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Detection, Quantitation, and Localization of Bovine Angiogenin by Immunological Assays
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
232, 323–327 (1997)
RC976280
Detection, Quantitation, and Localization of Bovine Angiogenin by Immunological Assays Soo-Ik Chang,*,1 Goo-Bo Jeong,† Seung-Ho Park,* Byung-Cheol Ahn,* Jung-Do Choi,* Quae Chae,* Sung Keon Namgoong,‡ and Soo-Il Chung§ *Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea; †Department of Anatomy, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea; ‡Department of Chemistry, Seoul Woman’s University, Seoul 139-774, Korea; and §Mogam Biotechnology Research Institute, Yongin-Kun 441-910, Korea
Received February 3, 1997
Bovine angiogenin (bAng) is a potent blood vessel inducing protein found in bovine serum and milk. Antisera have been raised against bAng. Western blot analysis for bAng indicated that the polyclonal antibody recognized bAng specifically, and no cross-reactivity with bovine RNase A, a protein homologous to bAng, was observed. The sandwich enzyme-linked immunosorbent assay for bAng was sensitive to 10 pg of bAng, and this assay was able to quantitate bAng in bovine serum (100–180 ng/ mL) and milk (4–8 mg/mL). Strong positive immunohistochemical reactions were detected in alveolar cells, the secretion of alveolar cells and excretory ducts in sections of cow mammary gland, epithelial cells of visceral peritoneum and bile-duct in sections of cow liver, and epithelial cells and mucous glands in sections of cow gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis. q 1997 Academic Press
Angiogenin is a potent blood vessel inducing protein originally purified from the conditioned media of cultured human colon adenocarcinoma cells (HT-29) [1]. It was later detected and purified from normal human serum [2], and bovine serum [3–4] and milk [5–6], and its mRNA is expressed in both normal and tumor cells [7–10]. The mechanism of action of angiogenin on angiogenesis is complex, and a dual-site model for the action of angiogenin has been suggested [11–12]. In addition, it has been suggested that nuclear translocation of angio1 To whom correspondence should be addressed. Fax: 82-431-672306. E-mail: [email protected]. Abbreviations used: hAng, human angiogenin; bAng, bovine angiogenin; RNase A, bovine pancreatic ribonuclease A; CM, carboxymethyl; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PRI, placental ribonuclease inhibitor.
genin is a critical step in the process of angiogenesis [13]. In order to elucidate its biological mechanism, further studies of angiogenin on its localization in various organs and tissues as well as understanding the relationship between structure and function are needed. It has been reported that some enzymatic and biological activities of bovine angiogenin (bAng) are identical with those of human angiogenin (hAng) [3]. bAng has 64% sequence identity to hAng [4–5]. Recently, the crystal structure of bAng has been determined and is closely similar to that of hAng [14–15]. Bovine milk is a readily accessible source of bAng, and it is easier and more economical to obtain bAng than hAng. Therefore, bAng could be used as an alternative material for further studies. Various assays for angiogenin have been developed since its first isolation using the chicken embryo chorioallantoic membrane (CAM) assay which monitors new blood vessel growth in vivo. Among these are assays based on i) inhibition of protein synthesis or degradation of reticulocyte RNA [16], ii) cleavage of 28 and 18S rRNA or tRNA [16–17], and iii) the capacity of angiogenin to bind placental ribonuclease inhibitor [18]. The routine detection and sensitive quantitation of angiogenin is necessary to elucidate the mechanism of action of this protein. The purpose of this study is to develop simple and sensitive immunoassays that could be used not only to detect and quantitate angiogenin, but which could also be used to localize angiogenin in tumor and normal organs and tissues. We report here sensitive Western blot analysis and sandwich enzyme-linked immunosorbent assay (ELISA) for bAng, which showed no crossreactivity with bovine pancreatic ribonuclease A (RNase A), a protein homologous to bAng, and thus provides a means of detecting and quantitating angio-
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genin in both unfractionated samples and chromatographic fractions which contain RNases [19]. We also report the first immunohistochemical localization of angiogenin in various organs and tissues. Rybak et al. [7] reported the distribution of angiogenin transcripts in human cells representative of different tumor and normal phenotypes by using both human cDNAs for angiogenin and the corresponding RNA antisense probes. The presence of angiogenin transcripts, however, does not ensure that the protein is synthesized, and thus it is necessary to further examine the distribution of angiogenin itself in tumor and normal cells. MATERIALS AND METHODS Materials. An immuno-blot assay kit and prestained molecular weight standards were obtained from Bio-Rad Laboratories. An enhanced chemiluminescence Western blot analysis kit (RPN 2108) and biotinylated molecular weight markers (RPN 2107) were from Amersham. NHS-LC-biotin, o-phenylenediamine, and streptavidin horseradish peroxidase were from Pierce Chemical Co.. Bovine pancreatic ribonuclease A, bovine serum albumin (Fr.V), Freund’s adjuvants (complete and incomplete) were from Sigma Chemical Co. Fetal calf serum was from GIBCO Co.. bAng was purified from milk as described [3]. All other chemicals used were of analytical grade. Immunization procedure. Rabbits were injected subcutaneously with 100 mg of bAng emulsified in Freund’s complete adjuvant. Booster injections of bAng (100 mg) in Freund’s incomplete adjuvant were given at 10-day intervals. After the third immunization, blood was collected and the antiserum was isolated. The IgG fraction of anti-bAng antisera was purified by protein A-Sepharose chromatography. A portion of the rabbit anti-bAng