Clin. exp. Immunol. (1976) 26, 267-272.
Antigenicity of janin, a new protein from smooth muscle P. T R E N C H E V MRC Rheumatism Research Unit, Canadian Red Cross Memorial Hospital, Taplow, Berkshire (Received 25 February 1976)
SUMMARY
The exact nature of smooth muscle autoantibodies is still unclear. The antigen(s) which they are directed against remain unknown. Janin, a so far unknown smooth muscle protein, was obtained from smooth muscle tissue extracts and purified by means of three different procedures. Its mol. w. was estimated at 60,000 daltons. It stimulated a specific anti-janin antibody in rabbits as shown by tube test, double diffusion in agar and indirect immunofluorescence. The new protein absorbed eighteen out of 104 human smooth muscle positive sera that were not absorbed with smooth muscle actin, myosin, heavy meromyosin, light meromyosin, tropomyosin and brain tubulin. This would vindicate a view that smooth muscle autoantibodies differ from patient to patient with regard to the autoantigen(s) involved.
INTRODUCTION Smooth muscle autoantibodies were first discovered in sera from patients with chronic active hepatitis (Johnson, Holborow & Glynn, 1965). Since then they have been found in other diseases (Holborow, 1972; Holborow, Hemsted & Mead, 1973; Wilson et al., 1975), and in some healthy people. Their role however remains unclear. Smooth muscle autoantibodies are defined as antibodies which in immunofluorescent tests stain smooth muscle cells. The antigens in the smooth muscle cell which may be involved in the reaction are probably contractile proteins typical for muscle cells and believed to be present in non-muscle cells (Pollard & Weihing, 1974; Trenchev, Sneyd & Holborow, 1974; Holborow et al., 1975) as well. In some sera from patients with chronic active hepatitis an anti-smooth muscle antibody is detected which can be completely absorbed with actin (Gabbiani et al., 1973). This led to the conclusion that some human smooth muscle autoantibodies are anti-actin. However, comparatively few positive sera can be absorbed in this way and almost invariably these are from patients with chronic active hepatitis. Work done in this laboratory (Trenchev et al., manuscript in preparation) would indicate that various sera contain antibodies against different contractile proteins or constellations of them. Even so some smooth muscle autoantibodies cannot be absorbed with any known contractile protein. The possibility arises that these are antibodies directed against non-contractile proteins or unknown contractile proteins. In this report evidence is presented that janin, a new smooth muscle protein, is the autoantigen for smooth muscle autoantibodies in 17% of positive human sera. This protein was found in the material usually discarded when purifying smooth muscle contractile proteins. MATERIALS AND METHODS Smooth muscle tissue was obtained from human uteri, fresh or kept at - 20'C. The tissue was homogenized and washed twice with cold phosphate-buffered saline pH 7-4 prior to the extraction. Purification ofjanin obtained from the myosin containing extract. The homogenate was extracted with Guba-Staub (Offer, Moos & Starr, 1973) solution containing 03 M KCl, 01 M KH2PO4, 005 M K2HPO4. The myosin from this extract was precipitated by fourteen-fold dilution with cold distilled water pH 7.5. The precipitate was separated by centrifugation for Correspondence: Dr P. Trenchev, MRC Rheumatism Research Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire.
267
P. Trenchev
268
10 min at 38,000 g. It consisted of myosin with some actin, tropomysin and other unidentified proteins. The supernatant, usually discarded, is called here S fraction and was used to obtain janin. The pH of the mixture was brought down to 7 0 and the resulting precipitate separated by centrifugation and discarded. It contained small amounts of janin, actin, myosin, tropomyosin and other unidentified proteins. The pH of the supernatant was then further lowered with N HC1 to 5 2. At this point a large precipitate was formed consisting mainly of actin and less janin, tropomyosin, myosin and other proteins. The precipitate was spun down and discarded. The supernatant (Fig. la) was adjusted to pH 4-2. The resulting precipitate contained mainly janin, but small quantities TA
J
b~~~~~~~~~~~~~~~~~~~~~
of actin, tropomyosin and other proteins were also present. This precipitate was discarded. The remaining solution contained mainly janin, although other proteins were seen when SDS gels were overloaded with 30-40.pg of protein per gel. The whole procedure of isoelectric point precipitation was repeated and the isolated janin was further purified by means of two different techniques. (a) Chromatography on a Sephadex G-200 column equilibrated with the actin buffer janin is obtained in the first peak as monitored by absorbency at 280 nm. (b) Solid ammonium sulphate was added to 1-4 m final concentration (186 g/l). The precipitate, containing actin, tropomyosin and some other proteins, was discarded. The supernatant contained inC. almost pure janin (Fig. 2). It was dialysed against actin buffer and stored at Purtfication ofjanin from the actin containing extracts. Acetone-dried tissue powder was prepared from the homogenate as
(16 /l.Th pecptaecotinngati, rooyoinan
_.
