Molecular cloning of liver-kidney-microsomal (LKM-1) autoantigen
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107 mcnm~ ~-,:.c~ oF HBV-m~ ~EC~-~ON ~ PER~HER~ BLeD ~O~t~R cats ~ qJ~_crs ~'~ o m m c HBV A~ HDV/~V L~,~a:rE~. S.Ha~rin,A.Cr,~C..Tar-ini°,p.Colclrt~:~,F.Di Bl,'~i ¢ ,G..CTJint"l H':LG.AntonPI I i§,C.~iP] Io°°,C.Fzk)i~n'), F.~§,L.P~liaro. CHni_ca Y~'dica R , P a l e ~ - o ; ° C ~ di AI lergologia f, Inrnrlolcg'ia CI inica. Rom;':'Dip.Biol.Celhdape,Palerrro;~Cattec~a di Virolqgia.Pc~l;°°I.-,'t. CNR.L'Aqttila; IT_AI..Y. AI~ per'~l blood ~lear" cells (F'B~) fi~ction.a~ m-.a, aaa-d by intPr'lakin-2 (1l~2) ~/~m~t- ~e~n (X-II~) synt~s and by 1L-2 ~ (TI~2R) e x f I ~ i o n , i s d ~ T v e d in HBV-ml;Cxqd chrmic liver di sea_~e (HBV/OJ)). HBV-I~ int~ration within FBvC~ cause s~h ahnonnaLiti~, lhr effect of ~ .-ao-'rinff~-'tion on the ~ is tdax:x~. To assess ~ a ~ ~ d e f ~ t of PBVEallo~-; tlY. l:X"~i.'Co'cP of It3V ~ HDV,v,e.,-,lat died ~ i n i n ( r ~ ) and staphy]c~_a] ~.odn B(.'~)-ir~ n-2,rL-2R,~-r~ pr~cion,ard HBV-mA in sE~'u'nand in ~ fr,cm 44 patients with biopsy-pmvm O.I),16 of ~hcrnwith damnic ~V .,.aCx~.infection (I-[N/CLD). F ~ t~dthy HSV negative controls were also eval~ated.N~ patierts with HSV replication (.~nm HBV41tAix~i tire) had lower amunt of TL-2R on ~ and pmducE~l si/~ficar~ly l~s ~-IFN after ~ stimalation ~ ounlxar~ to the 25 pati~s withaat HBV replication (serun HBV-II~ negat/ve) and to controls (p<0.01 by ,maly~i.~ of va~i;~ oe). IL--2 s5¢¢/',~i~ ~ simLlar" in the ~/~04:~. I-BV~ ~ , i n inte~ated or" free form,wrre ~ in 44~ o f HBV r e p l i ~ and in 2890of r~m-repli~.No correlations with ft]¢C functions ~as fand./~ive HDV inf~ ction,st-om by H[~ in the liver,vos poes~fc in 16 patients (12 of ~ HBV-[IqA seana'~ative), hn:rlg ~ IL-2R,1I-2 ~d ~-IFNwer~ ~ in arctr/cs ~ ] e to P~a]thy onntrDls,~ile HDV-Lrdnfe~t~d sabjects had lome TI-2R levels (p<O.O~ by ar~ysis of v~). HBV--n~ was rr~smt in PB~ fr~rn 3~ HDV/C~D and from 3~ HBV/C~D.Ct~ re ~a.dts st~oest that,in the onua'se of 1-15V/C/.D or" I[N/C1/), I-BV-t~ in RPE does not interfere, with ~ fix~ions. HBV-DNA may be found in PB~I= ir~ive of H~V r~pl/caLive staO.ts and of HDV st~aerinfection. Its ~ is her~ not r~Y~,ined for ,,~,'~teiname of d'nx~c viral infection. 108 MOLECULAR CLONINGOF LIVER-KIDNEY-MICROSOMAI (LKM-I) AUTOANTIGEN M. Manns~ E.F. dohnson v K.d. v Griffin v E.M. Tan~ K.F. Sullivan Dept. of Basic and Clinical Research, Scripps Clinic and Research Foundation, La dolla, USA LKM-I autoantibodies characterize a subgroup of autoimmunetype chronic active hepat- itis (AI-CAH) and detect a 50 KD polypeptide present in liver microsomes (Hepatology 7, 1033, 1987). Six out of 17 sera exhibiting the LKM characteristic immunofluorescence (IF) pattern reacted with a 50 KD polypeptide, 4 with a 64 KD polypeptide and 2 with both. One high titer LKM serum from a patient with AI-CAH was used to screen a human liver ~l.gt-11 cDNA expression library. Several clones were identified, isolated and purified. All had