IgG was biotinylated using NHS-LC-biotin (Pierce) according to the manufacturer’s protocol. Biotinylated anti-bAng IgG was separated from unbound biotin using a Centricon 30 microconcentrator (Amicon). Western blot analysis. SDS-PAGE was performed under reducing conditions by using 15% gels as described by Laemmli [20]. Proteins were transferred electophoretically to 0.45-mm nitrocellulose membranes by using a Hoeffer TE22 mini transphor electrophoresis unit at a constant current of 200 mA for 2 h. Following transfer, sheets were incubated for 1 h with 20 mM Tris, 0.5 M NaCl, pH 7.5 (TBS) containing 3% gelatin to prevent nonspecific adsorption. Sheets were then washed with TBS containing 0.05% Tween 20 (TBS/Tween) and incubated for overnight at 47C with rabbit antiserum (dilution 1/200 in TBS containing 1% gelatin) raised against bAng as described above. In other experiments, the IgG fraction of anti-bAng antisera was used instead of the rabbit antiserum itself. After being washed with TBS/Tween, sheets were incubated for 2 h at room temperature with alkaline phosphatase conjugated goat anti-rabbit IgG (diluted 1/300 in TBS containing 1% gelatin). The sheets were washed with TBS/Tween and later with TBS. The proteins recognized by the primary antiserum were then detected by addition of color reagents A and B (Bio-Rad Laboratories). In other experiments, bAng was detected by enhanced chemiluminescence (ECL). Sandwich ELISA. Microplates were coated with 100 mL per well of anti-bAng IgG diluted to 24.2 mg/mL in 50 mM carbonate-bicarbonate buffer (pH 9.6) and incubated for 3 h at 377C. The plates were then washed three times with PBS containing 0.05% Tween-20 (PBS/ Tween-20). The plates were blocked with 200 mL per well of 1% BSA in PBS for 1 h at 377C. After the plates were washed three times with PBS/Tween-20, 100 mL per well of bAng standards and samples which had been diluted with 0.1% BSA in PBS were added to the
plates and incubated for 60 min at 377C. The plates were then washed three times with PBS/Tween-20, 100 mL per well of biotinylated rabbit anti-bAng IgG diluted to 5 mg/mL in 0.1% BSA in PBS was added, and the plates were incubated at 377C for 1 h, and again the plates were washed three times with PBS/Tween-20. 100 mL per well of Streptavidin-horseradish peroxidase diluted to 1 mg/mL in PBS/ Tween-20 were added, and the plates were incubated for 30 min at 377C. The plates were washed, and 100 mL per well of substrate solution (0.1% O-phenylenediamine, 0.02% H2O2 , 50 mM citrate, and 100 mM phosphate buffer, pH 5.0) was added. Color development was allowed to proceed for 15 min at room temperature and was stopped by adding 100 mL per well of 2 M H2SO4 . The absorbance was then measured at 492 nm on a microplate reader (Titertek Multiscan PLUS, Labsystems), and the concentration of bAng was calculated based on the standard curve of bAng. Immunohistochemistry. Tissues of cow liver and mammary gland freshly obtained surgically were directly fixed by treatment with phosphate-buffered saline (PBS) containing 4% paraformaldehyde overnight at 377C. The specimens were dehydrated through ethanol and xylene, and were embedded with paraffin until used. After cutting the formalin-fixed paraffin-embedded material at 4–5 mm, sections were de-waxed thoroughly in xylene and ethanol. The specimens were washed with PBS, blocked with 0.4% normal goat serum for 30 min, and then incubated for 1 h at 377C with PBS containing rabbit anti-bovine angiogenin IgG (dilution 1/200). The specimens were washed three times with PBS, and then incubated for 1 h at room temperature with biotinylated goat anti-rabbit IgG. The specimens were washed three times with PBS, and then incubated for 30 min at room temperature with avidin conjugated horseradish peroxidase. Localization of bAng was visualized by incubation of the slide for 3–5 min with a freshly prepared solution of 0.1% 3,3 *-diaminobenzidine (DAB) in PBS containing 0.05% hydrogen peroxide. The specimens were counterstained with Mayer’s hematoxylin. To confirm the specificity of the immunohistochemical signal, the specimens were incubated with preimmune rabbit IgG or without rabbit antibovine angiogenin IgG, and then stained as described above.
RESULTS Production of antisera to bAng. Rabbit anti-bAng antiserum was obtained by the immunization procedure described in MATERIALS AND METHODS. Ouchterlony double diffusion analysis with bAng and RNase A, a protein homologous to bAng, was performed with an agarose gel. Visible precipitates were formed with the antiserum and bAng but not with RNase A (data not shown). The same antiserum was used for all experiments carried out in this study. Western blot analysis. The specificity of binding of polyclonal anti-bAng antibodies to bAng was further investigated using Western blot analysis. As shown in Fig. 1, the polyclonal antibody recognized a single polypeptide band at 16 500Da, which corresponds to the molecular weight of bAng by SDS-PAGE [3], and no cross-reactivity with bovine RNase A was observed. The sensitivity of detection of bAng in this system is 50 ng (data not shown). Sandwich ELISA. A representative standard curve for bAng is shown in Fig. 2. The lower limit of sensitivity of the assay was 0.1 ng/mL. RNase A showed no reactivity even at a concentration of 1000 ng/mL in
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FIG. 1. Cross-reactivity of polyclonal rabbit anti-bovine angiogenin antibodies with bovine angiogenin and homologous bovine RNase A. Bovine angiogenin (1 mg) or bovine RNase A (1 mg) was electrophoresed on 15% polyacrylamide gels containing SDS followed by transfer to nitrocellulose sheets. Sheets were then reacted with rabbit antiserum (dilution 1/200) to bovine angiogenin, followed by treatment with alkaline phosphatase conjugated goat anti-rabbit IgG and the appropriate chromatogenous substrate. (A) polyacrylamide gel stained with coomassie blue. (B) Westen blot analysis of the same gel. (Lane 1) Bovine RNase A; (lane 2) Prestained protein molecular mass markers (Bio-Rad); (lane 3) Bovine angiogenin.
