Download Proteolytic and other metabolic pathways in lysosomes

608th Meeting
Held at the University of Keele on 1 I - 13 April 7 984
The Lysosome and its Membrane
Society Host Colloquium organized by J. B. Lloyd (Keele) and R. T. Dean (Brunei), and
edited by J. B. Lloyd
Proteolytic and other metabolic pathways in lysosomes
ALAN J. BARRETT
Biochemistry Department, Strangeways Research
Laboratory, Worts Causeway, Cambridge CBI 4RN, U.K.
It is obvious that the maintenance of the differentiated
structure of an organism must require approximately as
much breakdown of complex molecules as synthesis of
them. Moreover, the selectivity of the breakdown processes
must precisely complement that of biosynthesis. Discovering the ways in which this is achieved represents an
important and challenging aspect of biochemistry and cell
biology.
At one time, the lysosomal system tended to be thought of
primarily in connection with cell pathology. There was the
idea that the lysosomes represent a self-destruct system for
the cell, and much emphasis was put on the dire
consequences of release of the lysosomal enzymes into the
bulk of the cytoplasm, or into extracellular space. Direct
experiments cast doubt on this view, however, and today
most of us would tend to put more emphasis on the role of
the lysosomal system as an integral part of the normal
biochemical machinery of the cell.
It is now clear that both lysosomal and non-lysosomal
pathways make appreciable contributions to the intracellular degradation of complex molecules, including proteins,
polysaccharides, nucleic acids and lipids. The relative
importance of the lysosomal and non-lysosomal routes
undoubtedly varies between cell types, and different
metabolic conditions. Information about the size of the
lysosomal contribution can be obtained by determining the
effect of treating cultured cells with ammonium chloride, or
salts of other weak bases that accumulate in the lysosomes
and interfere with their function. In particular, the bases
interfere with the acidification of the lysosomal space,
which is necessary for the action of the lysosomal
hydrolases, essentially all of which have acid pH optima.
The acidification depends on ATP-dependent proton
transport, so lysosomal metabolic pathways are at least
partially ATP dependent. More specific ways of probing
lysosomal metabolism depend on the use of inhibitors of
particular enzymes, or the study of the effects of the many
known genetic deficiences of individual lysosomal enzymes.
About 50 lysosomal enzymes are known, and their
properties are reviewed by Barrett & Heath (1977). Table 1
is intended to indicate how these enzymes can be seen as
contributing to the pathways for catabolism of four major
classes of complex molecules.
I have called the four pathways the ‘proteolytic’‘glycanoVOl. 12
899
lytic’, ‘nuclease’ and ‘lipolytic’ pathways. The proteolytic
and nuclease pathways start with enzymes specialized for
action on the inner regions of the intact linear polymers that
are their substrates, the endopeptidases and endonucleases,
respectively. There are definite sequencesof reactions in the
other two pathways, too, although not arranged in quite the
same way. It is possible that some of the enzyme molecules
may be physically organized in the lysosomes in the order in
which they act, as has been found in some other organelles,
so that substrate can be passed from one to another in an
efficient process. There is little direct evidence for this, as
yet, although it has sometimes been found that intact
lysosomes provide more efficient catalysis than simple
mixtures of the appropriate enzymes in solution. Also, the
existence of the proton-transport system that acidifies the
lysosomal contents is a clear demonstration that vectoral
processes do occur in lysosomes.
The proteolytic pathway
The proteolytic system of the lysosome is one in which no
hereditary deficiency has yet been discovered. This may be
because most of the deficiencies that occur are lethal in
utero. Certainly, the lysosomal proteases must be extremely
active recycling proteins in the embryo, where remodelling
and growth of tissues are so rapid. Even in the adult, there is
no doubt that lysosomal proteolysis is a quantitatively
important aspect of metabolism. One tends to think of the
major site of proteolysis in the body being the digestive
system, but in fact far more protein is broken down in the
tissues in protein turnover, and half or more of this in the
lysosomal system. Correspondingly, the lysosomes are a
more important source of amino acids for new protein
synthesis than is the gut.
