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Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs

Animals and blood samples

Dogs were client owned or protection dogs brought to the Kagoshima University Veterinary Teaching Hospital for veterinary care. Residues of heparinized blood samples applied for biochemical

tests were used in this study. Information including the patient’s signalment, body condition score (5 points scale), diagnosis and undergoing treatment was obtained from the veterinary

medical record or from the veterinarians in charge.

Determination of canine SIRT1 mRNA nucleotide sequence

Total RNA was extracted from an EDTA-K2-treated blood sample from client owned dogs using a PureLink RNA Mini Kit (ThermoFisher Scientific, Waltham, MA, U.S.A.). Reverse transcription

polymerase chain reaction (PCR) was performed using Primescript One Step RT-PCR kit (Takara, Kusatsu, Japan). In a preliminary experiment, we could not amplify cDNA fragments encompassing

the whole predicted canine SIRT1 coding region (GenBank Accession No. XM_546130.4). Thus, primer pairs were designed to amplify two overlapping DNA fragments that cover the

whole SIRT1 coding region by nested and semi-nested PCR. In the first PCR, 5′-end and 3′-end DNA fragments were amplified by One-Step RT-PCR using the Sense_1-Reverse_1 and

Sense_2-Reverse_2 primer pairs, respectively (Table 1). PCR conditions were 94°C for 2 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 3 min. Then RT-PCR products were used as templates, nested and

semi-nested PCRs were conducted by GoTaq® Hot Start Colorless Master Mix (Promega, Madison, WI, U.S.A.), to amplify 5′ and 3′ cDNA fragments using the Sense_3-Reverse_3 and Sense_2-Reverse_1

primer pairs (Table 1). The PCR conditions were the same as the first PCR. The second set of PCR products were electrophoresed in 2% agarose. DNA

fragments were extracted from the gel and purified using High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany). The nucleotide sequence was determined by the dye

terminator method at a commercial laboratory, Fasmac (Atsugi, Japan). We also investigated canine SIRT1 transcript variants by 5′-terminal inverse PCR amplification. Total

RNA was extracted as mentioned above from blood samples obtained from a client owned Labrador Retriever. The 5′-end of canine SIRT1 cDNA was amplified using a 5′-Full RACE

Core Kit (Takara) according to the manufacturer’s instructions. The nucleotide sequence of the 5′-phosphorylated primer for reverse transcription (RT-primer) is shown in Table 1. Inverse nested PCRs were performed using GoTaq® Hot Start Colorless Master Mix. The nucleotide sequences of the primers for the first (S1-A1 primers) and

nested PCR (S2-A2 primers) are shown in Table 1. The PCR conditions for the first and second PCRs were 94°C for 2 min, followed by 30 cycles of

94°C for 30 sec, 55°C for 30 sec and 72°C for 3 min. The amplified PCR products were electrophoresed and purified as mentioned above, to determine the nucleotide sequences.

Western blot analysis of canine SIRT1 protein

We conducted to detect canine endogenous SIRT1 protein levels in PBMCs by western blotting. Heparinized blood sample from two client owned dogs was used for the analysis. The samples were

overlaid on Lympholyte-H specific gravity 1.077 (Cedarlane, Burlington, Ontario, Canada) and centrifuged at 900 × g for 30 min. PBMCs were collected and washed in phosphate-buffered saline

(PBS). Human embryonic kidney cells 293 (HEK293) and Madin-Darby canine kidney cells (MDCK) were also

used for western blotting as a source of human and canine SIRT1 protein. Cultured cells were resuspended in PBS and an equal volume of 2x sample buffer [0.125 M Tris-HCl, pH 6.8, 10% (v/v)

2-mercaptoethanol, 4% sodium dodecyl sulfate (SDS), 10% sucrose, 0.01% Bromophenol blue], and incubated at 95°C for 3 min. Samples were stored at −20°C until use. Twenty microliters of each

sample at the concentration of 2.5–5 mg/ml protein concentration (Bradford protein assay kit, Takara) were loaded on 8% polyacrylamide gels and electrophoresed. Samples were

transferred to nitrocellulose membranes (GE NitrocelluloseTM Pure Unsupported Nitrocellulose Membranes, GVS Filter Technology, Emilia-Romagna, Italy) using Trans blot SD Semi-Dry