.:...
FIG. 2. SDS gels of purified
om ohe roeis ws isade. hespenaan onaie
If~~~~~~
janin from myosin extract. Gels loaded with 10 pg (a), 20 pg (b) and 40 pg (c).
Antigenicity ofjanin
269
previously described (Trenchev et al., 1974; Holborow et al., 1975). The powder was extracted with actin buffer containing 0-08 M ATP, 0-1 mm CaCl2, 0-01 mm dithiotreitol, pH 8-2. The extract was clarified by centrifugation for 20 min at 38,000 g. The supernate contains janin in addition to actin (Fig. lb). Janin was separated from the mixture by isoelectric point precipitation at pH 4-1. The twice-washed precipitate consisted mainly of janin with traces of actin and tropomyosin. It was redissolved in 0-02 M NaCl pH 7 0. Janin was precipitated from this solution by lowering the pH to 3-9 (Fig. 3).
FIG. 3. SDS gels of janin purified from actin extract. Gels loaded with 30 jug (a) and 60 pg (b). Immunization procedure. All rabbits were screened beforehand for spontaneous antibodies and only the negative ones used in the experiment. Nine of these were immunized by means of a single s.c. injection. Each rabbit received 1 mg of janin mixed with equal volume of Freund's complete adjuvant. The animals were bled 21 days after the injection. The double diffusion test in agar and the tube test were carried out as described (Trenchev et at., 1974; Holborow et at., were
1975). Absorption
was carried out by mixing janin with antiserum and allowing the mixture to stand for 30 min at room temperature. Immunofluorescent microscopy was performed as described, by means of indirect staining (Trenchev et at., 1974). Goat anti-rabbit gammaglobulin serum was obtained from Nordic Laboratories, Maidenhead, Berkshire. SDS electrophoresis was carried out in 7.5¶y4 gels according to Weber & Osborn (1969). Smooth muscle myosin, heavy meromyosin, light meromyosin, actin and tropomyosin were prepared as previously described (Trenchev et at., 1974; Holborow et at., 1975). Human smooth muscle positive sera were selected for test at random. They included 104 sera from patients suffering from rheumatoid arthritis (forty-eight), Still's disease (thirty-six), breast cancer (seven) and thirteen healthy persons. All these sera contained anti-smooth muscle antibodies as judged by immunofluorescent microscopy.
RESULTS
Purity ofianin Preparations This was checked on SDS polyacrylamide gels. All three procedures produced reasonably purified janin. It appeared as a homogeneous single band even in overloaded gels of up to 60yjg per gel (Fig. 3). Its molecular weight as estimated by this method was found to be 60,000 + 2000 with sample proteins ovalbumin (43,000), human albumin (68,000) and smooth muscle actin (42,000). Janin preparations were compared with bovine brain and guinea-pig platelet tubulins (a generous gift from Dr N. Crawford, F
270
P. Trenchev
Birmingham University). The tubulins gave a differently placed band at 55,000 daltons. A mixture of janin and tubulin produced two separate bands corresponding to those for tubulin and janin. Purified myosin preparations did not show any band similar to that of janin even after prolonged storage at 4VC up to 2 months.
Antigenicity ofjanin Janin appeared to have stimulated production of anti-janin antibodies in four out of nine rabbits. These antibodies precipitated with janin in tube test up to 1: 128 dilutions of a solution containing 0-85 mg/ml janin. No precipitate was seen with human smooth muscle actin, myosin, heavy meromyosin, light meromyosin, tropomyosin or the tubulin preparations. The pre-immune sera with janin gave negative results. In double diffusion in agar the anti-janin sera produced a single precipitin line against janin. This line gradually moved away from the well, containing the antiserum, when tested against dilutions of janin from 1: 1 to 1: 12. The pre-immune sera failed to give any detectable precipitation with janin.