Mego et al. (1972) isolated lysosomes from cells that had
been allowed to endocytose radiolabelled protein, and
showed that intracellular degradation of this exogenous
substrate proceeded in oitro. The stimulation of the process
by ATP and Mg2+ suggested that an active system for
acidification of the lysosomal contents made an important
contribution. Early evidence of the role of lysosomes in the
intracellular degradation of endogenousproteins came from
the work of Dean (1975) with liver cells, in which it was
shown that pepstatin partially inhibited the process. Since
pepstatin is a rather specific inhibitor of lysosomal
cathepsin D in cells, this result showed that there must be a
lysosomal pathway for degradation of endogenous proteins
too. Further work with inhibitors of the lysosomal cysteine
proteinases (Shaw & Dean, 1980) and with weak bases, has
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BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Enzymes of the lysosomal metabolic pathways
Proteolytic pathway
Cathepsin D
Cathepsin B
Cathepsin H
Cathepsin L
(Glycosidases and phosphoprotein
phosphatase also act
on intact proteins)
Tripeptidyl peptidase
Dipeptidyl peptidase I
Dipeptidyl peptidase I1
Arginyl aminopeptidase
(cathepsin H)
Peptidyl dipeptidase C
(cathepsin B)
Carboxypeptiase A
Carboxypeptidase B
Prolyl carboxypeptidase
Tyrosine carboxypeptidase
Dipeptidase I
Dipeptidase I1
Glycanolytic pathway
Nuclease pathway
Hyaluronidase
Ribonuclease I1
Heparin endoglucuronidase
Deoxyribonuclease I1
Heparan sulphate endoglycosidase
Lysozyme
Exonuclease (5’-terminal)
Acid phosphatase
a-L-Fucosidase
(There are additional enzymes
a-Galactosidase
with phosphodiesterase,
PGalactosidase
pyrophosphatase, nucleoside
a-Glucosidase
triphosphatase and
j%Glucosidase
phosphoamidase activities
a-N-Acetylgalactosaminidase
in lysosomes)
a-N-Acetylgluwsaminidase
B-N- Acetylglucosaminidase
&Glucuronidase
a-L-Iduronidase
a-Mannosidase
&Mannosidase
Neuraminidase
BAspartylglucosylaminase
Chondroitin bsulphatase
Heparin sulphamatase
Iduronosulphatase
Sulphatases A and B
more precisely defined the size of the lysosomal contribution. Often, it is in the order of 50% of the total intracellular
proteolysis.
The protein substrates for the lysosomal proteases may
originate mainly within the cell, or mainly outside,
depending upon the cell type. Proteolysis is initiated by the
proteinases (endopeptidases), which act in the middle part
of the polypeptide chains. The major lysosomal proteinases
are cathepsin D (an aspartic proteinase), and cathepsins B,
H and L (all cysteine proteinases homologous with papain).
They all have acid pH optima, but the acid pH of the
interior of the lysosomes facilitates proteolysis not only by
providing ideal conditions for the enzymes, but also by
providing an environment in which many substrate proteins
are partially unfolded. This is very important for collagen,
for example, as is shown by the fact that the pH optimum
for the degradation of this protein is distinctly lower than
can be accounted for by the pH dependence of the enzymes
themselves.
One reason for the effectiveness of the lysosomal system
in proteolysis is the very high concentration of some of the
enzymes. We have been abIe to calculate that both
cathepsin D and cathepsin B exist at about 1mM concentration in the lysosomesof human liver (Dean & Barrett, 1976);
this represents 25-40mg/ml, about IOOOO times more
concentrated than they would be in conventional test-tube
assays of proteolytic activity. For this rehson, the real-life
specificities of the enzymes may be much broader than
conventional tests with diIute systems would indicate. Such
breadth of specificity suggests another reason why deficiencies of individual proteinases have not been detected : most
of the enzymes are not indispensable. This situation could
have arisen in evolution to make the important system of
lysosomal proteolysis more robust in the face of genetic
variation. It may explain why the three homologous cysteine
proteinases exist, since copying of the original gene would
protect it against deletion. The enzymes have now diverged
so that each has its own specificity, but if necessary they
could perhaps substitute for one another quite well.
