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Unique properties of cathepsin D purified from alcoholic cirrhotic liver
International Hepatology Communications, 1993; 1: 152-157 © 1993 Elsevier Science Publishers B.V. All rights reserved 0928-4346193l$06.00
152
HEPCOM 00029
Unique properties of cathepsin D purified from alcoholic cirrhotic liver Motoyuki Ohhira, Minoru Ono, Masumi Ohhira, Hitoyoshi Ohta, Akinori Matsumoto, Chihiro Sekiya and Masayoshi Namiki The Department of Internal Medicine III, Asahikawa Medical College, Asahikawa, Japan (Received 30 September 1992; revised version received 15 March 1993; accepted 9 April 1993)
In order to show the possibility of the impaired function of Golgi apparatus in alcoholic cirrhotic liver, we studied the property of sugar chains on purified cathepsin D, which was an aspartic protease in lysosome. Alcoholic cirrhotic liver contained a large amount of cathepsin D which had no binding affinity for concanavalin A. On the other hand, no cathepsin D was detected in concanavalin A-unbound fractions from normal liver, viral cirrhotic liver and hepatoma tissues. In the treatment with exogenous hydrolases to cleave carbohydrate moieties, abnormal cathepsin D purified from alcoholic cirrhotic liver contained altered sugar chains. Since the processing of oligosaccharide moieties on lysosomal hydrolases are performed in Golgi apparatus, the alteration of sugar chains suggests that the abnormal intracellular processing is caused by the dysfunction of Golgi apparatus. Unique cathepsin D described here may support the hypothesis that the impaired function of Golgi apparatus is associated with the mechanism of alcoholic liver injury.
Key words': Lysosomal enzyme; Cathepsin D; Concanavalin A; Asparagine-linked carbohydrate moiety; Posttranslational processing; Liver cirrhosis
Lysosomal hydrolases are glycoproteins which have high-mannose-type c a r b o h y d r a t e moieties [1,2]. The lysosomal hydrolases undergo post-translational processing at c a r b o h y drate chains coupled with targeting to lysosomes via Golgi apparatus. The impairment o f Golgi apparatus function seems to be associated with the mechanism o f alcoholic liver injury [3]. Lysosomal hydrolases are possibly affected by the dysfunction o f Golgi apparatus in the alcoholic liver injury, but no studies are documented. In h u m a n tissues, the modifications o f c a r b o h y d r a t e chains on lysosomal enzymes are demonstrated in various cancers [4-8], but these are only in high-mannose-type oligosaccharides which have high affinity for concanavalin A. In the present study, we demonstrate
Correspondence to." Motoyuki Ohhira, M.D., the Department of Internal Medicine III, Asahikawa Medical College, 4-5-3 Nishikagura, Asahikawa 078, Japan.
CATHEPSIND IN ALCOHOLICCIRRHOTICLIVER
153
that cathepsin D (EC 3.4.23.5), which is a lysosomal aspartic protease, contains altered sugar chains which have no binding affinity for concanavalin A in alcoholic cirrhotic liver.