FIG. 4. Immunofluorescent micrograph of rat pancreas stained with rabbit anti-janin serum. Specific fluorescence in smooth muscle cells in arterial walls. (Magnification x 269.)
In the immunofluorescent test the anti-janin antibody gave strong specific fluorescence of all smooth muscle cells of stomach and blood vessel walls (Fig. 4), of the myoepithelial cells of salivary glandand of liver cells in a polygonal pattern (Trenchev et al., 1974). Specific fluorescence was obtained with serum dilutions up to 1: 80. The staining was confined to the cytoplasm of the cells. There was no nuclear staining. No species specificity was found since anti-janin sera reacted not only with human, but also with rat, mouse, rabbit and guinea-pig smooth muscle cells. The antisera however were tissue specific, no fluorescence being seen when skeletal or heart muscle tissues from the same animals were stained with anti-janin except for vascular staining. The pre-immune sera did not produce any staining of the smooth muscle cells. All specific fluorescence was successfully absorbed with as little janin as 8-5 pg/10 y1 antiserum, but not with tubulin, actin, myosin, heavy meromyosin, light meromyosin or tropomyosin of up to 150 pg/ 10 /1 antiserum.
Absorption of human autoantibodies by janin Successful absorbtion by janin of smooth muscle autoantibodies was established for eighteen out of
Antigenicity ofjanin
271
104 sera tested. The ratio was about 10 gg of janin for 10 pl of serum. Only the smooth muscle staining was absorbed and in liver the pattern of staining which corresponds to that seen with anti-janin rabbit sera. Nuclear or parietal cell fluorescence appeared unchanged after absorption with janin (Figs 5 and 6). The S fraction had a similar effect. The only difference was that 30 pg of S fraction protein or more was necessary for successful absorption of 10 p1 of a smooth muscle positive serum.
FIG. 5. Cryostat section of rat stomach stained with human smooth muscle positive serum. Specific fluorescence in muscularis mucosa, muscle fibres and parietal cells. (Magnification x 96.)
FIG. 6. Cryostat section of rat stomach treated with the same human serum as in Fig. 5, but absorbed for 30 min with 10 pg janin/10 ul of the serum. The staining of the muscularis mucosa and muscle fibres is absorbed. The staining of the parietal cells is not absorbed. (Magnification x 96.)
DISCUSSION These results suggest that about 17% ofthe total number of 104 human smooth muscle positive sera contain anti-janin antibody. Their smooth muscle activity was specifically absorbed with janin. Considerably
272
P. Trenchev
more protein was required to absorb the same sera with the S fraction. This contains janin as well as other proteins and the fact that nearly three times less purified janin was necessary to absorb the same sera would suggest that the autoantibody is specific for janin. It was not absorbed with actin, tubulin, myosin, heavy meromyosin, light meromyosin or tropomyosin. Apparently the so-called smooth muscle autoantibodies in different human sera are directed against different antigens including janin. Janin is most likely a smooth muscle protein since it is extracted from smooth muscle cells and the anti-janin antibody stains the cytoplasm of smooth muscle cells only. Janin differs from other contractile proteins in its molecular weight, electrophoretic mobility, isoelectric point and antigenic specificity. The estimated mol. wt., 60,000 daltons, is true only if janin is not a dimer of two chains of about 60,000 each. Janin is probably not a tubulin-like protein, since it showed different electrophoretic mobility in SDS gels. It also seems unlikely that janin is a breakdown product of myosin. Myosin preparations did not show in SDS gels any additional band in the 60,000 region even after 2 months storage at 4°C. It is not yet known whether janin is a contractile or non-contractile protein. In many ways it resembles other smooth muscle contractile proteins. Like these, janin is a highly acidic protein, forms filaments at low pH, is extracted with both actin and myosin, and is found in contractile structures. Electron microscopy studies with peroxidase-labelled anti-janin antibody (Trenchev et al., manuscript in preparation) gave a filamentous pattern throughout the smooth muscle cell cytoplasm. The exact nature of janin in relation to the function of smooth muscle cells is at present under study. It is hoped that more information about janin and other smooth muscle autoantigens will throw some light on the significance of such widespread autoantibodies as the smooth muscle autoantibodies. REFERENCES GABBIANI, G., RYAN, G.B., LAMELIN, J.P., VASSALI, P., OFFER, G., Moos, C. & STARR, R. (1973) A new protein of the thick filaments of vertebrae skeletal myofibrils. CRUCHAUD, A. & LUSCHER, E.F. (1973) Human smooth J. mol. Biol. 74, 653. muscle autoantibody: its identification as antiactin antibody and a study of its binding to 'nonmuscular' cells. POLLARD, T.D. & WEIHING, R.D. (1974) Cytoplasmic actin and myosin and cell movement. Crit. Rev. Biochem. Amer. ]. Path. 72, 473. 