605th MEETING, STRATHCLYDE
529
Intracellular turnover and secretion of lysosomal enzymes
WENDY JESSUP, JUDITH L. BODMER,
ROGER T. DEAN, VALERIE A. GREENAWAY
and PATRICIA LEON1
Cell Biology Research Group, Department of Applied
Biology, Brunel University, Uxbridge, Middx. UB8 3PH,
U .K .
Intracellular turnover
The degradation of lysosomal enzymes begins, strictly,
during their biosynthesis, since several controlled proteolytic cleavages occur during conversion of the newly
translated polypeptides to the mature proteins which form
the bulk of the catalytically active enzyme (Hasilik & von
Figura, 1983). These commence with signal-peptide cleavage following import of the enzyme protein into the endoplasmic reticulum. Additional trimming of the newly
synthesized enzyme protein occurs subsequent to the
removal of the signal-peptide sequence. So far, all lysosomal
enzymes studied have been found to be synthesized as larger
precursors or pro-enzymes. The kinetics of the first cleavage
of these pro-enzymes, which can involve removal of both
protein and carbohydrate, is quite rapid (half-lives of 1-5 h)
and consistent with proteolytic processing occurring during
and/or just after transport of precursors into the lysosomal
system (Hasilik & von Figura, 1983).
Once they reach the lysosomal system, lysosomal enzymes
undergo decay with first-order kinetics. Several studies have
been made of the turnover of lysosomal enzymes in a range
of cell types and using several different techniques (Touster,
1978). Differences in the absolute rates of degradation
measured for individual lysosomal enzymes probably reflect
both true variations between cell type or species, and
inaccuracies in measurement due to the limitations of the
methods employed. However, several characteristic features of lysosomal enzyme turnover appear common to all
systems studied. Firstly, turnover is heterogeneous, since in
all instances where several lysosomal enzymes have been
studied together in the same system, the measured degradation rates of the individual enzymes are different. Others
have found that both membrane and soluble fractions of
purified lysosomes are degraded at heterogenous rates
(Dean & Barrett, 1976). Secondly, no correlation between
half-life and molecular size has been observed for the
degradation of lysosomal enzymes, unlike those of many
other cellular structures. Finally, the turnover of lysosomal
hydrolases is relatively slow. This is particularly striking
when the rates of lysosomal enzyme degradation are compared with those of other proteins which have been endocytosed into the lysosomal system (Stahl et al., 1980).
Presumably lysosomal enzymes have evolved to maintain
stability at the acid pH of the lysosome and resistance to
degradation by lysosomal proteinases. This stability to
intra-lysomal degradation is observed both by endogenously
synthesized enzymes and by purified lysosomal enzymes
entering the cell by pinocytosis.
The data available for turnover of the carbohydrate
moiety of lysosomal enzymes are much more limited. In
general, protein and carbohydrate appear to turn over as a
unit. In experiments where dual-labelling of protein and
carbohydrate of macrophage glycoproteins using [35S]methionine and [3H]mannosewas performed, parallel loss
of isotope from both peptidyl and carbohydrate portions of
both P-glucuronidase and P-galactosidase was found (Skudlarek & Swank, 1981). These studies do not, however,
preclude the possibility that in lysosomal enzymes, like
other glycoproteins (Baumann & Doyle, 1982), peripheral
Abbreviation used : M6P, mannose 6-phosphate.
VOl.
12
sugars such as fucose and sialic acid may be more susceptible to cleavage than more proximal sugars such as
mannose.
Lysosomal storage diseases are metabolic disorders in
which one or more lysosomal enzymes are deficient or
absent. This defect usually leads to the accumulation of
undegraded macromolecular material in the lysosomes of
the affected tissues. The deficiency of a particular lysosomal
enzyme activity could be expected to arise from a
dysfunction in one of several events in its synthesis and
transport into the lysosomal system. Evidence has recently
appeared which shows that in several storage diseases the
enzyme deficiency is due rather to an accelerated rate of
degradation of the enzyme protein. Fibroblasts from
patients with combined fi-galactosidase/neuraminidasedeficiency, in which P-galactosidase levels are 5-10% of
normal, synthesize the enzyme at the normal rate but
degrade it 10-fold faster than normal fibroblasts (van
Diggelen et al., 1981). It has recently been shown that this
accelerated degradation is caused by the failure of the
affected cells to produce a ‘stabilizing’ protein which forms
a complex with fi-galactosidaseand neuraminidase, activating the latter and protecting the former against degradation
(d’Azzo et al., 1982). It will be interesting to see how many
other lysosomal enzymes are stablized in this way. In lateonset glycogenosis 11, a deficiency of a-glucosidase is
associated with the production of apparently normal
enzyme, but in reduced amounts. Recent labelling experiments have indicated that near normal amounts of enzyme
are synthesized, but that much of this enzyme is rapidly
degraded in a compartment proximal to the site of lysosoma1 hydrolase packaging (Steckel et al., 1982). However,
those molecules of a-glucosidase which enter the lysosomal
system appear stable.