Transfer Cell (Bio-rad, Hercules, CA, U.S.A.). The membranes were washed with Tris-buffered saline with 0.1% Tween (TBS-T: 0.05 M Tris, 0.138 M NaCl, 0.0027 M KCl and 0.1% Tween20), and then

incubated in 1 x TBS-T supplemented with 5% (w/v) Nonfat Dry milk (Cell Signaling Technology, Danvers, MA, U.S.A.). The membranes were washed with 1 x TBS-T, and then incubated with the

anti-human SIRT1 monoclonal antibody, 1F3 (Cell Signaling Technology), diluted in 1 x TBS-T (1:1,000) at 4°C overnight. The membranes were washed with 1 x TBS-T and incubated with diluted

(1:5,000) horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark) for 1 hr at room temperature. The membranes were washed with TBS-T and chemiluminescence was

detected with ECL Prime Western Blotting Detection System (GE Healthcare, Parsippany, NJ, U.S.A.) using the FUSION SOLO S imaging system (Vilber-Loumat, Collegien, France).

Expression of recombinant canine SIRT1

Canine SIRT1 expression plasmid was constructed to examine canine SIRT1 protein could be detected by flow cytometry using the monoclonal antibody 1F3 against human SIRT1. We detected three

canine SIRT1 mRNA transcript variants Variant1-3. The deduced amino acid sequence of Variant2 and Variant3 was the same . Thus we planned to express the protein coded by

Variant1 and Variant2 (Fig. 1). Nucleotides corresponding to Variant1 were synthesized at Fasmac, adding a Kozak sequence “ccacc” adjunct to the putative start codon. The codon was modified to optimize protein

expression in human cells. DNA fragments corresponding to Variant1 and Variant2 were amplified by PCR from the synthesized gene using KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan) and

sub-cloned into pcDNA3.1 (+), a mammalian expression plasmid. The Canine SIRT1 expression plasmids, named Dog_Sirt1-long pcDNA3.1, Dog_Sirt1-short pcDNA3.1 was transfected into 293T cells

[9] using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnotics, Basel, Switzerland) and cultured for 48 hr. Then, the culture medium was removed

and the cells were detached using TrypeLE express (Thermo Fisher Scientific). The washed cells were applied for flow cytometry analysis.

Flow cytometry analysis of canine SIRT1 protein

Canine SIRT1 expression was examined in SIRT1 expression plasmids transfected 293T cells and PBMCs. PBMCs were isolated by gradient centrifugation from heparinized blood samples using

Lympholyte-H as mentioned above. PBMCs were collected and washed with PBS. PBMCs and the transfected 293T cells were fixed and permeabilized using FIX & PERM® Cell Permeablization

Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. The cells were incubated with 1F3 monoclonal antibody (1:200), anti-β-actin monoclonal antibody (8224,

Abcam; 1:200) as a positive control, or mouse IgG1k isotype Ctrl (MOPC-21, BioLegend, CA, U.S.A.; 1:100) at 4°C for 30 min. The cells were washed and incubated with phycoerythrin conjugated

goat anti-mouse IgG (1: 1,000) for 20 min at room temperature. The cells were then washed with PBS and flow cytometry analysis was performed using FACSCalibur with CellQuest pro software

(Becton Dickinson, Franklin Lakes, NJ, U.S.A.). SIRT1 expression in lymphocytes was represented with the ratio of SIRT1 to β-actin mean fluorescence intensity.

Statistics

The relation between SIRT1 expression (SIRT1/β-actin ratio) and age, gender, degree of obesity was evaluated statistically. Pearson’s correlation coefficient was calculated between SIRT1

and age. Differences of SIRT1 expression among gender was statistically tested by one way analysis of variance with post-hoc Tukey honestly significant difference test. Spearman’s rank

correlation coefficient was calculated between SIRT1 and body condition score. Significant level less than 0.05 was considered statistically significant.

Nucleotide sequence of canine SIRT1 mRNA

In our preliminary experiment, we failed to amplify the cDNA fragment containing the entire coding region of the predicted canine SIRT1 mRNA (XM_546130.4). Thus, we

amplified a 5′-end- and a 3′-end cDNA fragment that covers the entire coding region. Though the 3′-end cDNA fragment was successfully amplified, the 5′-end DNA fragment was amplified only in

one of five blood samples examined. A schematic view of these DNA fragments is shown in Fig. 1. The amplified 5′-end fragment included the putative

initiation codon of the predicted canine SIRT1 mRNA, but nucleotides 108 to 434 of the predicted sequence were not present (named Variant 1, INSD accession No. LC342295).

