ADONIS 001928059100071Y
Immunology 1991 72 418-425
Identification of components of the endoplasmic reticulum and Golgi complex by murine autoreactive monoclonal antibodies J. KOOY, J. R. UNDERWOOD* & P. A. GLEESON Department of Pathology and Immunology, Monash Medical School and *Department of Biochemistry, Monash University, Melbourne, Australia
Acceptedfor publication 31 October 1990
SUMMARY have been produced by the fusion of spleen cells antibodies monoclonal A number of autoreactive from unprimed BALB/c mice. The specificities of two of these monoclonal autoantibodies, MUI 38 and MUI 100, have been further examined. By indirect immunofluorescence, monoclonal antibody MUI 38 showed discrete perinuclear staining of acetone-fixed murine 3T3 fibroblasts, which was similar to that obtained with the Golgi vital stain, C6-NBD-ceramide, and with rhodamine-labelled wheat germ agglutinin. Furthermore, the staining pattern with antibody MUI 38 in cells treated with either monensin, taxol or nocodazol was altered in a manner consistent with the known effects of these drugs on Golgi morphology. In contrast, monoclonal antibody MUI 100 showed a diffuse cytoplasmic staining pattern, similar to FITC-Con A, indicative of reactivity with the endoplasmic reticulum. At high dilutions antibody MUI 100 showed only a perinuclear staining pattern, indicating that MUI 100 reacted with the Golgi as well as the endoplasmic reticulum. Both monoclonal antibodies are IgM K class and both showed reactivity with acetone-fixed fibroblasts from a number of species, indicating that the antigens are highly conserved. By immunoblotting with total membranes of murine 3T3 cells under either reducing or non-reducing conditions, monoclonal antibody MUI 100 reacted with a number of components with apparent molecular weights (MW) from 27,000 to 63,000. This reactivity was abolished when the 3T3 membranes were treated with sodium periodate, indicating antibody MUI 100 may be specific for carbohydrate. In addition, MUI 100, but not MUI 38, possessed rheumatoid factor activity, reacting with IgG from normal sera of a number of different species. Furthermore, monoclonal antibody MUI 100 was shown to be specific for the Fc domain of IgG. Absorption of MUI 100 antibody with normal rabbit IgG-Sepharose reduced the anti-endoplasmic reticulum reactivity, therefore both activities are attributable to the same antibody.
INTRODUCTION Circulating autoantibodies can be detected in a wide variety of autoimmune diseases which specifically recognize various tissues, cells or intracellular organelles.' These autoantigens are not species-specific and are often well-conserved molecules with important biological functions, for example U 1 RNP, which plays a key role in RNA splicing, has been identified as an autoantigen in systemic lupus erythematosus and mixed connective tissue disease.' Autoantibodies which recognize a wide variety of common cellular antigens have also been found in the sera of healthy individuals.2 A number of investigators have reported the presence of B lymphocytes within the normal human and murine B-cell repertoire capable of producing autoreactive antibodies to intracellular structures.3'7 In a preCorrespondence: Dr P. Gleeson, Dept. of Pathology and Immunology, Monash Medical School, Commercial Road, Prahran, Victoria 3181, Australia.
vious study we produced a number of murine autoreactive monoclonal antibodies by the fusion of splenic B cells from unprimed BALB/c mice.8 The resulting hybridomas from these fusions were screened against murine cells and tissues and a high frequency of autoreactivity was detected. A number of these monoclonal autoantibodies were shown, by indirect immunofluorescence, to react with intracellular structures including the cytoskeleton, nuclei, mitochondria, cytoplasmic vesicles, cytoplasm and Golgi complex.8 Many autoantigens have been shown to be functionally important conserved molecules.' Autoreactive murine monoclonal antibodies, therefore, represent potentially valuable tools for the identification of important intracellular membrane components. The secretory pathway of the cell includes the endoplasmic reticulum, Golgi compex and transport vesicles.9 To date, only a limited number of membrane components from this pathway have been characterized, due largely to their low abundance in the cell. This problem is further exacerbated by the common finding that these components are poorly immuno-
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Monoclonal antibodies to endoplasmic reticulum and Golgi genic. '0 Here we have characterized two autoreactive antibodies, one which is specific for the Golgi complex (MUI 38) and one which recognizes antigens found in both the endoplasmic reticulum and Golgi complex (MUI 100). This latter monoclonal antibody is also a rheumatoid factor.
MATERIALS AND METHODS Sera and monoclonal antibodies The two murine autoreactive monoclonal antibodies, MUI 38 and MUI 100, were produced as previously described8 and the hybridomas cloned twice. The antibodies were obtained from hybridoma culture supernatants by precipitation with 50% ammonium sulphate, and the resulting precipitates redissolved at 1/10 original volume in phosphate-buffered saline (PBS), dialysed and stored at 700. Rabbit anti-mouse IgM antibodies were produced by standard immunization procedures, purified by 40% ammonium sulphate fractionation followed by affinity chromatography on a monoclonal IgM-Sepharose 4B column. The bound material was eluted with 0-05 M diethylamine, pH 11-5, immediately neutralized with 1 M Tris-HCl, pH 7-8, dialysed against PBS and stored at 40 in the presence of 0-02% sodium azide. -
Cell culture Murine 3T3 fibroblasts, normal rat kidney fibroblasts (NRK; CSL, Melbourne, Australia), Baby Hamster Kidney (BALB/c derived; Flow, Stanmore, NSW, Australia) fibroblasts (BHK21 C13; Flow, Irvine, Ayrshire, U.K.) and human foreskin fibroblasts (a gift from B. H. Toh, Alfred Hospital, Melbourne, Australia) were maintained in exponential growth in Dulbecco's modified Eagle's medium (DME) supplemented with 10% (v/v) foetal calf serum (FCS), 2 mm glutamine, 100 U/ml penicillin and 0-1% (w/v) streptomycin. Cells were cultured in a humidified 10% CO2 (90% air) atmosphere at 37°.
Immunofluorescence microscopy Cells were grown attached to 12-well glass microscope slides (ICN-Flow, Sydney, Australia) in normal medium for 24-48 hr before use. The cells were rinsed in PBS, fixed and permeabilized with cold acetone (-20°) for 30 s, air dried, and then incubated with either MUI 38, MUI 100 (10-fold concentrates of hybridoma supernatants) or isotype control antibodies at room temperature for 30 min under humidified conditions. After washing three times with PBS to remove unbound antibody, the cells were incubated for 30 min with fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse Ig (1:30 dilution) (Silenus Laboratories, Melbourne, Australia). Excess second antibody was removed by washing three times with PBS, and the slides were then mounted in phosphate-buffered glycerol and examined by incident fluorescence microscopy using an excitation wavelength of 510 nm for rhodamine and 495 nm for FITC. Control experiments were carried out by incubating fixed cells with FITC-sheep anti-mouse Ig alone. The Golgi complex of unfixed cells was stained using the fluorescent sphingolipid vital stain C6-NBD-ceramide (Molecular Probes, Eugene, OR) complexed to bovine serum albumin, as described by Lipsky and Pagano." Drug treatment Stock solutions (10 nM) of nocodazole (Sigma, St Louis, MO),
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monensin (Sigma) and taxol (National Cancer Institute, NIH, Bethesda, MD) were prepared in either ethanol (monensin) or dimethyl sulphoxide (taxol and nocodazole) and stored at -20°. Immediately prior to use 10 pM solutions were prepared in DME. For staining, cells were grown on coverslips and the medium overlying the cells was replaced with fresh medium (50 p1) containing 10 /M of the drug. Cells were treated either with monensin for 10 min, with nocodazole for 45 min or with taxol for 6 hr at 37°. In some experiments cells were also allowed to recover from the 45 min nocodazole treatment by replacing the drug-containing medium with nocodazole free-medium and incubating for a further 1, 2 and 3 hr, replacing the medium every 20 min. After drug treatment the cells were fixed and processed for immunofluorescence. Control experiments were carried out by incubating cells for the equivalent time in drugfree medium containing either 0-1% dimethylsulphoxide or 0-1 % ethanol. No effect on staining patterns was observed in these control experiments. Lectin staining Cells were incubated with 0-26 mg/ml FITC-Con A and 0 5 mg/ml tetramethylrhodamine isothiocyanate (TRITC)wheat germ agglutinin (TRITC-WGA; Sigma) for 30 min at room temperature under humidified conditions, and the cells washed and mounted as described above. Control experiments were carried out in the presence of the inhibitors 100 mm methylc-D-mannoside and 2 mm N', N-diacetylchitobiose (chitobiose) (Sigma). The lectins were incubated with the saccharides for 45 min before addition to the cells.