the ELISA (data not shown). We applied this sandwich ELISA method for the estimation of bAng levels in bovine serum and milk. The content of bAng in adult calf serum, fetal calf serum, and cow milk is 0.18, 0.10 and 3.8–7.5 mg/mL, respectively. Next, we used the immunological assay to detect bovine milk angiogenin in column fractions from the CM-cellulose chromatography step during isolation of the protein [3]. Bovine milk is applied to CM-52 cation-exchange resin, and the bound protein is then eluted with 1 M NaCl and designated CM2 (Fig. 3A). As shown in Fig. 3A, bAng was detected with fractions 4–7. The presence of bAng in these fractions was confirmed by Western blot analysis (Fig. 3B). Immunohistochemical localization of bAng. We carried out immunohistochemical localization of the bAng in
FIG. 2. Standard curve of the sandwich ELISA for bAng. Native and biotin-conjugated anti-bAng rabbit IgG were used as the catching and developing antibody, respectively. Detection was carried out by horseradish peroxidase conjugated streptavidin and appropriate chromatogeneous substrate as described under Materials and Methods. Increasing amounts of bovine angiogenin expressed in mg of protein/L are plotted against optical density at 492 nm. Each point indicates the mean of duplicate assays.
FIG. 3. CM-cellulose chromatography of cow milk, and sandwich ELISA and Western blot analysis of the CM2 fractions. (A) 5 mL of cow milk was centrifuged at 15600g, diluted with 10 mL of 50 mM sodium phosphate (pH 6.6) and applied to CM-cellulose chromatography (1 1 5 cm). Elution was accomplished with 1 M NaCl in 10 mM Tris, pH 8.0. Each fraction contains 200 mL. Sandwich ELISA was used to detect bovine angiogenin in the fractions as described under Materials and Methods. (B) Fractions (designated as CM2) were electrophoresed on 15% polyacrylamide gels containing SDS followed by transfer to nitrocellulose sheets. Sheets were then reacted with rabbit anti-bovine angiogenin IgG, followed by treatment with horseradish peroxidase conjugated donkey anti-rabbit IgG and the ECL substrate (Amersham). ECL was detected for 2 min. (Lane 1) 0.2 mg of purified bovine angiogenin; (lane 2) biotinylated molecular mass markers (Amersham); (lanes 3–9) CM2 fraction 2–8 from 5 mL of cow milk (see Fig. 3A). The positions of the molecular-mass markers (kDa) are also shown. Arrow indicates the 16.5 kDa band.
the sections of various organs and tissues of cow. Strong positive reactions were detected in alveolar cells and the secretion of alveolar cells (Fig. 4A) as well as excretory ducts in sections of mammary gland, and weak positive reactions were observed in endothelial cells, smooth muscle cells, fibroblast cells and myoepithelial cells in sections of mammary gland (data not shown). These immunohistochemical signals, however, were not detected when the specimens were stained with preimmune rabbit IgG or without pre-incubation with rabbit anti-bAng IgG (Fig. 4B). In addition, strong positive reactions were observed in epithelial cells of visceral peritoneum and bileduct in sections of liver (Fig. 4C), and a weak positive reaction was observed in hepatocytes, endothelial cells, and smooth muscle cells in sections of liver (data not shown). Finally, strong positive reactions were detected in epithelial cells and mucous glands in sections of gallbladder (data not shown), and weak positive reactions were found in endothelial cells, vascular smooth muscle
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FIG. 4. Immunohistochemical staining of mammary gland, cow liver, and gallbladder. Native polyclonal rabbit anti-bAng IgG was used as the first antibody, and detection was carried out by biotin conjugated goat anti-rabbit IgG, horseradish peroxidase conjugated avidin and 3,3*-diaminobenzidine as a chromatogeneous substrate. (A) Strong positive immunohistochemical reactions were detected in alveolar cells and the secretion of alveolar cells (F) in sections of mammary gland. (B) No immunohistochemical reactions were detected when the sections of mammary gland were stained without pre-incubation with rabbit anti-bAng IgG. (C) Strong positive immunohistochemical reactions were detected in epithelial cells of visceral peritoneum (F) in sections of cow liver. (D) Weak positive reactions were found in endothelial cells (F) and vascular smooth muscle cells (*) in sections of gallbladder.