Few observations are available concerning the effects of
altered carbohydrate structure on the turnover of lysosomal
enzymes. We have recently induced altered glycosylation
experimentally in cultured macrophages by incubating
them with the indolizidine alkaloid, swainsonine, and have
measured lysosomal enzyme activities in such cells (Greenaway et al., 1983). Swainsonine, purified from the plant
Swainsona canescens, is a potent inhibitor of lysosomal amannosidase, and causes symptoms of mannosidosis in
animals which ingest the plant, or which are fed the purified
alkaloid (Jolly et al., 1981). Our intention was to investigate
its potential as a means of producing an experimental and
reversible storage disease in cultured cells. However, swainsonine also strongly inhibits Golgi a-mannosidase I1
(Tulsiani et al., 1982). As a result, in swainsonine-treated
cells the production of complex N-linked glycoproteins is
blocked, and replaced with an increased proportion of
hybrid structures (Tulsiani & Touster, 1983~).In our
experiments, swainsonine inhibited lysosomal a-mannosidase both in cell extracts and in cells incubated with the
alkaloid. Interestingly, during prolonged exposure of
macrophages to 0.1 mM-swainsonine, significant increases
were measured in the specific activities and absolute
amounts of a-mannosidase and another lysosomal enzyme,
P-hexosaminidase, relative to control cultures (Table 1 ;
Greenaway et al., 1983). Tulsiani & Touster (19836) have
reported elevated levels of lysosomal a-mannosidasc in
tissues of rats exposed to swainsonine. The cause of the
elevated lysosomal enzyme activities in swainsonine-treated
cells is not known, though it seems more likely to result,
directly or indirectly, from alterations in glycoprotein
processing, rather than from the induced lysosomal mannosidosis. Such alterations might be expected to affect either
the intracellular transport or the degradation of lysosomal
530
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Effects of chronic swainsonine exposure on macrophage enzyme activities
Cultures (1.5 x lo6 cells) were incubated in Medium 199, 10% (v/v) alkaline-inactivated swine serum, 100 i.u./ml penicillin
and 100pg/ml streptomycin kO.1 mM-swainsonine for the periods indicated. The medium was changed every 48-72h. Cells
were washed extensively, lysed in 0.1% (v/v) Triton X-100 and assayed. S/C = ratio of swainsonine-treated to control
activity.
u-Mannosidase
(nmol/h per mg of protein)
Time
(days)
0
7
14
A
r
Control
553 k 38
544k44
171k17
Swainsonine
548k25
1178k89
1097k53
fi-Hexosaminidase
(pmol/h per mg of protein)
\
SjC
1.0
2.2
6.4
r
Control
3.82f0.59
10.53f2.85
22.61 f2.22
enzymes. It would be interesting to know whether lysosomal
enzymes in swainsonine-treated cells have abnormal carbohydrate structures, and if the observed accumulation of
lysosomal enzymes reflects a resistance to degradation,
consequent on the altered glycosylation,
Lysosomal enzyme secretion
The secretion of lysosomal enzymes occurs in many cell
types under various conditions (Jessup et al., 19836), and in
some instances can contribute significantly to lysosomal
enzyme turnover.
In the fibroblast, secretion induced by several agents can
conveniently be explained in terms of their interactions
with the M6P-dependent system for the packaging of lysosoma1 enzymes. Thus dysfunctions in the latter system due
to deficiency of M6P-ligands, M6P-receptors, amineinduced inhibition of M6P-receptor recycling and inhibition of glycosylation all cause diversion of newly synthesized
proenzymes to the exterior (Jessup et al., 19836).
Secretion of lysosomal enzymes by macrophages is a more
complex and less understood process. Firstly, the range of
materials which induce secretion is wider than for fibroblasts, including complement components, immune complexes, some particulate agents (e.g. zymosan and asbestos)
and pharmacological agents such as cytochalasin B and
ammonium chloride (Dean, 1979). Secondly, the kinetics of
macrophage lysosomal enzyme secretion are quite different
from those of fibroblasts (see below).