Iodination of proteins Proteins were iodinated to a specific activity of approximately 3-4 x 107 d.p.m./mg by the chloramine T method'2 using carrierfree Na 1251 (Amersham, Bucks, U.K.).
Immunoblotting Subconfluent 3T3 cells (approximately 5 x 106) were harvested in PBS by scraping with a rubber policeman and centrifuging at 300 g for 5 min. Total cellular proteins were extracted in reducing or non-reducing SDS electrophoresis sample buffer.'3 Alternatively, total membranes from 3T3 cells were prepared following homogenization of cells, by gentle passage through a 5-ml pipette (1-3 mm diameter) 30 times in 0-25 M sucrose in 5 mm Tris-HCl, pH 7 4, containing 1 mM EDTA, the cell nuclei and cellular debris removed by centrifugation at 1260 g for 15 min at 40, and then the microsomes collected by centrifugation at 50,000 g for 90 min at 20.14 Proteins in the cell extract or membrane preparation were separated by SDS-PAGE (7 5 or 10% polyacrylamide) and transferred to nitrocellulose (Schleicher and Schuell, Dassel, Germany) for 16 hr at 60 V and 4° according to Towbin et al.'5 Following transfer, the nitrocellulose was blocked with 3% casein (Sigma) in PBS for 2 hr. The nitrocellulose membrane was cut into strips and incubated for 1-1-5 hr with antibody MUI 38 or 100 diluted in PBS containing 3% casein. After three 10 min washes in PBS containing 0-05% Nonidet P-40 and 0-05% Tween 20, the strips were then incubated for 1-1-5 hr with peroxidase-conjugated rabbit anti-mouse-Ig (Dakopatts, Glostrup, Denmark), diluted 1:100 in PBS containing 3% casein, and the strips washed as before. Bound perioxidase was detected by incubation for 10 min with substrate solution 0-05%
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J. Kooy, J. R. Underwood & P. A. Gleeson
4-chloro-1-naphthol/0 02% H202. The stained strips were washed in PBS, and stored in the dark prior to photography. Molecular weight standards (Bio Rad, Richmond, CA), transferred to nitrocellulose strips, were stained with 1 % Ponceau S
(Sigma). Sodium periodate oxidation Periodate oxidation of 3T3 extracts or membrane preparations was carried out as described elsewhere. 16 As a control, BSA was similarly and probed with rabbit anti-BSA serum. Immunoprecipitation Murine 3T3 fibroblast cells were metabolically labelled for 5 hr with [35S]-methionine and extracted as described by Ralton et al.'7 The [35S]-labelled-extracts were initially precleared by addition of 20 PI of affinity purified rabbit anti-mouse IgM and incubation for 1 hr on ice; then by sequential additions of two aliquots of Staphylococcal aureus cells (50 jul and 100 ,ul of a 10% suspension), each of which was incubated on ice for 10 min and then removed by centrifugation. Pre-cleared extracts (300 PI) were then incubated with ammonium sulphate-concentrated hybridoma supernatants (20 pI) for 1 hr followed by 20 P1 of affinity-purified rabbit anti-mouse IgM for a further 1 hr on ice. The immune complexes were collected by addition of S. aureus cells as described above. The cell pellets were washed using the buffers described by Owen et al.'8 and the immune complexes solubilized in electrophoresis sample buffer at 1000 for 2 min, and analysed by SDS-PAGE.'3
Isolation of rabbit IgG Fc fragments IgG from normal rabbit sera was isolated by protein ASepharose chromatography,'9 and the purified rabbit IgG digested with papain as follows. A 1 mg/ml solution of papain, in 0-1 M Tris-HCl, pH 8-0, containing 2 mm EDTA and 0-1 mM dithiothreitol, was activated for 15 min at 30°. The activated papain (40 jg) was added to 1 ml of purified rabbit IgG (4 mg/ml) (enzyme: substrate ratio (1:100) in the same buffer, and incubated at 370 for 1 hr. After the incubation the papain was inactivated by the addition of iodoacetamide to a final concentration of 20 mm and left on ice for 1 hr. The digests were then dialysed overnight against PBS and aliquots analysed by SDS-PAGE. The Fc fragments from the papain digest were isolated by protein A-Sepharose chromatography. The protein A-bound fraction was dialysed against PBS and analysis by SDS-PAGE under non-reducing conditions revealed a major Coomassie blue-staining 50,000 MW component. Analysis of rheumatoid factor activity by immunoprecipitation and dot blot assay Purified rabbit IgG was coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) at a concentration of 10 mg/ ml according to the manufacturers instructions. Aliquots of '25l-labelled monoclonal antibody preparation were incubated for 1 hr at room temperature with 100 p1 of a 50% suspension (in PBS) of normal IgG-Sepharose or Sepharose 4B alone. The beads were washed three times with PBS and the bound material released by heating at 1000 for 2 min in reducing SDS electrophoresis sample buffer'3 and the samples analysed by SDS-PAGE. A dot blot assay was carried out by spotting 0-5 Ml aliquots
(containing 10 ng-l pg protein) of undigested IgG, Fc and F(ab')2 fragments on duplicate strips of nitrocellulose. The nitrocellulose membranes were then blocked with 3% BSA in PBS for 2 hr at room temperature and incubated with 10 ml of '251-labelled monoclonal antibody preparations (2 x 106 c.p.m./ ml) in PBS containing 3% BSA for 1 hr with agitation. The membranes were then briefly rinsed four times with PBS, washed once in PBS for 10 min, three times in PBS containing 0 05% Tween 20 and 0-05% Nonidet P-40 for 10 min each, and finally in PBS. The membranes were then dried and autoradiography performed at -70° using Dupont Lightning Plus intensifying screens and Agfa Curix X-ray film. RESULTS Intracellular specificity of monoclonal antibodies MUI 38 and 100 The intracellular specificities of monoclonal antibodies MUI 38 and 100 were analysed by indirect immunofluorescence on acetone-fixed murine 3T3 fibroblast cells. Immunofluorescence with antibody MUI 38 revealed a discrete perinuclear staining, characteristic of the Golgi complex (Fig. la, b). As positive controls for Golgi staining, 3T3 cells were treated with the fluorescent sphingolipid, C6-NBD-ceramide, that specifically stains Golgi of living cells, and also with TRITC-WGA (Fig. 2a, b). WGA binds to sialylated glycoproteins and, therefore, has a high affinity for the Golgi complex, but, in addition, also binds to cytoplasmic vesicles and the plasma membrane. Intense perinuclear staining, similar to antibody MUI 38, was observed with both markers. In control experiments the binding of WGA was significantly reduced in the presence of the saccharide inhibitor 2 mm chitobiose (Fig. 2c). Immunofluorescence with monoclonal antibody MUI 100 revealed a diffuse cytoplasmic staining pattern (Fig. lc, d), very similar to that found with FITC-Con A (Fig. 2d). This lectin can be used as a marker for the endoplasmic reticulum as it binds very strongly to high mannose N-glycans.20'2' However, in addition, Con A will bind glycoproteins of the Golgi, cytoplasmic vesicles and plasma membrane, as for WGA. Hence, the endoplasmic reticulum can be defined as the region stained by FITC-Con A but not by TRITC-WGA. At high dilutions of antibody MUI 100 the staining pattern was found to be restricted to a perinuclear 'Golgi' region, indicating that antibody MUI 100 reacts with components of both the endoplasmic reticulum and Golgi complex. At higher concentrations of this antibody the Golgi-related reactivity is presumably masked by the reactivity to the endoplasmic reticulum. Conjugate and isotype controls for monoclonal antibodies MUI 38 and 100 showed no staining, as did Con A in the presence of the inhibitor 100 mm methyl-a-D-mannoside. Effect of Golgi disruptive drugs on staining with monoclonal antibodies MUI 38 and 100 To confirm the specificity of monoclonal antibodies MUI 38 and 100, murine 3T3 cells were treated with drugs known to effect Golgi morphology. The ionophore monensin alters the ultrastructure of the Golgi complex in a wide variety of mammalian cells causing extensive swelling of the cisternae.22 Pre-treatment of cells with monensin resulted in a more diffuse perinuclear staining by antibody MUI 38, indicative of Golgi swelling (Fig. 3b). Taxol and nocodazole are anti-mitotic drugs
Monoclonal antibodies to endoplasmic reticulum and Golgi
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Figure 1. Indirect immunofluorescence of acetone-fixed murine 3T3 fibroblasts reacted with monoclonal antibody MUI 38 (a, b) and MUI 100 (c, d). Note perinuclear staining in (a) and (b), and more extensive cytoplasmic staining in (c) and (d). Magnification; (a) x 200; (c) x 400; (b, d) x 1140.
