knockout mice
All mice described in this work were generated on C57BL/6 background. Mice were treated in accordance with the Guide for the Care and Use of Laboratory Animals
(NIH publication 85–23, 1996), and the study was approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. MsrA transgenic mice were generated as described [14
]. MsrA residues 21–233 were included in the construct TgCyto_Myr for the cytosolic targeted transgenic mice, and the overexpressed MsrA was myristoylated in vivo
as in the Wt [15
]. Possible transgenic mice were screened for the presence of the transgene by PCR as described [14
], and the founders were crossed with wild type C57BL/6 mice to generate transgenic lines. The KO_A mice used in this study were originally generated from a 129/SvJ background and later backcrossed for 10 generations into the C57BL/6 background [16
]. Female mice heterozygous for the floxed exons 2 and 3 of the mice MsrB1 gene were generated by Ozgene (Bentley DC, WA, Australia) on a C57BL/6 background. Exon 2 and 3 of the MsrB1 gene were flanked by LoxP sites on chromosome 17 (). Genomic excision of exon 2 and 3 of MsrB1 gene was accomplished by crossing with male B6.C-Tg (CMV-cre)1Cgn/J Cre recombinase expressing mice (006054, The Jackson laboratory, Bar Harbor, ME, USA). Offspring were genotyped by PCR using 3 primers (Forward 5′ CAGGTTGATCTAGGCAAATCCTCAGC 3′, Reverse 5′ GTCTCTCCCTCCACTAGCAGACACAG 3′, Reverse 5′ ATGAGTGTGCGTACTTCGAGTGACTG 3′) to confirm genomic deletion of exon 2 and 3 of MsrB1 gene (). After that, KO_B1 mice were further bred with KO_A mice for generation of MsrA/MsrB1 double knockout mice. The levels of transgenic MsrA in the overexpression mice, and the absence of MsrA and/or MsrB1 in the knockout mice, were confirmed by Western blotting using brain, liver and kidney tissues.
Figure 2 Generation of MsrB1 and double knockout mice. A) Scheme of the mouse MsrB1 gene and its flanking regions on chromosome 17. A segment with two LoxP sites flanking exon 2 and 3 replaced the endogenous sequence through homologous recombination (arrows). (more ...)
Weanling mice growth and diets
The growth study on male C5BL/6 mice was performed in the NIH Building 50 shared mice facility. Mice were housed at 3–4 animals per cage, with room temperature maintained at 20–23°, 30–70% humidity, and a 14/10 h light/dark cycle. Mice were weaned at 24 days of age and were immediately placed on a custom-manufactured diet containing 0.1%, 0.15%, 0.20%, and 0.45% methionine respectively in otherwise unmodified TestDiet 578C (TestDiet, Richmond, IN, USA). Growth rate was monitored by measuring body weight 3 times a week for one month. At the end the one month on the defined diets, the mice were about 10 weeks of age. After an overnight fast they were anesthetized with 0.01ml/g body weight ketamine/xylazine cocktail (ketamine 80mg/ml, xylazine 6mg/ml, K4138, Sigma, MO, USA). Blood and tissue samples were quickly taken, snap-frozen in liquid nitrogen, and stored at −80° for further analyses. The first study employed only the Wt genotype, with 5 animals per diet. The methionine content of the diet was 0.10%, 0.15%, 0.20%, or 0.45%. The second study included all genotypes and the diets containing 0.10%, 0.15%, and 0.45% methionine. The number of animals on the 3 diets by genotype were: Wt - 12,19,13; KO_A - 8,7,3; KO_B1 - 4,3,4; KO_AB1 - 8,4,8; TgCyto_Myr - 6,5,2. Growth curves were empirically fit to second order polynomials and compared with correction for multiple comparisons using Prism software, version 5.03 (GraphPad Software, La Jolla, CA).
