Development and characterization of monospecific anti-Sgt1 antibodies

The method of de vel op ment and pu ri fi ca tion of monospecific polyclonal an ti bod ies against Sgt1 was de scribed and their char ac ter is tics were pre sented. Pro tein Sgt1 plays an im por tant role in the reg u la tion of cell cy cle, Hsp90-re lated proteasome deg ra da tion of pro teins and pro/anti-apoptotic sig nal ing along with other mo lec u lar chap er ons and co-chap er ons. The fur ther in ves ti ga tion of Sgt1 role and its func tion ing with other mem bers of Hsp90-chap er ons fam ily is im por tant for elu ci da tion of the cell sig nal ing reg u la tion, which is sig nif i cant for cardio-vas cu lar pa thol o gies pro gres sion, par tic u larly, for di lated cardiomyopathy.

In tro duc tion.The study of the mech a nisms of anti-stress re sponse is quite im por tant for eval u a tion of cardio-vas cu lar pa thol o gies pro gres sion and for de velop ment of new ef fec tive ther a peu tic tools based on the apoptotic sig nal ing block age.Spe cial ized fam ily of anti-stress pro teins in clud ing mo lec u lar chap er ons, their co-chap er ons, and tar get pro teins plays a crit i cal role in pro-and anti-apoptotic sig nal ing.Re cently discov ered pro tein Sgt1 is a po ten tial co-chap eron and/or tar get pro tein.
Sgt1 has been first de scribed in reg u la tion of cell cir cle in yeast [1] which sup presses G2 al lele of Skp1 pro tein, one of the kinetochore CBF3 core com plex [2].Re cently it has been shown that Sgt1 plays a role in Hsp90-re lated proteasome deg ra da tion of pro teins via in ter ac tion with com plex of Skp1-Cul1-F-box (SCF) E3 ubiquitin-ligase [3].Sgt1 ex pres sion has been observed in nu mer ous eukaryotes in clud ing plants and mam mals [4,5].How ever, mam ma lian Sgt1 cel lu lar dis tri bu tion and pe cu liar i ties are poorly un der stood.The in ves ti ga tion on Sgt1 ex pres sion in hu man tis sues re vealed the vari a tion of its ex pres sion in dif fer ent tissues with max i mum level in brain, kid ney, lung, in testines, and heart [6].The se quence-anal y sis of Sgt1-proteins pu ri fied from yeast, hu man cells, rat, rice, and Arabidopsis sug gests 3 con ser va tive do mains (tetratricopeptide re peat (TPR), CHORD-con tain ing pro teins and Sgt1 (CS), and Sgt1-spe cific do main (SGS), as well as two vari able re gions -VR1 and VR2 [7,8].TPR is known as HSP-bind ing do main, SGS-domain in ter acts with Ca 2+ -bind ing pro teins of S100-family [9], ànd CS-do main has a high se quence-homology with Hsp90-co-chap eron p23 pro tein [10].
The study on the role and func tion of Sgt1 in complexes with other mem bers of Hsp90 fam ily is im portant for elu ci da tion of the cell sig nal ing reg u la tion, which is suf fi cient for cardio-vas cu lar pa thol o gies progres sion, in par tic u lar, for di lated cardiomyopathy.The de vel op ment of monospecific an ti bod ies with high af fin ity is nec es sary for this pur pose al though it could be dif fi cult due to high con ser va tion of Sgt1 mol e cule struc ture which de ter mines its low immunogenicity.
Ma te ri als and meth ods.Re com bi nant hu man Sgt1 of 95% pu rity (trun cated C-end do main, mo lec u lar weight 20 kDa) is a kind gift of Prof. Anna Filipek (Nencki In sti tute of Ex per i men tal Bi ol ogy of Pol ish Acad emy of Sci ences, War saw, Po land).Hsp90 prep ara tion pu ri fied from bo vine brain is a kind gift of Prof. Jacek Kuznicki (In ter na tional In sti tute of Cell and Molec u lar Bi ol ogy, War saw, Po land).Re com bi nant GroEL was pu ri fied ac cord ing to [24].