which act directly on microtubules.2324 Both drugs resulted in loss of perinuclear staining by antibody MUI 38 (Fig. 3). Pretreatment of cells with taxol resulted in the staining by antibody MUI 38 of discrete structures well away from the nucleus close to the plasma membrane in approximately 10% of the cells (Fig. 3c). Nocodazole treatment resulted in the staining of cytoplasmic vesicles by this antibody (Fig. 3d). The altered staining pattern with nocodazole was reversed by removal of the drug over a 3 hr period and the perinuclear staining pattern gradually restored (Fig. 3e). These results are compatible with the known effects of taxol and nocodazole on the Golgi complex. In contrast, these drugs had little effect on the staining pattern generated by antibody MUI 100.
Isotype and species specificity of monoclonal antibodies MUI 38 and 100 The isotypes of MUI 38 and 100 antibodies were established by
immunoprecipitation and by ELISA. Both techniques indicated that the monoclonal antibodies were of IgM isotype, consistent with that originally reported for these monoclonal antibodies.8 The species specificity of the monoclonal antibodies were tested on rat, hamster and human fibroblast cells. Both monoclonal antibodies stained fibroblasts of all three species, in addition to mouse fibroblasts, with a similar staining pattern observed for each antibody. The antigens recognized by the monoclonal antibodies MUI 38 and 100 are, therefore, not species-specific. Monoclonal antibody MUI 100 recognizes multiple antigens of 27,000-63,000 MW Immunoblotting experiments were carried out to identify the antigens recognized by monoclonal antibodies MUI 38 and 100. Antibody MUI 100 reacted with a number of components using either total cellular or membrane extracts of 3T3 fibroblasts
J. Kooy, J. R. Underwood & P. A. Gleeson
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analysed under either reducing or non-reducing conditions. The apparent molecular weights of the major components were 63,000, 52,000, 48,000, 44,000 and 27,000 (Fig. 4). Similar results were obtained with whole cell extracts as with the extracts of membrane preparations. In contrast, antibody MUI 38 showed no reactivity by immunoblotting with these cell preparations under either non-reducing or reducing conditions. Immunoprecipitation experiements were also carried out using extracts of 3T3 cells metabolically labelled with [35S]methionine; the major 63,000 MW component was immunoprecipitated with monoclonal antibody MUI 100. Monoclonal antibody MUI 38 did not, however, precipitate a specific antigen. One possible explanation for the reactivity of MUI 100 with a number of components is that this monoclonal antibody is recognizing a carbohydrate epitope. To determine if carbohydrate is required for MUI 100 binding, we carried out experiments involving oxidation of the antigens with sodium periodate. Sodium periodate treatment of the membranes prior to immunoblotting resulted in a dramatic loss of MUI 100 reactivity with all the components (Fig. 4c). A control to assess the extent of protein degradation by this oxidation procedure was the reaction of the non-glycosylated protein BSA with an anti-BSA serum. No apparent loss of antibody binding to BSA was observed after sodium periodate treatment. Monoclonal antibody MUI 100 is a rheumatoid factor and binds to the Fc domain of normal IgG
"44
I Ft
_
.
Figure 2. Immunofluorescence of acetone-fixed murine 3T3 fibroblasts reacted with (a) the Golgi vital stain C6-NBD-ceramide, (b) TRITCWGA, (c) TRITC-WGA preincubated with 2 mM chitobiose and (d) FITC-Con A. Magnification x 400.
It has been reported that a high frequency of rheumatoid factor producing B cells persist in the spleens of normal mice.2526 Consequently, we examined monoclonal antibodies MUI 100 and MUI 38 for rheumatoid factor activity. Rheumatoid factor activity was detected by the ability of '25I-labelled MUI 100 to immunoprecipitate IgG from normal sera of a number of species. 125I-labelled MUI 100 was immunoprecipitated with normal rabbit, mouse and sheep sera, whereas 1251-labelled MUI 38 was only precipitated with specific anti-mouse IgM serum and not by normal serum. 1251-labelled MUI 100 did not react with S. aureus alone, therefore MUI 100 is reacting with a component of normal serum that can bind to S. aureus, most likely IgG. Purified IgG from normal rabbit serum was coupled to CNBr-activated Sepharose 4B and tested directly for reactivity with antibody MUI 100. '251I-labelled MUI 100 bound to IgG-Sepharose 4B but not to Sepharose 4B alone (Fig. 5), whereas antibody MUI 38 did not bind to IgG beads. The reactivity of MUI 100 antibody with normal IgG indicated it is a rheumatoid factor. To directly test whether antibody MUI 100 possessed both anti-endoplasmic reticulum and rheumatoid factor activity, the hybridoma supernatant was initially incubated with IgGSepharose 4B. The reactivity of the remaining supernatant with acetone-fixed cells was substantially reduced after absorption with IgG-Sepharose 4B, whereas incubation with Sepharose alone or BSA-Sepharose 4B showed no significant reduction in reactivity. Therefore, both activities are attributable to the same antibody. The specificity of antibody MUI 100 with normal IgG was further investigated. A dot blot analysis was carried out using intact IgG and purified Fc and Fab fragments. '251-labelled MUI 100 detected 250 ng of intact IgG and 100 ng of Fc fragment, but
Monoclonal antibodies to endoplasmic reticulum and Golgi
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I
Figure 3. Effect of Golgi disruptive drugs on the immunofluorescence staining pattern produced by monoclonal antibody MUI 38. Murine 3T3 fibroblasts were either (a) untreated, (b) treated with monensin for 10 min, (c) treated with taxol for 6 h, (d) treated with nocodazol for 45 min or (e) treated with nocodazol for 45 min then incubated for 1 hr with nocodazole-free medium to wash out the drug. The treated cells were acetone-fixed and reacted with MUI 38. Magnification x 1140.