cells, smooth muscle cells, and fibroblast cells in sections of gallbladder (Fig. 4D). DISCUSSION The sandwich ELISA developed in this study detects bAng in samples which contain significant amounts of RNase(s) since the polyclonal antibody used has no cross-reactivity with RNase A. In contrast, using the placental ribonuclease inhibitor (PRI) binding assay [18] that detects any molecule which bind PRI, assays of unfractionated bovine serum and milk would be uninformative since other PRI-binding proteins in the pancreatic RNase superfamily would also be detected. The sandwich ELISA for bAng established in this study is highly sensitive since the lower limit of sensitivity for the assay was found to be 0.1 ng/mL bAng. This immunological assay is 12- to 40-fold more sensitive than the PRI binding assay [18] and the CAM assay [1]. It is worthy to note that the sensitivity of the PRI binding assay is 8- to 80-fold higher than that of other assays for angiogenin based on cleavage of rRNA [16– 17] or inhibition of protein translation [16]. Recently,
Bla¨ser et al. [21] reported a highly sensitive immunoenzymometric assay for the determination of human angiogenin with 0.05 ng/mL of the lower limit of sensitivity. We have used the sandwich ELISA to measure bAng in bovine serum and milk. Previous studies [3] reported the isolation of bAng from bovine plasma with a yield of 30–80 ng/mL. In addition, Spik et al. [6] reported the isolation of bAng from bovine milk with a yield of 500 ng/mL. Thus, the present studies suggest that the actual content of this protein is higher than the amount isolated. We have used the sandwich ELISA to detect bAng in fractionated samples (CM2 fractions) as shown in Fig. 3. These results demonstrate that the sandwich ELISA for bAng could be used to monitor the purification of bAng from cow milk [19]. In this study, the tissue distribution of angiogenin was assessed by the first immunohistochemical analysis using the polyclonal rabbit anti-bAng IgG. Various organs and tissues of cow were examined (Fig. 4). Previously, expression of angiogenin mRNA was found in tumor cells as well as in normal epithelial, fibroblast and blood cells [7]. In addition, no increase in angiogenin mRNA expression is detected in SV40 trans-
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formed lung fibroblasts versus normal lung fibroblasts. Recently, Moenner et al. [22] investigated the expression of human angiogenin by various human cells in culture, and found that tumor epithelial cells, vascular endothelial cells, vascular muscle cells, and fibroblasts secreted angiogenin. In addition, Shimoyama et al. [10] investigated the relationship between the clinicopathological parameters of pancreatic cancer patients and the degree of human angiogenin mRNA expression, and reported that increased angiogenin mRNA expression was found in pancreatic cancer patients. Finally, Burgmann et al. [23] determined serum angiogenin concentrations in patients with peripheral arterial occlusive disease using ELISA, and found that serum angiogenin concentrations in patients with stage IV peripheral arterial occlusive disease are higher than normal subjects and patients without the disease. The bovine angiogenin used in this study was purified from cow milk and this protein was also found in calf serum but the origin of its synthesis is unknown. In this study, we found that bovine angiogenin protein is most abundant in the alveolar cells and excretory ducts of the mammary gland. These results account for the existence of bovine angiogenin in cow milk. In addition, we found that bovine angiogenin protein is most abundant in the epithelial cells and bile-duct of cow liver, and epithelial cells and mucous gland of gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis. In conclusion, various immunological assays developed in this study provided a useful means for detection, quantitation, and localization of angiogenin, and these assays should provide a means to elucidate the biological mechanism of action of angiogenin in normal physiological and pathological states. ACKNOWLEDGMENTS We thank Dr. Young-Soo Kim (Chungbuk National University) for help in production of polyclonal rabbit antisera to bovine angiogenin and Mr. Seung-Bum Paik and Mr. Byung-Yoon Whang for early purification of bovine angiogenin from cow milk. This work was supported by the Basic Science Research Institute Program, Ministry of Education, the Republic of Korea, 1994 (Project BSRI-94-3434) and in part by a grant from Ministry of Science and Technology, the Republic of Korea, 1995.