2, 1. HOLBOROW, E.J. (1972) Smooth muscle autoantibodies, viral infections and malignant disease. Proc. roy. Soc. Med. TRENcHEv, P., SNEYD, P. & HOLBOROW, E.J. (1974) Immunofluorescent tracing of smooth muscle contractile 65, 481. protein antigens in tissues other than smooth muscle. HOLBOROW, E.J., HEMsTED, E.H. & MAAD, S.W. (1973) Clin. exp. Immunol. 16, 125. Smooth muscle autoantibodies in infectious monoWEBER, K. & OsBoRN, M. (1969) The reliability of molecular nucleosis. Brit. med. ]. iii, 323. weight determinations by Dodecyl sulphate polyacrylHOLBOROW, E.J., TRENcHEv, P.S., DoRLING, J. & WEBB, amide gel electrophoresis. 5. biol. Chem. 277, 4406. H.J. (1975) Demonstration of smooth muscle contractile protein antigens in liver and epithelial cells. Ann. N.Y. WILSON, C., EADE, O.E., ELSTEIN, M., LLOYD, R. & WRIGHT, R. (1975) Smooth-muscle antibodies in infertility. Acad. Sci. 254, 489. Lancet, ii, 1238. JOHNSON, G.D., HOLBOROW, E.J. & GLYNN, L.E. (1965) Antibody to smooth muscle in patients with liver disease. Lancet, ii, 878.
Antigenicity of janin, a new protein from smooth muscle P. T R E N C H E V MRC Rheumatism Research Unit, Canadian Red Cross Memorial Hospital, Taplow, Berkshire (Received 25 February 1976)
SUMMARY
The exact nature of smooth muscle autoantibodies is still unclear. The antigen(s) which they are directed against remain unknown. Janin, a so far unknown smooth muscle protein, was obtained from smooth muscle tissue extracts and purified by means of three different procedures. Its mol. w. was estimated at 60,000 daltons. It stimulated a specific anti-janin antibody in rabbits as shown by tube test, double diffusion in agar and indirect immunofluorescence. The new protein absorbed eighteen out of 104 human smooth muscle positive sera that were not absorbed with smooth muscle actin, myosin, heavy meromyosin, light meromyosin, tropomyosin and brain tubulin. This would vindicate a view that smooth muscle autoantibodies differ from patient to patient with regard to the autoantigen(s) involved.
INTRODUCTION Smooth muscle autoantibodies were first discovered in sera from patients with chronic active hepatitis (Johnson, Holborow & Glynn, 1965). Since then they have been found in other diseases (Holborow, 1972; Holborow, Hemsted & Mead, 1973; Wilson et al., 1975), and in some healthy people. Their role however remains unclear. Smooth muscle autoantibodies are defined as antibodies which in immunofluorescent tests stain smooth muscle cells. The antigens in the smooth muscle cell which may be involved in the reaction are probably contractile proteins typical for muscle cells and believed to be present in non-muscle cells (Pollard & Weihing, 1974; Trenchev, Sneyd & Holborow, 1974; Holborow et al., 1975) as well. In some sera from patients with chronic active hepatitis an anti-smooth muscle antibody is detected which can be completely absorbed with actin (Gabbiani et al., 1973). This led to the conclusion that some human smooth muscle autoantibodies are anti-actin. However, comparatively few positive sera can be absorbed in this way and almost invariably these are from patients with chronic active hepatitis. Work done in this laboratory (Trenchev et al., manuscript in preparation) would indicate that various sera contain antibodies against different contractile proteins or constellations of them. Even so some smooth muscle autoantibodies cannot be absorbed with any known contractile protein. The possibility arises that these are antibodies directed against non-contractile proteins or unknown contractile proteins. In this report evidence is presented that janin, a new smooth muscle protein, is the autoantigen for smooth muscle autoantibodies in 17% of positive human sera. This protein was found in the material usually discarded when purifying smooth muscle contractile proteins. MATERIALS AND METHODS Smooth muscle tissue was obtained from human uteri, fresh or kept at - 20'C. The tissue was homogenized and washed twice with cold phosphate-buffered saline pH 7-4 prior to the extraction. Purification ofjanin obtained from the myosin containing extract. The homogenate was extracted with Guba-Staub (Offer, Moos & Starr, 1973) solution containing 03 M KCl, 01 M KH2PO4, 005 M K2HPO4. The myosin from this extract was precipitated by fourteen-fold dilution with cold distilled water pH 7.5. The precipitate was separated by centrifugation for Correspondence: Dr P. Trenchev, MRC Rheumatism Research Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire.