showed no reactivity with F(ab')2 at the highest amount (1 jg) applied. Therefore MUI 100 recognizes the Fc domain of IgG. To determine if the reactivity of MUI 100 with the Fc fragments involved the recognition carbohydrate, the blotting experiments were also carried out after periodate oxidation of intact IgG and Fc fragments. No loss in binding was observed after periodate oxidation. DISCUSSION Autoantibodies reacting with a range of cellular structures can be detected in the sera of individuals suffering from a variety of disorders apparently related to immune dysfunction. Autoantibodies with apparently similar specificity have also been detected in the serum of healthy individuals and animals. Furthermore, analysis of the immune repertoire of healthy animals and humans has revealed that a large proportion of the
B lymphocytes produces antibodies which react with intracellular organelles. A number of autoantibodies from both healthy individuals and patients with autoimmune disease have been shown to recognize biologically conserved molecules. Knowledge of the structures of these molecules have provided insights into important cellular processes, for example splicing of precursor messenger RNAs.' Thus, such antibodies represent potentially useful reagents for the identification of novel organelle-specific molecules. Although many so called naturally occurring autoantibodies derived from healthy animals and individuals have been described which react with intracellular organelles, very few of the corresponding autoantigens have been characterized. Here we have shown that two murine autoreactive monoclonal antibodies react with components of the endoplasmic reticulum and Golgi complex. One of these monoclonal antibodies, MUI 100, was shown to recognize a number of membrane components by immunoblotting and
J. Kooy, J. R. Underwood & P. A. Gleeson
424 (a)
(c)
MW
116,000-
92=0066,400
45,000-
30,000-
20,00
1
1 23
23
NdO4
-
+
Figure 4. Immunoblot analysis of 3T3 fibroblast extracts with monoclonal antibodies MUI 100 and 38. Cell extracts of murine 3T3 cells analysed under (a) reducing or (b) non-reducing conditions were reacted with monoclonal antibody MUI 100 (lane 1), monoclonal antibody MUI 38 (lane 2), or with conjugate alone (lane 3). (c) Proteins of murine 3T3 fibroblast membrane preparations were separated by SDS-PAGE, electroblotted onto a nitrocellulose membrane and incubated with 20 mm sodium periodate or with buffer alone, and the nitrocellulose probed with antibody MUI 100 followed by peroxidase-conjugated anti-immunoglobulin.
(a)
(b)
MW
200P0066,000-
45,00030,000-
20,0001
2
I 2 a
Figure 5. Interaction of monoclonal antibodies MUI 38 and 100 with normal rabbit IgG-Sepharose 4B. (a) 1251-labelled MUI 100 hybridoma supernatant and (b) 1251I-labelled MUI 38 supernatant were incubated with IgG-Sepharose 4B (lane 3) or Sepharose 4B alone (lane 2). Lane 1 shows the corresponding total 125I-labelled supernatants. The beads were washed and the bound material was released from the beads in electrophoresis sample buffer, and the labelled proteins separated on a 10% acrylamide gel under reducing conditions and detected by autoradiography at 700. Molecular mass standards are shown. -
preliminary data suggests that this multi-reactivity could be due to an anti-carbohydrate specificity. This monoclonal autoantibody also displayed rheumatoid factor activity. Both autoreactive monoclonal antibodies recognized conserved epitopes on intracellular molecules as they reacted with fibroblasts from a number of different species. The use of a Golgi-specific glycolipid vital stain and WGA, as well as drugs
known to perturb the typical morphology of the Golgi, strongly suggested that monoclonal antibody MUI 38 is specific for the Golgi. Antibody MUI 100, on the other hand, appeared to interact with both the endoplasmic reticulum and the Golgi complex as the staining pattern was similar to that obtained with FITC-Con A and, furthermore, juxtanuclear staining was clearly detected at high dilutions of the antibody. These studies were based on data collected by immunofluorescence microscopy and the conclusions are therefore dependent on the comparison of staining patterns and the effects of drug treatments. Confirmation of the intracellular specificity of both antibodies will require ultrastructural localisation studies. The reactivity of the monoclonal antibodies with cytoplasmic structures of acetone-treated cells suggested that the epitopes are either protein or carbohydrate in nature rather than glycolipid. The interaction of MUI 100 with both endoplasmic reticulum and Golgi would be consistent with the multiple specificity detected by immunoblotting of microsome preparations. The sensitivity of the epitopes to mild periodate oxidation suggests that the MUI 100 antibody is binding to the carbohydrate of these membrane glycoproteins. The endoplasmic reticulum is the site of N-linked glycosylation21. However, it seems unlikely that MUI 100 is interacting with the N-glycans of the endoplasmic reticulum and Golgi, as the synthesized MUI 100 immunoglobulin in the hybridoma cells would be capable of binding to endogenous epitopes in the endomembrane transport system, potentially precluding their secretion from the cell. Furthermore, treatment of the membrane glycoproteins with Nglycanase has been shown to have no effect on the binding of antibody MUI 100 (unpublished observations). Of particular interest are the recent reports of a novel type of glycosylation in which GlcNAc is 0-glycosidically linked to proteins localized to the cytoplasmic and nucleoplasmic compartments of the cells (reviewed by 27). Significantly the cytoplasmic O-GlcNAc modified proteins include membrane glycoproteins found in the endoplasmic reticulum and Golgi.28 The possibility that monoclonal antibody MUI 100 is binding to cytoplasmically orientated O-GlcNAc saccharides is currently being explored and a monoclonal antibody with this specificity would be very valuable in the characterization of this novel class of glycoproteins. The rheumatoid factor activity of antibody MUI 100 was not, however, destroyed by periodate, suggesting that this cross-reactivity was not due to shared carbohydrate epitopes, but possibly due to polyspecificity often displayed by IgM naturally occurring autoantibodies.29 It has been suggested that naturally occurring IgM autoantibodies have a low affinity for self-molecules." The successful detection of the solubilized antigens recognized by antibody MUI 100 may be associated with the anti-carbohydrate reactivity of this antibody and a high density of saccharide epitopes/ autoantigen molecule. Therefore, even though autoreactive anti-carbohydrate IgM may be of low intrinsic affinity, these antibodies may be very useful for the biochemical characterization of organelle-specific components due to their multivalency. The inability to identify the solubilized antigen recognized by MUI 38 by either immunoprecipitation or immunoblotting may be due to a low affinity interaction with a protein epitope or, alternatively, disruption of conformational epitopes by the detergent. Autoantibodies to the Golgi have been associated with autoimmune diseases, in particular Sjogren's syndrome.3'32 The
Monoclonal antibodies to endoplasmic reticulum and Golgi antigens recognized by these disease-associated autoantibodies have not been characterized. The observation that monoclonal autoantibodies derived from healthy animals can also react with components of the Golgi shows that this organelle-specificity is present in the normal B-cell repertoire. However, the relationship between the two sets of autoantibodies is, at this stage, not clear.
ACKNOWLEDGMENTS This work was supported by a project grant from the Australian Research Council and a Monash University Special Research Grant. We thank the Natural Products Branch, Division of Cancer Treatment, National Cancer Institute, NIH, for the gift of Taxol.