Materials and Methods
Chemicals Endo-fl-N-acetylglucosaminidase H (endo-H) [9] was purchased from Seikagaku Kogyo (Japan). N-glycanase (NGase) [10] and endo-]~-N-acetylglucosaminidase F (endo-F) [11] were from Genzyme (UK). Other reagents were of analytical grade. Human liver tissues Cirrhotic liver tissue was obtained at autopsy from a patient with alcoholic abuse. Anti-C100-3 antibody and HBs antigen, which were the markers of hepatitis virus infection, were not detected in the serum of the patient. Histological features of the liver were consistent with alcoholic liver cirrhosis. Normal liver of a patient, who died of acute myocardial infarction, cirrhotic liver of viral hepatitis and hepatoma tissues were also obtained at autopsy. All the tissues were obtained within 5 h after death and stored at -80°C until use. Preparation of cathepsin D from human liver tissues Cathepsin D was purified according to the method described previously [4,5] with some modifications. All procedures were carried at 4 ° C. Tissue homogenate was centrifuged at 5000 x g for 30 rain. Resultant supernatant was subjected to ammonium sulfate precipitation and the obtained pellet was dialyzed against 5 mM sodium acetate buffer (pH 5.2), containing 0.1 M NaC1 (buffer A). The solution was applied to concanavalin A-Sepharose column (Con A column) previously equilibrated by buffer A. The bound component was removed with 0.5 M ~-methylmannopyranoside in buffer A. Flow-through fractions and bound fractions containing cathepsin D activity were collected. Cathepsin D in these two preparations were purified with pepstatin-Sepharose chromatography and gel permeation chromatography described previously [4,5]. Protein concentration was determined by the method of Lowry et al [12] with bovine serum albumin as a standard. Treatment of cathepsin D with exogenous hydrolases Reaction mixture was 15/11 in volume, and the hydrolysis treatments were performed at 37°C for 16 h. The purified cathepsin D (3 ~g) was incubated with 5 mU of endo-H [9] in 15 mM citrate-phosphate buffer (pH 5.4), containing 10 ~M pepstatin A and 1 mM PMSF (protease inhibitors). In endo-F [11] treatment, 3 ~g of cathepsin was incubated with 1 mU of endo-F in 15 mM Tris-maleate buffer (pH 6.1), containing 30 mM EDTA, 1.25% NP-40 and protease inhibitors. NGase [10] treatment was performed as follow; 3/lg of cathepsin D was hydrolyzed by 250 mU NGase in 300 mM sodium-phosphate buffer (pH 8.6), containing protease inhibitors. The hydrolase-treated cathepsin D was subjected to electrophoresis.
154
M. OHHIRA et al.
Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was performed according to the method of Laemmli [13]. Gels were treated with Coomassie brilliant blue R-250 for protein staining.
Results
Binding profile of cathepsin D activity for Con A column In alcoholic cirrhotic liver, cathepsin D activity was demonstrated in both flow-through fractions and bound fractions on Con A-Sepharose chromatography (Table 1). On the other hand, in normal liver, viral cirrhotic liver and hepatoma tissues, cathepsin D activity was completely bound to the Con A column.
Purified cathepsin D From an alcoholic cirrhotic liver of 500 g, 9 mg cathepsin D in Con A-bound fractions and 14 mg protein in Con A-unbound fractions were purified, respectively (Table 1). The molecular mass of each enzyme was 43 kDa by gel permeation chromatography (data not shown). Specific enzymatic activity [4,5] had a similar level to the activities of the enzymes purified from normal liver, viral cirrhotic liver and hepatoma [4,5] (data not shown). Each enzyme preparation exhibited three major bands: one heavy chain (31 kDa) and two light chains (15 kDa and 14 kDa) were detected on polyacrylamide gel electrophoresis (Fig. 1). The electrophoretic patterns of these enzymes were similar to those purified from other human liver tissues [4,5].
Effects of exogenous hydrolases on cathepsin D from alcoholic cirrhotic liver (Fig. 2) Upon treatment with NGase of two cathepsin D preparations from alcoholic cirrhotic liver, a 31-kDa peptide was completely changed to 29 kDa and two light chains of 14 and 15 kDa were decreased in size to 13 kDa peptide. This result indicated that cathepsin D purified from Con A-unbound fractions also had asparagine-linked sugar moieties and the sugar chains were of a similar size to those from Con A-bound fractions. In both cathepsin D preparations, treatments with endo-H and endo-F showed the same electrophoretic manner. Most of the 31-kDa peptide and all of the 15-kDa peptide were changed to a 29-kDa peptide and a 13-kDa peptide, respectively. A part of the 31-kDa peptide and all of
TABLE 1 Purified cathepsin D from human liver tissues Purified cathepsin D (mg/500 g tissue) Human liver tissue
Con A-bound
Con A-flow through
Alcoholic cirrhotic liver Normal liver Viral cirrhotic liver Hepatoma
9 35 42 49
14 0 0 0
CATHEPSIND IN ALCOHOLICCIRRHOTICLIVER
155
MW 92.5K 66.2K 43.0K 31.OK 21.5K
LI.~ L2-~
14.4K
B
PT
Fig. 1. Electrophoretic pattern of purified cathepsin D from alcoholic cirrhotic liver. Lane B, cathepsin D purified from Con A-bound fractions. Lane PT, cathepsin D from Con A-flow through fractions. H 1, heavy chain of 31 kDa; H2, minor component of deglycosylated H 1; L1, light chain of 15 kDa; L2, light chain of 14 kDa.
