Autoantibodies against tyrosyl-tRNA synthetase and its separated domains at essential hypertension

M. Yu. Grom1, L. F. Yakovenko2, V. M. Granich3, A. S. Dobrohod3, O. O. Torbas3, G. D. Radchenko3, Yu. M. Sirenko3, L. L. Sidorik2, A. I. Kornelyuk2 1 Educational and Scientifi c Center "Institute of Biology" Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., Kyiv, Ukraine, 01601 2 Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 3 National Scientifi c Center "M. D. Strazhesko Institute of Cardiology, NAMS of Ukraine" 5, Narodnogo Opolchennya Str., Kyiv, Ukraine, 03680 grom.m.yu@gmail.com


Introduction
Tyrosyl-tRNA synthetase (TyrRS) is one of 20 conservative ancient enzymes that are critical at the initial stage of protein synthesis.The aminoacylation reaction, catalyzed by aminoacyl-tRNA synthetases, attaches each amino acid to its cognate tRNA [1,2].In addition to the aminoacylation, tRNA synthetases perform other non-canonical functions due to the interac-tions with various cellular partners [3].New missions can be associated with their cytoplasmic forms as well as with nuclear and secreted extracellular forms that affect the signaling, immune response, and pathways of the cardiovascular development [4,5].Considering the functional versatility of ARSs, their expanded functions and expression may be associated with the pathology of various human diseases [6].These enzymes are implicated into the neuronal diseases [7][8][9], tumorogene-sis, [10], autoimmune diseases, namely, antisynthetases syndrom [11][12][13], and heart failure [14].
The native mammalian TyrRS is a procytokine [15].Under apoptotic conditions, it can be secreted and split by leukocyte elastase into two fragments with distinct cytokine properties [16,17].The N-ter minal catalytic fragment (miniTyrRS) with a Glu-Leu-Arg (ELR) cytokine motif is a potent promoter of the angiogenesis [18] and polymorphonuclear leu kocytes recruitment [19,20].The C-terminal domain (CTD) has a high sequence similarity to the mature form of the pro-infl ammatory cytokine-like protein known as hu man Endothelial-Monocyte-Activating Polypep ti de (EMAP II) [21] endowed with angiogenic properties [22,23].
Considering that the functional diversity of ARSs is often associated with pathological conditions, and that the separated domains of TyrRS are actively involved in the angiogenesis, we inferred a role of TyrRS in the cardiovascular diseases.Moreover, taking into account the capacity of the enzyme fragments to endotheliocytes recruitment [24][25][26][27], probably TyrRS is pathologically associated with essential hypertension (EH).
The latest studies are focused on the activities of TyrRS domains in the treatment of cardiovascular diseases (CVD) [28][29][30], but they do not consider the importance of specifi c autoantibodies (aAbs) against TyrRS and its natural fragments.Meanwhile, the role of antibodies in the pathogenesis of CVD, including EH, is highlighted [31].Therefore, the investigation of aAbs against TyrRS and its natural fragments can improve our understanding of an importance of the enzyme and its separated domains in health and pathologies in general, EH in particular.The purpose of this study was to identify the autoantibodies against TyrRS and its individual modules in sera of the persons with EH, the healthy individuals with family history of EH, and the normal healthy subjects in a control group.

Patients and Sera
128 persons with EH were examined (35.3 % females and 64.7 % males; mean ± SD ages 48. 4 ± 27,6).All of them had high blood pressure from 7 to 20 years.Serum samples were selected from 25 well-charac-terized persons with EH and with or without target organ damage, 12 healthy individuals with fa mily history of EH, and 32 healthy volunteers.The males were recruited into all of the cohort studies in a higher proportion.This research was conducted in compliance with the declaration of Helsinki and was approved by the local ethics committee in the National Scientifi c Center «M.D. Strazhesko Institute of Car diology» of NAMS of Ukraine.All subjects were informed of the study purposes, and their informed consents were obtained.

Production and purifi cation of TyrRS and its separated domains
Generation of the recombinant full-length His-TyrRS (528 aa), His-miniTyrRS (362 aa), and His-CTD (166 aa) of Bos taurus has been performed according to our previous report [14].The use of the bovine proteins is valid because of a high identity to the human TyrRS and its natural fragments (≈95 %).The constructed plasmid vectors designated as pET-30a-TyrRS, pET-30a-miniTyrRS, and pET-30a-His-CTD were transformed into E. coli BL21 (DE3) pLysE cells (Novagen, Madison, WI) grown in Lysogeny broth (LB) at 37 C to optical density (OD) of 0.7-0.9(600 nm).The expression of recombinant proteins with 1 mM iso propyl-β-D(2)-thioga lacto pyra noside (IPTG, Fermentas, Cam bridge, United Kin gdom) was induced for 4 h.Affi nity purifi cation of the recombinant proteins from the cultural media using nickelnitriloacetic acid (Ni-NTA resin, Thermo Scientifi c, USA) was carried out according to the manufacturer's recommendations.The purity of His-TyrRS, His-mini-TyrRS, and His-CTD was confi r med by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a mixture of mar ker proteins (Fermentas, Lithuania).

