BIOCHEMICAL
Vol. 79, No. 3, 1977
CYSTIC FIBROSIS AND OF ALKALINE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
: LEAKAGE OF LYSOSOMAL ENZYMES
PHOSPHATASE INTO THE EXTRACELLULAR
SPACE
P. H&sli and E. Vogt Department of Molecular Biology Institut Pasteur 25, rue du Dr. Roux, 75015 Paris, France
Received
June 16, 1977 SUMMARY
Nine lysosomal enzymes and alkaline phosphatase have been assayed with two different ultramicro techniques in the intraand extracellular space of fibroblast cultures derived from the skin of cystic fibrosis patients, cystic fibrosis carriers, and normal controls, respectively. Evidence has been obtained for a multiple leakage of lysosomal enzymes and of alkaline phosphatase into the medium of fibroblast cultures from cystic fibrosis patients and carriers. The situation is comparable to a certain extent, to that observed in I-cell-disease (mucolipidosis II). This multiple leakage results in the decrease of intracellular activity of several lysosomal enzymes in cultures from cystic fibrosis patients and carriers and due to the coordinate regulation of the synthesis of the "leaky enzymes" in an overshooting of the intracellular alkaline phosphatase activity in cultures from cystic fibrosis patients. It also explains the retarded catabolism of certain molecules, such as the Tamm-Horsfall glycoprotein, in cystic fibrosis cells. It is speculated that the basic defect in cystic fibrosis leads to abnormal recognition sites on lysosomal enzymes and on alkaline phosphatase, and in consequence to the leakage of these enzymes into the extracellular space. The present findings allow one to develop methods for the pre- and postnatal diagnosis of cystic fibrosis with cell cultures, and for the detection of cystic fibrosis carriers with the peripheral blood. Cystic
fibrosis
of Caucasian important. in cystic methods
descent
and its
Many laboratories
are,
fibrosis, for
mutation
by a multiple culture
and trying
the detection
which
I-cell-disease.
the most
(I),
The present
the
(CF) is
paper affects
medium
(2),
social
hereditary
and economic
therefore,
to develop
in people
implications
searching diagnostic
disease
for
are very
the primary
procedures
defect
as well
as
of CF-carriers. demonstrates lysosomes
The phenotype leakage
frequent
that
in a way which
of cultured
of lysosomal
cystic
is
is
somewhat
a type
enzymes
into
of
is characterized
the extracellular increase
of
reminiscent
I-cell-fibroblasts
and by a compensatory
Copyright 0 1977 by Academic Press, Inc. At1 rights of reproduction in any form reserved.
fibrosis
of the
space,
i.e.
intracellular
741 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 3, 1977
activity the
of non-leaky
latter
level,
enzymes
phenomenon
experiments
and of alkaline
is due to a coordinate
of the replacement All
of these
have been
the expression
of enzyme activities
per-protein-basis.
They are,
very
fine
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fluctuations
(AP),
regulation,
enzymes
done with
phosphatase
(3).
ultramicro
techniques.
MATERIALS
They allow
instead
particularly
of enzyme activities
;
at the transcriptional
on a per-cell-basis
therefore,
respectively
well
in cultured
of on a
suited cells
to monitor (4,5).
AND METHODS
Cell culture conditions (6), ultramicro methods to measure enzyme activities at the single cell level (4,5,6), individual enzyme assays based on the use of artificial fluorogenic methylumbelliferone-coupled substrates (8), the isolation of urinary Tamm-Horsfall glycoprotein (TW ; 6) and the principle of AP-induction with THP (6) have been described elsewhere. For the present studies the AP-induction procedure has been modified and two different ultramicro techniques have been used. Technique I . Culture medium : HAM FlO with 15% FCS. Fibroblasts were seeded into5PZastic Film Dishes (PFD's ; 4,7) with an initial concentration of 1.5 x 10 cells per PFD .AP-induction : 24 hours after seeding, the fibroblast cultures were treated for 3 consecytive days with THP (100 ng/mJ medium) ; for the last 2 days of induction 1 x 10 -5M isoproterenol and I x 10 M theophylline and for the last day 1 x 10 M isoproterenol were given in addition into the THP-containing medium. Then the cells were washed, lyophilized, isolated from the PFD's and enzyme activities were assayed in ParafiZm Micro Cuvettes lPMC'.sl as described previously (3,4,5,6,8). Technique II . Culture medium : HAM FIO with 15% FCS. Fibroblasts were grown to confluency in PFD's and then induced for 3 consecutive days with THP as described Under-Technique I. Then the induction-medium was replaced for 24 hours by HAM FIO with 0.1% heat-inactivated FCS to collect extracellular enzymes ; this medium was cleared by centrifugation (10,000 g for 5 minutes) and lyophilized to concentrate the collected extracellular proteins. The cells, remaining in the PFD, were washed, trypsinized and diluted with saline to a final concentration of 50 cells per 0.1 ~1 ; the cells were then disrupted by 3 cycles of freezing and thawing and the 10,000 g supernatant fraction of this extract was used for the enzyme assays. For the determination of enzyme activities in the extracellular space the lyophilized medium was dissolved in water h d been conditioned by 50 cells. in such a way that 0.1 ~1 of this solution Enzyme activities are expressed in 10 -14 moles of substrate converted per cell per hour or as the percentage of the mean activities of controls.