REFERENCES 1. Fett, J. W., Strydom, D. J., Lobb, R. R., Alderman, E. M., Bethune, J. L., Riordan, J. F., and Vallee, B. L. (1985) Biochemistry 24, 5480–5486. 2. Shapiro, R., Strydom, D. J., Olson, K. A., and Vallee, B. L. (1987) Biochemistry 26, 5141–5146. 3. Bond, M. D., and Vallee, B. L. (1988) Biochemistry 27, 6282– 6287. 4. Bond, M. D., and Strydom, D. J. (1989) Biochemistry 28, 6110– 6113. 5. Maes, P., Damart, D., Rommens, C., Montreuil, J., Spik, G., and Tartar, A. (1988) FEBS Lett. 241, 41–45. 6. Spik, G, Tartar, A., and Montreuil, J. (19-11-1987) International extension of french patent no 8715984. 7. Rybak, S. M., Fett, J. W., Yao, Q.-Z., and Vallee, B. L. (1987) Biochem. Biophys. Res. Commun. 146, 1240–1248. 8. Weiner, H. L., Weiner, L. H., and Swain, J. L. (1987) Science 237, 280–282. 9. Li, D., Bell, J., Brown, A., and Berry, C. L. (1994) J. Pathol. 172(2), 171–175. 10. Shimoyama, S., Gansauge, F., Gansauge, S., Negri, G., Oohara, T., and Beger, H. G. (1996) Cancer Res. 56, 2703–2706. 11. Hallahan, T. W., Shapiro, R., and Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222–2226. 12. Hallahan, T. W., Shapiro, R., Strydom, D. J., and Vallee, B. L. (1992) Biochemistry 31, 8022–8029. 13. Moroianu, J., and Riordan, J. F. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1677–1681. 14. Acharya, K. R., Shapiro, R., Riordan, J. F., and Vallee, B. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2949–2953. 15. Acharya, K. R., Shapiro, R., Allen. S. C., Riordan, J. F., and Vallee, B. L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2915–2919. 16. St. Clair, D. K., Rybak, S. M., Riodan, J. F., and Vallee, B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8330–8334. 17. Shapiro, R., Riordan, J. F., and Vallee, B. L. (1986) Biochemistry 25, 3527–3532. 18. Bond, M. D. (1988) Anal. Biochem. 173, 166–173. 19. Chang, S.-I., Paik, S.-B., So, S.-H., and Ahn, B.-C. (1996) J. Biochem. Mol. Biol. 29, 353–358. 20. Laemmli, U. K. (1970) Nature 229, 680–685. 21. Bla¨ser, J., Triebel, S., Kopp, C., and Tschesche, H. (1993) Eur. J. Clin. Chem. Biochem. 31, 513–516. 22. Moenner, M., Gusse, M., Hatzi, E., and Badet, J. (1994) Eur. J. Biochem. 226, 483–490. 23. Burgmann, H., Hollenstein, U., Maca, T., Zedwitz-Liebenstein, K., Thalhammer, F., Koppensteiner, R., Ehringer, H., and Graninger, W. (1996) J. Clin. Pathol. 49, 508–510.
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232, 323–327 (1997)
RC976280
Detection, Quantitation, and Localization of Bovine Angiogenin by Immunological Assays Soo-Ik Chang,*,1 Goo-Bo Jeong,† Seung-Ho Park,* Byung-Cheol Ahn,* Jung-Do Choi,* Quae Chae,* Sung Keon Namgoong,‡ and Soo-Il Chung§ *Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea; †Department of Anatomy, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea; ‡Department of Chemistry, Seoul Woman’s University, Seoul 139-774, Korea; and §Mogam Biotechnology Research Institute, Yongin-Kun 441-910, Korea
Received February 3, 1997
Bovine angiogenin (bAng) is a potent blood vessel inducing protein found in bovine serum and milk. Antisera have been raised against bAng. Western blot analysis for bAng indicated that the polyclonal antibody recognized bAng specifically, and no cross-reactivity with bovine RNase A, a protein homologous to bAng, was observed. The sandwich enzyme-linked immunosorbent assay for bAng was sensitive to 10 pg of bAng, and this assay was able to quantitate bAng in bovine serum (100–180 ng/ mL) and milk (4–8 mg/mL). Strong positive immunohistochemical reactions were detected in alveolar cells, the secretion of alveolar cells and excretory ducts in sections of cow mammary gland, epithelial cells of visceral peritoneum and bile-duct in sections of cow liver, and epithelial cells and mucous glands in sections of cow gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis. q 1997 Academic Press
Angiogenin is a potent blood vessel inducing protein originally purified from the conditioned media of cultured human colon adenocarcinoma cells (HT-29) [1]. It was later detected and purified from normal human serum [2], and bovine serum [3–4] and milk [5–6], and its mRNA is expressed in both normal and tumor cells [7–10]. The mechanism of action of angiogenin on angiogenesis is complex, and a dual-site model for the action of angiogenin has been suggested [11–12]. In addition, it has been suggested that nuclear translocation of angio1 To whom correspondence should be addressed. Fax: 82-431-672306. E-mail: [email protected]. Abbreviations used: hAng, human angiogenin; bAng, bovine angiogenin; RNase A, bovine pancreatic ribonuclease A; CM, carboxymethyl; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PRI, placental ribonuclease inhibitor.
