/ . Biochem., 78, 1365-1367 (1975)
PRELIMINARY COMMUNICATION
Degradation of Rod Outer Segment Proteins by Cathepsin D
Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Miyagi 980 Received for publication, August 26, 1975
The degradation of proteins of the rod outer segment (ROS) fraction by partially purified cathepsin D [EC 3. 4. 23. 5] from the retinal pigment epithelium was studied. The ROS fraction, prepared from bovine eyes by sucrose density gradient centrifugation, had little cathepsin D activity. Partially purified cathepsin D, obtained from a crude extract of bovine retinal pigment epithelium using bovine serum albumin as a substrate, hydrolyzed the proteins of the ROS fraction. The rate of degradation of ROS proteins was proportional to both the enzyme concentration and the incubation time. With ROS proteins as substrate, the optimal pH of cathepsin D was about 3.5. The degradation of ROS proteins was inhibited by pepstatin.
Early autoradiographic studies ( / ) suggested that the photoreceptor outer segment is continuously renewed at its base, with concomitant removal of material at its apex. Recent studies (2) showed that the pigment epithelial cells must have a highly developed phago-lysosomal system capable of continuous digestion of large amounts of the rod outer segment. Little is known about the protease(s) in the lysosomal system which degrades the rhodopsin molecule. In previous studies {3,4) using bovine serum albumin as substrate, we found that among bovine eye tissues the retinal pigment epithelial cells have the highest specific activity of cathepsin D [EC 3.4.23.5], and we purified the enzyme about 25-fold from a crude extract of retinal pigment epithelial cells by acid treatment, ammonium sulfate fractionation, and Sephadex G-200 column chromatography. In the present work we studied the degradation of proteins of the rod outer segment by cathepsin D in vitro.
Vol. 78, No. 6, 1975
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1365/830062 by University of Otago user on 19 January 2019
Seiji HAYASAKA, Satoshi HARA, and Katsuyoshi MIZUNO
The rod outer segment fraction was prepared by the method of Heller ( 5 ) and purified further by the method of Pannbacker (6). Bovine eyes obtained at a slaughter house were kept at 4° until use. The neurosensory retina was gently removed in ice-cold 0.25 M sucrose, and homogenized using a Teflon homogenizer. The homogenate was centrifuged at 10,000 xg for 30 min and the precipitate was suspended in 1.02 M sucrose and centrifuged at 10,000X0 for 45 min. The resulting supernatant was diluted to 0.25 M sucrose. These centrifugation procedures were repeated 3 times. Then the suspension was layered over a continuous sucrose density gradient (24 to 41%), and centrifuged at 30,000 rpm for 3 hr. The main band was found by electron microscopy to contain the rod outer segments. In this work, the purified fraction of rod outer segments was used as substrate instead of their solubilized proteins, because membranestructures of the rod outer segments engulfed
1365
1366
PRELIMINARY COMMUNICATION
TABLE I. Degradations of bovine serum albumin and the rod outer segment fraction by cathepsin D on incubation at 37° for 60 min at pH 3.5. The averages of values in triplicate experiments are shown. Addition ROS fraction
(2.2 mg)
(2.2 mg)
Activity
Cathepsin D Crude (1.0 mg)
Partially purified (0.04 mg)
(fig tyrosine released)
+
0.0
+
0.0
+
0.1
+ + +
0.0
+
0.1
+
3.2
+
+
3.1
+
3.1 -f-