An i mal ex per i ments have been car ried out in ac cordance to the Ukrai nian Ethic Com mit tee Pro to col.An imals were de cap i tated un der es ter nar co sis, and the heart was stopped by per fu sion with cold phos phate buffer sa line (PBS).
Pro tein pu rity was checked by elec tro pho re sis in 12% PAGE at de na tur ing con di tions by the method of Laemmli [12].
Pro tein con cen tra tions were es ti mated by the Bradford's method [13].
Prep a ra tion of anti-Sgt1 monospecific polyclonal an ti bod ies.The im mu ni za tion of rab bits has been performed ac cord ing to the spe cific scheme de vel oped in our lab o ra tory re cently [14].In 7 days af ter the last immu ni za tion, the blood from rab bits was col lected, the se rum was tested for con tent of anti-Sgt1 an ti bod ies using ELISA method.Immunoglobulins were pre cip itated from the se rum with so dium sul phate buffer at 50% sat u ra tion.
Immunoglobulins were stored un der sat u rated sodium sul phate buffer with 0.3% NaN 3 be fore pu ri fi cation at 4°C.
Pu ri fi ca tion of an ti bod ies us ing DEAE-Toyopearl 650M col umn chro ma tog ra phy.DEAE-Toyopearl-650Ì col umn (ToyoSoda, Ja pan) was equil i brated with phos phate buffer sa line (PBS), pÍ 7.2.Pre cip i tate of immunoglobulins was dis solved in PBS buffer with 0.1 mM PMSF, loaded onto col umn (40-50 mg of to tal proteins per 2.5 ml of resin), and washed by 2-3 vol umes of PBS.Un bounded frac tions were col lected, and fur ther pu ri fi ca tion of an ti bod ies was con ducted us ing Pro tein G-Sepharose col umn (Pharmacia, Swe den), equil ibrated with PBS buffer.Pu ri fied on DEAE-Toyopearl immunoglobulins were ap plied on the col umn (1.5 mg of pro tein per 1 ml of resin) and in cu bated for 2-3 hours at room tem per a ture.The col umn was washed by PBS buffer, elu tion of Ig was done with 0.2 M glycine buffer (pH 2.5) with neu tral iza tion of eluate by Tris base.The pu rity of IgG ob tained was con trolled by SDS-PAGE elec tro pho re sis.IgG-con tain ing frac tions were di alyzed against PBS-buffer per night at 4°C, sat u rated by glyc erol up to 50% and stored at -20°C.
Af fine col umn syn the sis.Af fine col umn was prepared by cou pling highly pu ri fied an ti gen (trun cated C-end do main of Sgt1) to the CNBr-ac ti vated Sepharose 4B (Pharmacia, Swe den) as de scribed in [14].
Af fine pu ri fi ca tion of spe cific an ti bod ies.IgG , puri fied by the Pro tein G-Sepharose chro ma tog ra phy, were di a lyzed over night with PBS buffer, ðÍ 7.4 at +4 °Ñ, and ap plied to the col umn (10-12 mg per 2 ml of resin) with sub se quent in cu ba tion at room tem per a ture for 3-4 hours.Af ter wash ing with PBS/0.5 Ì NaCl buffer, ðÍ 7.4, and then with PBS, cou pled an ti bod ies were eluted by 0.2 Ì glycine buffer, ðÍ 2.5 with neutral iza tion by 1 M Tris-base, ðÍ 11.0.Com bined protein frac tions were di a lyzed over night in PBS buffer with PMSF, ðÍ 7.4 at +4°Ñ.
The con cen tra tion of an ti bod ies ob tained was assayed spec tro pho to met ri cally us ing the ex tinc tion co effi cient at 280 nm E= 1.35 cm -1 mg -1 ml and M r 150 000.
The spec i fic ity of an ti bod ies ob tained at each pu rifi ca tion step was con trolled by the ELISA method and their pu rity was es ti mated by 12% PAGE-SDS elec tropho re sis.

En zyme-linked immunosorbent as say(ELISA).