267
P. Trenchev
268
10 min at 38,000 g. It consisted of myosin with some actin, tropomysin and other unidentified proteins. The supernatant, usually discarded, is called here S fraction and was used to obtain janin. The pH of the mixture was brought down to 7 0 and the resulting precipitate separated by centrifugation and discarded. It contained small amounts of janin, actin, myosin, tropomyosin and other unidentified proteins. The pH of the supernatant was then further lowered with N HC1 to 5 2. At this point a large precipitate was formed consisting mainly of actin and less janin, tropomyosin, myosin and other proteins. The precipitate was spun down and discarded. The supernatant (Fig. la) was adjusted to pH 4-2. The resulting precipitate contained mainly janin, but small quantities TA
J
b~~~~~~~~~~~~~~~~~~~~~
of actin, tropomyosin and other proteins were also present. This precipitate was discarded. The remaining solution contained mainly janin, although other proteins were seen when SDS gels were overloaded with 30-40.pg of protein per gel. The whole procedure of isoelectric point precipitation was repeated and the isolated janin was further purified by means of two different techniques. (a) Chromatography on a Sephadex G-200 column equilibrated with the actin buffer janin is obtained in the first peak as monitored by absorbency at 280 nm. (b) Solid ammonium sulphate was added to 1-4 m final concentration (186 g/l). The precipitate, containing actin, tropomyosin and some other proteins, was discarded. The supernatant contained inC. almost pure janin (Fig. 2). It was dialysed against actin buffer and stored at Purtfication ofjanin from the actin containing extracts. Acetone-dried tissue powder was prepared from the homogenate as
(16 /l.Th pecptaecotinngati, rooyoinan
_.
.:...
FIG. 2. SDS gels of purified
om ohe roeis ws isade. hespenaan onaie
If~~~~~~
janin from myosin extract. Gels loaded with 10 pg (a), 20 pg (b) and 40 pg (c).
Antigenicity ofjanin
269
previously described (Trenchev et al., 1974; Holborow et al., 1975). The powder was extracted with actin buffer containing 0-08 M ATP, 0-1 mm CaCl2, 0-01 mm dithiotreitol, pH 8-2. The extract was clarified by centrifugation for 20 min at 38,000 g. The supernate contains janin in addition to actin (Fig. lb). Janin was separated from the mixture by isoelectric point precipitation at pH 4-1. The twice-washed precipitate consisted mainly of janin with traces of actin and tropomyosin. It was redissolved in 0-02 M NaCl pH 7 0. Janin was precipitated from this solution by lowering the pH to 3-9 (Fig. 3).