This deleted region is within exon 1 and does not cause a codon frame shift downstream of the transcript. Because this transcript was amplified only in one sample, 5′ inverse RT-PCR was

conducted to detect other transcript variants. Two DNA fragments were amplified: one variant starts at nucleotide 456 (named Variant 2, INSD accession No. LC342296) of the predicted canine

SIRT1 mRNA (XM_546130.4), and the other starts at nucleotide 626 (named Variant 3, INSD accession No. LC342297; Fig. 1). The

putative translation start codon was predicted using the ATGpr program (http://atgpr.dbcls.jp/), indicating that

the translation start at nucleotide 663 and the frame was the same as that of the predicted canine SIRT1 mRNA (XM_546130.4).

Detection of canine SIRT1 protein by Western blotting

Monoclonal antibodies against canine SIRT1 protein have not been reported. Based on the canine SIRT1 nucleotide sequence, we selected the monoclonal antibody 1F3 (Cell

Signaling Technology), which was produced using the C-terminal region of human SIRT1 as the immunogen. The deduced canine SIRT amino acid sequence (Variant 1) is identical to human SIRT1 in

189 out of 190 amino acids in this region. The deduced molecular size of human SIRT1 is 81.6 kDa. A single band of approximately 128 kDa was detected in HEK293 cells, which are

human-derived, by western blotting (Fig. 2). The deduced molecular size of canine SIRT1 was 70.9 kDa for Variant 1, and 62.1 kDa for Variants 2 and 3. In MDCK cells, which are dog-derived, and in canine PBMC, a single band of

approximately 120 kDa was detected in each cell line (Fig. 2).

Flow cytometry analysis of canine SIRT1 in PBMCs

To confirm the reactivity of 1F3 antibody with canine SIRT1 protein, the SIRT1 expression plasmids (Dog_Sirt1-long pcDNA3.1 and Dog_Sirt1-short pcDNA3.1) was transfected into 293T cells.

Canine SIRT1 expression was examined by flow cytometry after fixation and permeabilization. Though this antibody may react with endogenous human SIRT1, the fluorescence intensity increased

in cells transfected with the SIRT1 expression plasmids, compared with the mock-transfected cells (Fig. 3) indicating that 1F3 reacted to canine SIRT1. Next, canine SIRT1 in PBMCs was examined by flow cytometry. PBMCs were fixed and permeabilized, and then incubated with 1F3 as the

primary antibody. SIRT1 expression in representative samples is shown in Fig. 4. SIRT1 was detected in lymphocytes, monocytes and contaminated granulocytes. Though SIRT1 was detected in all the dogs tested, the fluorescence intensity varied among individuals

(Fig. 5). Relation of SIRT1 expression and age, gender, degree of obesity (body condition score) was examined. Correlation between SIRT1 expression and age or body condition score was not

statistically significant. SIRT1 expression was statistically different among genders (Fig. 6). SIRT1 expression in neutered female dogs was higher than that in intact- female and intact-male dogs (P<0.05).

DISCUSSION

In our knowledge, this is the first report that demonstrates SIRT1 transcription variants in dogs. In this study, three 5′ transcription variants were detected in PBMC from dogs. At least six

variants have been reported in both human and mouse (The AceView genes, NCBI). Most variants in human are shorter with a deletion at exon 1 or exon 2. Though we did not examine the function of

the three transcript variants in dogs, some studies have demonstrated that the SIRT1 transcript variants function differently from the full-length SIRT1,

especially at the interaction point with p53 [27, 45]. We detected three variants by an RT-PCR-based method and

only one band of approximately 120 kDa was observed by western blotting. The deduced molecular size of the canine SIRT1 protein was 70.9 kDa (Variant 1) and 62.1 kDa (Variants 2 and 3). The

deduced molecular size of human SIRT1 was 81.6 kDa. We detected a 128-kDa band using HEK293 cells, as shown in a previous study using muscle samples

and in the 1F3 antibody product data sheet supplied by the manufacturer (CST). The differences in observed SIRT1 molecular sizes are likely due to posttranslational glycosylation. The bands

detected by western blotting in canine PBMCs presumably derived from Variants 2 and 3 mainly because only these variants were detected by inverse RT-PCR. However, the molecular size of SIRT1

derived from each transcript variants should be clarified by the expression of recombinant SIRT1 in canine cells.