Proteinases are very dangerous enzymes to have at large
in the body, and effective systems always exist for keeping
them localized. The lysosomal proteinases are, of course,
confined by the lysosomal membrane, but additionally,
Lipolytic pathway
Triacylglycerol lipase
Phospholipase A ,
Phospholipase A2
Phosphatidate phosphatase
Acylsphingosine deacvlase
Sphinomykn phosphodiesterase
(Other enzymes including
glywsidases and sulphatases
also make important
contributions to the
lipolytic pathway. Moreover,
there are necessary
‘activator’ proteins)
cathepsin D undergoes a conformational change at neutral
pH that immediately inactivates it completely although
reversibly outside the lysosome. All three of the cysteine
proteinases are irreversibly denatured at neutral pH. This
process takes a few minutes, but there are also protein
inhibitors to regulate the cysteine proteinases. These are the
cystatins in the cytosol, and cystatins, a-cysteine proteinase
inhibitor and a,-macroglobulin in the plasma (Starkey &
Barrett, 1973; Barrett et al., 1984; Gounaris et al., 1984).
It is natural to wonder what interactions exist between
the lysosomal proteinases and the other Iysosomal enzymes.
There is evidence that the lysosomal proteinases bring about
proteolytic processing of a number of the lysosomal
enzymes, but it is obviously essential that this proteolysis is
strictly limited. Naturally, the other lysosomal proteins
would be expected to have evolved resistance to the
proteinases with which they have to co-exist. One way in
which deficiencies of lysosomal enzymes may arise is by a
breakdown of this resistance. There are also instances of
proteins being transported in the lysosomal system without
their being degraded. The mechanisms by which this occurs
are somewhat mysterious, but their existence is another
indication of the sophistication of the lysosomal proteolytic
system.
Intact proteins can be subject to the action of two other
types of lysosomal enzyme, in addition to the endopeptidases. These are the phosphoprotein phosphatases, which
remove phosphate ester groups from proteins such as
casein containing serine phosphate, and the glycosidases
which whittle down the carbohydrate prosthetic groups of
glycoproteins by stepwise attack from the non-reducing
termini. These enzymes form part of the glycanolytic
pathway of the lysosomes, of course.
The oligopeptides produced by the action of the
lysosomal endopeptidases are further degraded by exopeptidases. The activity of these enzymes should not be
underestimated. It is difficult to detect intermediates in
lysosomal proteolysis, and this indicates that the endopeptidases are rate limiting, and that once proteolysis has been
initiated, it is rapidly taken to completion. Many of the
lysosomal exopeptidases are very efficient catalysts. For
example, dipeptidyl peptidase I cleaves more bonds in the
B-chain of insulin than trypsin, chymotrypsin and pepsin
1984
608th MEETING, KEELE
together. Evidence is beginning to appear that the
sequential action of exopeptidases may become important
at an earlier stage in the degradation of many proteins than
has previously been thought. Thus, McDonald & Hoisington (1983) has recently shown the existence of a lysosomal
tripeptidyl peptidase that liberates N-terminal Gly-Pro-X
tripeptides. This is likely to be very effective in breaking up
the tripeptide repeating sequence of denatured collagen
chains. The Gly-Pro-X tripeptides would be favoured
substrates of the lysosomal dipeptidyl peptidase 11, and GlyPro produced by the action of this enzyme would be
expected to diffuse from the lysosomes and be converted
to amino acids by the cytosolic proline dipeptidase
(McDonald & Barrett, 1984).
Both dipeptides and amino acids escape from the
lysosomes to re-enter the metabolic pathways of the cell as a
whole. Cystine derived from disulphide bridges of the
substrate proteins requires a special system to handle it,
however, which fails in cystinosis.
90 1
The lysosomal membrane is permeable to monosaccharides, and undoubtedly the free sugars produced by the
lysosomal exoglycosidases return through it to the cytosolic
pools.
The nuclease pathway
RNA and DNA enter the lysosomal system as a result of
autophagy, and endocytosis of extracellular debris.
Sameshima et al. (1981) have published evidence for dual
pathways of RNA catabolism in a macrophage-like cell
line, one of which was sensitive to NH,Cl, and undoubtedly
lysosomal. It was of interest that the lysosomal pathway was
absent in I-cells, which have defective lysosomal activities.