the 14-kDa peptide remained unchanged, probably because the sugar chain structures of these peptides were resistant to hydrolysis by endo-H and endo-F. The electrophoretic patterns of deglycosylated cathepsin D preparations purified from normal liver, viral cirrhotic liver and hepatoma showed a similar electrophoretic pattern to that of cathepsin D from alcoholic cirrhotic liver (data not shown).
Discussion
In the present study, we demonstrated that alcoholic cirrhotic liver contained a large amount of unique cathepsin D which lost the binding affinity for Con A column although all peptides of cathepsin D had asparagine-linked sugar chains. In other human liver tissues, we could not detect the cathepsin D activity in Con-A-flow through fractions (Table 1). Since Con A has high affinity for high-mannose type sugar chains, the abnormal property of cathepsin D in alcoholic cirrhotic liver is probably caused by the alteration of carbohydrate moiety on cathepsin D enzyme. In the analysis of cDNA for human cathepsin D, only one site capable of linking with asparagine-linked oligosaccharide chains is recognized in both heavy and light chains [14]. In the treatment of abnormal cathepsin D from alcoholic cirrhotic liver with NGase, which
156
M. OHHIRAet al.
MW ~66k -,43k ~,31k -,21k ~,14k
L3 C
EH
EF
I
NG I
B
E
C
EH
EF
I
NG I
PT
Fig. 2. Effects of exogenous hydrolases on cathepsin D from alcoholic cirrhotic liver. Treated cathepsin D was subjected to polyacrylamidegel electrophoresis. H2 and L3, deglycosylatedheavy and light chains of cathepsin D. Lane C, untreated cathepsin D; EH, endo-H treatment; EF, endo-F treatment; NG, NGase treatment; E, stabilizer proteins in endo-H preparation. MW, molecular weight markers from Bio-Rad. cleaved all types of asparagine-linked oligosaccharide chains [11], both heavy (31 kDa) and light (15 and 14 kDa) chains were decreased in size to 29 kDa and 13 kDa, respectively. The peptides of 29 kDa and 13 kDa seem to be simple peptides which have no asparagine-linked carbohydrate chains, and the decrease of molecular mass with 1-2 kDa means that the peptide seems to have one asparagine-linked oligosaccharide unit. In the treatment with endo-H and endo-F, all of the 15-kDa and most of the 31-kDa peptides were changed to 13 kDa and 29 kDa, respectively, and the results suggested that these peptides contain altered high-mannose- or hybrid-type sugar chains [9,11]. All of the 14-kDa and a residual part of the 31-kDa peptides seem to have hybrid or complex type chains [9,11]. It seems to be essential that lysosomal hydrolases contain high-mannose-type oligosaccharide chains because mannose-6-phosphate residue on high-mannose-type sugar chains acts as a recognition marker for the targeting to lysosome via Golgi apparatus in the normal processing of lysosomal enzymes [1,2]. Con A affinity chromatography is widely used for the purification of lysosomal hydrolases because Con A has high affinity for high-mannose-type carbohydrate chains [6-8]. The lysosomal enzyme which has no binding affinity for Con A column seems to be extremely rare and unique, and we suggest that the processing of carbohydrate moiety on cathepsin D is altered by impaired Golgi function in alcoholic cirrhotic liver. The structure of carbohydrate moiety on cathepsin D and the changes of processing enzymes in Golgi apparatus are not yet clarified, thus further study of these problems in alcoholic liver injury is necessary. The abnormal cathepsin D described here may also represent abnormal function of Golgi apparatus in alcoholic liver injury.
CATHEPSIN D IN ALCOHOLIC CIRRHOTIC LIVER
157
Acknowledgement The authors are indebted to Ms Izumi Okumura for her help with this manuscript.