To confirm the specificity of 1F3 antibody to canine SIRT1 protein, recombinant canine SIRT1 was expressed in 293T cells. Because this cell line is derived from human, 1F3 also reacted with

the endogenous human SIRT1. However, a marked enhancement of the signal was demonstrated in the canine SIRT1 expression plasmid-transfected cells. These results indicated that intracellular

canine SIRT1 is detectable by flow cytometry using 1F3 antibody. Recently, compounds that activate SIRT1 enzymatic activity have been intensely investigated in association with lifespan

extension. Recombinant canine SIRT1 protein expressed in transfected cells may be useful for screening compounds by measuring the enzymatic activity of canine SIRT1 in cultured cell lines.

Compounds that possibly enhance SIRT1 expression have also been investigated intensively [6, 7, 14, 39, 41]. Though it has not been fully demonstrated that enhancement of

SIRT1 expression is associated with lifespan extension, if it comes to that, measurement of SIRT1 expression level may be valuable for monitoring the patient’s condition and for evaluating the

effect of interventions. In this study, we examined intracellular SIRT1 level in peripheral blood lymphocytes for ease of access in clinical cases. Though some studies measured the serum SIRT1

concentration to evaluate the SIRT1 expression level [10, 24, 29, 32, 52], the significance of the plasma SIRT1 concentration remains to be clarified because SIRT1 localizes mainly in

the nucleus or the cytoplasm. SIRT1 in the cellular lysate and in the serum can potentially be measured by enzyme linked immunosorbent assay (ELISA) in dogs. However, as only one antibody is

available at present, it may be difficult to establish sensitive and specific Sandwich ELISA to measure canine SIRT1. The information on the canine SIRT1 5′-end transcript

variants obtained in this study will be useful for the development of antibodies and ELISA for canine SIRT1 measurement.

Minimally invasive procedures for sample collection make it easier to monitor the fluctuation of SIRT1 expression. Flow cytometry analysis can evaluate SIRT1 expression levels in individual

cells, thus enabling comparison of the expression between leukocyte subsets. In this study, flow cytometry analysis was conducted using a blood sample residues (usually less than 1

ml of blood) obtained from animals in veterinary care. SIRT1 seemed to be expressed in all leukocyte subsets: lymphocytes, monocytes, and contaminated granulocytes. In human

diabetes patients, SIRT1 was detected in monocytes and granulocytes, but not in lymphocytes, by immunocytochemical staining. The discrepancy may be

because of species differences or the high sensitivity of flow cytometry applied in this study. Because the permeability of the cells may differ between the samples, we used β-actin as an

internal control for evaluating SIRT1 protein expression. The compensated SIRT1 expression level markedly varied among individual dogs. Though major factors that influence the SIRT1 expression

were not fully clarified in this study, the level in neutered female dogs was higher than intact-female and intact-male dogs. Variance of SIRT1 protein expression by age and gender has also

been reported in human [22, 25, 34]. But the trend of the variance in human

was study dependent probably reflecting the difference of sample tissue and method applied. While negative correlation between SIRT1 expression and obesity has also been demonstrated in human

[19, 31, 33, 48], significant

correlation between SIRT1 expression and body condition score of the dogs was not found in this study. Because the samples were obtained from dogs admitted for veterinary care, other factors

including disease, medication, as well as circadian change might influence the SIRT1 expression. Comparison of SIRT1 expression among species or

individuals in specific condition may contribute to further understanding the regulation of SIRT1 expression. Transcription factors including HIC1, E2F1 and FOXO are involved in the regulation

in response to environmental change in humans [5, 40, 46]. Most of the

responsive elements to these factors are located within 1kb upstream from SIRT1 initiation codon. Though the promoter/enhancer region of the canine SIRT1 gene

has not been completely analyzed, nucleotide sequence of the region is well conserved between two species. Future studies using a large number of samples in specific condition may clarify

major contributing factors that influence SIRT1 expression in dogs.

In conclusion, herein we report the nucleotide sequence of canine SIRT1 cDNA and the SIRT1 transcript variants in PBMCs. Canine SIRT1 were detectable using a

monoclonal antibody developed for human SIRT1. SIRT1 expression in peripheral leukocytes can be examined by flow cytometry using this antibody. This method is applicable for further research

evaluating the effect of intervention on potentially fluctuating SIRT1 expression in dogs.