Both the ribonuclease and deoxyribonuclease of lysosomes have acid pH optima. The lysosomal deoxyribonuclease, deoxyribonuclease 11, differs from the pancreatic
enzyme in that it creates new 3’-phosphate termini, rather
than 5’-termini.Oligonucleotides in the lysosome are further
degraded by the acid exonuclease that cleaves 3’-phosphomononucleotides from the 5’-termini. The lysosome contains several enzymes with acid phosphatase, phosphodiesterase and pyrophosphatase activities to complete the
hydrolysis of these and other phosphates.
The glycanolytic pathway
The lysosomal metabolic pathway that involves the
largest number of individual enzymes is that for degrading
polysaccharides and oligosaccharides. These polymers can The Iipolytic pathway
be referred to collectively as glycans, so I call this the
Ultrastructural features characteristic of lipid assemblies
glycanolytic pathway.
are
commonly seen in lysosomes, and at one time it was
The lysosomal degradation of glycosaminoglycans has
doubted
that lysosomes contain enzymes with significant
been studied by Rome & Crain (1981), with isolated
activity
against
lipids. It is now very clear, however, that
lysosomes containing 3SS-labelled substrate. The process
there
is
an
important
lipolytic pathway in the lysosome, and
was stimulated by ATP plus MgZ+,but the effect was
that
deficiency
of
enzymes
that contribute to it is a serious
abolished by the proton ionophore, nigericin. In the same
matter.
study, evidence consistent with the existence of an
Amongst the enzymes of the lipolytic pathway is
additional non-lysosomal glycanolytic pathway was
triacylglycerol
lipase, which cleaves long-chain fatty acids
obtained.
from
triglycerides
and cholesterol esters. Phospholipids are
Rather surprisingly, endoglycosidases are not prominent
degraded
by
phospholipases
A, and A 2 and by phosphatiin lysosomes, although large polysaccharide molecules
date
phosphatidase.
undoubtedly are degraded there. It seems that much
The complex lipids containing the amino-alcohol sphindegradation occurs by the sequential removal of sugars from
gosine
have several enzymes for their catabolism in
the non-reducing terminal positions of the polymers. Thus,
lysosomes.
0-linked phosphocholine is liberated by sphinit has been shown that the concerted action of P-N-acetylgomyelin
phosphodiesterase,
and 0-linked galactose and
glucosaminidase and fi-glucuronidase can lead to the
degradation of chondroitin sulphate, which is formed of glucose residues are removed by the sequential action of the
alternating residues of N-acetylgalactosamine (the links of appropriate exoglycosidases. Cerebroside 3-sulphate is a
which are also cleaved by the glucosaminidase), and substrate for sulphatase A. Ceramide (N-acylsphingosine) is
glucuronic acid. Similarly, the complex and often branched broken down to the free sphingosine and fatty acid by
structures of glycoprotein carbohydrate groups are degrad- acylsphingosine deacylase, an enzyme deficient in Farber’s
ed by sequential removal of the terminal residues, which disease (Barrett & Heath, 1977).
An especially interesting aspect of the action of the
will include any sialic acid and fucose present, followed by
lysosomal
glycosidases on glycosphingolipids is the existhe intermediate residues, often galactose, mannose and the
N-acetylhexosamines. The final links to the protein may be tence of a number of activator proteins that dramatically
0-glycosidic links to serine or threonine, or N-glycosidic enhance the action of certain of the enzymes on the lipid
links to asparagine. The 0-glycosidic links are hydrolysed substrates. These proteins may well be involved as
by the appropriate exoglycosidases without any require- specialized detergents, bringing the enzymes into contact
ment for degradation of the protein. In contrast, hydrolysis with the hydrophobic substrates, but they show a much
of the N-glycosidic link by P-aspartylglucosylaminase higher degree of specificity than one would normally expect
requires the aspargine to be free, although the hexosamine for such a mechanism of action.
can be substituted.