ELISA assay
Specifi c binding of serum IgG aAbs to the recombinant enzyme or its separated domains was analy zed using direct solidphase ELISA.TyrRS, miniTyrRS, or CTD (1 μg/well) in a phosphate-buffered saline (PBS, pH 7.4) were incubated in 96-well polystyrene plates.Then the plates were washed ten ti mes with PBS containing 0.1 % Tween-20 (PBS-T), and, in order to block non-specifi c binding, the samples were incubated for 1 h at 37 C with 100 μl of PBS-T added to each well.Subsequently, the wells loaded with 1 : 50 diluted aliquots of sera were incubated for 18 h at 4 C after washing with PBS-T. 100 μl of horseradish peroxidase (HRP)-conjugated goat antihuman IgG antibodies (Sigma, USA) were added to each well and incubated for 1 h at 37 C.The plates were washed again with PBS-T, then with the substrate solution, containing 0.02 % H 2 O 2 , 0.1 M citrate-phosphate buffer (pH 5.8), and 0.5 mg/mL 2,2'azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) sodium salt (Sigma, USA), was added to each well.After 20 min of incubation at 37 C, the absorbance was measured at 405 nm in ELISA reader.PBS, preimmune serum of rabbit, and secondary antibodies served as negative controls.The sera from immunized by the full-length TyrRS rabbits were used as positive controls.For each sample, the OD of the uncoated well was subtracted from the OD of the coated well and then subjected to the data analysis.The OD values greater than the mean + 2 SD values of the normal controls were considered as positive.

Western blot analysis of recombinant peptides
The bacterially expressed His-TyrRS, His-miniTyrRS, and His-CTD recombinant proteins were boiled, resolved by 12 % SDS-PAGE, and electrotransferred to the nitrocellulose membrane (Amersham Bios cience, Germany).The membrane was divided into strips and blocked by 5 % non-fat milk in PBS-T for 1 h at room temperature followed by a triple wash with PBS containing 0.1 % Tween-20.The strips with PBS-T, immunized by TyrRS rabbit serum (1 : 75), and sera of the control healthy individuals, or the healthy subjects with family history of CVD, or the patients with EH (1:100) were incubated for 18 h at 4 C.The peroxidase-conjugated secondary antibodies (Sigma, USA) to the strips after washing and incubated were added for 1 h at a room temperature.The strips were washed, and the immunoreactivity was detected by ChemiDoc System (Bio-Rad Laboratories, USA).

Statistical analysis
All statistical analyses were performed using the Statistica software, version 7.0.Differences in non-par-ametric data were tested by the Mann-Whitney test.A P-value less than 0.05 was considered as statistically signifi cant.

Production and characterization of recombinant TyrRS, miniTyrRS, and CTD
To generate the recombinant full-length TyrRS, mi ni-TyrRS, and CTD, we expressed them in the E. coli BL21 (DE3) pLysE cells using the pET-30a expression system and His-tag sequence in order to facilitate the purifi cation of the recombinant proteins.After induction with IPTG we observed high levels of the His-TyrRS, His-miniTyrRS, and His-CTD expression in bacterial cells.Then recombinant proteins were purifi ed on Ni-NTA agarose under denaturing conditions.The required purity (more than 95 %) of the proteins was confi rmed by SDS-PAGE (data not shown).
Anti-TyrRS positive sera in 12 % (3 of 25) of the persons with EH were observed (Fig. 1).The elevated levels of anti-miniTyrRS antidobies were detected in 52 % (13 of 25) of the persons with EH (Fig. 2, A).Anti-CTD positive sera were shown in 60 % (15 of 25) of the persons with EH and 50 % (6 of 12) of the individuals with family history of EH (Fig. 2, B).The serum anti-miniTyrRS concentrations were signifi cantly higher in the persons with EH compared with those in the Ab-negative normal healthy volunteers (p < 0.001).The serum levels of anti-CTD aAbs were elevated not only among the persons with EH (p = 0.002), but also among the healthy individuals with family history of the pathology (p = 0.037).

Detection of anti-TyrRS, anti-mini-TyrRS, and anti-CTD aAbs by Western blotting
The ELISA representative serum samples (positive, poorly and moderate reactive) of each group were subsequently confi rmed by Western blotting against the full-length TyrRS (Fig. 3, A) and its natural fragments (Fig. 3, B).

