179
Biochimica et Biophysica Acta, 583 ( 1 9 7 9 ) 1 7 9 - - 1 8 8 @ E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28827
E F F E C T OF LECTINS ON ENDOCYTOSIS AND SECRETION OF LYSOSOMAL ENZYMES BY C U L T U R E D F I B R O B L A S T S
H A N N E L O R E BEECK, K U R T U L L R I C H and K U R T V O N F I G U R A *
Physiologisch-Chemisches Institut der Universit~'t Miinster, Waldeyerstrasse 15, D-4400 Miinster (F.R.G.) (Received J u n e 2 7 t h , 1978)
Key words: Lectin; Endocytosis; Enzyme secretion; Concanavalin A; Agglutinin; (Cultured fibroblast, Lysosome)
Summary 1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of ~-N-acetylglucosaminidase and increased extracellular accumulation of/3-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl ~-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited ~-N-acetylglucosaminidase endocytosis, b u t had no effect on extracellular ~-N-acetylglucosaminidase accumulation. 4. Concanavalin A-~-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.
Introduction Cultured human skin fibroblasts release lysosomal enzymes by secretion [ 1] and internalize secreted lysosomal enzymes, or those that have been added to the culture medium, b y receptor mediated endocytosis [2,3]. It has been proposed that in fibroblasts the transport of lysosomal enzymes from the site of * To whom reprint requests should be addressed.
180 their synthesis to the lysosomes involves secretion and recapture of lysosomal enzymes [4]. Recent studies suggest that this transfer does not necessarily involve secretion but cycling of lysosomal enzymes via the cell surface in a receptor-bound form [5]. The genetic deficiency of the recognition marker on the lysosomal enzymes is supposed to cause the intracellular deficiency and extracellular accumulation of several lysosomal enzymes in cultured I-cell (mucolipidoses II) fibroblasts [4]. Increased extracellular accumulation and decreased endocytosis of lysosomal enzymes has been observed after treatment of h u m a n skin fibroblasts with cytochalasin B [3,6] and antimicrotubular drugs [7]. The present study describes the effect of polyvalent lectins such as Concanavalin A and wheat germ agglutinin, and lectin derivatives such as divalent succinylated Concanavalin A and monovalent succinylated Concanavalin A on secretion and endocytosis of lysosomal enzymes, a-N-Acetylglucosaminidase was chosen as marker enzyme for endocytosis [8] and fl-N-acetylglucosaminidase as that for secretion [ 1]. Materials and Methods Concanavalin A and wheat germ agglutinin were obtained from Sigma Chemical Co. (St. Louis) and Boehringer Mannheim (Mannheim), p-nitrophenyl glucosides from Koch-Light (Colnbrook). a-N-Acetylglucosaminidase (EC 3.2.1.50) from h u m a n urine was obtained after purification step 2 of Ref. 9. Cell culture. Human skin fibroblasts were grown in plastic dishes with Eagle's minimal essential medium supplemented with 10% foetal calf serum (LS-Labor Service, Mfinchen) exactly as described before [ 10]. Preparation of Concanavalin A, succinylated Concanavalin A and monovalent succinylated Concanavalin A. Concanavalin A was purified according to Ref. 11 and succinylated with succinic anhydride [12]. At pH 7.0 the succinylated Concanavalin A eluted in a single protein peak from a Biogel P-200 column. Monovalent succinylated Concanavalin A was prepared by photoaffinity labelling of succinylated Concanavalin A with p-azidophenyl-a-D-mannopyranoside exactly as described for Concanavalin A [13], except that ultraviolet irradiation was done at 253.7 nm for 30 min at a distance of 5 cm (approx. 