RESULTS Table cell
1 reviews
strains
controls.
from
the
intracellular
5 patients
The intracellular
a-galactosidase,
cL-mannosidase
with activities
enzyme activity I-cell-disease
profiles
as compared
of S-galactosidase,
and S-glucuronidase
742
are
in fibroblast to 16 normal
hexosaminidase, significantly
(i.e.
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 : Intracellular enzyme activity profiles, relative values, fibroblast cell strains from 5 patients with I-cell-disease and from 16 normal controls. Technique cf. methods. Each determination was carried out in five-fold with 20 cells per incubation.
Controls, n = 16 mean + SD%
pat.1
b-galactosidase
100 k 9.8% ( 4.3)%
hexosaminidase
100 ?13.4%
a-galactosidase
100 + 8.3%
pat.2
pat.3
pat.4
I,
pat.5
8%
6%
6%
9%
6%
(28.5)
17%
19%
15%
25%
15%
( 0.5)
19%
22%
9%
45%
8%
cl-mannosidase
100 +_ 8.3% ( 0.6)
27%
29%
29%
30%
36%
@-glucuronidase
100 f 9.1% ( 0.7)
31%
20%
8%
105%
15%
a-glucosidase
100 * 7.0% ( 0.6)
218%
238%
157%
625%
153%
S-glucosidase
100 + 6.9% ( 0.8)
168%
171%
174%
350%
184%
acid
100 + 8.5% (19.5)
199%
234%
217%
299%
195%
100 +_ll.S%
415%
465%
696%
2461%
335%
indicate
mean enzyme activities
phosphatase
alkaline
phosphatase
e values in brackets moles of substrate
below the
-3 S.D.)
and alkaline +3 S.D.)
column
the exception
of b-glucuronidase
of ct-glucosidase,
phosphatase,
on the other
B-glucosidase,
hand,
in patient acid
are significately(i.e.
in
4 ;
phosphatase to over
increased.
in fibroblast
cell
Table
B-galactosidase, decreased
as compared
the -3 S.D.
; o-galactosidase phosphatase
is displayed
to a dramatic
from
family
1 with
level, is
are
only
of the
enzyme activity cystic
increased
is
AP-activities
743
phosphatase
while
are
clearly
of -3 to
a-glucosidase
the +3 S.D.
CF-heterozygotes.
intracellular
(2 CF-homozygotes
on the border
decreased, to or over
profiles
In the CF-homozygotes
and acid
B-glucosidase slightly
fibrosis
controls.
cr-mannosidase
by the obligate
increase
intracellular
to 16 normal
hexosaminidase,
below
and alkaline
2 presents
strains
2 CF-heterozygotes)
trend
with
activities
In comparison,
-2 S.D.
in the control converted/cell/hour.
decreased,
intracellular
( 0.5)
Induction
level. with
The same THF' leads
in the CF-children
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 2 : Intracellular enzyme activity profiles, relative values, fibroblast cell strains from family 1 with cystic fibrosis (2 CF-homozygous sibs, 2 CF-heterozygous parents) and from 16 normal controls. Technique I, cf. methods. Alkaline phosphatase without and with THP-induction (cf. methods). Each determination was carried out in five-fold with 20 cells per incubation. Controls, n = 16 mean * SD%
sib
I
sib
?
2d)
father
mother
S-galactosidase
100 i: 9.8% ( 4.3)*
58%
63%
67%
69%
hexosaminidase
100 +13.4%
(28.5)
53%
58%
73%
79%
a-galactosidase
100 f 8.3%
( 0.5)
80%
89%
81%
79%
a-mannosidase
100 k 8.3%
( 0.6)
55%
65%
77%
75%
S-glucuronidase
100 f 9.1% ( 0.7)
110%
120%
101%
107%
a-glucosidase
100 f 7.0% ( 0.6)
165%
120%
91%
90%
S-glucosidase
100 t 6.9% ( 0.8)
69%
89%
83%
87%
acid
100 2 8.5% (19.5)
50%
66%
87%
83%
( 0.5)
210%
180%
105%
99%
alkaline phosphatase THP-induced
100 +-18.0% ( 0.7)
380%
430%
120%
130%
values in brackets :,-n emoles of substance
in the control converted/cell/hour.