genin is a critical step in the process of angiogenesis [13]. In order to elucidate its biological mechanism, further studies of angiogenin on its localization in various organs and tissues as well as understanding the relationship between structure and function are needed. It has been reported that some enzymatic and biological activities of bovine angiogenin (bAng) are identical with those of human angiogenin (hAng) [3]. bAng has 64% sequence identity to hAng [4–5]. Recently, the crystal structure of bAng has been determined and is closely similar to that of hAng [14–15]. Bovine milk is a readily accessible source of bAng, and it is easier and more economical to obtain bAng than hAng. Therefore, bAng could be used as an alternative material for further studies. Various assays for angiogenin have been developed since its first isolation using the chicken embryo chorioallantoic membrane (CAM) assay which monitors new blood vessel growth in vivo. Among these are assays based on i) inhibition of protein synthesis or degradation of reticulocyte RNA [16], ii) cleavage of 28 and 18S rRNA or tRNA [16–17], and iii) the capacity of angiogenin to bind placental ribonuclease inhibitor [18]. The routine detection and sensitive quantitation of angiogenin is necessary to elucidate the mechanism of action of this protein. The purpose of this study is to develop simple and sensitive immunoassays that could be used not only to detect and quantitate angiogenin, but which could also be used to localize angiogenin in tumor and normal organs and tissues. We report here sensitive Western blot analysis and sandwich enzyme-linked immunosorbent assay (ELISA) for bAng, which showed no crossreactivity with bovine pancreatic ribonuclease A (RNase A), a protein homologous to bAng, and thus provides a means of detecting and quantitating angio-
323
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genin in both unfractionated samples and chromatographic fractions which contain RNases [19]. We also report the first immunohistochemical localization of angiogenin in various organs and tissues. Rybak et al. [7] reported the distribution of angiogenin transcripts in human cells representative of different tumor and normal phenotypes by using both human cDNAs for angiogenin and the corresponding RNA antisense probes. The presence of angiogenin transcripts, however, does not ensure that the protein is synthesized, and thus it is necessary to further examine the distribution of angiogenin itself in tumor and normal cells. MATERIALS AND METHODS Materials. An immuno-blot assay kit and prestained molecular weight standards were obtained from Bio-Rad Laboratories. An enhanced chemiluminescence Western blot analysis kit (RPN 2108) and biotinylated molecular weight markers (RPN 2107) were from Amersham. NHS-LC-biotin, o-phenylenediamine, and streptavidin horseradish peroxidase were from Pierce Chemical Co.. Bovine pancreatic ribonuclease A, bovine serum albumin (Fr.V), Freund’s adjuvants (complete and incomplete) were from Sigma Chemical Co. Fetal calf serum was from GIBCO Co.. bAng was purified from milk as described [3]. All other chemicals used were of analytical grade. Immunization procedure. Rabbits were injected subcutaneously with 100 mg of bAng emulsified in Freund’s complete adjuvant. Booster injections of bAng (100 mg) in Freund’s incomplete adjuvant were given at 10-day intervals. After the third immunization, blood was collected and the antiserum was isolated. The IgG fraction of anti-bAng antisera was purified by protein A-Sepharose chromatography. A portion of the rabbit anti-bAng IgG was biotinylated using NHS-LC-biotin (Pierce) according to the manufacturer’s protocol. Biotinylated anti-bAng IgG was separated from unbound biotin using a Centricon 30 microconcentrator (Amicon). Western blot analysis. SDS-PAGE was performed under reducing conditions by using 15% gels as described by Laemmli [20]. Proteins were transferred electophoretically to 0.45-mm nitrocellulose membranes by using a Hoeffer TE22 mini transphor electrophoresis unit at a constant current of 200 mA for 2 h. Following transfer, sheets were incubated for 1 h with 20 mM Tris, 0.5 M NaCl, pH 7.5 (TBS) containing 3% gelatin to prevent nonspecific adsorption. Sheets were then washed with TBS containing 0.05% Tween 20 (TBS/Tween) and incubated for overnight at 47C with rabbit antiserum (dilution 1/200 in TBS containing 1% gelatin) raised against bAng as described above. In other experiments, the IgG fraction of anti-bAng antisera was used instead of the rabbit antiserum itself. After being washed with TBS/Tween, sheets were incubated for 2 h at room temperature with alkaline phosphatase conjugated goat anti-rabbit IgG (diluted 1/300 in TBS containing 1% gelatin). The sheets were washed with TBS/Tween and later with TBS. The proteins recognized by the primary antiserum were then detected by addition of color reagents A and B (Bio-Rad Laboratories). In other experiments, bAng was detected by enhanced chemiluminescence (ECL). Sandwich ELISA. Microplates were coated with 100 mL per well of anti-bAng IgG diluted to 24.2 mg/mL in 50 mM carbonate-bicarbonate buffer (pH 9.6) and incubated for 3 h at 377C. The plates were then washed three times with PBS containing 0.05% Tween-20 (PBS/ Tween-20). The plates were blocked with 200 mL per well of 1% BSA in PBS for 1 h at 377C. After the plates were washed three times with PBS/Tween-20, 100 mL per well of bAng standards and samples which had been diluted with 0.1% BSA in PBS were added to the
plates and incubated for 60 min at 377C. The plates were then washed three times with PBS/Tween-20, 100 mL per well of biotinylated rabbit anti-bAng IgG diluted to 5 mg/mL in 0.1% BSA in PBS was added, and the plates were incubated at 377C for 1 h, and again the plates were washed three times with PBS/Tween-20. 100 mL per well of Streptavidin-horseradish peroxidase diluted to 1 mg/mL in PBS/ Tween-20 were added, and the plates were incubated for 30 min at 377C. The plates were washed, and 100 mL per well of substrate solution (0.1% O-phenylenediamine, 0.02% H2O2 , 50 mM citrate, and 100 mM phosphate buffer, pH 5.0) was added. Color development was allowed to proceed for 15 min at room temperature and was stopped by adding 100 mL per well of 2 M H2SO4 . The absorbance was then measured at 492 nm on a microplate reader (Titertek Multiscan PLUS, Labsystems), and the concentration of bAng was calculated based on the standard curve of bAng. Immunohistochemistry. Tissues of cow liver and mammary gland freshly obtained surgically were directly fixed by treatment with phosphate-buffered saline (PBS) containing 4% paraformaldehyde overnight at 377C. The specimens were dehydrated through ethanol and xylene, and were embedded with paraffin until used. After cutting the formalin-fixed paraffin-embedded material at 4–5 mm, sections were de-waxed thoroughly in xylene and ethanol. The specimens were washed with PBS, blocked with 0.4% normal goat serum for 30 min, and then incubated for 1 h at 377C with PBS containing rabbit anti-bovine angiogenin IgG (dilution 1/200). The specimens were washed three times with PBS, and then incubated for 1 h at room temperature with biotinylated goat anti-rabbit IgG. The specimens were washed three times with PBS, and then incubated for 30 min at room temperature with avidin conjugated horseradish peroxidase. Localization of bAng was visualized by incubation of the slide for 3–5 min with a freshly prepared solution of 0.1% 3,3 *-diaminobenzidine (DAB) in PBS containing 0.05% hydrogen peroxide. The specimens were counterstained with Mayer’s hematoxylin. To confirm the specificity of the immunohistochemical signal, the specimens were incubated with preimmune rabbit IgG or without rabbit antibovine angiogenin IgG, and then stained as described above.