References 1 Sly WS, Fisher HD. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem 1982;18:67-85. 2 yon Figura K, Hasilik A. Lysosomal enzyme and their receptors. Annu Rev Biochem 1986;55:167-193. 3 Baraona E, Leo MA, Borowsky SA, Lieber CS. Alcoholic hepatomegaly: Accumulation of protein in the liver. Science 1975;190:794-795. 4 Maguchi S, Taniguchi N, Makita A. Elevated activity and increased mannose-6-phosphate in the carbohydrate moiety of cathepsin D from human hepatoma. Cancer Res 1988;48:362-367. 5 0 h h i r a M, Gasa S, Makita A, Sekiya C, Namiki M. Elevated carbohydrate phosphotransferase activity in human hepatoma and phosphorylation of cathepsin D. Br J Cancer 1991;63:905-908. 6 0 n o M, Taniguchi N, Makita A, Fujita M, Sekiya C, Namiki Y. Phosphorylation of betaglucuronidase from human normal liver and hepatoma by cAMP-dependent protein kinase. J Biol Chem 1988;263:5884-5889. 7 Gasa S, Makita A, Kameya T, et al. Elevated activities and properties of arylsulfatase A and B and B-variants in human lung tumors. Cancer Res 1980;40:3804-3809. 8 Fujita M, Taniguchi N, Makita A, Oikawa K. Cancer-associated alteration of beta-glucuronidase in human lung cancer: Elevated activity and increased phosphorylation. Gann 1984;75:508-517. 9 Tarentino AL, Plummer TH Jr, Maley F. The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H. J Biol Chem 1974;249:818-824. 10 Tarentino AL, G6mez CM, Plummer TH Jr. Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry 1985;24:46654671. 11 Elder JH, Alexander S. Endo-beta-N-acetylglucosaminidase F: Endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci 1982;79:45404544. 12 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 13 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;224:149-154. 14 Faust PL, Kornfeld S, Chirgwin JM. Cloning and sequence analysis of eDNA for human cathepsin D. Proc Natl Acad Sei USA 1985;82:49104914.
152
HEPCOM 00029
Unique properties of cathepsin D purified from alcoholic cirrhotic liver Motoyuki Ohhira, Minoru Ono, Masumi Ohhira, Hitoyoshi Ohta, Akinori Matsumoto, Chihiro Sekiya and Masayoshi Namiki The Department of Internal Medicine III, Asahikawa Medical College, Asahikawa, Japan (Received 30 September 1992; revised version received 15 March 1993; accepted 9 April 1993)
In order to show the possibility of the impaired function of Golgi apparatus in alcoholic cirrhotic liver, we studied the property of sugar chains on purified cathepsin D, which was an aspartic protease in lysosome. Alcoholic cirrhotic liver contained a large amount of cathepsin D which had no binding affinity for concanavalin A. On the other hand, no cathepsin D was detected in concanavalin A-unbound fractions from normal liver, viral cirrhotic liver and hepatoma tissues. In the treatment with exogenous hydrolases to cleave carbohydrate moieties, abnormal cathepsin D purified from alcoholic cirrhotic liver contained altered sugar chains. Since the processing of oligosaccharide moieties on lysosomal hydrolases are performed in Golgi apparatus, the alteration of sugar chains suggests that the abnormal intracellular processing is caused by the dysfunction of Golgi apparatus. Unique cathepsin D described here may support the hypothesis that the impaired function of Golgi apparatus is associated with the mechanism of alcoholic liver injury.
