Bioscience Reports, Vol. 6, No. 11, 1986
Species Variations Amongst Proteinases in
Liver Lysosomes
Daniel Bi~ehet, Alain Obled and Christiane Deval
Received January 2, 1987
KEY WORDS: speciesvariation; cystein proteinases; cathepsin D; liver lysosome.
Cathepsin B, H, L and D activities in liver lysosomes were compared between species.
Although cathepsin B and D were detected in bovine, pig, chicken and rat liver,
striking species differences were evident for cathepsin H and L. Cathepsin L activity
was particularly high in chicken lysosomal extracts, but could not be detected in
bovine and pig extracts. Whereas there was no significant cathepsin H activity in
bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H
activity.
INTRODUCTION
To understand the mechanisms governing protein catabolism, we need to describe the
proteolytic enzymes involved in this process. Lysosomes are particularly rich in
exopeptidases and proteinases and are thus believed to play an important role in tissue
proteolysis (1). Four lysosomal proteinases are usually distinguished: three cystein
proteinases, namely cathepsins B, H and L, and the carboxyl proteinase, cathepsin D.
Amino acid sequences have recently been determined for rat liver cathepsins B and H
(2), human liver cathepsin B (3) and porcine spleen cathepsin D (4).
Different concentrations of cathepsins B, H and L exist in different tissues of the
rat (5, 6), and this might partly explain the differences in protein catabolism observed
in different tissues (7). Rates of protein turnover are also reported to differ between
species (8, 9), but there is yet no extensive data on whether this might be related to
different levels of lysosomal cathepsins. This is the first report showing large species
variations amongst cathepsins in liver lysosomes. Such species differences are likely to
have important implications when considering the role lysosomes and cathepsins play
in protein catabolism.
S.R.V., Unitb de Recherchessur les Prot~ines Musculaires, INRA Theix, 63122Ceyrat, France.
991
0144-8463/86/1100-0991505.00/0 9 1986 Plenum Publishing Corporation
992
B~chet, Obled and Deval
MATERIALS AND METHODS
Chemicals
Methylcoumarylamide substrates, benzyloxycarbonyl-Phe-Arg-4-methyl-7coumarylamide
(Z-Phe-Arg-NMec),
benzyl-oxycarbonyl-Arg-Arg-4-methyl-7coumarylamide (Z-Arg-Arg-NMec) and arginine-4-methyl-7-coumarylamide (ArgNMec) were purchased from Bachem Feinchemikalien A.G. (Bubendorf, Switzerland).
Benzyloxycarbonyl-Phe-Phe-CHN2 (Z-Phe-Phe-CHN2) was kindly provided by Dr
E. Shaw (Basel, Switzerland). Pepstatin was purchased from Serva-Tebu (France) and
Brij-35 from Merck (France). All other reagents were of analytical grade.
Preparation of Lysosomal Extracts
Livers from heifer, pig, chicken and rats were obtained rapidly after death from
the Institute slaughter house, and lysosomes were prepared mainly as described in (10).
Livers were minced and homogenized with a Potter glass-teflon homogenizer in 10
volumes of 10 mM sodium phosphate buffer, pH 7.4, containing 0.25 M sucrose and
1 mM EDTA. The tissue homogenate was centrifuged 10 min at 1000 x g and then at
4000 x g for another 10 rain. The supernatant was centrifuged at 20,000 x g for 15 min
and the lysosomal pellet homogenized in 30 mM sodium phosphate buffer, pH 5.8, and
frozen at -20~
The Iysosomal homogenate was thawed and dialysed overnight
against 30 mM sodium phosphate buffer, pH 5.8. The lysosomal extract was recovered
after 30rain centrifugation at 100,000 x 9. Aliquots of both tissue and lysosomal
homogenates were also made 0.2 % Triton X-100 and stored at - 2 0 ~ until further
analysis of N-acetyl-fl-D-glueosaminidase activity (11) and protein content (12).
According to N-acetyl-fl-D-glucosaminidase activity, yields of lysosomes were
44 _+2 % (n = 6), 44 _+ 15 % (n = 6), 40 ___10 % (n = 6) and 32 _+ 5 %'(n = 6) for bovine,
rat, pig and chicken liver, respectively.
Mono-S Chromatography and Gel Filtration Studies
A mono-S column from a Pharmacia FPLC system was pre-equilibrated with
30 mM sodium phosphate buffer, pH 5.8. When indicated, lysosomal extracts were
applied on the column and eluted with a linear gradient of NaC1 (0-0.8 M). The flow
rate was 0.8 ml. rain- t and 0.8 ml fractions were collected and studied for cathepsin
activities.
Assays of Cathepsins B, H and L
Incubation buffer for thiol proteinases was 100mM sodium acetate, 5 mM
dithiothreitol and 1raM EDTA, pH 5.5. Methylcoumarylamide substrates were
10 mM in dimethyl-sulfoxide and, before use, diluted to 40 #M with 0.1% (w/v) Brij35. Z-Arg-Arg-NMec and Arg-N-Mec were used as substrates for cathepsins B (13)
and H (14), respectively. Cathepsin L was detected using Z-Phe-Arg-NMec as
substrate (13).
