Acta histochem. (Jena) 93,333-340 (1992) Gustav Fischer Verlag lena· Stuttgart· New York
Institute of Biochemistry, Faculty of Medicine, University of Halle', Germany, Department of Cell Biology, University of Utrecht 2 , The Netherlands, Institute of Pathological Anatomy, University of Halle 3 , Germany
Immunoelectron microscopic localization of microsomal alanine aminopeptidase By URSULA KETTMANN', BRUNO HUMBEL>, HANS-JORGEN HOLZHAUSEN 3 , HANNES BAHN 3 , and HARALD AURICH' With 5 Figures (Received October 30, 1991 accepted April 4, 1992, in revised form)
Summary The localization of microsomal alanine aminopeptidase was investigated in the rat kidney. Resin embedding failed to demonstrate the localization of the enzyme by immunogold labelling. Using a cryo-ultramicrotomy method the enzyme could be detected on the luminal side of the brush border membrane of proximal tubular cells and to a lesser degree in their mitochondria. Furthermore, vesicular structures labelled with gold were found in the cytoplasma in the apical region of these cells.
1. Introduction Microsomal alanine aminopeptidase (mAAP, aminopeptidase MIN, EC 3.4.11.2.; Mr 280000) is a membrane-bound exopeptidase intensely investigated in its biochemical behaviour and orrurrence. mAAP is one of the major proteins of the microvillar membrane of small intestinal enterocytes and renal proximal tubular cells of mammals. It is also present in the plasma membrane of other tissues like liver, placenta, lunge, lymph nodes, blood vessels and brain (HOTTER et al. 1986; KENNY et al. 1987). According to KENNY and MAROUX (1982) mAAP is anchored by a membrane-spanning hydrophobic peptide (Mr = 3500) in the brush border membrane. A stalk segment of about 5 nm length (HUSSAIN 1985) connects the hydrophobic peptide to the main domain of the dimeric enzyme which is assumed to be exposed to the luminal side of the membrane. OLSEN et al. (1988) succeeded in deducing from cloned eDNA the complete amino acid sequence of human intestinal mAAP, thus confirming the statements of KENNY and MAROUX and HUSSAIN. The enzyme has a carbohydrate content of about 20%. Many membrane-bound peptidases could be localized in several tissues by enzyme histochemical methods (LOJDA et al. 1979) and also by immunostaining (KENNY et al. 1987) at the light microscopic level. It was the purpose of this study to visualize mAAP by immunoelectron microscopy in order to investigate whether mAAP is restricted to the brush border membrane of rat kidney tubular cells or whether is might be localized also in other compartments of these cells. For immuno-ultrastructural labelling it is essential to preserve the antigenicity of proteins throughout the whole preparation of ultrathin sections. Because of their exposed position on the external surface of the cell it is difficult to find out the localization of ectoenzymes such as mAAP with immuno-ultrastructural methods.