FIG. 3. SDS gels of janin purified from actin extract. Gels loaded with 30 jug (a) and 60 pg (b). Immunization procedure. All rabbits were screened beforehand for spontaneous antibodies and only the negative ones used in the experiment. Nine of these were immunized by means of a single s.c. injection. Each rabbit received 1 mg of janin mixed with equal volume of Freund's complete adjuvant. The animals were bled 21 days after the injection. The double diffusion test in agar and the tube test were carried out as described (Trenchev et at., 1974; Holborow et at., were
1975). Absorption
was carried out by mixing janin with antiserum and allowing the mixture to stand for 30 min at room temperature. Immunofluorescent microscopy was performed as described, by means of indirect staining (Trenchev et at., 1974). Goat anti-rabbit gammaglobulin serum was obtained from Nordic Laboratories, Maidenhead, Berkshire. SDS electrophoresis was carried out in 7.5¶y4 gels according to Weber & Osborn (1969). Smooth muscle myosin, heavy meromyosin, light meromyosin, actin and tropomyosin were prepared as previously described (Trenchev et at., 1974; Holborow et at., 1975). Human smooth muscle positive sera were selected for test at random. They included 104 sera from patients suffering from rheumatoid arthritis (forty-eight), Still's disease (thirty-six), breast cancer (seven) and thirteen healthy persons. All these sera contained anti-smooth muscle antibodies as judged by immunofluorescent microscopy.
RESULTS
Purity ofianin Preparations This was checked on SDS polyacrylamide gels. All three procedures produced reasonably purified janin. It appeared as a homogeneous single band even in overloaded gels of up to 60yjg per gel (Fig. 3). Its molecular weight as estimated by this method was found to be 60,000 + 2000 with sample proteins ovalbumin (43,000), human albumin (68,000) and smooth muscle actin (42,000). Janin preparations were compared with bovine brain and guinea-pig platelet tubulins (a generous gift from Dr N. Crawford, F
270
P. Trenchev
Birmingham University). The tubulins gave a differently placed band at 55,000 daltons. A mixture of janin and tubulin produced two separate bands corresponding to those for tubulin and janin. Purified myosin preparations did not show any band similar to that of janin even after prolonged storage at 4VC up to 2 months.
Antigenicity ofjanin Janin appeared to have stimulated production of anti-janin antibodies in four out of nine rabbits. These antibodies precipitated with janin in tube test up to 1: 128 dilutions of a solution containing 0-85 mg/ml janin. No precipitate was seen with human smooth muscle actin, myosin, heavy meromyosin, light meromyosin, tropomyosin or the tubulin preparations. The pre-immune sera with janin gave negative results. In double diffusion in agar the anti-janin sera produced a single precipitin line against janin. This line gradually moved away from the well, containing the antiserum, when tested against dilutions of janin from 1: 1 to 1: 12. The pre-immune sera failed to give any detectable precipitation with janin.
FIG. 4. Immunofluorescent micrograph of rat pancreas stained with rabbit anti-janin serum. Specific fluorescence in smooth muscle cells in arterial walls. (Magnification x 269.)
In the immunofluorescent test the anti-janin antibody gave strong specific fluorescence of all smooth muscle cells of stomach and blood vessel walls (Fig. 4), of the myoepithelial cells of salivary glandand of liver cells in a polygonal pattern (Trenchev et al., 1974). Specific fluorescence was obtained with serum dilutions up to 1: 80. The staining was confined to the cytoplasm of the cells. There was no nuclear staining. No species specificity was found since anti-janin sera reacted not only with human, but also with rat, mouse, rabbit and guinea-pig smooth muscle cells. The antisera however were tissue specific, no fluorescence being seen when skeletal or heart muscle tissues from the same animals were stained with anti-janin except for vascular staining. The pre-immune sera did not produce any staining of the smooth muscle cells. All specific fluorescence was successfully absorbed with as little janin as 8-5 pg/10 y1 antiserum, but not with tubulin, actin, myosin, heavy meromyosin, light meromyosin or tropomyosin of up to 150 pg/ 10 /1 antiserum.
Absorption of human autoantibodies by janin Successful absorbtion by janin of smooth muscle autoantibodies was established for eighteen out of
Antigenicity ofjanin
271
104 sera tested. The ratio was about 10 gg of janin for 10 pl of serum. Only the smooth muscle staining was absorbed and in liver the pattern of staining which corresponds to that seen with anti-janin rabbit sera. Nuclear or parietal cell fluorescence appeared unchanged after absorption with janin (Figs 5 and 6). The S fraction had a similar effect. The only difference was that 30 pg of S fraction protein or more was necessary for successful absorption of 10 p1 of a smooth muscle positive serum.
FIG. 5. Cryostat section of rat stomach stained with human smooth muscle positive serum. Specific fluorescence in muscularis mucosa, muscle fibres and parietal cells. (Magnification x 96.)