300 ergs • mm -2 • s-l). Treatment of fibroblasts with lectins. Lectins were dissolved in Hank's balanced salt solution [14] immediately prior to use and incubated for 15 min at 37°C with confluent fibroblast cultures. Controls were incubated without lectins or with lectins in the presence of either 55 mM methyl a-D-mannoside (Concanavalin A and concanavalin derivatives) or 55 mM N-acetylglucosamine (wheat germ agglutinin). Unbound lectins were removed by washing twice with Hank's solution. For determination of a-N-acetylglucosaminidase endocytosis fibroblasts deficient in a-N-acetylglucosaminidase were incubated for 4--6 h with 2 ml medium containing 4 mU a-N-acetylglucosaminidase. Cells were harvested with 0.1% trypsin containing either 55 mM methyl a-D-mannoside (Concanavalin A) or 55 mM N-acetylglucosamine (wheat germ agglutinin). Without inclusion of carbohydrates the fibroblasts were not detached by trypsin. The cells were
181 collected by centrifugation, washed twice with 0.15 M NaC1, suspended in 0.5 ml 0.15 M NaC1, subjected to ten freez-thaw cycles and assayed for a-N-acetylglucosaminidase activity [9] and protein [15]. For determination of fi-N-acetylglucosaminidase secretion, cultures were fed with 5 ml medium containing 10% heat inactivated foetal calf serum (heated to 70°C for 30 min) [1]. Activities of ~-N-acetylglucosaminidase activity and lactate dehydrogenase were determined in the medium after various periods of time and in the cell homogenate at the end of the experiment [1]. Secretion of ~-N-acetylglucosaminidase was expressed as extracellular enzyme activity in percent of the sum of extra- and intracellular activity. Preliminary experiments showed that presence of up to 100 pg lectin/ml did not affect the activities of a-N-acetylglucosaminidase and ~-N-acetylglucosaminidase. Preparation of lectin-a-N-acetylglucosaminidase complexes, a-N-Acetylglucosaminidase (12 mU/ml in 0.15 M NaC1) was mixed with an equal part of lectin dissolved in 0.15 M NaC1, incubated for 30 min at 37°C and then overnight at 4°C. In controls the lectin was omitted or 55 mM of either methyl a-D-ammoside {Concanavalin A) or N-acetylglucosamine (wheat germ agglutinin)were included. The incubation mixtures were centrifuged at 8000 X g for 10 min and a-N-acetylglucosaminidase activity was determined in the supernatant and the precipitate, washed and resuspended in 0.15 M NaC1. Prior to determination of endocytosis the lectin a-N-acetylglucosaminidase complexes were carefully suspended b y five strokes in a Potter-Elvehjem homogenizer. Results
Effect of lectins on a-N-acetylglucosaminidase endocy tosis Pretreatment of human skin fibroblasts with Concanavalin A and wheat germ agglutinin inhibited endocytosis of a-N-acetylglucosaminidase in a dose-dependent manner (Fig. 1). The inhibitory effect was n o t observed, when either methyl a-D-mannoside (Concanavalin A) or N-acetylglucosamine (wheat germ agglutinin) was present during the preincubation with the lectins. Treatment of fibroblasts with the haptenic carbohydrates alone had no effect on endocytosis. The inhibitory Concanavalin A effect on endocytosis was almost completely reversible within a few days (Fig. 2). Concanavalin A and wheat germ agglutinin are polyvalent lectins that reduce motility of cell membrane components after their binding to the cell surface [17]. A number of biological effects of Concanavalin A are not produced by mono- and divalent Concanavalin A derivatives that bind to same receptors on the cell surface, b u t seem n o t to alter motility of membrane components [12, 16--18]. Pretreatment of fibroblasts with mono- and divalent Concanavalin A derivatives inhibited endocytosis of a-N-acetylglucosaminidase. Both derivatives were, however, by two orders of magnitude less effective than polyvalent Concanavalin A (Fig. 3).