RESULTS Production of antisera to bAng. Rabbit anti-bAng antiserum was obtained by the immunization procedure described in MATERIALS AND METHODS. Ouchterlony double diffusion analysis with bAng and RNase A, a protein homologous to bAng, was performed with an agarose gel. Visible precipitates were formed with the antiserum and bAng but not with RNase A (data not shown). The same antiserum was used for all experiments carried out in this study. Western blot analysis. The specificity of binding of polyclonal anti-bAng antibodies to bAng was further investigated using Western blot analysis. As shown in Fig. 1, the polyclonal antibody recognized a single polypeptide band at 16 500Da, which corresponds to the molecular weight of bAng by SDS-PAGE [3], and no cross-reactivity with bovine RNase A was observed. The sensitivity of detection of bAng in this system is 50 ng (data not shown). Sandwich ELISA. A representative standard curve for bAng is shown in Fig. 2. The lower limit of sensitivity of the assay was 0.1 ng/mL. RNase A showed no reactivity even at a concentration of 1000 ng/mL in
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FIG. 1. Cross-reactivity of polyclonal rabbit anti-bovine angiogenin antibodies with bovine angiogenin and homologous bovine RNase A. Bovine angiogenin (1 mg) or bovine RNase A (1 mg) was electrophoresed on 15% polyacrylamide gels containing SDS followed by transfer to nitrocellulose sheets. Sheets were then reacted with rabbit antiserum (dilution 1/200) to bovine angiogenin, followed by treatment with alkaline phosphatase conjugated goat anti-rabbit IgG and the appropriate chromatogenous substrate. (A) polyacrylamide gel stained with coomassie blue. (B) Westen blot analysis of the same gel. (Lane 1) Bovine RNase A; (lane 2) Prestained protein molecular mass markers (Bio-Rad); (lane 3) Bovine angiogenin.
the ELISA (data not shown). We applied this sandwich ELISA method for the estimation of bAng levels in bovine serum and milk. The content of bAng in adult calf serum, fetal calf serum, and cow milk is 0.18, 0.10 and 3.8–7.5 mg/mL, respectively. Next, we used the immunological assay to detect bovine milk angiogenin in column fractions from the CM-cellulose chromatography step during isolation of the protein [3]. Bovine milk is applied to CM-52 cation-exchange resin, and the bound protein is then eluted with 1 M NaCl and designated CM2 (Fig. 3A). As shown in Fig. 3A, bAng was detected with fractions 4–7. The presence of bAng in these fractions was confirmed by Western blot analysis (Fig. 3B). Immunohistochemical localization of bAng. We carried out immunohistochemical localization of the bAng in
FIG. 2. Standard curve of the sandwich ELISA for bAng. Native and biotin-conjugated anti-bAng rabbit IgG were used as the catching and developing antibody, respectively. Detection was carried out by horseradish peroxidase conjugated streptavidin and appropriate chromatogeneous substrate as described under Materials and Methods. Increasing amounts of bovine angiogenin expressed in mg of protein/L are plotted against optical density at 492 nm. Each point indicates the mean of duplicate assays.
FIG. 3. CM-cellulose chromatography of cow milk, and sandwich ELISA and Western blot analysis of the CM2 fractions. (A) 5 mL of cow milk was centrifuged at 15600g, diluted with 10 mL of 50 mM sodium phosphate (pH 6.6) and applied to CM-cellulose chromatography (1 1 5 cm). Elution was accomplished with 1 M NaCl in 10 mM Tris, pH 8.0. Each fraction contains 200 mL. Sandwich ELISA was used to detect bovine angiogenin in the fractions as described under Materials and Methods. (B) Fractions (designated as CM2) were electrophoresed on 15% polyacrylamide gels containing SDS followed by transfer to nitrocellulose sheets. Sheets were then reacted with rabbit anti-bovine angiogenin IgG, followed by treatment with horseradish peroxidase conjugated donkey anti-rabbit IgG and the ECL substrate (Amersham). ECL was detected for 2 min. (Lane 1) 0.2 mg of purified bovine angiogenin; (lane 2) biotinylated molecular mass markers (Amersham); (lanes 3–9) CM2 fraction 2–8 from 5 mL of cow milk (see Fig. 3A). The positions of the molecular-mass markers (kDa) are also shown. Arrow indicates the 16.5 kDa band.