indicate
mean enzyme activities
phosphatase
alkaline
but
phosphatase
to only Fig.
a very moderate 1 reviews
THF'-induction (not
100 +11.8%
increase
with
the CF- homozygotes
cell
family the
a-mannosidase,
a-glucosidase
and S-glucosidase of the
and intracellular increased.
same
alkaline
from
activities
a-galactosidase,
enzymes
are below are
far
phosphatase
In the CF-heterozygotes
family
2) as compared
intracellular
the
parents.
enzyme activity
cultures
in Table
a-fucosidase,
activities
in the heterozygous
and extracellular
intra-
in fibroblast
identical
column
2 with
cystic
to 14 normal of acid
the -3 S.D.
values
are also
extracellular
after fibrosis
controls.
In
phosphatase,
b-galactosidase,
hexosaminidase,
level
above the +3 S.D.
744
profiles,
; the extracellular level
very
; the extra
significantly
enzyme activities
follow
in
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I r I
q
DC
100
100. .
a
b
0
o.
:
Fig.]. Intraand extracellular enzymes activity profiles, relative values, fibroblast cell strains from family 2 with cystic fibrosis and from 14 normal controls, after 'HP-induction. Technique II, cf. methods (a-mannosidase, a-galactosidase, a-glucosidase, B-glucosidase were incubated at 25'C, the other enzymes at 37°C). Each point is the mean of 5 determinations and corresponds to 50 cells or the medium conditioned by 50 cells. The horizontal 100% bar corresponds to the mean of 14 normal control cell strains with the following standart deviations (intracellular/extracellular) : alkaline phosphatase (8.7/11.5) ; acid phosphatase (6.9/22.8) ; o-fucosidase (5.3/20.3) cl-mannosidase (7.1/14.7) ; cr-galactosidase (8.4127.8) ; 8-galactosidase (6.9/12.3) ; hexosaminidase (6.618.2) ; cl-glucosidase (8.9125.6) ; b-glucosidase (4.6/18.9) ; @-glucuronidase (7.1/14.3). a. CF-children (9 ,c?) : A, intracellular ; A, extracellular b. Parents (obligate heterozygotes) : m, intracellular ; q , extracellular
the
same trend,
icant
activity
o-galactosidase,
with decrease
the exception is
of B-glucosidase
detectable
8-galactosidase
only
for
and o-glucosidase
745
; intracellularly
a-fucosidase,
a signif-
o-mannosidase,
; the intracellular
activity
;
Vol. 79, No. 3, 1977
of alkaline
BIOCHEMICAL
phosphatase
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in particular
is normal
in heterozygotes.
DISCUSSION This (6)
study
permits
CF-patients
confirms
one,
as a rule,
on the
To the present, with
into
only,
data
picture which
shown)
CF-cases
THP-induceable
that
will
where
different
be found
is
the
;
that
index-patient
the
is
This
or heterozygous and 15 CF-heterozygotes
the multiple
in the
enzyme
for
type
the studied
the
of cystic
other
lysosomal
it enzymes
clinical fibrosis,
enzymes,
Alternatively,
leakage.
none of
; in addition, rare
THP-
phosphatase
are not THP-induceable,
leaky
for
THP-induceable
or heterozygotes
display
syndrome.
leaky
treated
and of alkaline
CF-homo-
unusual.
nor
were
CF-homo-
show enzyme leakage is very
on the other. have been
14 CF-homozygotes
2 sibs
between
are THP-induceable.
by all
: all
where
cultures
controls
enzymes
index-case
or heterozygotes
completely
could is
be
possible
and induceable
substrates.
The multiple phosphatase
enzyme
(Table
of acid
results
phosphatase CF-heterozygotes (3,6,8).
leakage
2, Fig.
enzyme activities.
weaker
with
glycoprotein
The conclusion
in families
displayed
family,
a biochemically
this
is
not
CF-cases
of lysosomal
a THP-induceable
is neither
enzymes,
that
9 families
(data
of these
by different
diagnosed
leakage
So far
such types
and normal
not THP-induceable.
indicate
In the one exceptional the CF-homo-
were
space
have been analyzed with
fibroblast
51 of the CF-homozygotes
a multiple
families
CF-carriers
of clinically
members.
with
glycoprotein.