Endocy tosis of lectin-a-N-acety lglucosaminidase complexes Concanavalin A and wheat germ agglutinin precipitate a-N-acetylglucosaminidase (Fig. 4). The endocytosis rate of a-N-acetylglucosaminidase when
182
I00
~
,
~2 z
i
lb
1;0
1000
LECTIN (ffg/ml) Fig. 1. E f f e c t o f Coneanavalin A (o) and w h e a t germ ~ g l n t i n i n (a) t r e a t m e n t o f fibroblasts on ~-N-acetylglueosaminidase endoeytosis. Confluent fibroblasts were incubated for 15 rain in the presence of 1--1000 ttg l e c t i n / m l . E n d o c y t o s i s of ~-N-acetylglucosaminidase was measured during the following 4 h and expressed in percent of a lectin free control. When fibroblasts were incubated with either 500 #g Concanavalin A/ml in the presence of 55 mM methyl ~-D-mannoside or 100 Pg wheat germ agglutinin]ml in the presence of 55 mM N-acetylglucosamine, the ~-N-acetylglucosaminidase endocytosis was 105 and 97% t h a t o f t h e c o n t r o l , w h i c h h a d a n c ~ - N - a c e t y l g l u c o s a m i n i d a s e c l e a r a n c e o f 0 . 0 8 3 m l • h -1 • m g -1 cell p r o tein.
added to the culture medium as a complex with lectins, may be enhanced or reduced (Table I). Whereas mannose 6-phosphate was a potent inhibitor of endocytosis of free a-N-acetylglucosaminidase, it had no effect on the endocytosis of lectin-a-N-acetylglucosaminidase complexes (Table I). Mannose 6-phosphate inhibits lysosomal enzyme endocytosis by competing with the recogni-
)8O i
Z~ og
40
q 20Z i
I
2
3
INCUBATION (days) Fig. 2. Reversibility of Concanavalin A-mediated inhibition of c~-N-acetylglucosaminidase endocytosis. Fibroblasts were incubated for 15 m i n in the presence of 50 ~zg Concanavalin A/ml. Endocytosis during a 4-h period was determined directly after the removal of u n b o u n d Concanavalin A and 1--4 days later. Endocytosis is expressed in percent of that of lectin-free controls.
183 MONOVALENT SUCCINYLATED CONCANAVAL1N A (F:j/ml) "5 "6
2
20
200
i
I
[
WHEAT GERM A G G L U T I N I N (mg/rnl) 0.63
1.25
2.50
~ooq~
100
r-?
~ )z~
80
< o ~o~
60 ue D~
z
~
\
40 ~
S ~,
20 ~
/
,
Z
i
o~~ 40 tA~
~,z
z-. 20], L
i
10
"~'~ :r
100
• 0,019
,
I 0.30
f
0.075
C O N C A N A V A L I N A (mg/ml) LECTIN (#g/rnl)
F i g . 3. E f f e c t o f f i b r o b l a s t t r e a t m e n t w i t h m o n o - , d i - a n d p o l y v a l e n t C o n c a n a v a l i n A o n a - N - a c e t y l g l u c o s aminidase endocytosis. Fibzoblasts were incubated with 1--200 #g Concanavalin A (o) and succinylated Concanavalin A (e) or 2--400 pg monovalent s u c c i n y l a t e d C o n c a n a v a l i n A ( a ) a s d e s c r i b e d i n F i g . 1. When fibroblasts were incubated with Concanavalin A and the two Concanavalin A derivatives in the presence of 55 mM methyl a-n-mannoside cx-N-acetylglucosaminidase endocytosis was in the range of the controls. F i g . 4. P r e c i p i t a t i o n o f ~ - N - a c e t y l g l u c o s a m i n i d a s e For details see Methods.
by Concanavalin
A (c) and wheat
germ agglutinin (a).
tion marker on lysosomal enzymes for binding to cell surface receptors [19-23]. These results suggest therefore that the recognition marker on a-N-acetylglucosaminidase reacted with Concanavalin A is not accessible for the cell surface receptors as has been demonstrated for a-N-acetylglucosaminidase-antibody complexes [5]. The Concanavalin A~-N-acetylglucosaminidase complexes are recognized via the lectin. In various experiments (n = 5) the mean rate of Concanavalin A-a-N-acetylglucosaminidase complexes varied between TABLE
I
ENDOCYTOSIS
OF LECTIN-c~-N-ACETYLGLUCOSAMINIDASE
All values are the mean of triplicates (range in brackets). o f t h e c o n t r o l w a s 0 . 0 6 4 m l - h -1 • m g -1 c e l l p r o t e i n .