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13. LAEMMLI U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227, 680. 14. HARMS E., KERN H. & SCHNEIDER J.A. (1980) Human lysosomes can be purified from diploid fibroblasts by free-flow electrophoresis. Proc. nat!. Acad. Sci. U.S.A. 77, 6139. 15. TOWBIN H., STAEHIELIN T. & GORDON J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natl. Acad. Sci. U.S.A. 76, 4350. 16. GOLDKORN I., GLEESON P.A. & TOH B.H. (1989) Gastric parietal cell antigens of 60-90, 92 and 100- 120 kDa associated with autoimmune gastritis and pernicious anemia. J. biol. Chem. 264, 18768. 17. RALTON J.E., JACKSON H., ZANONI M. & GLEESON P.A. (1989) Effect of glycosylation inhibitors on the structure and function of the transferrin receptor. Eur. J. Biochem. 186, 637. 18. OWEN M.J., KIssoNERGHIs A.-M. & LODISH H.F. (1980) Biosynthesis of HLA-A and HLA-B antigens in vivo. J. biol. Chem. 255, 9678. 19. GODING J.W. (1983) Monoclonal Antibodies: Principles and Practice, pp. 119-122, Academic Press, London. 20. CARVER, J.P., MACKENZIE A.E. & HARDMAN K.D. (1985) Molecular model for the complex between Concanavalin A and a biantennarycomplex class glycopeptide. Biopolymers, 24, 49. 21. KORNFELD R. & KORNFELD S. (1985) Assembly of asparagine-linked oligosaccharides. Ann. Rev. Biochem. 54,631. 22. TARTAKOFF A.H. (1983) Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell, 32, 1026. 23. WEHLAND J., HENKART M., KAUSNER R. & SANDOVAL V. (1983) Role of microtubules in the distribution of the Golgi apparatus: Effect of taxol and microinjected anti-a-tubulin antibodies. Proc. natl. Acad. Sci. U.S.A. 80,4286. 24. TASSIN A.M., PAINTRAND M., BERGER E.G. & BORNENS M. (1985) The Golgi apparatus remains associated with microtubule organising centers during myogenesis. J. Cell Biol. 101, 630. 25. DRESSER D.W. (1978) Most IgM-producing cells in the mouse secrete auto-antibodies (rheumatoid factor). Nature, 274, 480. 26. DZIARSKI R. (1982) Preferential induction of autoantibody secretion in polyclonal activation by peptidoglycan and lipopolysaccharide 1. In vitro studies. J. Immunol. 128, 1018. 27. HART G.W., HALTIWANGER R.S., HOLT G.D. & KELLY W.G. (1989) Glycosylation in the nucleus and cytoplasm. Ann. Rev. Biochem. 58, 841. 28. CAPASSO J.M., ABEIION C. & HIRSCHBERG C.B. (1988) An intrinsic membrane glycoprotein of the Golgi apparatus with O-linked Nacetyglucosamine facing the cytosol. J. biol. Chem. 263, 19778. 29. TERNYNCK T. & AVRAMEAS S. (1986) Murine natural monoclonal autoantibodies; A study of their polyspecificities and their affinities. Immunol. Rev. 94, 99. 30. McHEYZER-WILLIAMS M.G. & NOSSSAL G.J.V. (1988) Clonal analysis of autoantibody-producing cell precursors in the preimmune B cell repertoire. J. Immunol. 141, 4118. 31. FRITZLER M.J., ETHERINGTON J., SOKOLUK C., KINSELLA T.D. & VALENCIA D. W. (1984) Antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the Golgi apparatus. J. Immunol. 132, 2904. 32. RODRIGUEZ J.L., GELPI C., THOMPSON T.M., REAL F.J. & FERNANDEZ J. (1982) Anti-Golgi complex autoantibodies in a patient with Sjogren's syndrome and lymphoma. Clin. exp. Immunol. 49, 579.
Immunology 1991 72 418-425
Identification of components of the endoplasmic reticulum and Golgi complex by murine autoreactive monoclonal antibodies J. KOOY, J. R. UNDERWOOD* & P. A. GLEESON Department of Pathology and Immunology, Monash Medical School and *Department of Biochemistry, Monash University, Melbourne, Australia
Acceptedfor publication 31 October 1990
SUMMARY have been produced by the fusion of spleen cells antibodies monoclonal A number of autoreactive from unprimed BALB/c mice. The specificities of two of these monoclonal autoantibodies, MUI 38 and MUI 100, have been further examined. By indirect immunofluorescence, monoclonal antibody MUI 38 showed discrete perinuclear staining of acetone-fixed murine 3T3 fibroblasts, which was similar to that obtained with the Golgi vital stain, C6-NBD-ceramide, and with rhodamine-labelled wheat germ agglutinin. Furthermore, the staining pattern with antibody MUI 38 in cells treated with either monensin, taxol or nocodazol was altered in a manner consistent with the known effects of these drugs on Golgi morphology. In contrast, monoclonal antibody MUI 100 showed a diffuse cytoplasmic staining pattern, similar to FITC-Con A, indicative of reactivity with the endoplasmic reticulum. At high dilutions antibody MUI 100 showed only a perinuclear staining pattern, indicating that MUI 100 reacted with the Golgi as well as the endoplasmic reticulum. Both monoclonal antibodies are IgM K class and both showed reactivity with acetone-fixed fibroblasts from a number of species, indicating that the antigens are highly conserved. By immunoblotting with total membranes of murine 3T3 cells under either reducing or non-reducing conditions, monoclonal antibody MUI 100 reacted with a number of components with apparent molecular weights (MW) from 27,000 to 63,000. This reactivity was abolished when the 3T3 membranes were treated with sodium periodate, indicating antibody MUI 100 may be specific for carbohydrate. In addition, MUI 100, but not MUI 38, possessed rheumatoid factor activity, reacting with IgG from normal sera of a number of different species. Furthermore, monoclonal antibody MUI 100 was shown to be specific for the Fc domain of IgG. Absorption of MUI 100 antibody with normal rabbit IgG-Sepharose reduced the anti-endoplasmic reticulum reactivity, therefore both activities are attributable to the same antibody.
INTRODUCTION Circulating autoantibodies can be detected in a wide variety of autoimmune diseases which specifically recognize various tissues, cells or intracellular organelles.' These autoantigens are not species-specific and are often well-conserved molecules with important biological functions, for example U 1 RNP, which plays a key role in RNA splicing, has been identified as an autoantigen in systemic lupus erythematosus and mixed connective tissue disease.' Autoantibodies which recognize a wide variety of common cellular antigens have also been found in the sera of healthy individuals.2 A number of investigators have reported the presence of B lymphocytes within the normal human and murine B-cell repertoire capable of producing autoreactive antibodies to intracellular structures.3'7 In a preCorrespondence: Dr P. Gleeson, Dept. of Pathology and Immunology, Monash Medical School, Commercial Road, Prahran, Victoria 3181, Australia.
vious study we produced a number of murine autoreactive monoclonal antibodies by the fusion of splenic B cells from unprimed BALB/c mice.8 The resulting hybridomas from these fusions were screened against murine cells and tissues and a high frequency of autoreactivity was detected. A number of these monoclonal autoantibodies were shown, by indirect immunofluorescence, to react with intracellular structures including the cytoskeleton, nuclei, mitochondria, cytoplasmic vesicles, cytoplasm and Golgi complex.8 Many autoantigens have been shown to be functionally important conserved molecules.' Autoreactive murine monoclonal antibodies, therefore, represent potentially valuable tools for the identification of important intracellular membrane components. The secretory pathway of the cell includes the endoplasmic reticulum, Golgi compex and transport vesicles.9 To date, only a limited number of membrane components from this pathway have been characterized, due largely to their low abundance in the cell. This problem is further exacerbated by the common finding that these components are poorly immuno-
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Monoclonal antibodies to endoplasmic reticulum and Golgi genic. '0 Here we have characterized two autoreactive antibodies, one which is specific for the Golgi complex (MUI 38) and one which recognizes antigens found in both the endoplasmic reticulum and Golgi complex (MUI 100). This latter monoclonal antibody is also a rheumatoid factor.
MATERIALS AND METHODS Sera and monoclonal antibodies The two murine autoreactive monoclonal antibodies, MUI 38 and MUI 100, were produced as previously described8 and the hybridomas cloned twice. The antibodies were obtained from hybridoma culture supernatants by precipitation with 50% ammonium sulphate, and the resulting precipitates redissolved at 1/10 original volume in phosphate-buffered saline (PBS), dialysed and stored at 700. Rabbit anti-mouse IgM antibodies were produced by standard immunization procedures, purified by 40% ammonium sulphate fractionation followed by affinity chromatography on a monoclonal IgM-Sepharose 4B column. The bound material was eluted with 0-05 M diethylamine, pH 11-5, immediately neutralized with 1 M Tris-HCl, pH 7-8, dialysed against PBS and stored at 40 in the presence of 0-02% sodium azide. -
Cell culture Murine 3T3 fibroblasts, normal rat kidney fibroblasts (NRK; CSL, Melbourne, Australia), Baby Hamster Kidney (BALB/c derived; Flow, Stanmore, NSW, Australia) fibroblasts (BHK21 C13; Flow, Irvine, Ayrshire, U.K.) and human foreskin fibroblasts (a gift from B. H. Toh, Alfred Hospital, Melbourne, Australia) were maintained in exponential growth in Dulbecco's modified Eagle's medium (DME) supplemented with 10% (v/v) foetal calf serum (FCS), 2 mm glutamine, 100 U/ml penicillin and 0-1% (w/v) streptomycin. Cells were cultured in a humidified 10% CO2 (90% air) atmosphere at 37°.