334
U. KETTMANN et al.
In this study we worked out suitable conditions for tissue fixation and embedding to preserve the antigenicity of mAAP. 2. Materials and Methods 2.1. Antisera: Polyclonal monospecific rabbit anti-rat-AAP antisera prepared in our laboratory were used (KETTMANN, in prep.). The specificity of sera was proved by 2-dimensional immunoelectrophoresis according to LAURELL (1965) and by Western blot. For both tests brush border vesicles prepared from rat kidneys were used. For immunoelectrophoresis these vesicles were solubilized with 0.1 % Triton X-100 (Serva, Heidelberg) and separated in the 1st dimension in agarose (Standard EEO, Serva, Heidelberg) for 2 h (5 V/cm). Electrophoresis in 1% agarose containing polyclonal specific mAAP-antiserum or polyvalent antiserum against microvillar proteins as control respectively was carried out in the 2nd dimension (4 h; I V/cm). Antigenantibody precipiation was detected with Coomassie Blue R-250 (Serva, Heidelberg) after extensive washing in 115 mmol NaCl to remove unbound antibodies or antigen respectively. For Western blots vesicles were boiled with SDS/mercaptoethanole/bromphenole blue according to LAMMLI (1970) before SDS-polyacrylamide electrophoresis in 10% gels. After blotting to 2 I-lm pore size nitrocellulose membrane (Serva, Heidelberg) the enzyme was monitored by specific antibodies and secondary antibody complexed to PAP and visualization of peroxidase activity with HzOz/diaminobenzidine. 2.2. Test of different fixation protocols: To evaluate fixation conditions the influence of different fixation protocols on the antigenicity of the mAAP was studied on Western blots of the isolated enzyme. After blocking unspecific protein binding sites on nitrocellulose, fixation was performed for 30 min at room temperature. The Western blots were reated with the following fixatives: 2% glutaraldehyde [(GA); (EM grade, Serva, Heidelberg)], 3% formaldehyde, freshly prepared from paraformaldehyde (FA); 2% FA; I % FA containing 0.1 % GA; I % FA containing 0.02% GA; 1% OS04 in veronal-acetate buffer according to PALADE (1952). The blots were extensively washed with buffer (50 mmol Tris-HCI, ISO mmol NaCl, 0.02% Triton X-lOO, pH = 7.4) prior to the overnight incubation with specific antiserum diluted 1120000 in the same buffer. Binding of primary antibodies was monitored as described above. Additionally, the influence of fixatives on antigenicity was tested on cryostat sections. Pieces of rat kidney cortex were fixed with 4% FA (unbuffered) or 2% FA containing 0.1 % GA in PBS, pH =7.4 for different time intervals, i.e. 5 min to 120 min at room temperature. Cryostat sections of unfixed and fixed material were incubated with rabbit anti-mAAP antiserum. The reaction was detected with FITC labelled goat anti-rabbit Igfraction (Sifin, Berlin) or FITC-protein A (Serva, Heidelberg). The fluorescence intensity of unfixed tissue served as a control. 2.3. EM embedding techniques: Rats were killed in ether narcosis and small cubes « 5 mm 3) of kidney cortex were excised and washed in ice-cold PBS. The cubes were either fixed with 2 % FA at room temperature or with I % FA containing 0.02% GA at 4°C for 30 min. To control the influence of different embedding procedures the tissue blocks were either conventionally dehydrated with acetone and embedded in Araldite (Serva, Heidelberg) or dehydrated with methanol and embedded in Lowicryl K4M (Lowi, Waldkraiburg) using the PLT technique introduced by CARLEMALM et al. (1982). Alternatively they were low-temperature-embedded in Lowicryl HM20 (Lowi, Waldkraiburg) using freeze-substitution to dehydration. The resin embeddings were compared with cryosections prepared according to TOKUYASU (1973). Fixed samples were infiltrated with 1.6 mol sucrose containing 10% polyvinylpyrrolidone (PVP KI5, Fluka, Buchs) and sectioned on a ReichertJung FC 4/D cryoultramicrotome according to TOKUYASU (1973, 1989). 2. 4. Immunolabelling: The immunogold labelling of ultrathin sections was done on formvar coated nickels grids (Balzer, Liechtenstein). Inactivation of free aldehyde groups of the fixatives was achieved by incubation with PBS (pHJ = 7.4) containing 50 mmol glycine. Unspecific protein binding sites were blocked with PBS containing 0.3 % HSA (Serumwerk, Dessau) for sections embedded in Araldite and Lowicryl K4M, or with PBS containing 0.5% BSA (Sigma, Munich) and 0.2% gelatin (Merck, Darmstadt) for sections embedded in Lowicryl HM20 and for cryosections. To prevent background staining these protein containing buffers were used for all dilutions and washings. The grids were incubated with 1/250 diluted specific antiserum at room temperature over night in a moist chamber. The reaction with gold labelled secondary antibody or protein A was done for I h at room temperature. Protein A (Serva, Heidelberg) was labelled with 16 nm gold particles; goat anti-rabbit IgG (Janssen, Beerse, Belgium) was labelled with 10 nm gold particles. After washing in tri-distilled water Araldite and Lowicryl sections were stained with uranyl acetate. Ultrathin cryosections cut according to TOKUYASU (1973,1989) were placed on nickel grids, thawed and thoroughly washed in PBS to remove sucrose and polyvinylpyrrolidone. Immunogold labelling was carried out as described above. After the last washing
Immunoelectron microscopy of alanine aminopeptidase
335
procedure in tri-distilled water the sections were embedded in 1.1 % tylose (MH300, Fluka, Buchs) containing 0.5% uranyl acetate (GRIFFITH et al. 1984). All described procedures were performed with the respective preinmmune serum as a control.