COMPLEXES
The mean a-N-acetylglucosaminidase
clearance
Enyzme
Mannose
Endocytosis
(1 mU/ml)
6-phosphate (0.5 mM)
(% o f c o n t r o l )
a-N-A cet ylglucosaminidase
-+
I00 9
-+
178 (174--189) 187 (179--193)
~-N-Acetylglucosaminidase-concanavalin
A complex
~-N-Acetylglucosaminidase-wheat g e r m a g g l u t i n i n c o m p l e x
--
48
(93--104) (9--10)
(47--50)
184
12
_~
-
10
zb 5~ ~o
6
u~
4
2 •
J I0
100
}000
LECTIN (pg/rnl)
Fig. 5. E f f e c t o f C o n c a n a v a l i n A ( o ) a n d w h e a t g e r m a g g l u t i n i n ( o ) t r e a t m e n t o f f i b r o b l a s t s o n e x t r a c e l l u lax f l - N - a c e t y l g l u c o s a m i n i d a s e a c c u m u l a t i o n . F i b r o b l a s t s w e r e t r e a t e d w i t h u p to 5 0 0 # g l e c t i n / m l as d e s c r i b e d in Fig. 1. E x t r a c e l l u l a x ~ - N - a c e t y l g l u c o s a m i n i d a s e a c t i v i t y is e x p r e s s e d in p e r c e n t in i n t r a - a n d e x t r a c e U u l a r e n z y m e a c t i v i t y . T h e p e r c e n t a g e o f e x t r a e e l l u l a r l a c t a t e d e h y d r o g e n a s e a c t i v i t y is s h o w n f o r the e x p e r i m e n t with Concanavalin A (e). T r e a t m e n t with up to 200 pg w h e a t g e r m agglutinin/ml had no e f f e c t o n t h e release o f l a c t a t e d e h y d r o g e n a s e .
178 and 303% of that of controls. In one experiment the endocytosis rate was even reduced to 40% of that of the controls. This great variation may reflect the variable size of the Concanavalin A-enzyme complexes that are endocytosed.
300 T
200
~
loo
D N
1 INCUBATION (days)
F i g . 6. R e v e r s i b i l i t y o f t h e C o n c a n a v a l i n A m e d i a t e d i n c r e a s e o f e x t r a e e l l u l a x f l - N - a c e t y l g l u c o s a m i n i d a s e accumulation. Fibroblasts were treated with 50/zg Coneanavalin A/ml, and extraceUular accumulation of ~ - N - a c e t y l g l u c o s a m i n i d a s e d u r i n g a 24-h p e r i o d w a s d e t e r m i n e d d i r e c t l y a f t e r t h e C o n c a n a v a l i n A t r e a t m e n t a n d 1--3 d a y s l a t e r a n d c o m p a r e d to t h a t o f c o n t r o l s .
185
T
i
24
36
2
O'~
I 12
INCUBATION (hours')
Fig. 7. T i m e c o u r s e o f e x t r a c e l l u l a r ~-N-acetylglueosaminidase a c c u m u l a t i o n . Cells w e r e t r e a t e d w i t h 10 /zg C o n c a n a v a l i n A / m l ( e ) , 1 0 0 / ~ g C o n c a n a v a l i n A/m1 (D) or w i t h 1 0 0 Mg C o n c a n a v a l i n A / m l in the prese n c e o f 55 m M m e t h y l ~ - D - m a n n o s i d e (o). All v a l u e s are t h e m e a n o f d u p l i c a t e s .