ELISA as say was per formed as de scribed in [15] with mod i fi ca tions.An ti gen (re com bi nant Sgt1, C-end truncated form, 10 µg/ml) was ad sorbed to the wells of 96-well plates (Nunc) in 0.1 M car bon ate buffer, pH 9.0, over night at 4°Ñ.The re main ing ad sorp tion sites were blocked with PBS, con tain ing 0.1% Tween-20 and 0.5% gel a tine (PBS-T-Gel); an ti sera or spe cific anti bod ies in prop erly con cen tra tions/di lu tions were added in 0.1 ml of PBS-T-Gel to each well; in cu ba tion with an ti bod ies lasted 2 h at 37°Ñ.All fur ther pro cedures were car ried out at 37°Ñ .Then, wells were washed with PBS-T, and sec ond ary an ti bod ies of gout against whole mol e cules of rab bit IgG con ju gated with horse-rad ish peroxidase (Promega) were added to the wells and in cu bated for 1 h.Af ter wash ing with PBS-T, the re ac tion prod uct was vi su al ized by add ing ABTS (0.5 mg/ml, Sigma) to 50 mM so dium ci trate buffer, pH 5.0, and 0.05% Í 2 Î 2 .The re ac tion was stopped by adding of 0.5 ml ci trate buffer con tain ing 0.3% NaN 3 , and the re sult of re ac tion was as sayed quan ti ta tively on BioLab rid der (UK).
West ern-blot anal y sis.The spec i fic ity of anti-Sgt1 an ti bod ies and their pos si ble cross-re ac tiv ity was revealed us ing West ern-blot anal y sis as de scribed in [16] with some mod i fi ca tions.
Af ter sep a ra tion by elec tro pho re sis polypeptides were electrotransferred onto nitrocellulose mem brane (Protran, USA) in 20 mM Tris/100 mM glycine/20% meth a nol buffer at 200 mA/20 V dur ing 2 h.Pro tein trans fer onto nitrocellulose mem brane un der pres sure ("pas sive") was done over night in PBS buffer.Wash ing and block age of the mem brane was done in PBS-T buffer (80 mM Na 2 HPO 4 ; 20 mM NaH 2 PO 4 ; 100 mM NaCL pH 7.5; 0.1% Tween-20) con tain ing 5% of fatty-free dry milk (PBS-T-M) at room tem per a ture.All fur ther pro ce dures in clud ing the in cu ba tion with specific an ti sera or pu ri fied an ti bod ies were car ried out in PBS-T-M buffer at room tem per a ture.Af ter wash ing with PBS-T, sec ond ary an ti bod ies of gout against whole mol e cules of rab bit IgG con ju gated with horse-rad ish peroxidase (Promega) were added to the mem brane and in cu bated dur ing 2 h.Af ter ex ten sive wash ing with PBS-T, the fluorogene ECL (Amersham, UK) was added to the mem brane (ac cord ing to the manu fac turer's pro to col) and ex posed on the X-ray film (Ko dak) for vi su al iza tion of the sig nals.
Re sults and dis cus sion.The study of apoptotic signal ing reg u la tion re vealed the im por tant role of mo lecu lar chap er ons Hsp90 fam ily in the mech a nisms of ac tiva tion and sup pres sion of such sig nal path ways [17,18].It has been shown re cently that the Hsp90 is one of the ma jor fac tors in the mech a nism of qual ity con trol of pro teins via poly-ubiquitination and proteasome substrate deg ra da tion [19].In ves ti ga tion of Hsp90 structure re vealed the C-ter mi nal do main of dimerization and ATPase N-ter mi nal do main, which in ter act with dif fer ent sub strates and nu mer ous co hort co-chap er ons [20].The se quenc ing of re cently dis cov ered pro tein Sgt1 [6], par tic i pat ing in anti-mi cro bial in nate im mu - nity, iden ti fied that both plant and mam ma lian Sgt1 pro tein are com posed of 3 do mains.Sgt1 cen tral CS-do main was iden ti fied as Hsp90-linked pro tein p23-like struc ture [7,8] ca pa ble of in ter act ing with Hsp90 [21].Ñ-ter mi nal do main of Sgt1 mol e cule (SGS) in ter acts with Ñà 2+ -bind ing S100 pro teins [22], such as annexins, psoriasin, calcyclin, parvalbumin, calmodulin, and troponins which may sug gest the impor tant role of Sgt1 in con trac til ity of mus cle cells, and cardiomyocytes in par tic u lar [23].