the sections of various organs and tissues of cow. Strong positive reactions were detected in alveolar cells and the secretion of alveolar cells (Fig. 4A) as well as excretory ducts in sections of mammary gland, and weak positive reactions were observed in endothelial cells, smooth muscle cells, fibroblast cells and myoepithelial cells in sections of mammary gland (data not shown). These immunohistochemical signals, however, were not detected when the specimens were stained with preimmune rabbit IgG or without pre-incubation with rabbit anti-bAng IgG (Fig. 4B). In addition, strong positive reactions were observed in epithelial cells of visceral peritoneum and bileduct in sections of liver (Fig. 4C), and a weak positive reaction was observed in hepatocytes, endothelial cells, and smooth muscle cells in sections of liver (data not shown). Finally, strong positive reactions were detected in epithelial cells and mucous glands in sections of gallbladder (data not shown), and weak positive reactions were found in endothelial cells, vascular smooth muscle
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FIG. 4. Immunohistochemical staining of mammary gland, cow liver, and gallbladder. Native polyclonal rabbit anti-bAng IgG was used as the first antibody, and detection was carried out by biotin conjugated goat anti-rabbit IgG, horseradish peroxidase conjugated avidin and 3,3*-diaminobenzidine as a chromatogeneous substrate. (A) Strong positive immunohistochemical reactions were detected in alveolar cells and the secretion of alveolar cells (F) in sections of mammary gland. (B) No immunohistochemical reactions were detected when the sections of mammary gland were stained without pre-incubation with rabbit anti-bAng IgG. (C) Strong positive immunohistochemical reactions were detected in epithelial cells of visceral peritoneum (F) in sections of cow liver. (D) Weak positive reactions were found in endothelial cells (F) and vascular smooth muscle cells (*) in sections of gallbladder.
cells, smooth muscle cells, and fibroblast cells in sections of gallbladder (Fig. 4D). DISCUSSION The sandwich ELISA developed in this study detects bAng in samples which contain significant amounts of RNase(s) since the polyclonal antibody used has no cross-reactivity with RNase A. In contrast, using the placental ribonuclease inhibitor (PRI) binding assay [18] that detects any molecule which bind PRI, assays of unfractionated bovine serum and milk would be uninformative since other PRI-binding proteins in the pancreatic RNase superfamily would also be detected. The sandwich ELISA for bAng established in this study is highly sensitive since the lower limit of sensitivity for the assay was found to be 0.1 ng/mL bAng. This immunological assay is 12- to 40-fold more sensitive than the PRI binding assay [18] and the CAM assay [1]. It is worthy to note that the sensitivity of the PRI binding assay is 8- to 80-fold higher than that of other assays for angiogenin based on cleavage of rRNA [16– 17] or inhibition of protein translation [16]. Recently,
Bla¨ser et al. [21] reported a highly sensitive immunoenzymometric assay for the determination of human angiogenin with 0.05 ng/mL of the lower limit of sensitivity. We have used the sandwich ELISA to measure bAng in bovine serum and milk. Previous studies [3] reported the isolation of bAng from bovine plasma with a yield of 30–80 ng/mL. In addition, Spik et al. [6] reported the isolation of bAng from bovine milk with a yield of 500 ng/mL. Thus, the present studies suggest that the actual content of this protein is higher than the amount isolated. We have used the sandwich ELISA to detect bAng in fractionated samples (CM2 fractions) as shown in Fig. 3. These results demonstrate that the sandwich ELISA for bAng could be used to monitor the purification of bAng from cow milk [19]. In this study, the tissue distribution of angiogenin was assessed by the first immunohistochemical analysis using the polyclonal rabbit anti-bAng IgG. Various organs and tissues of cow were examined (Fig. 4). Previously, expression of angiogenin mRNA was found in tumor cells as well as in normal epithelial, fibroblast and blood cells [7]. In addition, no increase in angiogenin mRNA expression is detected in SV40 trans-
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formed lung fibroblasts versus normal lung fibroblasts. Recently, Moenner et al. [22] investigated the expression of human angiogenin by various human cells in culture, and found that tumor epithelial cells, vascular endothelial cells, vascular muscle cells, and fibroblasts secreted angiogenin. In addition, Shimoyama et al. [10] investigated the relationship between the clinicopathological parameters of pancreatic cancer patients and the degree of human angiogenin mRNA expression, and reported that increased angiogenin mRNA expression was found in pancreatic cancer patients. Finally, Burgmann et al. [23] determined serum angiogenin concentrations in patients with peripheral arterial occlusive disease using ELISA, and found that serum angiogenin concentrations in patients with stage IV peripheral arterial occlusive disease are higher than normal subjects and patients without the disease. The bovine angiogenin used in this study was purified from cow milk and this protein was also found in calf serum but the origin of its synthesis is unknown. In this study, we found that bovine angiogenin protein is most abundant in the alveolar cells and excretory ducts of the mammary gland. These results account for the existence of bovine angiogenin in cow milk. In addition, we found that bovine angiogenin protein is most abundant in the epithelial cells and bile-duct of cow liver, and epithelial cells and mucous gland of gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis. In conclusion, various immunological assays developed in this study provided a useful means for detection, quantitation, and localization of angiogenin, and these assays should provide a means to elucidate the biological mechanism of action of angiogenin in normal physiological and pathological states. ACKNOWLEDGMENTS We thank Dr. Young-Soo Kim (Chungbuk National University) for help in production of polyclonal rabbit antisera to bovine angiogenin and Mr. Seung-Bum Paik and Mr. Byung-Yoon Whang for early purification of bovine angiogenin from cow milk. This work was supported by the Basic Science Research Institute Program, Ministry of Education, the Republic of Korea, 1994 (Project BSRI-94-3434) and in part by a grant from Ministry of Science and Technology, the Republic of Korea, 1995.
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