the extracellular
family
to discriminate
Tamm-Horsfall
and 25 CF-heterozygotes
The present induceable,
with
53 CF-homozygotes
sibs
majority
the AP-induction
one hand,
Tamm-Horsfall
2 CF-homozygous vast
that
I),
results,
in a decrease
The coordinate
regulation
and of alkaline
but This
here would
of the (Fig.
the
la).
of alkaline
of the corresponding
(3,6,8)
intracellular
signal
the absence
746
of lysosomal the
activity
for
intracellular
counteracts
The same regulation
stimulatory
explain
the exception
of the synthesis
phosphatase
in an overshooting in CF-homozygotes
with
leakage
of alkaline takes
place
the enzyme induction
of an overshooting
of intra-
in is
;
BIOCHEMICAL
Vol. 79, No. 3, 1977
cellular
alkaline
phosphatase
of the intracellular
activity
activities
-heterozygotes
(Fig.
The primary
defect
in cystic
fibrosis
and alkaline
This
reminiscent
of I-cell-disease
cystic
(lysosomal It
their
suggested
secretion
receptors
specific
affect
would
the normal
likely
qualify
in these
syndromes
multiple
enzyme leakage.
enzyme leakage likely
that
Different
mutations
recognition
site.
in the population the
leakage
(Table
could, This
leaky
as the
explain
the very
of mutants)
enzymes.
in family
2 (Fig.
high
(6).
747
would
explain
why
display
(IO)
different frequency
and also
1) ; this
fibrosis
we found
complex
with
a-glucosidase
on the
the
a multiple ; it
seems
mutant.
relatively
interfere
sites
homozygotes
the heterozygotes
could
; the one would
cystic
This
syndrome
therefore,
might
study,
hand.
which
granules
mutants.
a receptor
are probably
(heterogeneity
of specific
2) but
with
not
on the other
the recognition
the present
lysosomes,
on the one hand,
of mutations
the other
; that
such as primary
of digestive
site
hydrolases
and uptake
enzymes types
as well
but
between
be quantitative
of lysosomal
of the granules
In the Chediak-Higashi
sites
possibly
granules,
as recognition
we are dealing
The difference
recognition
two different
the heterozygotes
Enzyme recognition
no data
on the corresponding
in the homozygotes
there
in I-cell-disease
(phosphatases).
or matrix
and following
of lysosomal
though
by specific
in the granules,
to a loss
compartment.
the packaging
self-assembly
I-cell-disease,
most
that
followed
to find
the receptors
enzymes.
(9)
sites
expect
with
in the CF-
the extracellular
therefore,
of enzyme containing
recognition
interfere
normalization
into
as qualitative
on the membrane
One would
enzymes
of phosphatases. could,
as well
the self-assembly
needs
towards
seems to lead
(2),
and I-cell-disease
has been
leaky
phosphatase
a leakage
hydrolases)
requires is,
concerning
fibrosis
tendency
lb).
and of acid
are available
and the
of the other
enzymes is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is,
e.g.,
configurations. parts
of CF-carriers
the variability not
of the
leaky
has been observed
concerning in family previously
I
BIOCHEMICAL
Vol. 79, No. 3, 1977
This
first
evidence
"processing
mutation"
Preliminary
studies
amounts screen
in
also
the primary
allows
indicate
the blood
populations
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
defect
one to design the
leaky
of CF-heterozygotes for
in cystic
fibrosis
new diagnostic
enzymes
and that
are it
will
present
is
a
strategies. in
increased
become possible
to
CF-carriers. ACKNOWLEDGEMENTS
This DGRST (Nr.
study
has been
75 70 004)
supported
by grants
and the Fondation
from
INSERM (ATP 36 76 68),
de France.
REFERENCES 1. Di Sant'Agnese, P.A., and Davis, P.B. (1976) New Engl. J. Med. 295, 481-485. 2. Lightbody, J., Wiesmann, U., Hadorn, B., and Herschkowitz, N. (1971) Lancet r, 451 only. 3. Hi;sli, P. (1976) Adv. Exp. Med. Biol. 68, I-13. 4. HEsli, P. (1972) Tissue cultivation on plastic films, Tecnomara, Zirich. Defects, pp. 226-233, Excerpta Medica, Amsterdam. 5. H&li, P. (1974) Birth 6. Hgsli, P., Erickson, R.P., and Vogt, E. (1976) Biochem. Biophys. Res. Commun. 73, 209-216. (1977) Adv. Exp. Med. Biol. s, 591-596. 7. H&.li, P., and de Bruyn, C.H.M.M. 8. Hcsli, P. (1977) Clin. Chem. 3, p. 14761484. 9. Hickman, S., and Neufeld, E.F. (1972) Biochem. Biophys. Res. Commun. 2, 992-999. 10. HEsli, P., Griscelli, C., and Good, R.A., manuscript in preparation.