The in ves ti ga tion of abovementioned prob lems depends on the avail abil ity of spe cific anti-Sgt1 an ti bodies with high af fin ity.The de vel op ment of such an tibod ies is com pli cated due to high evo lu tion ary con serva tive struc ture and low immunogenicity of Sgt1 mol e cule.Thus, the first stage of such an ti bod ies gen era tion was the de vel op ment of spe cific scheme of an imals (rab bit) im mu ni za tion.We used the C-ter mi nal frag ment (mo lec u lar weight ~ 20 kDa) of re com bi nant hu man Sgt1 with 95% of pu rity (Fig. 1).We ob tained anti-Sgt1 im mune sera with suf fi cient ti ter (more than 10 -4 ) only af ter 5 im mu ni za tion steps al though we used only 300 µg of an ti gen in all pro ce dures us ing unique scheme of im mu ni za tion, pre vi ously de vel oped in our lab o ra tory.
Af ter all the pro ce dures of an ti bod ies pu ri fi ca tion, their af fin ity, spec i fic ity and pos si ble cross-re ac tiv ity were iden ti fied.The re sults of iden ti fi ca tion of the ti ter of spe cific anti-Sgt1 an ti sera and anti-Sgt an ti bod ies at dif fer ent pu ri fi ca tion steps on the dif fer ent ma trixes are pre sented in Fig. 2. The af fin ity of the spe cific an ti bodies ob tained in creased with ev ery step of pu ri fi ca tion.
The prob lem of the de vel op ment of spe cific an tibod ies di rected against re com bi nant an ti gens is com plicated by the fact that the an ti bod ies ob tained de novo are very of ten ca pa ble of rec og niz ing the re com bi nant an ti gen only, but not the na tive one (in an i mal cells and tis sue lysates), even at de tected ex pres sion of gene for pro tein in ves ti gated.The mech a nism of such pro cess is not known but could be linked with dif fer ences in recom bi nant and na tive pro teins fold ing.There fore, the char ac ter iza tion of new an ti bod ies spec i fic ity and cross-re ac tiv ity by West ern-blot anal y sis is im por tant.The re sults pre sented in Fig. 3 dem on strate spe cific inter ac tion of anti-Sgt1 an ti bod ies with re com bi nant an tigen as well as cor re spond ing polypeptide with mo lec ular weight ~ 40 kDa in lysates of mouse cardiomyocytes.More over, anti-Sgt1 an ti bod ies obtained rec og nized na tive Hsp90 pu ri fied from bo vine brain, re com bi nant GroEL (prokaryotic an a log of eukaryotic Hsp60) [24], and na tive Hsp60 in mouse cardiomyocytes lysate.We sup posed that Sgt1 has a com mon do main(s) with Hsp60 sup ported by anti-Sgt1 cross-re ac tiv ity in West ern-blot anal y sis.In this case our fur ther in ves ti ga tions should be con cen trated on com mon do mains search ing with se quenc ing of such do mains, and eval u a tion of pos si ble Sgt1 act ing like co-chap eron or tar get pro tein.This is suf fi cient to under stand the role of Sgtg1 and other CHORD-con taining pro teins in cardiomyocytes func tion ing and anti-stress re sponse, whose disregulation is man i fested in car dio vas cu lar pa thol o gies pro gres sion.
Con clu sions.Anti-Sgt1 monospecific polyclonal an ti bod ies with high pu rity and af fin ity have been first de vel oped.Anti-Sgt1 ob tained can be used in ELISA, West ern-blot anal y sis and immunoprecipitation.
We ob served the cross-re ac tiv ity of anti-Sgt1 an tibod ies with chap er ons Hsp90 and Hsp60 for the first time.

Fig. 1 Fig. 2
Fig.1 Elec tro pho retic anal y sis of the pu rity of hu man re com bi nant Sgt1 (DC frag ment), used as an ti gen for im mu ni za tion of rab bits: 1 -hu man re com bi nant Sgt1 (DC frag ment); 2 -mix ture of stan dard pro tein mark ers (Sigma)