2! ×~
~N
Biochimica et Biophysica Acta, 583 ( 1 9 7 9 ) 1 7 9 - - 1 8 8 @ E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28827
E F F E C T OF LECTINS ON ENDOCYTOSIS AND SECRETION OF LYSOSOMAL ENZYMES BY C U L T U R E D F I B R O B L A S T S
H A N N E L O R E BEECK, K U R T U L L R I C H and K U R T V O N F I G U R A *
Physiologisch-Chemisches Institut der Universit~'t Miinster, Waldeyerstrasse 15, D-4400 Miinster (F.R.G.) (Received J u n e 2 7 t h , 1978)
Key words: Lectin; Endocytosis; Enzyme secretion; Concanavalin A; Agglutinin; (Cultured fibroblast, Lysosome)
Summary 1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of ~-N-acetylglucosaminidase and increased extracellular accumulation of/3-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl ~-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited ~-N-acetylglucosaminidase endocytosis, b u t had no effect on extracellular ~-N-acetylglucosaminidase accumulation. 4. Concanavalin A-~-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.
Introduction Cultured human skin fibroblasts release lysosomal enzymes by secretion [ 1] and internalize secreted lysosomal enzymes, or those that have been added to the culture medium, b y receptor mediated endocytosis [2,3]. It has been proposed that in fibroblasts the transport of lysosomal enzymes from the site of * To whom reprint requests should be addressed.
180 their synthesis to the lysosomes involves secretion and recapture of lysosomal enzymes [4]. Recent studies suggest that this transfer does not necessarily involve secretion but cycling of lysosomal enzymes via the cell surface in a receptor-bound form [5]. The genetic deficiency of the recognition marker on the lysosomal enzymes is supposed to cause the intracellular deficiency and extracellular accumulation of several lysosomal enzymes in cultured I-cell (mucolipidoses II) fibroblasts [4]. Increased extracellular accumulation and decreased endocytosis of lysosomal enzymes has been observed after treatment of h u m a n skin fibroblasts with cytochalasin B [3,6] and antimicrotubular drugs [7]. The present study describes the effect of polyvalent lectins such as Concanavalin A and wheat germ agglutinin, and lectin derivatives such as divalent succinylated Concanavalin A and monovalent succinylated Concanavalin A on secretion and endocytosis of lysosomal enzymes, a-N-Acetylglucosaminidase was chosen as marker enzyme for endocytosis [8] and fl-N-acetylglucosaminidase as that for secretion [ 1]. Materials and Methods Concanavalin A and wheat germ agglutinin were obtained from Sigma Chemical Co. (St. Louis) and Boehringer Mannheim (Mannheim), p-nitrophenyl glucosides from Koch-Light (Colnbrook). a-N-Acetylglucosaminidase (EC 3.2.1.50) from h u m a n urine was obtained after purification step 2 of Ref. 9. Cell culture. Human skin fibroblasts were grown in plastic dishes with Eagle's minimal essential medium supplemented with 10% foetal calf serum (LS-Labor Service, Mfinchen) exactly as described before [ 10]. Preparation of Concanavalin A, succinylated Concanavalin A and monovalent succinylated Concanavalin A. Concanavalin A was purified according to Ref. 11 and succinylated with succinic anhydride [12]. At pH 7.0 the succinylated Concanavalin A eluted in a single protein peak from a Biogel P-200 column. Monovalent succinylated Concanavalin A was prepared by photoaffinity labelling of succinylated Concanavalin A with p-azidophenyl-a-D-mannopyranoside exactly as described for Concanavalin A [13], except that ultraviolet irradiation was done at 253.7 nm for 30 min at a distance of 5 cm (approx. 