Key words': Lysosomal enzyme; Cathepsin D; Concanavalin A; Asparagine-linked carbohydrate moiety; Posttranslational processing; Liver cirrhosis
Lysosomal hydrolases are glycoproteins which have high-mannose-type c a r b o h y d r a t e moieties [1,2]. The lysosomal hydrolases undergo post-translational processing at c a r b o h y drate chains coupled with targeting to lysosomes via Golgi apparatus. The impairment o f Golgi apparatus function seems to be associated with the mechanism o f alcoholic liver injury [3]. Lysosomal hydrolases are possibly affected by the dysfunction o f Golgi apparatus in the alcoholic liver injury, but no studies are documented. In h u m a n tissues, the modifications o f c a r b o h y d r a t e chains on lysosomal enzymes are demonstrated in various cancers [4-8], but these are only in high-mannose-type oligosaccharides which have high affinity for concanavalin A. In the present study, we demonstrate
Correspondence to." Motoyuki Ohhira, M.D., the Department of Internal Medicine III, Asahikawa Medical College, 4-5-3 Nishikagura, Asahikawa 078, Japan.
CATHEPSIND IN ALCOHOLICCIRRHOTICLIVER
153
that cathepsin D (EC 3.4.23.5), which is a lysosomal aspartic protease, contains altered sugar chains which have no binding affinity for concanavalin A in alcoholic cirrhotic liver.
Materials and Methods
Chemicals Endo-fl-N-acetylglucosaminidase H (endo-H) [9] was purchased from Seikagaku Kogyo (Japan). N-glycanase (NGase) [10] and endo-]~-N-acetylglucosaminidase F (endo-F) [11] were from Genzyme (UK). Other reagents were of analytical grade. Human liver tissues Cirrhotic liver tissue was obtained at autopsy from a patient with alcoholic abuse. Anti-C100-3 antibody and HBs antigen, which were the markers of hepatitis virus infection, were not detected in the serum of the patient. Histological features of the liver were consistent with alcoholic liver cirrhosis. Normal liver of a patient, who died of acute myocardial infarction, cirrhotic liver of viral hepatitis and hepatoma tissues were also obtained at autopsy. All the tissues were obtained within 5 h after death and stored at -80°C until use. Preparation of cathepsin D from human liver tissues Cathepsin D was purified according to the method described previously [4,5] with some modifications. All procedures were carried at 4 ° C. Tissue homogenate was centrifuged at 5000 x g for 30 rain. Resultant supernatant was subjected to ammonium sulfate precipitation and the obtained pellet was dialyzed against 5 mM sodium acetate buffer (pH 5.2), containing 0.1 M NaC1 (buffer A). The solution was applied to concanavalin A-Sepharose column (Con A column) previously equilibrated by buffer A. The bound component was removed with 0.5 M ~-methylmannopyranoside in buffer A. Flow-through fractions and bound fractions containing cathepsin D activity were collected. Cathepsin D in these two preparations were purified with pepstatin-Sepharose chromatography and gel permeation chromatography described previously [4,5]. Protein concentration was determined by the method of Lowry et al [12] with bovine serum albumin as a standard. Treatment of cathepsin D with exogenous hydrolases Reaction mixture was 15/11 in volume, and the hydrolysis treatments were performed at 37°C for 16 h. The purified cathepsin D (3 ~g) was incubated with 5 mU of endo-H [9] in 15 mM citrate-phosphate buffer (pH 5.4), containing 10 ~M pepstatin A and 1 mM PMSF (protease inhibitors). In endo-F [11] treatment, 3 ~g of cathepsin was incubated with 1 mU of endo-F in 15 mM Tris-maleate buffer (pH 6.1), containing 30 mM EDTA, 1.25% NP-40 and protease inhibitors. NGase [10] treatment was performed as follow; 3/lg of cathepsin D was hydrolyzed by 250 mU NGase in 300 mM sodium-phosphate buffer (pH 8.6), containing protease inhibitors. The hydrolase-treated cathepsin D was subjected to electrophoresis.
154
M. OHHIRA et al.
Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was performed according to the method of Laemmli [13]. Gels were treated with Coomassie brilliant blue R-250 for protein staining.
Results
Binding profile of cathepsin D activity for Con A column In alcoholic cirrhotic liver, cathepsin D activity was demonstrated in both flow-through fractions and bound fractions on Con A-Sepharose chromatography (Table 1). On the other hand, in normal liver, viral cirrhotic liver and hepatoma tissues, cathepsin D activity was completely bound to the Con A column.