748
Vol. 79, No. 3, 1977
CYSTIC FIBROSIS AND OF ALKALINE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
: LEAKAGE OF LYSOSOMAL ENZYMES
PHOSPHATASE INTO THE EXTRACELLULAR
SPACE
P. H&sli and E. Vogt Department of Molecular Biology Institut Pasteur 25, rue du Dr. Roux, 75015 Paris, France
Received
June 16, 1977 SUMMARY
Nine lysosomal enzymes and alkaline phosphatase have been assayed with two different ultramicro techniques in the intraand extracellular space of fibroblast cultures derived from the skin of cystic fibrosis patients, cystic fibrosis carriers, and normal controls, respectively. Evidence has been obtained for a multiple leakage of lysosomal enzymes and of alkaline phosphatase into the medium of fibroblast cultures from cystic fibrosis patients and carriers. The situation is comparable to a certain extent, to that observed in I-cell-disease (mucolipidosis II). This multiple leakage results in the decrease of intracellular activity of several lysosomal enzymes in cultures from cystic fibrosis patients and carriers and due to the coordinate regulation of the synthesis of the "leaky enzymes" in an overshooting of the intracellular alkaline phosphatase activity in cultures from cystic fibrosis patients. It also explains the retarded catabolism of certain molecules, such as the Tamm-Horsfall glycoprotein, in cystic fibrosis cells. It is speculated that the basic defect in cystic fibrosis leads to abnormal recognition sites on lysosomal enzymes and on alkaline phosphatase, and in consequence to the leakage of these enzymes into the extracellular space. The present findings allow one to develop methods for the pre- and postnatal diagnosis of cystic fibrosis with cell cultures, and for the detection of cystic fibrosis carriers with the peripheral blood. Cystic
fibrosis
of Caucasian important. in cystic methods
descent
and its
Many laboratories
are,
fibrosis, for
mutation
by a multiple culture
and trying
the detection
which
I-cell-disease.
the most
(I),
The present
the
(CF) is
paper affects
medium
(2),
social
hereditary
and economic
therefore,
to develop
in people
implications
searching diagnostic
disease
for
are very
the primary
procedures
defect
as well
as
of CF-carriers. demonstrates lysosomes
The phenotype leakage
frequent
that
in a way which
of cultured
of lysosomal
cystic
is
is
somewhat
a type
enzymes
into
of
is characterized
the extracellular increase
of
reminiscent
I-cell-fibroblasts
and by a compensatory
Copyright 0 1977 by Academic Press, Inc. At1 rights of reproduction in any form reserved.
fibrosis
of the
space,
i.e.
intracellular
741 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 3, 1977
activity the
of non-leaky
latter
level,
enzymes
phenomenon
experiments
and of alkaline
is due to a coordinate
of the replacement All
of these
have been
the expression
of enzyme activities
per-protein-basis.
They are,
very
fine
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fluctuations
(AP),
regulation,
enzymes
done with
phosphatase
(3).
ultramicro
techniques.
MATERIALS
They allow
instead
particularly
of enzyme activities
;
at the transcriptional
on a per-cell-basis
therefore,
respectively
well
in cultured
of on a
suited cells
to monitor (4,5).
AND METHODS
Cell culture conditions (6), ultramicro methods to measure enzyme activities at the single cell level (4,5,6), individual enzyme assays based on the use of artificial fluorogenic methylumbelliferone-coupled substrates (8), the isolation of urinary Tamm-Horsfall glycoprotein (TW ; 6) and the principle of AP-induction with THP (6) have been described elsewhere. For the present studies the AP-induction procedure has been modified and two different ultramicro techniques have been used. Technique I . Culture medium : HAM FlO with 15% FCS. Fibroblasts were seeded into5PZastic Film Dishes (PFD's ; 4,7) with an initial concentration of 1.5 x 10 cells per PFD .AP-induction : 24 hours after seeding, the fibroblast cultures were treated for 3 consecytive days with THP (100 ng/mJ medium) ; for the last 2 days of induction 1 x 10 -5M isoproterenol and I x 10 M theophylline and for the last day 1 x 10 M isoproterenol were given in addition into the THP-containing medium. Then the cells were washed, lyophilized, isolated from the PFD's and enzyme activities were assayed in ParafiZm Micro Cuvettes lPMC'.sl as described previously (3,4,5,6,8). Technique II . Culture medium : HAM FIO with 15% FCS. Fibroblasts were grown to confluency in PFD's and then induced for 3 consecutive days with THP as described Under-Technique I. Then the induction-medium was replaced for 24 hours by HAM FIO with 0.1% heat-inactivated FCS to collect extracellular enzymes ; this medium was cleared by centrifugation (10,000 g for 5 minutes) and lyophilized to concentrate the collected extracellular proteins. The cells, remaining in the PFD, were washed, trypsinized and diluted with saline to a final concentration of 50 cells per 0.1 ~1 ; the cells were then disrupted by 3 cycles of freezing and thawing and the 10,000 g supernatant fraction of this extract was used for the enzyme assays. For the determination of enzyme activities in the extracellular space the lyophilized medium was dissolved in water h d been conditioned by 50 cells. in such a way that 0.1 ~1 of this solution Enzyme activities are expressed in 10 -14 moles of substrate converted per cell per hour or as the percentage of the mean activities of controls.