+ in phagolysosomes were often observed by electron microscopy. Crude and partially purified cathepsin D were prepared frombovine retinal pigment epithelium as described previously (4). Cathepsin D [EC 3.4.23.5] activity was •determined as follows; 0.1 to 0.3 ml of the enzyme solution was incubated in a total volume of 1 ml for 1 hr at 37°C in 0.1 M citrate buffer at pH 3.5 with bovine serum albumin •or the rod outer segment fraction as substrate. The reaction was stopped by adding 1 ml of 10% trichloroacetic acid, and the tyrosine released was determined spectrophotometrically "by the method of Ceriotti and Spandrio (7). The protein content was determined by the method of Lowry et al. (8). Crude and partially purified cathepsin D "hydrolyzed both bovine serum albumin and rod outer segment proteins (Table I). These resuts suggest that cathepsin D in the retinal pigment epithelium may split proteins of the rod outer segment non-specifically. When the rod outer •segment fraction was used as substrate, the •enzyme activity in the Sephadex G-200 fraction was proportional to both the enzyme concentration and the incubation time, and the optimal pH was about 3.5. The degradation of rod outer segment proteins by cathepsin D was strongly inhibited by pepstatin (Fig. 1). Pep-
2.9
100O
0
001
002
003
0.04
Concentration of pepstatin
005 (jjg/ml)
Fig. 1. Effect of pepstatin on cathepsin D activity. 40 fig protein of partially purified cathepsin D were incubated at 37° for 60 min at pH 3.5 with 2.2 mg protein of ROS fraction and various amounts of pepstatin. Methanol was added at a final concentration of 1% to each incubation mixture, because pepstatin is soluble in methanol.
statin, which was first isolated from Streptomyces and found to be a pepsin inhibitor by Umezawa (9), is known to inhibit pepsin [EC 3. 4. 23.1] and cathepsin D in animals. The catheptic endopeptidases cathepsin D and E are believed to be the most important enzymes in turnover and degradation of general proteins in lysosomes of animal tissues, and / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1365/830062 by University of Otago user on 19 January 2019
Bovine serum albumin
DEGRADATION OF ROD OUTER SEGMENT PROTEINS
Vol. 78, No. 6, 1975
REFERENCES 1. Young, R.W. & Bok, D. (1969) / . Cell Biol. 42, 392-403 2. Feeney, L. (1973) Invest. Ophthalmol. 12, 635-638 3. Hayasaka, S., Hara, S., & Mizuno, K. (1975) Exp. Eye Res. 21, 307-313 4. Hayasaka, S., Hara, S., & Mizuno, K. (1975) Invest. Ophthalmol. 14, 617-620 5. Heller, J. (1968) Biochemistry 7, 2906-2913 6. Pannbacker, R.G. (1974) Invest. Ophthalmol. 13, 535-538 7. Ceriotti, G. & Spandrio, L. (1957) Biochem. J. 66, 607-610 8. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 9. Umezawa, H. (1972) in Enzyme Inhibitors of Microbial Origin (Umezawa, H.) p. 34, University of Tokyo Press, Tokyo, 1972 10. Hollyfield, J.G. & Ward, A. (1974) Invest. Ophthalmol. 13, 1016-1023 11. Burden, E.M., Yates, CM., & Reading, H.W. (1971) Exp. Eye Res. 12, 159-165 12. Herron, W.L., Riegel, B.W., & Myers, D.E. (1969) Invest. Ophthalmol. 8, 595-604
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1365/830062 by University of Otago user on 19 January 2019
the present results suggest that cathepsin D in the retinal pigment epithelium may split rod outer segment proteins non-specifically. This is supported by the electron microscopic findings of Hollyfield and Ward (70) that pigment •epithelial cells phagocytose latex beads introduced into the interphotoreceptor space showing that the phagocytic mechanism of the retinal pigment epithelium is not specific for outer segment disks. Pigmentary retinal dystrophy must be considered in relation to lysosomal enzymes. Burden et al. (11) and Herron et al. (12) proposed that retinal dystrophies are due to some abnormality of the photoreceptor renewal-removal mechanism of the pigment epithelium. Therefore, cathepsin D may participate not only in the mechanism of removal of disks in the normal retina, but also in the pathogenesis of retinal dysrophy. In other words, genetic anomalies resulting in either decrease or increase of the enzyme may cause pigmentary retinal dystrophy in man.