Immunofluorescence microscopy Cells were grown attached to 12-well glass microscope slides (ICN-Flow, Sydney, Australia) in normal medium for 24-48 hr before use. The cells were rinsed in PBS, fixed and permeabilized with cold acetone (-20°) for 30 s, air dried, and then incubated with either MUI 38, MUI 100 (10-fold concentrates of hybridoma supernatants) or isotype control antibodies at room temperature for 30 min under humidified conditions. After washing three times with PBS to remove unbound antibody, the cells were incubated for 30 min with fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse Ig (1:30 dilution) (Silenus Laboratories, Melbourne, Australia). Excess second antibody was removed by washing three times with PBS, and the slides were then mounted in phosphate-buffered glycerol and examined by incident fluorescence microscopy using an excitation wavelength of 510 nm for rhodamine and 495 nm for FITC. Control experiments were carried out by incubating fixed cells with FITC-sheep anti-mouse Ig alone. The Golgi complex of unfixed cells was stained using the fluorescent sphingolipid vital stain C6-NBD-ceramide (Molecular Probes, Eugene, OR) complexed to bovine serum albumin, as described by Lipsky and Pagano." Drug treatment Stock solutions (10 nM) of nocodazole (Sigma, St Louis, MO),
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monensin (Sigma) and taxol (National Cancer Institute, NIH, Bethesda, MD) were prepared in either ethanol (monensin) or dimethyl sulphoxide (taxol and nocodazole) and stored at -20°. Immediately prior to use 10 pM solutions were prepared in DME. For staining, cells were grown on coverslips and the medium overlying the cells was replaced with fresh medium (50 p1) containing 10 /M of the drug. Cells were treated either with monensin for 10 min, with nocodazole for 45 min or with taxol for 6 hr at 37°. In some experiments cells were also allowed to recover from the 45 min nocodazole treatment by replacing the drug-containing medium with nocodazole free-medium and incubating for a further 1, 2 and 3 hr, replacing the medium every 20 min. After drug treatment the cells were fixed and processed for immunofluorescence. Control experiments were carried out by incubating cells for the equivalent time in drugfree medium containing either 0-1% dimethylsulphoxide or 0-1 % ethanol. No effect on staining patterns was observed in these control experiments. Lectin staining Cells were incubated with 0-26 mg/ml FITC-Con A and 0 5 mg/ml tetramethylrhodamine isothiocyanate (TRITC)wheat germ agglutinin (TRITC-WGA; Sigma) for 30 min at room temperature under humidified conditions, and the cells washed and mounted as described above. Control experiments were carried out in the presence of the inhibitors 100 mm methylc-D-mannoside and 2 mm N', N-diacetylchitobiose (chitobiose) (Sigma). The lectins were incubated with the saccharides for 45 min before addition to the cells.
Iodination of proteins Proteins were iodinated to a specific activity of approximately 3-4 x 107 d.p.m./mg by the chloramine T method'2 using carrierfree Na 1251 (Amersham, Bucks, U.K.).
Immunoblotting Subconfluent 3T3 cells (approximately 5 x 106) were harvested in PBS by scraping with a rubber policeman and centrifuging at 300 g for 5 min. Total cellular proteins were extracted in reducing or non-reducing SDS electrophoresis sample buffer.'3 Alternatively, total membranes from 3T3 cells were prepared following homogenization of cells, by gentle passage through a 5-ml pipette (1-3 mm diameter) 30 times in 0-25 M sucrose in 5 mm Tris-HCl, pH 7 4, containing 1 mM EDTA, the cell nuclei and cellular debris removed by centrifugation at 1260 g for 15 min at 40, and then the microsomes collected by centrifugation at 50,000 g for 90 min at 20.14 Proteins in the cell extract or membrane preparation were separated by SDS-PAGE (7 5 or 10% polyacrylamide) and transferred to nitrocellulose (Schleicher and Schuell, Dassel, Germany) for 16 hr at 60 V and 4° according to Towbin et al.'5 Following transfer, the nitrocellulose was blocked with 3% casein (Sigma) in PBS for 2 hr. The nitrocellulose membrane was cut into strips and incubated for 1-1-5 hr with antibody MUI 38 or 100 diluted in PBS containing 3% casein. After three 10 min washes in PBS containing 0-05% Nonidet P-40 and 0-05% Tween 20, the strips were then incubated for 1-1-5 hr with peroxidase-conjugated rabbit anti-mouse-Ig (Dakopatts, Glostrup, Denmark), diluted 1:100 in PBS containing 3% casein, and the strips washed as before. Bound perioxidase was detected by incubation for 10 min with substrate solution 0-05%
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4-chloro-1-naphthol/0 02% H202. The stained strips were washed in PBS, and stored in the dark prior to photography. Molecular weight standards (Bio Rad, Richmond, CA), transferred to nitrocellulose strips, were stained with 1 % Ponceau S
(Sigma). Sodium periodate oxidation Periodate oxidation of 3T3 extracts or membrane preparations was carried out as described elsewhere. 16 As a control, BSA was similarly and probed with rabbit anti-BSA serum. Immunoprecipitation Murine 3T3 fibroblast cells were metabolically labelled for 5 hr with [35S]-methionine and extracted as described by Ralton et al.'7 The [35S]-labelled-extracts were initially precleared by addition of 20 PI of affinity purified rabbit anti-mouse IgM and incubation for 1 hr on ice; then by sequential additions of two aliquots of Staphylococcal aureus cells (50 jul and 100 ,ul of a 10% suspension), each of which was incubated on ice for 10 min and then removed by centrifugation. Pre-cleared extracts (300 PI) were then incubated with ammonium sulphate-concentrated hybridoma supernatants (20 pI) for 1 hr followed by 20 P1 of affinity-purified rabbit anti-mouse IgM for a further 1 hr on ice. The immune complexes were collected by addition of S. aureus cells as described above. The cell pellets were washed using the buffers described by Owen et al.'8 and the immune complexes solubilized in electrophoresis sample buffer at 1000 for 2 min, and analysed by SDS-PAGE.'3
Isolation of rabbit IgG Fc fragments IgG from normal rabbit sera was isolated by protein ASepharose chromatography,'9 and the purified rabbit IgG digested with papain as follows. A 1 mg/ml solution of papain, in 0-1 M Tris-HCl, pH 8-0, containing 2 mm EDTA and 0-1 mM dithiothreitol, was activated for 15 min at 30°. The activated papain (40 jg) was added to 1 ml of purified rabbit IgG (4 mg/ml) (enzyme: substrate ratio (1:100) in the same buffer, and incubated at 370 for 1 hr. After the incubation the papain was inactivated by the addition of iodoacetamide to a final concentration of 20 mm and left on ice for 1 hr. The digests were then dialysed overnight against PBS and aliquots analysed by SDS-PAGE. The Fc fragments from the papain digest were isolated by protein A-Sepharose chromatography. The protein A-bound fraction was dialysed against PBS and analysis by SDS-PAGE under non-reducing conditions revealed a major Coomassie blue-staining 50,000 MW component. Analysis of rheumatoid factor activity by immunoprecipitation and dot blot assay Purified rabbit IgG was coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) at a concentration of 10 mg/ ml according to the manufacturers instructions. Aliquots of '25l-labelled monoclonal antibody preparation were incubated for 1 hr at room temperature with 100 p1 of a 50% suspension (in PBS) of normal IgG-Sepharose or Sepharose 4B alone. The beads were washed three times with PBS and the bound material released by heating at 1000 for 2 min in reducing SDS electrophoresis sample buffer'3 and the samples analysed by SDS-PAGE. A dot blot assay was carried out by spotting 0-5 Ml aliquots