Proteinases of Liver Lysosomes
993
For each assay, 0.75 ml of incubation buffer was pre-incubated 5 min at 37~ with
5-20 #1 of enzyme sample. Assays were started by adding 0.25 ml of 40 #M substrate
solution, were incubated 5-15 min at 37~ and reactions were stopped by introducing
3 ml of 10 mM sodium acetate, 25 mM acetic acid buffer, pH 4.3, containing 30 mM
sodium chloroacetate. The concentration of product was measured in a Perkin-Elmer
LS-5 spectrofluorimeter, with excitation at 360 nm and emission at 460 nm, using
aminomethylcoumarin solutions as standards. The volume of enzyme sample and the
time of incubation were settled so that less than 10% substrate (1/~M) was hydrolysed.
Titration of Cathepsin D
Cathepsin D was titrated at pH 3.5 as described by Knight and Barrett (15), using
hemoglobin as substrate and pepstatin as inhibitor.
RESULTS AND DISCUSSION
When liver lysosomal extracts from different species were initially studied by
HPLC chromatography on mono-S column, distinct elution profiles of cystein
proteinases were evident (Fig. 1). Cathepsin B was identified with its specific substrate
Z-Arg-Arg-NMec (13) and was for all 4 species, pig, heifer, chicken and rat, not
retained on mono-S column at pH 5.8. However, pig liver lysosomal extracts also
exhibited a second form of cathepsin B which was only eluted with 0.2 M NaC1 (Fig.
1B).
Cathepsin H activity was detected using Arg-NMec as specific substrate (14). Like
cathepsin B, Arg-NMec activity was directly eluted unretarded from mono-S column.
Cathepsin H was however evident only for pig, chicken and rat lysosomal extracts (Fig.
1B, C, D). This Arg-NMec activity corresponded also to a peak of 25-30 Kdaltons by
gel filtration on Sephadex G-75, and was therefore not due to any high-Mr aminopeptidase (not shown).
Like cathepsin B, cathepsin g hydrolyses Z-Phe-Arg-NMec, but in contrast with
cathepsin B, has no activity on Z-Arg-Arg-NMec (13). A cathepsin L peak of Z-PheArg-NMec activity with no concomitant Z-Arg-Arg-NMec activity was, on mono-S
column, only evident for rat and chicken extracts, and was eluted with 0.4 M NaCI and
0.6 M NaC1, respectively (Fig. 1C, D). Cathepsin L activity recovered from mono-S
column was also shown to be inhibited by Z-Phe-Phe-CHN 2 and inactivated by a
treatment at neutral pH (see Fig. 2). No significant peak of cathepsin L activity could
be observed for bovine and pig liver lysosomal extracts by mono-S chromatography at
pH 5.8 (Fig. 1A, B).
These preliminary studies therefore suggested that bovine liver contained only
cathepsin B activity. Pig liver lysosomal extracts presented additional cathepsin H
activity. In contrast, rat and chicken liver lysosomal extracts revealed not only
cathepsins B and H, but also cathepsin L activity. At this stage, it was important to
verify that different profiles of cystein proteinases in different species were not related
to any loss of activity due to HPLC chromatography on mono-S columns. A better
994
Bechet, Obled and Deval
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Fig. 1. FPLC chromatography on Mono-S
column of lysosomal extract from (A) bovine, (B)
pig, (C) chicken and (D) rat liver. Activities against
Z-Phe-Arg-NMec ([~), Z-Arg-Arg-NMec (O) and
Arg-NMec (0) were measured as described in
Materials and Methods section. (...... ) NaC1
concentration.
comparison between species also required to develop a methodology allowing
proteinase activities to be directly measured in unfractionated lysosomal extracts.
The levels of cathepsin B and H activities could be measured directly in lysosomal
extracts using the specific substrates Z-Arg-Arg-NMec and Arg-NMec, respectively.
The lysosomal carboxyl proteinase cathepsin D could also be titrated at pH 3.5 using
hemoglobin as substrate and pepstatin as specific inhibitor (15). However, no specific
substrate has yet been described for cathepsin L. We therefore compared two methods
classically used to discriminate Z-Phe-Arg-NMec activity of cathepsin L from that of
cathepsin B (Fig. 2). The first one involves specific inhibition of cathepsin L by Z-PhePhe-CHN z (16). Low concentrations of this compound strongly inhibited cathepsin L
activity recovered from mono-S chromatography (Fig. 2A). It also appeared that,
under our assay conditions, chicken liver cathepsin B was significantly inhibited even
by low concentrations of Z-Phe-Phe-CHN2. Therefore, the use of this inhibitor did not
allow an unambiguous distinction of cathepsin L from cathepsin B Z-Phe-Arg-NMec
activity in unfractionated lysosomal extracts, at least with chicken liver.