an insert size of 1.2 Kb. To verify the immunological identity of the cloned cDNA antibodies were affinity purified from the autoantiserum using fusion proteins prepared from LKM clones or from an unrelated cDNA. Affinity purified antibodies from the immuno- positive clones significantly reacted on Western blots with the 50 KD microsomal protein and stained appropriate renal tubulus epithelia of mouse tissue in IF. Comparisonof the LKM-cDNA sequence with published nucleic acid sequences revealed extensive homologywith human cytochrome P-450 dbl which is known to metabolize a number of widely used drugs (Nature 331, 442, 1988). The LKM-cDNAwas also subcloned into plasmid pATH11 to obtain fused polypeptides as diagnostic reagents. All 14 sera from patients with LKM positive liver disease but not 3 LKM sera from patients with non-hepatic diseases nor other sera reacted with the LKM fusion protein. Conclusions: We describe the molecular cloning of LKM-I autoantigen which is a main target antigen in autoimmune hepatitis. Preliminary analysis demonstrates the utility of the cloned protein as diagnostic reagent. Isolation of this cDNA-clone will now enable detailed analysis of B and T cell immune responses in autoimmune liver diseases. It has to be evaluated whether the genetic background of pat- ients with LKM positive liver disease is linked to the genetic polymorphism known for db1. $56
Transcript of Molecular cloning of liver-kidney-microsomal (LKM-1) autoantigen
1 0 7 m c n m ~ ~-, : .c~ oF HBV-m~ ~EC~-~ON ~ PER~HER~ BLeD ~ O ~ t ~ R c a t s ~ qJ~_crs ~ ' ~
o m m c HBV A~ HDV/~V L~,~a:rE~. S.Ha~rin,A.Cr,~C..Tar-ini°,p.Colclrt~:~,F.Di Bl,'~i ¢ ,G..CTJint"l H':LG.AntonPI I i§,C.~iP] Io°°,C.Fzk)i~n'), F . ~ § , L . P ~ l i a r o . CHni_ca Y~'dica R , P a l e ~ - o ; ° C ~ di AI lergologia f, Inrnrlolcg'ia CI inica. Rom;':'Dip.Biol.Celhdape,Palerrro;~Cattec~a di Virolqgia.Pc~l;°°I.-,'t. CNR.L'Aqttila; IT_AI..Y.
A I ~ p e r ' ~ l blood ~ l e a r " cel ls (F'B~) fi~ction.a~ m-.a, aaa-d by intPr'lakin-2 (1l~2) ~ / ~ m ~ t - ~ e ~ n (X-II~) s y n t ~ s and by 1L-2 ~ (TI~2R) e x f I ~ i o n , i s d~Tved in HBV-ml;Cxqd chrmic liver di sea_~e (HBV/OJ)). HBV-I~ in t~ra t ion within FBvC ~ cause s ~ h ahnonnaLiti~, lhr effect of ~ .-ao-'rinff~-'tion on the ~ i s tdax:x~. To assess ~ a ~ ~ d e f ~ t of PBVE allo~-; tlY. l:X"~i.'Co'cP of It3V ~ HDV,v,e .,-,lat died ~ i n i n ( r ~ ) and staphy]c~_a] ~ . o d n B ( . ' ~ ) - i r ~ n -2 , rL -2R,~ - r~ p r ~ c i o n , a r d HBV-mA in sE~'u'n and in ~ fr,cm 44 patients with biopsy-pmvm O.I),16 of ~hcrn with damnic ~V .,.aCx~.infection (I-[N/CLD). F ~ t~dthy HSV negative controls were also e v a l ~ a t e d . N ~ patierts with HSV replication (.~nm HBV41tA ix~i tire) had lower amunt of TL-2R on ~ and pmducE~l s i /~f ica r~ ly l ~ s ~-IFN after ~ stimalation ~ ounlxar~ to the 25 p a t i ~ s withaat HBV replication (serun HBV-II~ negat/ve) and to controls (p<0.01 by ,maly~i.