Purified cathepsin D From an alcoholic cirrhotic liver of 500 g, 9 mg cathepsin D in Con A-bound fractions and 14 mg protein in Con A-unbound fractions were purified, respectively (Table 1). The molecular mass of each enzyme was 43 kDa by gel permeation chromatography (data not shown). Specific enzymatic activity [4,5] had a similar level to the activities of the enzymes purified from normal liver, viral cirrhotic liver and hepatoma [4,5] (data not shown). Each enzyme preparation exhibited three major bands: one heavy chain (31 kDa) and two light chains (15 kDa and 14 kDa) were detected on polyacrylamide gel electrophoresis (Fig. 1). The electrophoretic patterns of these enzymes were similar to those purified from other human liver tissues [4,5].
Effects of exogenous hydrolases on cathepsin D from alcoholic cirrhotic liver (Fig. 2) Upon treatment with NGase of two cathepsin D preparations from alcoholic cirrhotic liver, a 31-kDa peptide was completely changed to 29 kDa and two light chains of 14 and 15 kDa were decreased in size to 13 kDa peptide. This result indicated that cathepsin D purified from Con A-unbound fractions also had asparagine-linked sugar moieties and the sugar chains were of a similar size to those from Con A-bound fractions. In both cathepsin D preparations, treatments with endo-H and endo-F showed the same electrophoretic manner. Most of the 31-kDa peptide and all of the 15-kDa peptide were changed to a 29-kDa peptide and a 13-kDa peptide, respectively. A part of the 31-kDa peptide and all of
TABLE 1 Purified cathepsin D from human liver tissues Purified cathepsin D (mg/500 g tissue) Human liver tissue
Con A-bound
Con A-flow through
Alcoholic cirrhotic liver Normal liver Viral cirrhotic liver Hepatoma
9 35 42 49
14 0 0 0
CATHEPSIND IN ALCOHOLICCIRRHOTICLIVER
155
MW 92.5K 66.2K 43.0K 31.OK 21.5K
LI.~ L2-~
14.4K
B
PT
Fig. 1. Electrophoretic pattern of purified cathepsin D from alcoholic cirrhotic liver. Lane B, cathepsin D purified from Con A-bound fractions. Lane PT, cathepsin D from Con A-flow through fractions. H 1, heavy chain of 31 kDa; H2, minor component of deglycosylated H 1; L1, light chain of 15 kDa; L2, light chain of 14 kDa.
the 14-kDa peptide remained unchanged, probably because the sugar chain structures of these peptides were resistant to hydrolysis by endo-H and endo-F. The electrophoretic patterns of deglycosylated cathepsin D preparations purified from normal liver, viral cirrhotic liver and hepatoma showed a similar electrophoretic pattern to that of cathepsin D from alcoholic cirrhotic liver (data not shown).
Discussion
In the present study, we demonstrated that alcoholic cirrhotic liver contained a large amount of unique cathepsin D which lost the binding affinity for Con A column although all peptides of cathepsin D had asparagine-linked sugar chains. In other human liver tissues, we could not detect the cathepsin D activity in Con-A-flow through fractions (Table 1). Since Con A has high affinity for high-mannose type sugar chains, the abnormal property of cathepsin D in alcoholic cirrhotic liver is probably caused by the alteration of carbohydrate moiety on cathepsin D enzyme. In the analysis of cDNA for human cathepsin D, only one site capable of linking with asparagine-linked oligosaccharide chains is recognized in both heavy and light chains [14]. In the treatment of abnormal cathepsin D from alcoholic cirrhotic liver with NGase, which