3. Results The antiserum was found to be monospecific in immunoelectrophoresis (Fig. 1) as well as in Western blot (not shown). The specific antigen-antibody reaction could be detected in Western blots up to a dilution of 1/40000 by the secondary antibody method described in Materials and Methods.
a
Fig. I. Specificity control of anti-mAAP antiserum in the 2-dimensional immunelectrophoresis. 1st dimension: electrophoresis of microvillar proteins (as antigen) in 1% agarose. 2nd dimension: electrophoresis of the separated microvillar proteins. a. in agarose containing a polyvalent antiserum against microvillar proteins or b. in agarose containing the specific mAAP-antiserum; protein staining with Coomassie R-250.
Table I. Staining intensity of blotted AAP on nitrocellulose after 30 min incubation with fixatives. The staining procedures are described in Materials and Methods. Fixative
Relative Antigenicity
control
+++++
1% 2% 3% 2% 1% 1%
+ ++++ +++++ ++++ +++++
OsOq glutaraldehyde formaldehyde formaldehyde formaldehyde/O.I % glutaraldehyde formaldehyde/0.02% glutaraldehyde
On cryostat sections immunostained with mAAP-antiserum und protein A-FITe specific fluorescence was found only within the tubuli. There was no background and no fluorescence of glomerular basement membrane (Fig. 2). These results were in accordance with the findings
336
U.
KETTMANN
et al.
Fig. 2. Cryostat sections of rat kidney incubated with rabbit-mAAP antiserum and FITC-protein-A. a. unfixed tissue, b. tissue fixed with 4% FA for 30 min. X 375.
Immunoelectron microscopy of alanine aminopeptidase
337
Fig. 3. Ultrathin cryosection of brush border of rat kidney tubule immunolabelled with polyclonal rabbit-" mAAP-antiserum and goat-anti-rabbit-lgGIlO nm gold. Q. microvilli cross-sectioned, b. microvilli .longitudinale sectioned, c. labelling of brush border and single mitochondria (arrow), d. control labelling usihg the respective preimmunserum. x 43200. 22
Acta histochem .. Bd. 93. I
338
U.
KETTMANN
et al.
Fig. 4. Mitochondria of rat kidney tubulular cells. a. embedded in Lowicryl HM20, immunolabelled as in Fig. 3, b. embedded in Araldite, immunolabelled with the specific mAAP-antiserum and protein A with 16 nm gold, c. cryosection, immunolabelled as in Fig. 3, d) cryosection as control, incubation with preimmunserum instead of specific antiserum. a, c. d, X 5600, b. x 45000.