~ of va~i;~ oe). IL--2 s5¢¢/',~i~ ~ simLlar" in the ~ / ~ 0 4 : ~ . I - B V ~ ~ , i n inte~ated or" free form,wrre ~ in 44~ of HBV r e p l i ~ and in 2890 of r ~ m - r e p l i ~ . N o correlations with ft]¢C functions ~as f a n d . / ~ i v e HDV inf~ ction,st-om by H[~ in the liver,vos poes~fc in 16 patients (12 of ~ HBV-[IqA seana'~ative), hn:rlg ~ IL-2R,1I-2 ~ d ~-IFN wer~ ~ in arctr/cs ~ ] e to P~a]thy onntrDls,~ile HDV-Lrdnfe~t~d sabjects had lome TI-2R levels (p<O.O~ by a r~ys i s of v ~ ) . HBV--n~ was rr~smt in PB~ fr~rn 3~ HDV/C~D and from 3~ HBV/C~D. Ct~ re ~a.dts st~oest tha t , in the onua'se of 1-15V/C/.D or" I[N/C1/), I-BV-t~ in RPE does not interfere, with ~ f ix~ions . HBV-DNA may be found in PB~I= ir~ive of H~V r~pl/caLive staO.ts and of HDV st~aerinfection. I t s ~ is he r~ not r~Y~,ined for ,,~,'~teiname of d 'nx~c viral infection.
108 MOLECULAR CLONING OF LIVER-KIDNEY-MICROSOMAI (LKM-I) AUTOANTIGEN M. Manns~ E.F. dohnson v K.d. v Gri f f in v E.M. Tan~ K.F. Sullivan Dept. of Basic and Clinical Research, Scripps Clinic and Research Foundation, La dolla, USA
LKM-I autoantibodies characterize a subgroup of autoimmune type chronic active hepat- i t i s (AI-CAH) and detect a 50 KD polypeptide present in l iver microsomes (Hepatology 7, 1033, 1987). Six out of 17 sera exhibiting the LKM characteristic immunofluorescence (IF) pattern reacted with a 50 KD polypeptide, 4 with a 64 KD polypeptide and 2 with both. One high t i t e r LKM serum from a patient with AI-CAH was used to screen a human l iver ~l.gt-11 cDNA expression l ibrary. Several clones were identif ied, isolated and purified. All had an insert size of 1.2 Kb. To verify the immunological ident i ty of the cloned cDNA antibodies were a f f i n i t y purified from the autoantiserum using fusion proteins prepared from LKM clones or from an unrelated cDNA. Af f in i ty purified antibodies from the immuno- positive clones signif icant ly reacted on Western blots with the 50 KD microsomal protein and stained appropriate renal tubulus epithelia of mouse tissue in IF. Comparison of the LKM-cDNA sequence with published nucleic acid sequences revealed extensive homology with human cytochrome P-450 dbl which is known to metabolize a number of widely used drugs (Nature 331, 442, 1988). The LKM-cDNA was also subcloned into plasmid pATH11 to obtain fused polypeptides as diagnostic reagents. All 14 sera from patients with LKM positive l i ver disease but not 3 LKM sera from patients with non-hepatic diseases nor other sera reacted with the LKM fusion protein. Conclusions: We describe the molecular cloning of LKM-I autoantigen which is a main target antigen in autoimmune hepatitis. Preliminary analysis demonstrates the u t i l i t y of the cloned protein as diagnostic reagent. Isolation of this cDNA-clone wi l l now enable detailed analysis of B and T cell immune responses in autoimmune l iver diseases. I t has to be evaluated whether the genetic background of pat- ients with LKM positive l i ver disease is linked to the genetic polymorphism known for db1.
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