RESULTS Table cell
1 reviews
strains
controls.
from
the
intracellular
5 patients
The intracellular
a-galactosidase,
cL-mannosidase
with activities
enzyme activity I-cell-disease
profiles
as compared
of S-galactosidase,
and S-glucuronidase
742
are
in fibroblast to 16 normal
hexosaminidase, significantly
(i.e.
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 : Intracellular enzyme activity profiles, relative values, fibroblast cell strains from 5 patients with I-cell-disease and from 16 normal controls. Technique cf. methods. Each determination was carried out in five-fold with 20 cells per incubation.
Controls, n = 16 mean + SD%
pat.1
b-galactosidase
100 k 9.8% ( 4.3)%
hexosaminidase
100 ?13.4%
a-galactosidase
100 + 8.3%
pat.2
pat.3
pat.4
I,
pat.5
8%
6%
6%
9%
6%
(28.5)
17%
19%
15%
25%
15%
( 0.5)
19%
22%
9%
45%
8%
cl-mannosidase
100 +_ 8.3% ( 0.6)
27%
29%
29%
30%
36%
@-glucuronidase
100 f 9.1% ( 0.7)
31%
20%
8%
105%
15%
a-glucosidase
100 * 7.0% ( 0.6)
218%
238%
157%
625%
153%
S-glucosidase
100 + 6.9% ( 0.8)
168%
171%
174%
350%
184%
acid
100 + 8.5% (19.5)
199%
234%
217%
299%
195%
100 +_ll.S%
415%
465%
696%
2461%
335%
indicate
mean enzyme activities
phosphatase
alkaline
phosphatase
e values in brackets moles of substrate
below the
-3 S.D.)
and alkaline +3 S.D.)
column
the exception
of b-glucuronidase
of ct-glucosidase,
phosphatase,
on the other
B-glucosidase,
hand,
in patient acid
are significately(i.e.
in
4 ;
phosphatase to over
increased.
in fibroblast
cell
Table
B-galactosidase, decreased
as compared
the -3 S.D.
; o-galactosidase phosphatase
is displayed
to a dramatic
from
family
1 with
level, is
are
only
of the
enzyme activity cystic
increased
is
AP-activities
743
phosphatase
while
are
clearly
of -3 to
a-glucosidase
the +3 S.D.
CF-heterozygotes.
intracellular
(2 CF-homozygotes
on the border
decreased, to or over
profiles
In the CF-homozygotes
and acid
B-glucosidase slightly
fibrosis
controls.
cr-mannosidase
by the obligate
increase
intracellular
to 16 normal
hexosaminidase,
below
and alkaline
2 presents
strains
2 CF-heterozygotes)
trend
with
activities
In comparison,
-2 S.D.
in the control converted/cell/hour.
decreased,
intracellular
( 0.5)
Induction
level. with
The same THF' leads
in the CF-children
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 2 : Intracellular enzyme activity profiles, relative values, fibroblast cell strains from family 1 with cystic fibrosis (2 CF-homozygous sibs, 2 CF-heterozygous parents) and from 16 normal controls. Technique I, cf. methods. Alkaline phosphatase without and with THP-induction (cf. methods). Each determination was carried out in five-fold with 20 cells per incubation. Controls, n = 16 mean * SD%
sib
I
sib
?
2d)
father
mother
S-galactosidase
100 i: 9.8% ( 4.3)*
58%
63%
67%
69%
hexosaminidase
100 +13.4%
(28.5)
53%
58%
73%
79%
a-galactosidase
100 f 8.3%
( 0.5)
80%
89%
81%
79%
a-mannosidase
100 k 8.3%
( 0.6)
55%
65%
77%
75%
S-glucuronidase
100 f 9.1% ( 0.7)
110%
120%
101%
107%
a-glucosidase
100 f 7.0% ( 0.6)
165%
120%
91%
90%
S-glucosidase
100 t 6.9% ( 0.8)
69%
89%
83%
87%
acid
100 2 8.5% (19.5)
50%
66%
87%
83%
( 0.5)
210%
180%
105%
99%
alkaline phosphatase THP-induced
100 +-18.0% ( 0.7)
380%
430%
120%
130%
values in brackets :,-n emoles of substance
in the control converted/cell/hour.