Like other cell types, macrophage lysosomal enzymes are
synthesized as higher molecular weight pro-enzymes and
which undergo proteolytic cleavage to mature enzyme with
similar half-lives (Skudlarek & Swank, 1981). There is
evidence for a M6P-dependent sorting system, in that
macrophage lysosomal enzymes contain the M6P-ligand
(Jessup & Dean, 1982). We are not aware of any direct
demonstration of M6P-receptors in macrophages to date. In
addition, macrophages have a mannose/N-acetylglucosamine receptor which can mediate clearance of mannosecontaining glycoproteins from extracellular fluid (Stahl et
al., 1980). It is possible that this system may also function
intracellularly in retention and transport of lysosomal
enzymes.
Exposure of macrophages to zymosan particles causes a
dose- and timedependent secretion of lysosomal enzymes
(Dean et al., 1979) which comprises: (i) rapid release (in 14h) of a large proportion of the cellular content, independent of protein synthesis; (ii) sustained, linear rate of release
of enzymes on prolonged (days) exposure, requiring
continued protein synthesis (Dean et al., 1979, McCarthy et
al., 1982). A similar pattern of release is seen in response to
asbestos (Dean e f al., 1979).
Ammonium chloride also causes a dose-dependent secretion of lysosomal enzymes (Jessup et al., 1982). At a
concentration of 50 mM it produces a rapid, cycloheximideindependent discharge, analogous to that produced by
L
Swainsonine
4.92k0.45
28.62k 2.64
86.61 f 1.04
Lactate dehydrogenase
@mol/min per mg of protein)
\
SIC
1.3
2.7
3.8
r
Control
844k14
1231k62
1165k85
A
Swainsonine
1005k64
1611k168
2020k110
\
S/C
1.2
1.3
1.7
zymosan. At a concentration of lOmM the release is slower
(Jessup et al., 1983~).We have preliminary evidence that
the secretion induced by 10mM-ammonium chloride is
cycloheximide-sensitive after 4h. Brown et al. (1981) have
reported that ammonium chloride-elicited secretion of
macrophage lysosomal enzymes causes release of both proenzyme and mature forms of lysosomal B-hexosaminidase.
In summary, macrophages can be considered to secrete
lysosomal enzymes with two distinct kinetics. The first is a
rapid discharge, apparently from a preformed intracellular
store, which can be triggered by a range of stimuli. The
mechanisms by which these agents operate is not yet understood, though they are quite distinct from the calciummediated system in neutrophils. We have shown that
zymosan and ammonium chloride function additively in
inducing macrophage secretion (Jessup et al., 1982),
implying that they affect distinct aspects of the secretory
mechanism. The second mechanism of macrophage lysosoma1 enzyme secretion is a slower and more sustained
process, dependent on protein synthesis. It is possible that
this mechanism involves re-routing of newly synthesized
lysosomal enzymes directly to the exterior, by interference
either in the packaging at the Golgi, or in the intracellular
transit of nascent prelysosomal vesicles. Such a proposal
requires explanation of how materials as diverse as
zymosan, asbestos and ammonium chloride could interact
in such a scheme. We have some evidence that zymosan
may initiate secretion by macrophages via interaction with
the mannose-receptor (Bodmer & Dean, 1983). Similarly,
zymosan-induced lysosomal enzyme secretion in human
monocyte-derived macrophages is enhanced when the cells
are matured in the presence of mixed lymphocyte reaction
(Leoni & Dean, 1983), a treatment which also seems to
increase the number of available surface mannose receptors
(P. Leoni & R.T. Dean, unpublished work). However, the
intracellular signal which is induced by zymosan binding
and internalization, and the subsequent sequence of events
leading to lysosomal enzyme release, are not yet known.
d’Azzo, A., Hoogeveen, A., Reuser, A. J . J . , Robinson, D. &
Galjaard, H. (1982) Proc. Nail. Acod. Sci. U.S.A.,79,4535-4539
Baumann, H. & Doyle, D. (1982) in The Glycoconjugaies
(Horowitz, M. I., ed.), vol. IV, pp. 105-153, Academic Press,
London
Bodmer, J . L. & Dean, R. T. (1983) Biochem. Biophys. Res.
Commun. 113, 192-198
Brown, J. A., Skudlarek, M . D., Jahreis, G. P. & Swank, R. T.
(1981) J . Cell Biol. 91, 246a
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Dean, R. T. & Barrett, A. J . (1976) Essays Biochem. 12, 1-40
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1984
531
605th MEETING, STRATHCLYDE
Greenaway, V. A., Jessup, W., Dean, R. T. &Dorling, P. R. (1983)
Biochim. Biophys. Acta 762, 569-576
Hasilik, A. & von Figura, K. (1983) in Lysosomes in Biology and
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