Biol Cell (1992) 75, 253-256 © Elsevier, Paris
253
Letter to the Editor
Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts Nathalie G6rard, Jacqueline Thirion, Simone Wattiaux-De Coninck, Robert Wattiaux
*
Laboratoire de Chimie Physiologique, Facultds Universitaires Notre-Dame de la Paix, 61, rue de Bruxelles, B-5000, Namur, Belgium (Received 11 March 1992; accepted 16 July 1992)
Summary - The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis. carboxypeptidase / lysosomes / rat liver / human fibroblasts / subcellular fractionation / galactosialidosis
Introduction Galactosialidosis is a rare inherited lysosomal disease characterized by a combined deficiency of/3-galactosidase and c¢-neuraminidase. Galjaard and coworkers have shown that the genetic defect concerns a 'protective protein' which is either absent or abnormal in galactosialidosis patients. This protein is required for the conversion of immature ~neuraminidase into a functional enzyme and for the protection of/3-galactosidase against an excessive proteolysis in the lysosomes [2, 12]. The three components, o~neuraminidase,/3-galactosidase and the protective protein, are associated in a multienzymic complex bound to the lysosomal membrane by ~-neuraminidase [13]. By studying the complex purified from h u m a n placenta, Tranchemontagne et al [10], found that the protective protein is endowed with a carboxypeptidase activity. Obviously, the analogy between carboxypeptidase and the protective protein requires that the enzymatic activity measured exhibits a lysosomal localization. This is demonstrated in the work reported here which consists of fractionation experiments done on rat liver and on h u m a n fibroblasts, upon which biochemical detection of the diseases is generally performed.
Materials .and methods Human skin fibroblasts (CRL 1501), lung fibroblasts (WI-38) and transformed lung fibroblasts (WI-38 VA 13) were obtained from the American Type Culture Collection. Galactosialidosis skin fibroblasts (GM 00806) were provided by the Human Genetic Mutant Cell Repository. Cultures were performed at 37°C in minimum essential medium (MEM) supplemented with NaHCO 3 (2.18 mg/ml), penicillin (100 U/ml) and streptomycin (0.1 mg/ml), 10 070 foetal calf serum and adjusted to pH 7.4. The livers originated from male Wistar rats. Fibroblasts were homogenized in 0.25 mol/l cold sucrose with a tight Dounce
* Correspondence and reprints
homogenizer. Liver homogenization was achieved in the same medium by means of a small glass tube fitted with a Teflon pestle rotating at 2500 rev/min. Differential centrifugation was done according to de Duve et al [3] with, for fibroblasts, the modifications proposed by Remacle et al [9]. A nuclear fraction N, a heavy mitochondrial fraction M, a light mitochondrial fraction L, a microsomal fraction P and an unsedimentable fraction S were isolated. In gradient experiments, performed according to Beaufay et al [1] a total mitochondrial fraction (M + L fractions) was used. Enzymatic determinations were carried out on cell homogenates or on subcellular fractions, according to the following references:/3-galactosidase and ~-neuraminidase [7], cathepsin C [5] with glycyl-L-arginine 2-naphthylamide as substrate, carboxypeptidase [10] with N-Cbz-L-phenylalanyl-L-leucine as substrate. Proteins were measured by the method of Lowry et al [6].
Results Differential centrifugation Figure 1 illustrates the distribution pattern of carboxypeptidase and cathepsin C, a lysosomal enzyme, after differential centrifugation according to de Duve et al [3]. Experiments were performed on rat liver, normal (CRL 1501) and galactosialidosis (GM 00806) skin fibroblasts, and normal (WI-38) and transformed (WI-38 VA13) lung fibroblasts. In each case, the distribution of carboxypeptidase is similar to that of cathepsin C. The enzyme is mostly recovered in the mitochondrial fractions and exhibits a peak of relative specific activity in the light fraction L. It is to be noted that carboxypeptidase activity in G M 00806 is only 1.4°70 of that found in C R L 1501 (table I).
Isopycnic centrifugation To obtain more accurate information on the components of the mitochondrial fractions with which carboxypeptidase is associated, the total mitochondrial fraction (M + L) originating f r o m rat-liver and f r o m fibroblasts C R L 1501
254
N G 6 r a r d et al
ratliver
l°r 9_
][
8L
_
CRL 1501
GM 00S06
WI-35
WI-3S VA 13
5
OL c.=
/h
t,
'U ¢) .-.