300 ergs • mm -2 • s-l). Treatment of fibroblasts with lectins. Lectins were dissolved in Hank's balanced salt solution [14] immediately prior to use and incubated for 15 min at 37°C with confluent fibroblast cultures. Controls were incubated without lectins or with lectins in the presence of either 55 mM methyl a-D-mannoside (Concanavalin A and concanavalin derivatives) or 55 mM N-acetylglucosamine (wheat germ agglutinin). Unbound lectins were removed by washing twice with Hank's solution. For determination of a-N-acetylglucosaminidase endocytosis fibroblasts deficient in a-N-acetylglucosaminidase were incubated for 4--6 h with 2 ml medium containing 4 mU a-N-acetylglucosaminidase. Cells were harvested with 0.1% trypsin containing either 55 mM methyl a-D-mannoside (Concanavalin A) or 55 mM N-acetylglucosamine (wheat germ agglutinin). Without inclusion of carbohydrates the fibroblasts were not detached by trypsin. The cells were
181 collected by centrifugation, washed twice with 0.15 M NaC1, suspended in 0.5 ml 0.15 M NaC1, subjected to ten freez-thaw cycles and assayed for a-N-acetylglucosaminidase activity [9] and protein [15]. For determination of fi-N-acetylglucosaminidase secretion, cultures were fed with 5 ml medium containing 10% heat inactivated foetal calf serum (heated to 70°C for 30 min) [1]. Activities of ~-N-acetylglucosaminidase activity and lactate dehydrogenase were determined in the medium after various periods of time and in the cell homogenate at the end of the experiment [1]. Secretion of ~-N-acetylglucosaminidase was expressed as extracellular enzyme activity in percent of the sum of extra- and intracellular activity. Preliminary experiments showed that presence of up to 100 pg lectin/ml did not affect the activities of a-N-acetylglucosaminidase and ~-N-acetylglucosaminidase. Preparation of lectin-a-N-acetylglucosaminidase complexes, a-N-Acetylglucosaminidase (12 mU/ml in 0.15 M NaC1) was mixed with an equal part of lectin dissolved in 0.15 M NaC1, incubated for 30 min at 37°C and then overnight at 4°C. In controls the lectin was omitted or 55 mM of either methyl a-D-ammoside {Concanavalin A) or N-acetylglucosamine (wheat germ agglutinin)were included. The incubation mixtures were centrifuged at 8000 X g for 10 min and a-N-acetylglucosaminidase activity was determined in the supernatant and the precipitate, washed and resuspended in 0.15 M NaC1. Prior to determination of endocytosis the lectin a-N-acetylglucosaminidase complexes were carefully suspended b y five strokes in a Potter-Elvehjem homogenizer. Results
Effect of lectins on a-N-acetylglucosaminidase endocy tosis Pretreatment of human skin fibroblasts with Concanavalin A and wheat germ agglutinin inhibited endocytosis of a-N-acetylglucosaminidase in a dose-dependent manner (Fig. 1). The inhibitory effect was n o t observed, when either methyl a-D-mannoside (Concanavalin A) or N-acetylglucosamine (wheat germ agglutinin) was present during the preincubation with the lectins. Treatment of fibroblasts with the haptenic carbohydrates alone had no effect on endocytosis. The inhibitory Concanavalin A effect on endocytosis was almost completely reversible within a few days (Fig. 2). Concanavalin A and wheat germ agglutinin are polyvalent lectins that reduce motility of cell membrane components after their binding to the cell surface [17]. A number of biological effects of Concanavalin A are not produced by mono- and divalent Concanavalin A derivatives that bind to same receptors on the cell surface, b u t seem n o t to alter motility of membrane components [12, 16--18]. Pretreatment of fibroblasts with mono- and divalent Concanavalin A derivatives inhibited endocytosis of a-N-acetylglucosaminidase. Both derivatives were, however, by two orders of magnitude less effective than polyvalent Concanavalin A (Fig. 3).