made by enzyme activity investigations on unfixed cryostat sections using alanine-4-methoxy2-naphthylamide as substrate according to LoJDA et al. (1979). These findings confirm also the specificity of the antiserum. The influence of fixatives on the antigenicity of the isolated and blotted enzyme is summarized in Table 1. 2% FA or 1% FA with 0.02% GA gave the best results. Immunohistochemical investigations of fixation conditions showed a similar intensive FITC-fluorescence in the renal tubuli on cryostat sections whether unfixed or treated with 4 % FA for 30 min (Fig. 2). After fixation with 2% FA containing 0.1 % GA the fluorescence in the tubules was remarkebly diminished. On the basis of these results 2% to 3% FA or 1% FA with 0.02% GA were used for the
Inmwlllleledron
II; \:r~)s
Institute of Biochemistry, Faculty of Medicine, University of Halle', Germany, Department of Cell Biology, University of Utrecht 2 , The Netherlands, Institute of Pathological Anatomy, University of Halle 3 , Germany
Immunoelectron microscopic localization of microsomal alanine aminopeptidase By URSULA KETTMANN', BRUNO HUMBEL>, HANS-JORGEN HOLZHAUSEN 3 , HANNES BAHN 3 , and HARALD AURICH' With 5 Figures (Received October 30, 1991 accepted April 4, 1992, in revised form)
Summary The localization of microsomal alanine aminopeptidase was investigated in the rat kidney. Resin embedding failed to demonstrate the localization of the enzyme by immunogold labelling. Using a cryo-ultramicrotomy method the enzyme could be detected on the luminal side of the brush border membrane of proximal tubular cells and to a lesser degree in their mitochondria. Furthermore, vesicular structures labelled with gold were found in the cytoplasma in the apical region of these cells.
1. Introduction Microsomal alanine aminopeptidase (mAAP, aminopeptidase MIN, EC 3.4.11.2.; Mr 280000) is a membrane-bound exopeptidase intensely investigated in its biochemical behaviour and orrurrence. mAAP is one of the major proteins of the microvillar membrane of small intestinal enterocytes and renal proximal tubular cells of mammals. It is also present in the plasma membrane of other tissues like liver, placenta, lunge, lymph nodes, blood vessels and brain (HOTTER et al. 1986; KENNY et al. 1987). According to KENNY and MAROUX (1982) mAAP is anchored by a membrane-spanning hydrophobic peptide (Mr = 3500) in the brush border membrane. A stalk segment of about 5 nm length (HUSSAIN 1985) connects the hydrophobic peptide to the main domain of the dimeric enzyme which is assumed to be exposed to the luminal side of the membrane. OLSEN et al. (1988) succeeded in deducing from cloned eDNA the complete amino acid sequence of human intestinal mAAP, thus confirming the statements of KENNY and MAROUX and HUSSAIN. The enzyme has a carbohydrate content of about 20%. Many membrane-bound peptidases could be localized in several tissues by enzyme histochemical methods (LOJDA et al. 1979) and also by immunostaining (KENNY et al. 1987) at the light microscopic level. It was the purpose of this study to visualize mAAP by immunoelectron microscopy in order to investigate whether mAAP is restricted to the brush border membrane of rat kidney tubular cells or whether is might be localized also in other compartments of these cells. For immuno-ultrastructural labelling it is essential to preserve the antigenicity of proteins throughout the whole preparation of ultrathin sections. Because of their exposed position on the external surface of the cell it is difficult to find out the localization of ectoenzymes such as mAAP with immuno-ultrastructural methods.