(containing 10 ng-l pg protein) of undigested IgG, Fc and F(ab')2 fragments on duplicate strips of nitrocellulose. The nitrocellulose membranes were then blocked with 3% BSA in PBS for 2 hr at room temperature and incubated with 10 ml of '251-labelled monoclonal antibody preparations (2 x 106 c.p.m./ ml) in PBS containing 3% BSA for 1 hr with agitation. The membranes were then briefly rinsed four times with PBS, washed once in PBS for 10 min, three times in PBS containing 0 05% Tween 20 and 0-05% Nonidet P-40 for 10 min each, and finally in PBS. The membranes were then dried and autoradiography performed at -70° using Dupont Lightning Plus intensifying screens and Agfa Curix X-ray film. RESULTS Intracellular specificity of monoclonal antibodies MUI 38 and 100 The intracellular specificities of monoclonal antibodies MUI 38 and 100 were analysed by indirect immunofluorescence on acetone-fixed murine 3T3 fibroblast cells. Immunofluorescence with antibody MUI 38 revealed a discrete perinuclear staining, characteristic of the Golgi complex (Fig. la, b). As positive controls for Golgi staining, 3T3 cells were treated with the fluorescent sphingolipid, C6-NBD-ceramide, that specifically stains Golgi of living cells, and also with TRITC-WGA (Fig. 2a, b). WGA binds to sialylated glycoproteins and, therefore, has a high affinity for the Golgi complex, but, in addition, also binds to cytoplasmic vesicles and the plasma membrane. Intense perinuclear staining, similar to antibody MUI 38, was observed with both markers. In control experiments the binding of WGA was significantly reduced in the presence of the saccharide inhibitor 2 mm chitobiose (Fig. 2c). Immunofluorescence with monoclonal antibody MUI 100 revealed a diffuse cytoplasmic staining pattern (Fig. lc, d), very similar to that found with FITC-Con A (Fig. 2d). This lectin can be used as a marker for the endoplasmic reticulum as it binds very strongly to high mannose N-glycans.20'2' However, in addition, Con A will bind glycoproteins of the Golgi, cytoplasmic vesicles and plasma membrane, as for WGA. Hence, the endoplasmic reticulum can be defined as the region stained by FITC-Con A but not by TRITC-WGA. At high dilutions of antibody MUI 100 the staining pattern was found to be restricted to a perinuclear 'Golgi' region, indicating that antibody MUI 100 reacts with components of both the endoplasmic reticulum and Golgi complex. At higher concentrations of this antibody the Golgi-related reactivity is presumably masked by the reactivity to the endoplasmic reticulum. Conjugate and isotype controls for monoclonal antibodies MUI 38 and 100 showed no staining, as did Con A in the presence of the inhibitor 100 mm methyl-a-D-mannoside. Effect of Golgi disruptive drugs on staining with monoclonal antibodies MUI 38 and 100 To confirm the specificity of monoclonal antibodies MUI 38 and 100, murine 3T3 cells were treated with drugs known to effect Golgi morphology. The ionophore monensin alters the ultrastructure of the Golgi complex in a wide variety of mammalian cells causing extensive swelling of the cisternae.22 Pre-treatment of cells with monensin resulted in a more diffuse perinuclear staining by antibody MUI 38, indicative of Golgi swelling (Fig. 3b). Taxol and nocodazole are anti-mitotic drugs
Monoclonal antibodies to endoplasmic reticulum and Golgi
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_
Figure 1. Indirect immunofluorescence of acetone-fixed murine 3T3 fibroblasts reacted with monoclonal antibody MUI 38 (a, b) and MUI 100 (c, d). Note perinuclear staining in (a) and (b), and more extensive cytoplasmic staining in (c) and (d). Magnification; (a) x 200; (c) x 400; (b, d) x 1140.
which act directly on microtubules.2324 Both drugs resulted in loss of perinuclear staining by antibody MUI 38 (Fig. 3). Pretreatment of cells with taxol resulted in the staining by antibody MUI 38 of discrete structures well away from the nucleus close to the plasma membrane in approximately 10% of the cells (Fig. 3c). Nocodazole treatment resulted in the staining of cytoplasmic vesicles by this antibody (Fig. 3d). The altered staining pattern with nocodazole was reversed by removal of the drug over a 3 hr period and the perinuclear staining pattern gradually restored (Fig. 3e). These results are compatible with the known effects of taxol and nocodazole on the Golgi complex. In contrast, these drugs had little effect on the staining pattern generated by antibody MUI 100.
Isotype and species specificity of monoclonal antibodies MUI 38 and 100 The isotypes of MUI 38 and 100 antibodies were established by
immunoprecipitation and by ELISA. Both techniques indicated that the monoclonal antibodies were of IgM isotype, consistent with that originally reported for these monoclonal antibodies.8 The species specificity of the monoclonal antibodies were tested on rat, hamster and human fibroblast cells. Both monoclonal antibodies stained fibroblasts of all three species, in addition to mouse fibroblasts, with a similar staining pattern observed for each antibody. The antigens recognized by the monoclonal antibodies MUI 38 and 100 are, therefore, not species-specific. Monoclonal antibody MUI 100 recognizes multiple antigens of 27,000-63,000 MW Immunoblotting experiments were carried out to identify the antigens recognized by monoclonal antibodies MUI 38 and 100. Antibody MUI 100 reacted with a number of components using either total cellular or membrane extracts of 3T3 fibroblasts
J. Kooy, J. R. Underwood & P. A. Gleeson
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analysed under either reducing or non-reducing conditions. The apparent molecular weights of the major components were 63,000, 52,000, 48,000, 44,000 and 27,000 (Fig. 4). Similar results were obtained with whole cell extracts as with the extracts of membrane preparations. In contrast, antibody MUI 38 showed no reactivity by immunoblotting with these cell preparations under either non-reducing or reducing conditions. Immunoprecipitation experiements were also carried out using extracts of 3T3 cells metabolically labelled with [35S]methionine; the major 63,000 MW component was immunoprecipitated with monoclonal antibody MUI 100. Monoclonal antibody MUI 38 did not, however, precipitate a specific antigen. One possible explanation for the reactivity of MUI 100 with a number of components is that this monoclonal antibody is recognizing a carbohydrate epitope. To determine if carbohydrate is required for MUI 100 binding, we carried out experiments involving oxidation of the antigens with sodium periodate. Sodium periodate treatment of the membranes prior to immunoblotting resulted in a dramatic loss of MUI 100 reactivity with all the components (Fig. 4c). A control to assess the extent of protein degradation by this oxidation procedure was the reaction of the non-glycosylated protein BSA with an anti-BSA serum. No apparent loss of antibody binding to BSA was observed after sodium periodate treatment. Monoclonal antibody MUI 100 is a rheumatoid factor and binds to the Fc domain of normal IgG
"44
I Ft
_
.
Figure 2. Immunofluorescence of acetone-fixed murine 3T3 fibroblasts reacted with (a) the Golgi vital stain C6-NBD-ceramide, (b) TRITCWGA, (c) TRITC-WGA preincubated with 2 mM chitobiose and (d) FITC-Con A. Magnification x 400.
It has been reported that a high frequency of rheumatoid factor producing B cells persist in the spleens of normal mice.2526 Consequently, we examined monoclonal antibodies MUI 100 and MUI 38 for rheumatoid factor activity. Rheumatoid factor activity was detected by the ability of '25I-labelled MUI 100 to immunoprecipitate IgG from normal sera of a number of species. 125I-labelled MUI 100 was immunoprecipitated with normal rabbit, mouse and sheep sera, whereas 1251-labelled MUI 38 was only precipitated with specific anti-mouse IgM serum and not by normal serum. 1251-labelled MUI 100 did not react with S. aureus alone, therefore MUI 100 is reacting with a component of normal serum that can bind to S. aureus, most likely IgG. Purified IgG from normal rabbit serum was coupled to CNBr-activated Sepharose 4B and tested directly for reactivity with antibody MUI 100. '251I-labelled MUI 100 bound to IgG-Sepharose 4B but not to Sepharose 4B alone (Fig. 5), whereas antibody MUI 38 did not bind to IgG beads. The reactivity of MUI 100 antibody with normal IgG indicated it is a rheumatoid factor. To directly test whether antibody MUI 100 possessed both anti-endoplasmic reticulum and rheumatoid factor activity, the hybridoma supernatant was initially incubated with IgGSepharose 4B. The reactivity of the remaining supernatant with acetone-fixed cells was substantially reduced after absorption with IgG-Sepharose 4B, whereas incubation with Sepharose alone or BSA-Sepharose 4B showed no significant reduction in reactivity. Therefore, both activities are attributable to the same antibody. The specificity of antibody MUI 100 with normal IgG was further investigated. A dot blot analysis was carried out using intact IgG and purified Fc and Fab fragments. '251-labelled MUI 100 detected 250 ng of intact IgG and 100 ng of Fc fragment, but
Monoclonal antibodies to endoplasmic reticulum and Golgi
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I
Figure 3. Effect of Golgi disruptive drugs on the immunofluorescence staining pattern produced by monoclonal antibody MUI 38. Murine 3T3 fibroblasts were either (a) untreated, (b) treated with monensin for 10 min, (c) treated with taxol for 6 h, (d) treated with nocodazol for 45 min or (e) treated with nocodazol for 45 min then incubated for 1 hr with nocodazole-free medium to wash out the drug. The treated cells were acetone-fixed and reacted with MUI 38. Magnification x 1140.