indicate
mean enzyme activities
phosphatase
alkaline
but
phosphatase
to only Fig.
a very moderate 1 reviews
THF'-induction (not
100 +11.8%
increase
with
the CF- homozygotes
cell
family the
a-mannosidase,
a-glucosidase
and S-glucosidase of the
and intracellular increased.
same
alkaline
from
activities
a-galactosidase,
enzymes
are below are
far
phosphatase
In the CF-heterozygotes
family
2) as compared
intracellular
the
parents.
enzyme activity
cultures
in Table
a-fucosidase,
activities
in the heterozygous
and extracellular
intra-
in fibroblast
identical
column
2 with
cystic
to 14 normal of acid
the -3 S.D.
values
are also
extracellular
after fibrosis
controls.
In
phosphatase,
b-galactosidase,
hexosaminidase,
level
above the +3 S.D.
744
profiles,
; the extracellular level
very
; the extra
significantly
enzyme activities
follow
in
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I r I
q
DC
100
100. .
a
b
0
o.
:
Fig.]. Intraand extracellular enzymes activity profiles, relative values, fibroblast cell strains from family 2 with cystic fibrosis and from 14 normal controls, after 'HP-induction. Technique II, cf. methods (a-mannosidase, a-galactosidase, a-glucosidase, B-glucosidase were incubated at 25'C, the other enzymes at 37°C). Each point is the mean of 5 determinations and corresponds to 50 cells or the medium conditioned by 50 cells. The horizontal 100% bar corresponds to the mean of 14 normal control cell strains with the following standart deviations (intracellular/extracellular) : alkaline phosphatase (8.7/11.5) ; acid phosphatase (6.9/22.8) ; o-fucosidase (5.3/20.3) cl-mannosidase (7.1/14.7) ; cr-galactosidase (8.4127.8) ; 8-galactosidase (6.9/12.3) ; hexosaminidase (6.618.2) ; cl-glucosidase (8.9125.6) ; b-glucosidase (4.6/18.9) ; @-glucuronidase (7.1/14.3). a. CF-children (9 ,c?) : A, intracellular ; A, extracellular b. Parents (obligate heterozygotes) : m, intracellular ; q , extracellular
the
same trend,
icant
activity
o-galactosidase,
with decrease
the exception is
of B-glucosidase
detectable
8-galactosidase
only
for
and o-glucosidase
745
; intracellularly
a-fucosidase,
a signif-
o-mannosidase,
; the intracellular
activity
;
Vol. 79, No. 3, 1977
of alkaline
BIOCHEMICAL
phosphatase
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in particular
is normal
in heterozygotes.
DISCUSSION This (6)
study
permits
CF-patients
confirms
one,
as a rule,
on the
To the present, with
into
only,
data
picture which
shown)
CF-cases
THP-induceable
that
will
where
different
be found
is
the
;
that
index-patient
the
is
This
or heterozygous and 15 CF-heterozygotes
the multiple
in the
enzyme
for
type
the studied
the
of cystic
other
lysosomal
it enzymes
clinical fibrosis,
enzymes,
Alternatively,
leakage.
none of
; in addition, rare
THP-
phosphatase
are not THP-induceable,
leaky
for
THP-induceable
or heterozygotes
display
syndrome.
leaky
treated
and of alkaline
CF-homo-
unusual.
nor
were
CF-homo-
show enzyme leakage is very
on the other. have been
14 CF-homozygotes
2 sibs
between
are THP-induceable.
by all
: all
where
cultures
controls
enzymes
index-case
or heterozygotes
completely
could is
be
possible
and induceable
substrates.
The multiple phosphatase
enzyme
(Table
of acid
results
phosphatase CF-heterozygotes (3,6,8).
leakage
2, Fig.
enzyme activities.
weaker
with
glycoprotein
The conclusion
in families
displayed
family,
a biochemically
this
is
not
CF-cases
of lysosomal
a THP-induceable
is neither
enzymes,
that
9 families
(data
of these
by different
diagnosed
leakage
So far
such types
and normal
not THP-induceable.
indicate
In the one exceptional the CF-homo-
were
space
have been analyzed with
fibroblast
51 of the CF-homozygotes
a multiple
families
CF-carriers
of clinically
members.
with
glycoprotein.
the extracellular
family
to discriminate
Tamm-Horsfall
and 25 CF-heterozygotes
The present induceable,
with
53 CF-homozygotes
sibs
majority
the AP-induction
one hand,
Tamm-Horsfall
2 CF-homozygous vast
that
I),
results,
in a decrease
The coordinate
regulation
and of alkaline
but This
here would
of the (Fig.