334
U. KETTMANN et al.
In this study we worked out suitable conditions for tissue fixation and embedding to preserve the antigenicity of mAAP. 2. Materials and Methods 2.1. Antisera: Polyclonal monospecific rabbit anti-rat-AAP antisera prepared in our laboratory were used (KETTMANN, in prep.). The specificity of sera was proved by 2-dimensional immunoelectrophoresis according to LAURELL (1965) and by Western blot. For both tests brush border vesicles prepared from rat kidneys were used. For immunoelectrophoresis these vesicles were solubilized with 0.1 % Triton X-100 (Serva, Heidelberg) and separated in the 1st dimension in agarose (Standard EEO, Serva, Heidelberg) for 2 h (5 V/cm). Electrophoresis in 1% agarose containing polyclonal specific mAAP-antiserum or polyvalent antiserum against microvillar proteins as control respectively was carried out in the 2nd dimension (4 h; I V/cm). Antigenantibody precipiation was detected with Coomassie Blue R-250 (Serva, Heidelberg) after extensive washing in 115 mmol NaCl to remove unbound antibodies or antigen respectively. For Western blots vesicles were boiled with SDS/mercaptoethanole/bromphenole blue according to LAMMLI (1970) before SDS-polyacrylamide electrophoresis in 10% gels. After blotting to 2 I-lm pore size nitrocellulose membrane (Serva, Heidelberg) the enzyme was monitored by specific antibodies and secondary antibody complexed to PAP and visualization of peroxidase activity with HzOz/diaminobenzidine. 2.2. Test of different fixation protocols: To evaluate fixation conditions the influence of different fixation protocols on the antigenicity of the mAAP was studied on Western blots of the isolated enzyme. After blocking unspecific protein binding sites on nitrocellulose, fixation was performed for 30 min at room temperature. The Western blots were reated with the following fixatives: 2% glutaraldehyde [(GA); (EM grade, Serva, Heidelberg)], 3% formaldehyde, freshly prepared from paraformaldehyde (FA); 2% FA; I % FA containing 0.1 % GA; I % FA containing 0.02% GA; 1% OS04 in veronal-acetate buffer according to PALADE (1952). The blots were extensively washed with buffer (50 mmol Tris-HCI, ISO mmol NaCl, 0.02% Triton X-lOO, pH = 7.4) prior to the overnight incubation with specific antiserum diluted 1120000 in the same buffer. Binding of primary antibodies was monitored as described above. Additionally, the influence of fixatives on antigenicity was tested on cryostat sections. Pieces of rat kidney cortex were fixed with 4% FA (unbuffered) or 2% FA containing 0.1 % GA in PBS, pH =7.4 for different time intervals, i.e. 5 min to 120 min at room temperature. Cryostat sections of unfixed and fixed material were incubated with rabbit anti-mAAP antiserum. The reaction was detected with FITC labelled goat anti-rabbit Igfraction (Sifin, Berlin) or FITC-protein A (Serva, Heidelberg). The fluorescence intensity of unfixed tissue served as a control. 2.3. EM embedding techniques: Rats were killed in ether narcosis and small cubes « 5 mm 3) of kidney cortex were excised and washed in ice-cold PBS. The cubes were either fixed with 2 % FA at room temperature or with I % FA containing 0.02% GA at 4°C for 30 min. To control the influence of different embedding procedures the tissue blocks were either conventionally dehydrated with acetone and embedded in Araldite (Serva, Heidelberg) or dehydrated with methanol and embedded in Lowicryl K4M (Lowi, Waldkraiburg) using the PLT technique introduced by CARLEMALM et al. (1982). Alternatively they were low-temperature-embedded in Lowicryl HM20 (Lowi, Waldkraiburg) using freeze-substitution to dehydration. The resin embeddings were compared with cryosections prepared according to TOKUYASU (1973). Fixed samples were infiltrated with 1.6 mol sucrose containing 10% polyvinylpyrrolidone (PVP KI5, Fluka, Buchs) and sectioned on a ReichertJung FC 4/D cryoultramicrotome according to TOKUYASU (1973, 1989). 2. 4. Immunolabelling: The immunogold labelling of ultrathin sections was done on formvar coated nickels grids (Balzer, Liechtenstein). Inactivation of free aldehyde groups of the fixatives was achieved by incubation with PBS (pHJ = 7.4) containing 50 mmol glycine. Unspecific protein binding sites were blocked with PBS containing 0.3 % HSA (Serumwerk, Dessau) for sections embedded in Araldite and Lowicryl K4M, or with PBS containing 0.5% BSA (Sigma, Munich) and 0.2% gelatin (Merck, Darmstadt) for sections embedded in Lowicryl HM20 and for cryosections. To prevent background staining these protein containing buffers were used for all dilutions and washings. The grids were incubated with 1/250 diluted specific antiserum at room temperature over night in a moist chamber. The reaction with gold labelled secondary antibody or protein A was done for I h at room temperature. Protein A (Serva, Heidelberg) was labelled with 16 nm gold particles; goat anti-rabbit IgG (Janssen, Beerse, Belgium) was labelled with 10 nm gold particles. After washing in tri-distilled water Araldite and Lowicryl sections were stained with uranyl acetate. Ultrathin cryosections cut according to TOKUYASU (1973,1989) were placed on nickel grids, thawed and thoroughly washed in PBS to remove sucrose and polyvinylpyrrolidone. Immunogold labelling was carried out as described above. After the last washing
Immunoelectron microscopy of alanine aminopeptidase
335
procedure in tri-distilled water the sections were embedded in 1.1 % tylose (MH300, Fluka, Buchs) containing 0.5% uranyl acetate (GRIFFITH et al. 1984). All described procedures were performed with the respective preinmmune serum as a control.