FIG. 6. Cryostat section of rat stomach treated with the same human serum as in Fig. 5, but absorbed for 30 min with 10 pg janin/10 ul of the serum. The staining of the muscularis mucosa and muscle fibres is absorbed. The staining of the parietal cells is not absorbed. (Magnification x 96.)
DISCUSSION These results suggest that about 17% ofthe total number of 104 human smooth muscle positive sera contain anti-janin antibody. Their smooth muscle activity was specifically absorbed with janin. Considerably
272
P. Trenchev
more protein was required to absorb the same sera with the S fraction. This contains janin as well as other proteins and the fact that nearly three times less purified janin was necessary to absorb the same sera would suggest that the autoantibody is specific for janin. It was not absorbed with actin, tubulin, myosin, heavy meromyosin, light meromyosin or tropomyosin. Apparently the so-called smooth muscle autoantibodies in different human sera are directed against different antigens including janin. Janin is most likely a smooth muscle protein since it is extracted from smooth muscle cells and the anti-janin antibody stains the cytoplasm of smooth muscle cells only. Janin differs from other contractile proteins in its molecular weight, electrophoretic mobility, isoelectric point and antigenic specificity. The estimated mol. wt., 60,000 daltons, is true only if janin is not a dimer of two chains of about 60,000 each. Janin is probably not a tubulin-like protein, since it showed different electrophoretic mobility in SDS gels. It also seems unlikely that janin is a breakdown product of myosin. Myosin preparations did not show in SDS gels any additional band in the 60,000 region even after 2 months storage at 4°C. It is not yet known whether janin is a contractile or non-contractile protein. In many ways it resembles other smooth muscle contractile proteins. Like these, janin is a highly acidic protein, forms filaments at low pH, is extracted with both actin and myosin, and is found in contractile structures. Electron microscopy studies with peroxidase-labelled anti-janin antibody (Trenchev et al., manuscript in preparation) gave a filamentous pattern throughout the smooth muscle cell cytoplasm. The exact nature of janin in relation to the function of smooth muscle cells is at present under study. It is hoped that more information about janin and other smooth muscle autoantigens will throw some light on the significance of such widespread autoantibodies as the smooth muscle autoantibodies. REFERENCES GABBIANI, G., RYAN, G.B., LAMELIN, J.P., VASSALI, P., OFFER, G., Moos, C. & STARR, R. (1973) A new protein of the thick filaments of vertebrae skeletal myofibrils. CRUCHAUD, A. & LUSCHER, E.F. (1973) Human smooth J. mol. Biol. 74, 653. muscle autoantibody: its identification as antiactin antibody and a study of its binding to 'nonmuscular' cells. POLLARD, T.D. & WEIHING, R.D. (1974) Cytoplasmic actin and myosin and cell movement. Crit. Rev. Biochem. Amer. ]. Path. 72, 473. 2, 1. HOLBOROW, E.J. (1972) Smooth muscle autoantibodies, viral infections and malignant disease. Proc. roy. Soc. Med. TRENcHEv, P., SNEYD, P. & HOLBOROW, E.J. (1974) Immunofluorescent tracing of smooth muscle contractile 65, 481. protein antigens in tissues other than smooth muscle. HOLBOROW, E.J., HEMsTED, E.H. & MAAD, S.W. (1973) Clin. exp. Immunol. 16, 125. Smooth muscle autoantibodies in infectious monoWEBER, K. & OsBoRN, M. (1969) The reliability of molecular nucleosis. Brit. med. ]. iii, 323. weight determinations by Dodecyl sulphate polyacrylHOLBOROW, E.J., TRENcHEv, P.S., DoRLING, J. & WEBB, amide gel electrophoresis. 5. biol. Chem. 277, 4406. H.J. (1975) Demonstration of smooth muscle contractile protein antigens in liver and epithelial cells. Ann. N.Y. WILSON, C., EADE, O.E., ELSTEIN, M., LLOYD, R. & WRIGHT, R. (1975) Smooth-muscle antibodies in infertility. Acad. Sci. 254, 489. Lancet, ii, 1238. JOHNSON, G.D., HOLBOROW, E.J. & GLYNN, L.E. (1965) Antibody to smooth muscle in patients with liver disease. Lancet, ii, 878.