Discussion and Conclusion
A lot of studies postulate a role of autoantibodies in the pathogenesis of hypertension [32].For several decades it was known that EH is associated with the elevated serum levels of IgG and IgM autoantibodies [33,34].They can be involved into pathogenic reactions by binding to antigens expressed on the surface of endogenous cells, that leads to the destruction of cells via complement-or leukocyte-dependent interactions (type II hypersensitivity reaction) [35].aAbs may form «immune complexes», that can be deposited in various tissues and cause the local infl ammatory responses (type III hypersensitivity) [36].They also can act as non-immunogenic agonists to the receptors (so-called «type V hypersensitivity»).
EH is a multifactorial disease with indefi nite etiology, its pathogenesis is clearly associated with the development of vascular endothelial dysfunction, characterized by pro-trombotic, pro-infl ammatory and pro-constrictive vessel status [45].Meanwhile, the endotheliocytes recruitment and the pro-infl ammatory effects are the key properties of both distinct domains of TyrRS.[5,17].Considering all these facts, the involvement of natural fragments of the enzyme into pathogenesis of EH is quite possible.This is the fi rst investigation of the serum immunoreactivity of the persons with EH, the healthy individuals with family history of EH, and the healthy subjects against TyrRS and its separated domains.The study demonstrates the presence of persons with elevated levels of autoantibodies against the fulllength enzyme, miniTyrRS, and CTD in the normal healthy cohort.Such kind of immunoreactivity can be stipulated by some undetected infl ammatory process that resulted in the secretion of TyrRS or the appearance of the full-length protein in the intercellular space due to apoptosis.Our data demonstrate the signifi cant immunoreactivity against mini-TyrRS (52 %) and CTD (60 %) among the persons with EH. Surprisingly, we found out the increased levels of autoantibodies against CTD in sera of 50 % of the healthy individuals with family history of the pathology.
Difference in the values of aAbs against the fulllength enzyme and its natural fragments can be explained by the potential existence of the epitopes which are responsible for the cytokine activities and are sequestered in a native form by each other.The regions of cytokine activity -ELR on miniTyrRS and heptapeptide (Arg13-Thr19) on the N-terminus of CTD -in the full-length TyrRS have «face-toface» orientation, conditioned by the electrostatic interactions [46].Under the apoptotic conditions, specifi cally infl ammation, the native enzyme is secreted into intercellular space and can be cleaved by a extracellular protease such as leukocyte elastase.Moreover, the regions of cytokine activity can be un-masked in a cell via tRNA connection [46].As a result, both sequences providing the cytokine activity, become available and two distinct fragments obtain new cytokine functions [16].It seems absolutely logically to assume normally hidden sequences to be extremely immunogenic.The immunoreactivity of the persons with EH as well as of the individuals with family history of pathology suggests the presence of natural fragments of TyrRS, the formation of which is possible in terms of infl ammation [17], known to be a component of the EH pathogenesis.
Earlier we have demonstrated the TyrRS and its separated domains as autoantigens in heart failure caused by the dilated cardiomyopathy, myocarditis and ischemic heart disease [14].The highest immunoreactivity for the full-length enzyme and the lowest for CTD were revealed.According to our recent fi ndings in sera of the patients with hypertension the highest levels of aAbs were found against the CTD and the lowest against the full-length enzyme.The data comparison suggests potential involvement of aAbs against CTD and mini-TyrRS into the EH pathogenesis.
A practical signifi cance of TyrRS and its separated domains is a «double-edged weapon».On the one hand, TyrRS is one of eight aminoacyl-tRNA synthetases involved into the antisynthetase syndrome [12], its mutant form occurs in Charcot-Marie-Tooth hereditary neuropathy [47].On the other hand, mini-TyrRs is a potential drug in the myocardial ischemia treatment [30].Its particular role also was demonstrated in the platelet recovery in the patients suffering from the life-threatening thrombocytopenia or the bone marrow failure [28].For recovery of cardiac function after myocardial infarction [29] CTD can be applied as an antiangiogenic stimulus [27].
However, the infl uence of aAbs on TyrRS and its separated domains in the disease pathogenesis as well as in the treatment strategies remains to be elucidated.Potentially, the elevated levels of autoantibodies against mini-TyrRS and CTD in sera of the persons with EH may be used as prognostic markers of the EH severity or the therapy effectiveness.Moreover, the immunoreactivity of healthy individuals with family history of EH against CTD may be an early marker of hypertension.

Fig. 1 .
Fig. 1.Anti-TyrRS aAb levels in sera from persons with EH, he althy individuals with family history of EH and healthy controls shown as OD values.The line shows the cut-off value and means median +2SD values for the healthy controls

Fig. 2 .
Fig. 2. Anti-mini-TyrRS (A, C, E) and anti-CTD (B, D, F) aAb levels in sera from persons with EH, healthy individuals wit family history of EH and healthy controls shown as OD values.The lines (A, B) show the cut-off values and mean median +2SD values for the healthy controls.Data (D-F) are presented as box plots, where the boxes represent the 25 th to 75 th percentiles, the points within the boxes represent the median, and the lines outside the boxes represent the minimum and maximum values.Differences were analyzed by the Mann-Whitney U-test

Fig. 3 .
Fig. 3. Immunoreactivity of healthy subjects, individuals with family history of EH, and persons with EH against TyrRS (40 μg for each protein) (A), miniTyrRS (B), and CTD (C), obtained by Western blotting.Lines 1-3 represent the high, moderate, and low reactive sera of healthy volunteers against relevant protein according to ELISA; lines 4-6, by analogy, for individuals with family history of EH; lines 7-9 for persons with hypertension.C is control line, incubated with serum of rabbit immunized by full-length TyrRS; M is protein MW marker