1367
PRELIMINARY COMMUNICATION
Degradation of Rod Outer Segment Proteins by Cathepsin D
Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Miyagi 980 Received for publication, August 26, 1975
The degradation of proteins of the rod outer segment (ROS) fraction by partially purified cathepsin D [EC 3. 4. 23. 5] from the retinal pigment epithelium was studied. The ROS fraction, prepared from bovine eyes by sucrose density gradient centrifugation, had little cathepsin D activity. Partially purified cathepsin D, obtained from a crude extract of bovine retinal pigment epithelium using bovine serum albumin as a substrate, hydrolyzed the proteins of the ROS fraction. The rate of degradation of ROS proteins was proportional to both the enzyme concentration and the incubation time. With ROS proteins as substrate, the optimal pH of cathepsin D was about 3.5. The degradation of ROS proteins was inhibited by pepstatin.
Early autoradiographic studies ( / ) suggested that the photoreceptor outer segment is continuously renewed at its base, with concomitant removal of material at its apex. Recent studies (2) showed that the pigment epithelial cells must have a highly developed phago-lysosomal system capable of continuous digestion of large amounts of the rod outer segment. Little is known about the protease(s) in the lysosomal system which degrades the rhodopsin molecule. In previous studies {3,4) using bovine serum albumin as substrate, we found that among bovine eye tissues the retinal pigment epithelial cells have the highest specific activity of cathepsin D [EC 3.4.23.5], and we purified the enzyme about 25-fold from a crude extract of retinal pigment epithelial cells by acid treatment, ammonium sulfate fractionation, and Sephadex G-200 column chromatography. In the present work we studied the degradation of proteins of the rod outer segment by cathepsin D in vitro.
Vol. 78, No. 6, 1975
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1365/830062 by University of Otago user on 19 January 2019
Seiji HAYASAKA, Satoshi HARA, and Katsuyoshi MIZUNO
The rod outer segment fraction was prepared by the method of Heller ( 5 ) and purified further by the method of Pannbacker (6). Bovine eyes obtained at a slaughter house were kept at 4° until use. The neurosensory retina was gently removed in ice-cold 0.25 M sucrose, and homogenized using a Teflon homogenizer. The homogenate was centrifuged at 10,000 xg for 30 min and the precipitate was suspended in 1.02 M sucrose and centrifuged at 10,000X0 for 45 min. The resulting supernatant was diluted to 0.25 M sucrose. These centrifugation procedures were repeated 3 times. Then the suspension was layered over a continuous sucrose density gradient (24 to 41%), and centrifuged at 30,000 rpm for 3 hr. The main band was found by electron microscopy to contain the rod outer segments. In this work, the purified fraction of rod outer segments was used as substrate instead of their solubilized proteins, because membranestructures of the rod outer segments engulfed
1365
1366
PRELIMINARY COMMUNICATION
TABLE I. Degradations of bovine serum albumin and the rod outer segment fraction by cathepsin D on incubation at 37° for 60 min at pH 3.5. The averages of values in triplicate experiments are shown. Addition ROS fraction
(2.2 mg)
(2.2 mg)
Activity
Cathepsin D Crude (1.0 mg)
Partially purified (0.04 mg)
(fig tyrosine released)
+
0.0
+
0.0
+
0.1
+ + +
0.0
+
0.1
+
3.2
+
+
3.1
+
3.1 -f-