the
la).
of alkaline
of the corresponding
(3,6,8)
intracellular
signal
the absence
746
of lysosomal the
activity
for
intracellular
counteracts
The same regulation
stimulatory
explain
the exception
of the synthesis
phosphatase
in an overshooting in CF-homozygotes
with
leakage
of alkaline takes
place
the enzyme induction
of an overshooting
of intra-
in is
;
BIOCHEMICAL
Vol. 79, No. 3, 1977
cellular
alkaline
phosphatase
of the intracellular
activity
activities
-heterozygotes
(Fig.
The primary
defect
in cystic
fibrosis
and alkaline
This
reminiscent
of I-cell-disease
cystic
(lysosomal It
their
suggested
secretion
receptors
specific
affect
would
the normal
likely
qualify
in these
syndromes
multiple
enzyme leakage.
enzyme leakage likely
that
Different
mutations
recognition
site.
in the population the
leakage
(Table
could, This
leaky
as the
explain
the very
of mutants)
enzymes.
in family
2 (Fig.
high
(6).
747
would
explain
why
display
(IO)
different frequency
and also
1) ; this
fibrosis
we found
complex
with
a-glucosidase
on the
the
a multiple ; it
seems
mutant.
relatively
interfere
sites
homozygotes
the heterozygotes
could
; the one would
cystic
This
syndrome
therefore,
might
study,
hand.
which
granules
mutants.
a receptor
are probably
(heterogeneity
of specific
2) but
with
not
on the other
the recognition
the present
lysosomes,
on the one hand,
of mutations
the other
; that
such as primary
of digestive
site
hydrolases
and uptake
enzymes types
as well
but
between
be quantitative
of lysosomal
of the granules
In the Chediak-Higashi
sites
possibly
granules,
as recognition
we are dealing
The difference
recognition
two different
the heterozygotes
Enzyme recognition
no data
on the corresponding
in the homozygotes
there
in I-cell-disease
(phosphatases).
or matrix
and following
of lysosomal
though
by specific
in the granules,
to a loss
compartment.
the packaging
self-assembly
I-cell-disease,
most
that
followed
to find
the receptors
enzymes.
(9)
sites
expect
with
in the CF-
the extracellular
therefore,
of enzyme containing
recognition
interfere
normalization
into
as qualitative
on the membrane
One would
enzymes
of phosphatases. could,
as well
the self-assembly
needs
towards
seems to lead
(2),
and I-cell-disease
has been
leaky
phosphatase
a leakage
hydrolases)
requires is,
concerning
fibrosis
tendency
lb).
and of acid
are available
and the
of the other
enzymes is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is,
e.g.,
configurations. parts
of CF-carriers
the variability not
of the
leaky
has been observed
concerning in family previously
I
BIOCHEMICAL
Vol. 79, No. 3, 1977
This
first
evidence
"processing
mutation"
Preliminary
studies
amounts screen
in
also
the primary
allows
indicate
the blood
populations
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
defect
one to design the
leaky
of CF-heterozygotes for
in cystic
fibrosis
new diagnostic
enzymes
and that
are it
will
present
is
a
strategies. in
increased
become possible
to
CF-carriers. ACKNOWLEDGEMENTS
This DGRST (Nr.
study
has been
75 70 004)
supported
by grants
and the Fondation
from
INSERM (ATP 36 76 68),
de France.
REFERENCES 1. Di Sant'Agnese, P.A., and Davis, P.B. (1976) New Engl. J. Med. 295, 481-485. 2. Lightbody, J., Wiesmann, U., Hadorn, B., and Herschkowitz, N. (1971) Lancet r, 451 only. 3. Hi;sli, P. (1976) Adv. Exp. Med. Biol. 68, I-13. 4. HEsli, P. (1972) Tissue cultivation on plastic films, Tecnomara, Zirich. Defects, pp. 226-233, Excerpta Medica, Amsterdam. 5. H&li, P. (1974) Birth 6. Hgsli, P., Erickson, R.P., and Vogt, E. (1976) Biochem. Biophys. Res. Commun. 73, 209-216. (1977) Adv. Exp. Med. Biol. s, 591-596. 7. H&.li, P., and de Bruyn, C.H.M.M. 8. Hcsli, P. (1977) Clin. Chem. 3, p. 14761484. 9. Hickman, S., and Neufeld, E.F. (1972) Biochem. Biophys. Res. Commun. 2, 992-999. 10. HEsli, P., Griscelli, C., and Good, R.A., manuscript in preparation.
748