The second method involves preliminary inactivation of cathepsin L at neutral
pH (1). As shown in Fig. 2B, chicken and rat cathepsin L were rapidly inactivated at
pH 7.3. Cathepsin B from rat, bovine or pig liver was resistant, up to 40 min at neutral
pH, and chicken cathepsin B revealed significant inhibition but only after 20 min at pH
7.3. Therefore, cathepsin L activity could be directly assessed in liver lysosomal
Proteinases of Liver Lysosomes
995
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Fig. 2. Inhibition of cathepsin B and cathepsin L Z-Phe-Arg-NMec activity
by Z-Phe-Phe-CHN2 or neutral pH. Cathepsin B and cathepsin L fractions
recovered from Mono-S chromatography were either (A) preincubated with
different concentrations of Z-Phe-Phe-CHNz at 37~ for 5 rain, or (B) adjusted
to pH 7.3 and pre-incubated at 37~ for the indicated times. The residual ZPhe-Arg-NMec activity was then assessed as described in Materials and
Methods section. Rat cathepsin L (O); chicken cathepsin L (A); cathepsin B
from rat (O); chicken (A); pig (E]) and heifer (0).
extracts by comparing Z-Phe-Arg-NMec activity, with or without prior inhibition at
pH 7.3 for 20 min. For all four species, this pretreatment induced no more than 5
inhibition of cathepsin B activity, while inactivating rat and chicken cathepsin L by
70 ~ and 85 ~o, respectively.
Using these procedures , the levels of cathepsin B, H and L activities and the
concentration of active cathepsin D in liver lysosomal extracts from heifer, pig, chicken
or rat were measured and the results are presented in Fig. 3. For all 4 species, lysosomal
extracts contained at least cathepsin B and D activities. In fact, for bovine liver these
were the only activities which were detected, and their levels were the lowest when
compared with those of other species. Pig liver lysosomes were particularly rich in
cathepsin B activity, whereas cathepsin D was highly concentrated in both pig and
chicken liver lysosomes. Striking species differences amongst proteinase activity in
liver lysosomes were evident, mostly for cathepsin L and H. Very high levels of
cathepsin H activity were observed only in rat liver lysosomes, and very high levels of
cathepsin L activity only in chicken liver lysosomes. Moreover, using the same
experimental procedures, no significant cathepsin H and L activity could be detected in
bovine liver lysosomes and no significant cathepsin L activity in pig liver lysosomes.
The cathepsins H and/or L cannot be expressed in any tissue of bovine and pig
does not however seem to be the case. There are indeed indications that cathepsins H
and L are present in porcine spleen (17) and bovine spleen (18), as welt as at early stages
of bovine muscle differentiation (B~chet, D and colleagues, unpublished observations).
Therefore, cathepsin H and L genes not only exist in bovine and pig genomes, but also
can be transcribed, and result in expression of active enzymes at least in some tissues or
at some stage of development. Several mechanisms could possibly explain the absence
ofcathepsin H and L activities in bovine liver lysosomes and the absence ofcathepsin L
activity in pig liver lysosomes. Genes for cathepsins H and/or L might not be
996
B~het, Obled and Deval
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Fig. 3. Comparisonbetweenspeciesofcathepsin B, H, L and
D activities from liver lysosomes. Unfractionated lysosomal
extracts were studied for cathepsin B, cathepsin H and
cathepsin D activity as described in Materials and Methods
section. Cathepsin L activity was estimated as the difference
betweenZ-Phe-Arg-NMecactivitymeasuredwithout and that
measured with prior incubation for 20 min at pH 7.3. Vertical
bars represent standard deviations of mean values from 6
animals.
transcribed, or the mRNA encoding the proteinase might be unstable or not
translated, cDNA probes, not yet isolated for cathepsins H or L, should help to resolve
these points. Alternatively, these cathepsins might be present, eventually as precursor
forms and elsewhere other than in lysosomes, or exist in lysosomes but inactivated by
endogenous inhibitors (19).
Whatever the exact mechanism, activities of cathepsins in lysosomal extracts are
likely to reflect the proteolytic potential of lysosomes. In this regard, it is interesting to
emphasize that full activity of all four cathepsins, B, H, L and D, does not seem to be an
absolute requirement for basal proteolysis in liver of all species. According to our
results, liver protein catabolism occurs even with a limited pattern of active cystein
proteinases in lysosomes. One suggestion would be that the role lysosomes play in
protein catabolism varies from one species to another. Another likely explanation
would be that only one type of cathepsin, if sufficiently concentrated inside lysosomes
(20), exhibits a specificity large enough to hydrolyse most protein substrates.
Proteinases of Liver Lysosomes
997
ACKNOWLEDGEMENT
We are most grateful to F. Thomas for typing the manuscript.
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