5
OL
N
M L P
6
~b
S
N 100
0
M
L P 50
5
N 100
0
M L P 50
S
N 100
0
M
L 50
P
$ 100
N O
MLP S 50
100
Protein (%) Fig 1. Distribution patterns of carboxypeptidase and cathepsin C after differential centrifugation. Fractionations were performed
on rat liver, human fibroblasts (CRL 1501, galactosialidosis: GM 00806) and lung fibroblasts (WI-38, WI-38 VA 13). Ordinate: relative specific activity (percentage of total recovered activity/percentage of total recovered protein); abscissa, relative protein content of fraction (cumulatively from left to right); N, nuclear fraction; M, heavy mitochondrial fraction; L, light mitochondrial fraction; P, microsomal fraction; S, soluble fraction.
Table I. Cathepsin C and carboxypeptidase activities of fibro-
blasts. Absolute values (means _+ SD) are given in nmol naphthylamine released/min/mg protein for cathepsin C and nmol leucine released/min/mg proteins for carboxypeptidase.
Fibroblasts
Cathepsin C
Carboxypeptidase
CRL 1501
44.8 +__ 17.9 (n = 14) 81.7 _+ 14.1 (n = 11) 450 -e 118 (n = 12) 192.6 _ 30.2 (n = 10)
30.1 + 5.7 (n = 5) 0.43 _+ 0.40 (n = 3) 30.0 ___ 5.4 (n = 4) 25.5 _ 3.9 (n = 3)
GM 00806 WI-38 WI-38 VA 13
was analyzed by isopycnic centrifugation in a sucrose gradient. Results are presented in figure 2. The similarity of carboxypeptidase and cathepsin C distribution is evident. In the case of rat-liver, the same localization of the two enzymes is made more obvious by the density shift of the distributions caused by Triton WR-1339 injected to animal. This non-ionic detergent is a very specific density perturbant of lysosomes [14]. As illustrated in figure 2, it causes a striking decrease of lysosome density (cathepsin C); carboxypeptidase distribution is also subjected to the same shift towards low densities. Osmotic activation
A simple experimental approach that is frequently used to distinguish whether an enzyme is bound to the lysosomal membrane or is free in the organelle matrix is to subject lysosomes to an hypotonic shock and to measure the
release of the enzyme in the medium. In one experiment, the results of which are presented in figure 3, CRL 1501 fibroblasts were homogenized in 0.25 mol/1 sucrose or in 0.025 mol/l sucrose. Following this, the homogenates were centrifuged at 30 000 rev/min for 60 min and the amount of carboxypeptidase, ~-neuraminidase,/3-galactosidase and cathepsin C found in the pellet and the supernatant were measured. In the hypotonic medium, carboxypeptidase, ~-galactosidase and cathepsin C were released to the same extent while ct-neuraminidase remained located in the pellet. A comparison of carboxypeptidase, ~-galactosidase and cathepsin C release as a function of the medium tonicity is given in figure 4. Experiments were performed with normal fibroblasts (CRL 1501) and galactosialidosis fibroblasts (GM 00806). It is obvious that the three enzymes exhibit the same behavior and that there is no significant difference between the normal and the galactosialidosis cells.
Discussion
Our centrifugation results unambiguously show that carboxypeptidase measured in the conditions described by Tranchemontagne et al [10] is a lysosomal enzyme. Even the very low activity that remains in galactosialidosis fibroblasts belongs to lysosomes. Somewhat surprising is the fact that the enzyme is released to the same extent as cathepsin C by an hypotonic treatment. Owing to its association within a complex that is bound to the membrane, it would have been expected that carboxypeptidase would be retained by the lysosomal m e m b r a n e to a larger degree than cathepsin C. The same is true for fl-galactosidase. According to Potier et al [8], a dynamic equilibrium exists between the free and bound forms of protective protein and/3-galactosidase; moreover, as described by Van der H orst et al [11], in unconcentrated preparations, the corn-
Acid carboxypeptidase: subcellular localization
A
25
Carboxypeptidase
255
B
25
20 r...
O__
C'
15
15
10
10
5
o
]----'-n
C~
~22
25 5
20
t2o
25
Cathepsin C
20 r..~
15 i
~C
~.
• ,r-° 1.1 I 1.11
112 1.20
Density (~rnl) Fig 2. Density distribution histograms of carboxypeptidase and cathepsin C after isopycnic centrifugation of ML fraction in a sucrose gradient. The ML fraction was isolated from human skin fibroblasts (A), from normal rat liver ( B , - - - - ) and from liver of rat treated with Triton WR 1339 ( B , - - - - ) . Centrifugation was performed at 39 000 rev/min for 16 h in a SW-65 Beckman rotor. The sucrose gradient extended from 1.09 to 1.26 g/ml density. In ordinate, the frequency: Q/EQ.Ap where Q represents the activity found in the fraction, EQ the total recovered activity and Ap the increment of density from top to bottom of the fraction. The shaded block represents the amount present in the top and the bottom subfractions to scale; in order to facilitate comparison, identical abscissa values have been chosen arbitrarily in all the gradients. Arrows indicate the median equilibrium density. Cathepsin
> ~a
I~.
"-z.