Endocy tosis of lectin-a-N-acety lglucosaminidase complexes Concanavalin A and wheat germ agglutinin precipitate a-N-acetylglucosaminidase (Fig. 4). The endocytosis rate of a-N-acetylglucosaminidase when
182
I00
~
,
~2 z
i
lb
1;0
1000
LECTIN (ffg/ml) Fig. 1. E f f e c t o f Coneanavalin A (o) and w h e a t germ ~ g l n t i n i n (a) t r e a t m e n t o f fibroblasts on ~-N-acetylglueosaminidase endoeytosis. Confluent fibroblasts were incubated for 15 rain in the presence of 1--1000 ttg l e c t i n / m l . E n d o c y t o s i s of ~-N-acetylglucosaminidase was measured during the following 4 h and expressed in percent of a lectin free control. When fibroblasts were incubated with either 500 #g Concanavalin A/ml in the presence of 55 mM methyl ~-D-mannoside or 100 Pg wheat germ agglutinin]ml in the presence of 55 mM N-acetylglucosamine, the ~-N-acetylglucosaminidase endocytosis was 105 and 97% t h a t o f t h e c o n t r o l , w h i c h h a d a n c ~ - N - a c e t y l g l u c o s a m i n i d a s e c l e a r a n c e o f 0 . 0 8 3 m l • h -1 • m g -1 cell p r o tein.
added to the culture medium as a complex with lectins, may be enhanced or reduced (Table I). Whereas mannose 6-phosphate was a potent inhibitor of endocytosis of free a-N-acetylglucosaminidase, it had no effect on the endocytosis of lectin-a-N-acetylglucosaminidase complexes (Table I). Mannose 6-phosphate inhibits lysosomal enzyme endocytosis by competing with the recogni-
)8O i
Z~ og
40
q 20Z i
I
2
3
INCUBATION (days) Fig. 2. Reversibility of Concanavalin A-mediated inhibition of c~-N-acetylglucosaminidase endocytosis. Fibroblasts were incubated for 15 m i n in the presence of 50 ~zg Concanavalin A/ml. Endocytosis during a 4-h period was determined directly after the removal of u n b o u n d Concanavalin A and 1--4 days later. Endocytosis is expressed in percent of that of lectin-free controls.
183 MONOVALENT SUCCINYLATED CONCANAVAL1N A (F:j/ml) "5 "6
2
20
200
i
I
[
WHEAT GERM A G G L U T I N I N (mg/rnl) 0.63
1.25
2.50
~ooq~
100
r-?
~ )z~
80
< o ~o~
60 ue D~
z
~
\
40 ~
S ~,
20 ~
/
,
Z
i
o~~ 40 tA~
~,z
z-. 20], L
i
10
"~'~ :r
100
• 0,019
,
I 0.30
f
0.075
C O N C A N A V A L I N A (mg/ml) LECTIN (#g/rnl)
F i g . 3. E f f e c t o f f i b r o b l a s t t r e a t m e n t w i t h m o n o - , d i - a n d p o l y v a l e n t C o n c a n a v a l i n A o n a - N - a c e t y l g l u c o s aminidase endocytosis. Fibzoblasts were incubated with 1--200 #g Concanavalin A (o) and succinylated Concanavalin A (e) or 2--400 pg monovalent s u c c i n y l a t e d C o n c a n a v a l i n A ( a ) a s d e s c r i b e d i n F i g . 1. When fibroblasts were incubated with Concanavalin A and the two Concanavalin A derivatives in the presence of 55 mM methyl a-n-mannoside cx-N-acetylglucosaminidase endocytosis was in the range of the controls. F i g . 4. P r e c i p i t a t i o n o f ~ - N - a c e t y l g l u c o s a m i n i d a s e For details see Methods.
by Concanavalin
A (c) and wheat
germ agglutinin (a).
tion marker on lysosomal enzymes for binding to cell surface receptors [19-23]. These results suggest therefore that the recognition marker on a-N-acetylglucosaminidase reacted with Concanavalin A is not accessible for the cell surface receptors as has been demonstrated for a-N-acetylglucosaminidase-antibody complexes [5]. The Concanavalin A~-N-acetylglucosaminidase complexes are recognized via the lectin. In various experiments (n = 5) the mean rate of Concanavalin A-a-N-acetylglucosaminidase complexes varied between TABLE
I
ENDOCYTOSIS
OF LECTIN-c~-N-ACETYLGLUCOSAMINIDASE
All values are the mean of triplicates (range in brackets). o f t h e c o n t r o l w a s 0 . 0 6 4 m l - h -1 • m g -1 c e l l p r o t e i n .