The glycosidases make an important contribution to the Free-radical reactions
lipolytic pathway through their action on the glycolipids too
Apart from the specialized function of myeloperoxidase
(see below).
in the killing of bacteria in the lysosomal system of
Most of the hereditary deficiencies of lysosomal enzymes leucocytes, rather little is known about oxidative reactions
concern the exoglycosidases, and the nature of the interme- in lysosomes, and yet there are preliminary indications that
diates that accumulate has given much information about such reactions do occur more generally and that free radicals
the pathways for catabolism of carbohydrates. This will be are produced. It is becoming apparent that such radicals
dealt with in detail later in the present Colloquium.
have the capacity to depolymerize proteins, polysaccharides
As a footnote to the degradation of the carbohydrate and polynucleotides. One might therefore speculate that,
polymers, we should notice that several of them occur as under some circumstances at least, free radicals may act in
sulphate esters, and lysosomal sulphatases are available to concert with hydrolytic enzymes in the catabolic pathways
hydrolyse these.
of lysosomes.
VOI.
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BIOCHEMICAL SOCIETY TRANSACTIONS
Barrett, A. J. & Heath, M. F. (1977) in Lysosomes: a Laboratory
H a n d b k (Dingle, J. T., ed.), 2nd edn., pp. 19-145, NorthHolland Publishing Co., Amsterdam
Barrett, A. J., Davies, M. E.& Grubb, A. (1984)Biochem. Biophys.
Res. Commun. 120, 631-636
Dean, R. T. (1975) Nature (London) 257,414-416
Dean, R. T. & Barrett, A. J. (1976) Essays Biochem. 12, 1-40.
Gounaris, A. D., Brown, M. A. & Barrett, A. J. (1984)Biochem. J .
221,445-452
McDonald,'J. K.& Barrett, A. J. (1984) Mammalian Proteases: a
Glossary and Bibliography, vol. 2., Exopeptidases, Academic
Press, London
McDonald, J. K.& Hoisington, A. R. (1983) Fed. Proc. Fed. Am.
Soc. Exp. Bwl. 42, 1781
Mego, J.. Farb, R. M. &Barnes,J. (1972)Biochem. J . 12s. 763-770
Rome,L. H.&Crain,L. R. (1981)J.Bio1. Chem.256,10763-10768
Sameshima, M.,Liebhaber, S. A. & Schlessinger, D. (1981) Mol.
CeII. BWI. 1, 75-81
Shaw, E. & Dean. R.7.(1980) Biochem. J . 186, 385-390
Starkey, P. M. & Barrett, A. J. (1973) B k h e m . J . 131, 823-831
Metabolic consequences of genetic defects in lysosomes
JOSEPH M. TAGER,* LISBETH V. M. JONSSON,*
JOHANNES M. F. G. AERTS,* RONALD P.J. OUDE
ELFERINK,* ANDRe W. SCHRAM,* ANN H.
ERICKSONt and JOHN A. BARRANGERS
*Laboratory of Biochemistry, B.C.P. Jansen Institute,
University of Amsterdam. PO Box 20151, lo00 HD
Amsterdam, The Netherlands, tbboratory of Cell Bwlogy,
The Rockefeller University, New York, NY 10021, U.S.A.,
and $Developmental and Metabolic Neurology Branch,
Natwnal Institute of Neurological and Communicative
Disorders and Stroke, Natwnal Institutes of Health,
Bethesda, MD 20205, U.S.A.
The lysosomal apparatus functions as an intracellular
digestive system, in which hydrolytic enzymes with an acid
pH optimum bring about the breakdown of biological
macromolecules to their low-molecular-weight building
blocks. The digestion ofmacromolecules in the lysosomesis,
in general, a stepwise process: each step is catalysed by a
specific enzyme, and the product of one reaction forms the
substrate for the next reaction in the catabolic pathway.
A deficiency of a lysosomal enzyme leads to accumulation
of the substrates for that enzyme in the lysosomes, and to the
specific biochemical changes and characteristic clinical
symptomsof a lysosomal storage disease. In man, more than
30 hereditary disorders are at present known in which a
lysosomal enzyme, or in some cases more than one enzyme,
is deficient. Within each disease entity in the lysosomal
storage diseases there is considerable heterogeneity.