CathepsinC
o ~
O
253
Letter to the Editor
Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts Nathalie G6rard, Jacqueline Thirion, Simone Wattiaux-De Coninck, Robert Wattiaux
*
Laboratoire de Chimie Physiologique, Facultds Universitaires Notre-Dame de la Paix, 61, rue de Bruxelles, B-5000, Namur, Belgium (Received 11 March 1992; accepted 16 July 1992)
Summary - The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis. carboxypeptidase / lysosomes / rat liver / human fibroblasts / subcellular fractionation / galactosialidosis
Introduction Galactosialidosis is a rare inherited lysosomal disease characterized by a combined deficiency of/3-galactosidase and c¢-neuraminidase. Galjaard and coworkers have shown that the genetic defect concerns a 'protective protein' which is either absent or abnormal in galactosialidosis patients. This protein is required for the conversion of immature ~neuraminidase into a functional enzyme and for the protection of/3-galactosidase against an excessive proteolysis in the lysosomes [2, 12]. The three components, o~neuraminidase,/3-galactosidase and the protective protein, are associated in a multienzymic complex bound to the lysosomal membrane by ~-neuraminidase [13]. By studying the complex purified from h u m a n placenta, Tranchemontagne et al [10], found that the protective protein is endowed with a carboxypeptidase activity. Obviously, the analogy between carboxypeptidase and the protective protein requires that the enzymatic activity measured exhibits a lysosomal localization. This is demonstrated in the work reported here which consists of fractionation experiments done on rat liver and on h u m a n fibroblasts, upon which biochemical detection of the diseases is generally performed.
Materials .and methods Human skin fibroblasts (CRL 1501), lung fibroblasts (WI-38) and transformed lung fibroblasts (WI-38 VA 13) were obtained from the American Type Culture Collection. Galactosialidosis skin fibroblasts (GM 00806) were provided by the Human Genetic Mutant Cell Repository. Cultures were performed at 37°C in minimum essential medium (MEM) supplemented with NaHCO 3 (2.18 mg/ml), penicillin (100 U/ml) and streptomycin (0.1 mg/ml), 10 070 foetal calf serum and adjusted to pH 7.4. The livers originated from male Wistar rats. Fibroblasts were homogenized in 0.25 mol/l cold sucrose with a tight Dounce
* Correspondence and reprints
homogenizer. Liver homogenization was achieved in the same medium by means of a small glass tube fitted with a Teflon pestle rotating at 2500 rev/min. Differential centrifugation was done according to de Duve et al [3] with, for fibroblasts, the modifications proposed by Remacle et al [9]. A nuclear fraction N, a heavy mitochondrial fraction M, a light mitochondrial fraction L, a microsomal fraction P and an unsedimentable fraction S were isolated. In gradient experiments, performed according to Beaufay et al [1] a total mitochondrial fraction (M + L fractions) was used. Enzymatic determinations were carried out on cell homogenates or on subcellular fractions, according to the following references:/3-galactosidase and ~-neuraminidase [7], cathepsin C [5] with glycyl-L-arginine 2-naphthylamide as substrate, carboxypeptidase [10] with N-Cbz-L-phenylalanyl-L-leucine as substrate. Proteins were measured by the method of Lowry et al [6].
Results Differential centrifugation Figure 1 illustrates the distribution pattern of carboxypeptidase and cathepsin C, a lysosomal enzyme, after differential centrifugation according to de Duve et al [3]. Experiments were performed on rat liver, normal (CRL 1501) and galactosialidosis (GM 00806) skin fibroblasts, and normal (WI-38) and transformed (WI-38 VA13) lung fibroblasts. In each case, the distribution of carboxypeptidase is similar to that of cathepsin C. The enzyme is mostly recovered in the mitochondrial fractions and exhibits a peak of relative specific activity in the light fraction L. It is to be noted that carboxypeptidase activity in G M 00806 is only 1.4°70 of that found in C R L 1501 (table I).
Isopycnic centrifugation To obtain more accurate information on the components of the mitochondrial fractions with which carboxypeptidase is associated, the total mitochondrial fraction (M + L) originating f r o m rat-liver and f r o m fibroblasts C R L 1501
254
N G 6 r a r d et al
ratliver
l°r 9_
][
8L
_
CRL 1501
GM 00S06
WI-35
WI-3S VA 13
5
OL c.=
/h
t,
'U ¢) .-.
5
OL
N
M L P
6
~b
S
N 100
0
M
L P 50
5
N 100
0
M L P 50
S
N 100
0
M
L 50
P
$ 100
N O
MLP S 50
100
Protein (%) Fig 1. Distribution patterns of carboxypeptidase and cathepsin C after differential centrifugation. Fractionations were performed
on rat liver, human fibroblasts (CRL 1501, galactosialidosis: GM 00806) and lung fibroblasts (WI-38, WI-38 VA 13). Ordinate: relative specific activity (percentage of total recovered activity/percentage of total recovered protein); abscissa, relative protein content of fraction (cumulatively from left to right); N, nuclear fraction; M, heavy mitochondrial fraction; L, light mitochondrial fraction; P, microsomal fraction; S, soluble fraction.