COMPLEXES
The mean a-N-acetylglucosaminidase
clearance
Enyzme
Mannose
Endocytosis
(1 mU/ml)
6-phosphate (0.5 mM)
(% o f c o n t r o l )
a-N-A cet ylglucosaminidase
-+
I00 9
-+
178 (174--189) 187 (179--193)
~-N-Acetylglucosaminidase-concanavalin
A complex
~-N-Acetylglucosaminidase-wheat g e r m a g g l u t i n i n c o m p l e x
--
48
(93--104) (9--10)
(47--50)
184
12
_~
-
10
zb 5~ ~o
6
u~
4
2 •
J I0
100
}000
LECTIN (pg/rnl)
Fig. 5. E f f e c t o f C o n c a n a v a l i n A ( o ) a n d w h e a t g e r m a g g l u t i n i n ( o ) t r e a t m e n t o f f i b r o b l a s t s o n e x t r a c e l l u lax f l - N - a c e t y l g l u c o s a m i n i d a s e a c c u m u l a t i o n . F i b r o b l a s t s w e r e t r e a t e d w i t h u p to 5 0 0 # g l e c t i n / m l as d e s c r i b e d in Fig. 1. E x t r a c e l l u l a x ~ - N - a c e t y l g l u c o s a m i n i d a s e a c t i v i t y is e x p r e s s e d in p e r c e n t in i n t r a - a n d e x t r a c e U u l a r e n z y m e a c t i v i t y . T h e p e r c e n t a g e o f e x t r a e e l l u l a r l a c t a t e d e h y d r o g e n a s e a c t i v i t y is s h o w n f o r the e x p e r i m e n t with Concanavalin A (e). T r e a t m e n t with up to 200 pg w h e a t g e r m agglutinin/ml had no e f f e c t o n t h e release o f l a c t a t e d e h y d r o g e n a s e .
178 and 303% of that of controls. In one experiment the endocytosis rate was even reduced to 40% of that of the controls. This great variation may reflect the variable size of the Concanavalin A-enzyme complexes that are endocytosed.
300 T
200
~
loo
D N
1 INCUBATION (days)
F i g . 6. R e v e r s i b i l i t y o f t h e C o n c a n a v a l i n A m e d i a t e d i n c r e a s e o f e x t r a e e l l u l a x f l - N - a c e t y l g l u c o s a m i n i d a s e accumulation. Fibroblasts were treated with 50/zg Coneanavalin A/ml, and extraceUular accumulation of ~ - N - a c e t y l g l u c o s a m i n i d a s e d u r i n g a 24-h p e r i o d w a s d e t e r m i n e d d i r e c t l y a f t e r t h e C o n c a n a v a l i n A t r e a t m e n t a n d 1--3 d a y s l a t e r a n d c o m p a r e d to t h a t o f c o n t r o l s .
185
T
i
24
36
2
O'~
I 12
INCUBATION (hours')
Fig. 7. T i m e c o u r s e o f e x t r a c e l l u l a r ~-N-acetylglueosaminidase a c c u m u l a t i o n . Cells w e r e t r e a t e d w i t h 10 /zg C o n c a n a v a l i n A / m l ( e ) , 1 0 0 / ~ g C o n c a n a v a l i n A/m1 (D) or w i t h 1 0 0 Mg C o n c a n a v a l i n A / m l in the prese n c e o f 55 m M m e t h y l ~ - D - m a n n o s i d e (o). All v a l u e s are t h e m e a n o f d u p l i c a t e s .
2! ×~
~N