3. Results The antiserum was found to be monospecific in immunoelectrophoresis (Fig. 1) as well as in Western blot (not shown). The specific antigen-antibody reaction could be detected in Western blots up to a dilution of 1/40000 by the secondary antibody method described in Materials and Methods.
a
Fig. I. Specificity control of anti-mAAP antiserum in the 2-dimensional immunelectrophoresis. 1st dimension: electrophoresis of microvillar proteins (as antigen) in 1% agarose. 2nd dimension: electrophoresis of the separated microvillar proteins. a. in agarose containing a polyvalent antiserum against microvillar proteins or b. in agarose containing the specific mAAP-antiserum; protein staining with Coomassie R-250.
Table I. Staining intensity of blotted AAP on nitrocellulose after 30 min incubation with fixatives. The staining procedures are described in Materials and Methods. Fixative
Relative Antigenicity
control
+++++
1% 2% 3% 2% 1% 1%
+ ++++ +++++ ++++ +++++
OsOq glutaraldehyde formaldehyde formaldehyde formaldehyde/O.I % glutaraldehyde formaldehyde/0.02% glutaraldehyde
On cryostat sections immunostained with mAAP-antiserum und protein A-FITe specific fluorescence was found only within the tubuli. There was no background and no fluorescence of glomerular basement membrane (Fig. 2). These results were in accordance with the findings
336
U.
KETTMANN
et al.
Fig. 2. Cryostat sections of rat kidney incubated with rabbit-mAAP antiserum and FITC-protein-A. a. unfixed tissue, b. tissue fixed with 4% FA for 30 min. X 375.
Immunoelectron microscopy of alanine aminopeptidase
337
Fig. 3. Ultrathin cryosection of brush border of rat kidney tubule immunolabelled with polyclonal rabbit-" mAAP-antiserum and goat-anti-rabbit-lgGIlO nm gold. Q. microvilli cross-sectioned, b. microvilli .longitudinale sectioned, c. labelling of brush border and single mitochondria (arrow), d. control labelling usihg the respective preimmunserum. x 43200. 22
Acta histochem .. Bd. 93. I
338
U.
KETTMANN
et al.
Fig. 4. Mitochondria of rat kidney tubulular cells. a. embedded in Lowicryl HM20, immunolabelled as in Fig. 3, b. embedded in Araldite, immunolabelled with the specific mAAP-antiserum and protein A with 16 nm gold, c. cryosection, immunolabelled as in Fig. 3, d) cryosection as control, incubation with preimmunserum instead of specific antiserum. a, c. d, X 5600, b. x 45000.
made by enzyme activity investigations on unfixed cryostat sections using alanine-4-methoxy2-naphthylamide as substrate according to LoJDA et al. (1979). These findings confirm also the specificity of the antiserum. The influence of fixatives on the antigenicity of the isolated and blotted enzyme is summarized in Table 1. 2% FA or 1% FA with 0.02% GA gave the best results. Immunohistochemical investigations of fixation conditions showed a similar intensive FITC-fluorescence in the renal tubuli on cryostat sections whether unfixed or treated with 4 % FA for 30 min (Fig. 2). After fixation with 2% FA containing 0.1 % GA the fluorescence in the tubules was remarkebly diminished. On the basis of these results 2% to 3% FA or 1% FA with 0.02% GA were used for the
Inmwlllleledron
II; \:r~)s