Table I. Cathepsin C and carboxypeptidase activities of fibro-
blasts. Absolute values (means _+ SD) are given in nmol naphthylamine released/min/mg protein for cathepsin C and nmol leucine released/min/mg proteins for carboxypeptidase.
Fibroblasts
Cathepsin C
Carboxypeptidase
CRL 1501
44.8 +__ 17.9 (n = 14) 81.7 _+ 14.1 (n = 11) 450 -e 118 (n = 12) 192.6 _ 30.2 (n = 10)
30.1 + 5.7 (n = 5) 0.43 _+ 0.40 (n = 3) 30.0 ___ 5.4 (n = 4) 25.5 _ 3.9 (n = 3)
GM 00806 WI-38 WI-38 VA 13
was analyzed by isopycnic centrifugation in a sucrose gradient. Results are presented in figure 2. The similarity of carboxypeptidase and cathepsin C distribution is evident. In the case of rat-liver, the same localization of the two enzymes is made more obvious by the density shift of the distributions caused by Triton WR-1339 injected to animal. This non-ionic detergent is a very specific density perturbant of lysosomes [14]. As illustrated in figure 2, it causes a striking decrease of lysosome density (cathepsin C); carboxypeptidase distribution is also subjected to the same shift towards low densities. Osmotic activation
A simple experimental approach that is frequently used to distinguish whether an enzyme is bound to the lysosomal membrane or is free in the organelle matrix is to subject lysosomes to an hypotonic shock and to measure the
release of the enzyme in the medium. In one experiment, the results of which are presented in figure 3, CRL 1501 fibroblasts were homogenized in 0.25 mol/1 sucrose or in 0.025 mol/l sucrose. Following this, the homogenates were centrifuged at 30 000 rev/min for 60 min and the amount of carboxypeptidase, ~-neuraminidase,/3-galactosidase and cathepsin C found in the pellet and the supernatant were measured. In the hypotonic medium, carboxypeptidase, ~-galactosidase and cathepsin C were released to the same extent while ct-neuraminidase remained located in the pellet. A comparison of carboxypeptidase, ~-galactosidase and cathepsin C release as a function of the medium tonicity is given in figure 4. Experiments were performed with normal fibroblasts (CRL 1501) and galactosialidosis fibroblasts (GM 00806). It is obvious that the three enzymes exhibit the same behavior and that there is no significant difference between the normal and the galactosialidosis cells.
Discussion
Our centrifugation results unambiguously show that carboxypeptidase measured in the conditions described by Tranchemontagne et al [10] is a lysosomal enzyme. Even the very low activity that remains in galactosialidosis fibroblasts belongs to lysosomes. Somewhat surprising is the fact that the enzyme is released to the same extent as cathepsin C by an hypotonic treatment. Owing to its association within a complex that is bound to the membrane, it would have been expected that carboxypeptidase would be retained by the lysosomal m e m b r a n e to a larger degree than cathepsin C. The same is true for fl-galactosidase. According to Potier et al [8], a dynamic equilibrium exists between the free and bound forms of protective protein and/3-galactosidase; moreover, as described by Van der H orst et al [11], in unconcentrated preparations, the corn-
Acid carboxypeptidase: subcellular localization
A
25
Carboxypeptidase
255
B
25
20 r...
O__
C'
15
15
10
10
5
o
]----'-n
C~
~22
25 5
20
t2o
25
Cathepsin C
20 r..~
15 i
~C
~.
• ,r-° 1.1 I 1.11
112 1.20
Density (~rnl) Fig 2. Density distribution histograms of carboxypeptidase and cathepsin C after isopycnic centrifugation of ML fraction in a sucrose gradient. The ML fraction was isolated from human skin fibroblasts (A), from normal rat liver ( B , - - - - ) and from liver of rat treated with Triton WR 1339 ( B , - - - - ) . Centrifugation was performed at 39 000 rev/min for 16 h in a SW-65 Beckman rotor. The sucrose gradient extended from 1.09 to 1.26 g/ml density. In ordinate, the frequency: Q/EQ.Ap where Q represents the activity found in the fraction, EQ the total recovered activity and Ap the increment of density from top to bottom of the fraction. The shaded block represents the amount present in the top and the bottom subfractions to scale; in order to facilitate comparison, identical abscissa values have been chosen arbitrarily in all the gradients. Arrows indicate the median equilibrium density. Cathepsin
> ~a
I~.
"-z.
CathepsinC
o ~
O