All lysosomal enzymes that have been studied so far are
synthesized as precursors with molecular masses that are
higher than those of the mature products. All are glycoproteins and it is now clear that the oligosaccharide moiety
plays an essential role in directing lysosomal enzymes to the
primary lysosomes.
The stages in the synthesis and maturation of lysosomal
enzymes may be summarized as follows (Hasilik, 1980;
Hasilik & Neufeld, 1980a, b ; Tager et al., 1981; Sly &
Fischer, 1982; Von Figura & Hasifik, 1984). The synthesisof
the precursor polypeptidesoccurs on membrane-bound ribosomes; a signal sequence (Erickson et al., 1981) and the
signal recognition particle (Erickson et al., 1983) provide a
mechanism for translocation of the nascent polypeptides
across the membrane of the endoplasmic reticulum, where
glycosylation of the polypeptides is initiated. Asparagine
residues occurring in the tripeptide sequence -Asn-X-Ser-or
-Am-X-Thr- serve as acceptors for a complex oligosaccharide containing two N-acetylglucosamine, nine mannose and
three glucose residues which is transferred en bloc from a
lipid carrier to the polypeptide chain. In the third stage,
processing of the oligosaccharide occurs, which involves
stepwise cleavage of glucose and some mannose residues.
So far, the events that occur are common to all Nglycosidicallylinked glycoproteins. However, in the case of
lysosomal enzymes, the processing includes formation of
mannose 6-phosphate groups in the glycoprotein so that a
precursor molecule is formed with oligosaccharide chains
'that contain mannose &phosphate. This occurs in two
steps. Firstly, a specific N-acetylglucosaminylphosphotransferase catalyses the transfer of N-acetylglucosaminylphosphate from UDP-N-acetylglucosamine to
specific mannose residues in the oligosaccharide moiety of
lysosomal enzymes (Hasilik et al., 1981; Reitman &
Kornfeld, 1981). Secondly, a specific N-acetylglucosaminylphosphodiesterase catalyses the hydrolysis of the Nacetylglucosamine groups, so that mannose &phosphate
groups are exposed (Varki & Kornfeld, 1980; Waheed et ul.,
1981).
The fourth stage involves binding of the precursor
molecule to mannose 6-phosphate-specific receptors in the
Golgi apparatus; Brown & Farquar (1984) have recently
presented evidence suggesting that this occurs in the cis
Golgi cisternae. Finally, the precursor is transferred into
vesicles destined to become primary lysosomes; this is
accompanied by cleavage of the polypeptide chain. The
proteolytic cleavage may occur in several steps, some in
prelysosomal compartments. Further processing of the
oligosaccharide moieties, including removal of phosphate,
may occur.
Thus several genes are involved in the formation of a
mature lysosomal enzyme: the structural genes for the
polypeptides and the genes coding for enzymes involved in
the post-translation modifications of the polypeptides
which lead to the formation of precursors that can be
transported to the lysosomes.
Our present knowledge of how genetic defects lead to
deficiencies of lysosomal enzymes and hence to lysosomal
storage diseases is summarized in Table 1.
Consequences of a deficiency of N-acetylglucosaminylphosphotransferase :studies with cultured fibroblasts
Mucolipidosis 11, also known as I-cell disease, and
muco1,ipidosis 111, or pseudo-Hurler ieukodystrophy, were
the first lysosomal storage diseases to be described in which
defective processing occurs. These diseases are due to a
deficiency of the enzyme responsible for transferring
N-acetylglucosaminylphosphate from UDP-N-acetylglucosamine to mannose residues in the glycoprotein; the
deficiency is complete in mucolipidosis I1 (Hasilik et al.,
1981; Reitman et al., 1981) and partial in mucolipidosis
111 (Reitman et al., 1981; Hasilik et al., 1982). Thus, the
precursors of the lysosomal enzymes lack the mannose 6phosphate recognition marker and cannot be transported to
the lysosomes. Instead, they are secreted into the medium.
In fibroblasts from patients with these diseases several
lysosomal hydrolases are deficient (Leroy et al., 1972; Miller
et al., 1981). Different forms of mucolipidosis I11 can be
distinguished biochemically (Varki et al., 1981) and the
results of complementation studies indicate that at least
three genes are involved in the expression of N-acetyl-
1984