showed no reactivity with F(ab')2 at the highest amount (1 jg) applied. Therefore MUI 100 recognizes the Fc domain of IgG. To determine if the reactivity of MUI 100 with the Fc fragments involved the recognition carbohydrate, the blotting experiments were also carried out after periodate oxidation of intact IgG and Fc fragments. No loss in binding was observed after periodate oxidation. DISCUSSION Autoantibodies reacting with a range of cellular structures can be detected in the sera of individuals suffering from a variety of disorders apparently related to immune dysfunction. Autoantibodies with apparently similar specificity have also been detected in the serum of healthy individuals and animals. Furthermore, analysis of the immune repertoire of healthy animals and humans has revealed that a large proportion of the
B lymphocytes produces antibodies which react with intracellular organelles. A number of autoantibodies from both healthy individuals and patients with autoimmune disease have been shown to recognize biologically conserved molecules. Knowledge of the structures of these molecules have provided insights into important cellular processes, for example splicing of precursor messenger RNAs.' Thus, such antibodies represent potentially useful reagents for the identification of novel organelle-specific molecules. Although many so called naturally occurring autoantibodies derived from healthy animals and individuals have been described which react with intracellular organelles, very few of the corresponding autoantigens have been characterized. Here we have shown that two murine autoreactive monoclonal antibodies react with components of the endoplasmic reticulum and Golgi complex. One of these monoclonal antibodies, MUI 100, was shown to recognize a number of membrane components by immunoblotting and
J. Kooy, J. R. Underwood & P. A. Gleeson
424 (a)
(c)
MW
116,000-
92=0066,400
45,000-
30,000-
20,00
1
1 23
23
NdO4
-
+
Figure 4. Immunoblot analysis of 3T3 fibroblast extracts with monoclonal antibodies MUI 100 and 38. Cell extracts of murine 3T3 cells analysed under (a) reducing or (b) non-reducing conditions were reacted with monoclonal antibody MUI 100 (lane 1), monoclonal antibody MUI 38 (lane 2), or with conjugate alone (lane 3). (c) Proteins of murine 3T3 fibroblast membrane preparations were separated by SDS-PAGE, electroblotted onto a nitrocellulose membrane and incubated with 20 mm sodium periodate or with buffer alone, and the nitrocellulose probed with antibody MUI 100 followed by peroxidase-conjugated anti-immunoglobulin.
(a)
(b)
MW
200P0066,000-
45,00030,000-
20,0001
2
I 2 a
Figure 5. Interaction of monoclonal antibodies MUI 38 and 100 with normal rabbit IgG-Sepharose 4B. (a) 1251-labelled MUI 100 hybridoma supernatant and (b) 1251I-labelled MUI 38 supernatant were incubated with IgG-Sepharose 4B (lane 3) or Sepharose 4B alone (lane 2). Lane 1 shows the corresponding total 125I-labelled supernatants. The beads were washed and the bound material was released from the beads in electrophoresis sample buffer, and the labelled proteins separated on a 10% acrylamide gel under reducing conditions and detected by autoradiography at 700. Molecular mass standards are shown. -
preliminary data suggests that this multi-reactivity could be due to an anti-carbohydrate specificity. This monoclonal autoantibody also displayed rheumatoid factor activity. Both autoreactive monoclonal antibodies recognized conserved epitopes on intracellular molecules as they reacted with fibroblasts from a number of different species. The use of a Golgi-specific glycolipid vital stain and WGA, as well as drugs
known to perturb the typical morphology of the Golgi, strongly suggested that monoclonal antibody MUI 38 is specific for the Golgi. Antibody MUI 100, on the other hand, appeared to interact with both the endoplasmic reticulum and the Golgi complex as the staining pattern was similar to that obtained with FITC-Con A and, furthermore, juxtanuclear staining was clearly detected at high dilutions of the antibody. These studies were based on data collected by immunofluorescence microscopy and the conclusions are therefore dependent on the comparison of staining patterns and the effects of drug treatments. Confirmation of the intracellular specificity of both antibodies will require ultrastructural localisation studies. The reactivity of the monoclonal antibodies with cytoplasmic structures of acetone-treated cells suggested that the epitopes are either protein or carbohydrate in nature rather than glycolipid. The interaction of MUI 100 with both endoplasmic reticulum and Golgi would be consistent with the multiple specificity detected by immunoblotting of microsome preparations. The sensitivity of the epitopes to mild periodate oxidation suggests that the MUI 100 antibody is binding to the carbohydrate of these membrane glycoproteins. The endoplasmic reticulum is the site of N-linked glycosylation21. However, it seems unlikely that MUI 100 is interacting with the N-glycans of the endoplasmic reticulum and Golgi, as the synthesized MUI 100 immunoglobulin in the hybridoma cells would be capable of binding to endogenous epitopes in the endomembrane transport system, potentially precluding their secretion from the cell. Furthermore, treatment of the membrane glycoproteins with Nglycanase has been shown to have no effect on the binding of antibody MUI 100 (unpublished observations). Of particular interest are the recent reports of a novel type of glycosylation in which GlcNAc is 0-glycosidically linked to proteins localized to the cytoplasmic and nucleoplasmic compartments of the cells (reviewed by 27). Significantly the cytoplasmic O-GlcNAc modified proteins include membrane glycoproteins found in the endoplasmic reticulum and Golgi.28 The possibility that monoclonal antibody MUI 100 is binding to cytoplasmically orientated O-GlcNAc saccharides is currently being explored and a monoclonal antibody with this specificity would be very valuable in the characterization of this novel class of glycoproteins. The rheumatoid factor activity of antibody MUI 100 was not, however, destroyed by periodate, suggesting that this cross-reactivity was not due to shared carbohydrate epitopes, but possibly due to polyspecificity often displayed by IgM naturally occurring autoantibodies.29 It has been suggested that naturally occurring IgM autoantibodies have a low affinity for self-molecules." The successful detection of the solubilized antigens recognized by antibody MUI 100 may be associated with the anti-carbohydrate reactivity of this antibody and a high density of saccharide epitopes/ autoantigen molecule. Therefore, even though autoreactive anti-carbohydrate IgM may be of low intrinsic affinity, these antibodies may be very useful for the biochemical characterization of organelle-specific components due to their multivalency. The inability to identify the solubilized antigen recognized by MUI 38 by either immunoprecipitation or immunoblotting may be due to a low affinity interaction with a protein epitope or, alternatively, disruption of conformational epitopes by the detergent. Autoantibodies to the Golgi have been associated with autoimmune diseases, in particular Sjogren's syndrome.3'32 The
Monoclonal antibodies to endoplasmic reticulum and Golgi antigens recognized by these disease-associated autoantibodies have not been characterized. The observation that monoclonal autoantibodies derived from healthy animals can also react with components of the Golgi shows that this organelle-specificity is present in the normal B-cell repertoire. However, the relationship between the two sets of autoantibodies is, at this stage, not clear.
ACKNOWLEDGMENTS This work was supported by a project grant from the Australian Research Council and a Monash University Special Research Grant. We thank the Natural Products Branch, Division of Cancer Treatment, National Cancer Institute, NIH, for the gift of Taxol.
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