156
M. OHHIRAet al.
MW ~66k -,43k ~,31k -,21k ~,14k
L3 C
EH
EF
I
NG I
B
E
C
EH
EF
I
NG I
PT
Fig. 2. Effects of exogenous hydrolases on cathepsin D from alcoholic cirrhotic liver. Treated cathepsin D was subjected to polyacrylamidegel electrophoresis. H2 and L3, deglycosylatedheavy and light chains of cathepsin D. Lane C, untreated cathepsin D; EH, endo-H treatment; EF, endo-F treatment; NG, NGase treatment; E, stabilizer proteins in endo-H preparation. MW, molecular weight markers from Bio-Rad. cleaved all types of asparagine-linked oligosaccharide chains [11], both heavy (31 kDa) and light (15 and 14 kDa) chains were decreased in size to 29 kDa and 13 kDa, respectively. The peptides of 29 kDa and 13 kDa seem to be simple peptides which have no asparagine-linked carbohydrate chains, and the decrease of molecular mass with 1-2 kDa means that the peptide seems to have one asparagine-linked oligosaccharide unit. In the treatment with endo-H and endo-F, all of the 15-kDa and most of the 31-kDa peptides were changed to 13 kDa and 29 kDa, respectively, and the results suggested that these peptides contain altered high-mannose- or hybrid-type sugar chains [9,11]. All of the 14-kDa and a residual part of the 31-kDa peptides seem to have hybrid or complex type chains [9,11]. It seems to be essential that lysosomal hydrolases contain high-mannose-type oligosaccharide chains because mannose-6-phosphate residue on high-mannose-type sugar chains acts as a recognition marker for the targeting to lysosome via Golgi apparatus in the normal processing of lysosomal enzymes [1,2]. Con A affinity chromatography is widely used for the purification of lysosomal hydrolases because Con A has high affinity for high-mannose-type carbohydrate chains [6-8]. The lysosomal enzyme which has no binding affinity for Con A column seems to be extremely rare and unique, and we suggest that the processing of carbohydrate moiety on cathepsin D is altered by impaired Golgi function in alcoholic cirrhotic liver. The structure of carbohydrate moiety on cathepsin D and the changes of processing enzymes in Golgi apparatus are not yet clarified, thus further study of these problems in alcoholic liver injury is necessary. The abnormal cathepsin D described here may also represent abnormal function of Golgi apparatus in alcoholic liver injury.
CATHEPSIN D IN ALCOHOLIC CIRRHOTIC LIVER
157
Acknowledgement The authors are indebted to Ms Izumi Okumura for her help with this manuscript.
References 1 Sly WS, Fisher HD. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem 1982;18:67-85. 2 yon Figura K, Hasilik A. Lysosomal enzyme and their receptors. Annu Rev Biochem 1986;55:167-193. 3 Baraona E, Leo MA, Borowsky SA, Lieber CS. Alcoholic hepatomegaly: Accumulation of protein in the liver. Science 1975;190:794-795. 4 Maguchi S, Taniguchi N, Makita A. Elevated activity and increased mannose-6-phosphate in the carbohydrate moiety of cathepsin D from human hepatoma. Cancer Res 1988;48:362-367. 5 0 h h i r a M, Gasa S, Makita A, Sekiya C, Namiki M. Elevated carbohydrate phosphotransferase activity in human hepatoma and phosphorylation of cathepsin D. Br J Cancer 1991;63:905-908. 6 0 n o M, Taniguchi N, Makita A, Fujita M, Sekiya C, Namiki Y. Phosphorylation of betaglucuronidase from human normal liver and hepatoma by cAMP-dependent protein kinase. J Biol Chem 1988;263:5884-5889. 7 Gasa S, Makita A, Kameya T, et al. Elevated activities and properties of arylsulfatase A and B and B-variants in human lung tumors. Cancer Res 1980;40:3804-3809. 8 Fujita M, Taniguchi N, Makita A, Oikawa K. Cancer-associated alteration of beta-glucuronidase in human lung cancer: Elevated activity and increased phosphorylation. Gann 1984;75:508-517. 9 Tarentino AL, Plummer TH Jr, Maley F. The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H. J Biol Chem 1974;249:818-824. 10 Tarentino AL, G6mez CM, Plummer TH Jr. Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry 1985;24:46654671. 11 Elder JH, Alexander S. Endo-beta-N-acetylglucosaminidase F: Endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci 1982;79:45404544. 12 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 13 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;224:149-154. 14 Faust PL, Kornfeld S, Chirgwin JM. Cloning and sequence analysis of eDNA for human cathepsin D. Proc Natl Acad Sei USA 1985;82:49104914.