+ in phagolysosomes were often observed by electron microscopy. Crude and partially purified cathepsin D were prepared frombovine retinal pigment epithelium as described previously (4). Cathepsin D [EC 3.4.23.5] activity was •determined as follows; 0.1 to 0.3 ml of the enzyme solution was incubated in a total volume of 1 ml for 1 hr at 37°C in 0.1 M citrate buffer at pH 3.5 with bovine serum albumin •or the rod outer segment fraction as substrate. The reaction was stopped by adding 1 ml of 10% trichloroacetic acid, and the tyrosine released was determined spectrophotometrically "by the method of Ceriotti and Spandrio (7). The protein content was determined by the method of Lowry et al. (8). Crude and partially purified cathepsin D "hydrolyzed both bovine serum albumin and rod outer segment proteins (Table I). These resuts suggest that cathepsin D in the retinal pigment epithelium may split proteins of the rod outer segment non-specifically. When the rod outer •segment fraction was used as substrate, the •enzyme activity in the Sephadex G-200 fraction was proportional to both the enzyme concentration and the incubation time, and the optimal pH was about 3.5. The degradation of rod outer segment proteins by cathepsin D was strongly inhibited by pepstatin (Fig. 1). Pep-
2.9
100O
0
001
002
003
0.04
Concentration of pepstatin
005 (jjg/ml)
Fig. 1. Effect of pepstatin on cathepsin D activity. 40 fig protein of partially purified cathepsin D were incubated at 37° for 60 min at pH 3.5 with 2.2 mg protein of ROS fraction and various amounts of pepstatin. Methanol was added at a final concentration of 1% to each incubation mixture, because pepstatin is soluble in methanol.
statin, which was first isolated from Streptomyces and found to be a pepsin inhibitor by Umezawa (9), is known to inhibit pepsin [EC 3. 4. 23.1] and cathepsin D in animals. The catheptic endopeptidases cathepsin D and E are believed to be the most important enzymes in turnover and degradation of general proteins in lysosomes of animal tissues, and / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1365/830062 by University of Otago user on 19 January 2019
Bovine serum albumin
DEGRADATION OF ROD OUTER SEGMENT PROTEINS
Vol. 78, No. 6, 1975
REFERENCES 1. Young, R.W. & Bok, D. (1969) / . Cell Biol. 42, 392-403 2. Feeney, L. (1973) Invest. Ophthalmol. 12, 635-638 3. Hayasaka, S., Hara, S., & Mizuno, K. (1975) Exp. Eye Res. 21, 307-313 4. Hayasaka, S., Hara, S., & Mizuno, K. (1975) Invest. Ophthalmol. 14, 617-620 5. Heller, J. (1968) Biochemistry 7, 2906-2913 6. Pannbacker, R.G. (1974) Invest. Ophthalmol. 13, 535-538 7. Ceriotti, G. & Spandrio, L. (1957) Biochem. J. 66, 607-610 8. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 9. Umezawa, H. (1972) in Enzyme Inhibitors of Microbial Origin (Umezawa, H.) p. 34, University of Tokyo Press, Tokyo, 1972 10. Hollyfield, J.G. & Ward, A. (1974) Invest. Ophthalmol. 13, 1016-1023 11. Burden, E.M., Yates, CM., & Reading, H.W. (1971) Exp. Eye Res. 12, 159-165 12. Herron, W.L., Riegel, B.W., & Myers, D.E. (1969) Invest. Ophthalmol. 8, 595-604
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1365/830062 by University of Otago user on 19 January 2019
the present results suggest that cathepsin D in the retinal pigment epithelium may split rod outer segment proteins non-specifically. This is supported by the electron microscopic findings of Hollyfield and Ward (70) that pigment •epithelial cells phagocytose latex beads introduced into the interphotoreceptor space showing that the phagocytic mechanism of the retinal pigment epithelium is not specific for outer segment disks. Pigmentary retinal dystrophy must be considered in relation to lysosomal enzymes. Burden et al. (11) and Herron et al. (12) proposed that retinal dystrophies are due to some abnormality of the photoreceptor renewal-removal mechanism of the pigment epithelium. Therefore, cathepsin D may participate not only in the mechanism of removal of disks in the normal retina, but also in the pathogenesis of retinal dysrophy. In other words, genetic anomalies resulting in either decrease or increase of the enzyme may cause pigmentary retinal dystrophy in man.
1367