Liver, kidney and brain samples were weighed and homogenized with a hand-held micro grinder (Kontes Pellet Pestle) in ten volumes of RIPA lysis buffer (R0278, Sigma, MO, USA) supplemented with a 1:1,000 dilution of protease inhibitor cocktail (P8340, Sigma, MO, USA). Protein concentration was determined by the BCA method (Pierce-Thermo Scientific, USA). SDS-PAGE was performed on 10–20% gradient gels (EC61385, Invitrogen, CA, USA). Proteins were transferred onto 0.45μm nitrocellulose membrane (LC2001, Invitrogen, CA, USA) and then incubated in Odyssey blocking buffer (927–40000, Li-Cor, NE, USA) for 30 min. Overnight incubations with the primary antibodies were performed at 4° with a 1:25,000 dilution of polyclonal rabbit anti-MsrA (this laboratory), or a 1:20,000 dilution of monoclonal mouse anti-GAPDH (G8795, Sigma, MO, USA), or a 1:200 dilution of polyclonal rabbit anti-MsrB1 (LF-PA 0088, Abfrontier, Seoul, Korea), or a 1:1000 dilution of polyclonal rabbit anti-GNMT (sc-68871, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a 1:500 dilution of goat polyclonal anti-CBS (sc-46830 (K14), Santa Cruz Biotechnology, Santa Cruz, CA, USA). Incubations with the secondary antibodies were done at room temperature for 1 h with a 1:10,000 dilution of Dylight™ 680 goat anti-rabbit IgG (072-06-15-06, KPL, Gaithersburg, MD, USA) or IRDye 800CW goat anti-mouse IgG (926–32210, Li-Cor, Lincoln, NE, USA), or a1:20,000 dilution of IRDye 800CW donkey anti-goat IgG (926–32214, Li-Cor, Lincoln, NE, USA). For quantitation purpose, membranes were scanned with Li-Cor Odyssey Infrared Imager (Li-Cor, Lincoln, NE, USA) in the 700 nm and 800 nm channels.
Measurement of plasma methionine and tissue S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)
Blood was collected from the orbital sinus into a Vacutainer heparinized tube (BD, Franklin Lakes, NJ, USA) after removal of the eyeball. Plasma was immediately separated by centrifugation at 960 g
for 5 min at 4°. Ten μl plasma was mixed with 10 μl 20% trichloroacetic acid, and centrifuged at 20,800 g
for 3 min at 4°. Ten μl supernatant was mixed with 40 μl 0.05% trifluoroacetic acid and the pH was adjusted to 7.5 with 6 M NaOH. Amino acid analysis was performed as described [2
]. Brain, liver and kidney SAM and SAH levels were also measured as previously described [17
]. Briefly, 80–100 mg of tissue was homogenized in 4 volumes of 0.4M HClO4
and centrifuged at 20,800 g
for 15 min at 4°. The supernatant was filtered through 0.45μm syringe filter (SVJH004NS, Millipore, Billerica, MA, USA ) and 25 μl was injected onto an Eclipse 3 μm, 4.6 × 75mm C18
column (Agilent, Santa Clara, CA, USA) in an Agilent 1200 HPLC system. The initial mobile phase was 8mM 1-octanesulfonic acid sodium salt (O0133, Sigma, MO, USA) in 50mM NaH2
, pH3.0. Analytes were separated by developing a gradient of 20 to 45% methanol over 7.5 min. SAM and SAH were quantitated from the area of their peaks compared to that of SAM and SAH standards (A9384 and A4377, Sigma, St. Louis, MO, USA).
Determination of glycine N-methyltransferase (GNMT)
Liver tissue GNMT activity was determined with 3
H labeled SAM as described by Cook and Wagner with minor modifications [18
]. Lysate containing 200 μg protein was mixed with 20μl 0.5 M Tris HCl pH7.4, 20μl 10 mM glycine, and 20μl 1 mM adenosyl-L-methionine S-[methyl-3
H] (1 μCi/μmol) (NET155050UC, PerkinElmer, Waltham, MA, USA ) and brought to a final volume of 100μl. It was incubated at 25° for 15min, and the reaction stopped by addition of 50 μl ice-cold 10% trichloroacetic acid followed by 250μl 80 mg/ml acid washed charcoal. The tube was held on ice for 15min and then centrifuged at 20,800 g
for 2min. Radioactivity in the supernatant was measured in a liquid scintillation analyzer (TRI-CARB 2900TR, PerkinElmer, Waltham, MA, USA ). Blank values were obtained by omission of glycine in the reaction mixture. The GNMT specific activity was expressed as nmol sarcosine formed per min per milligram protein.