Download Allelic Amino Acid Substitutions Affect the

CLIN.
CHEM.
38/12,
2493-2500
(1992)
Allelic Amino Acid Substitutions
Germ-Cell
Alkaline Phosphatase
Marc
F. Hoylaerts,”2
Thomas
Affect the Conformation
Phenotypes
Manes,’
and
Jos#{233}
Luis
gene
(PLAP)
gene
encoding
placental
alkaline
phosphatase
displays a well-documented
allelic polymorphism.
Likewise,
different phenotypes
exist for the PLAP-related
germ-cell
alkaline phosphatase
(GCAP). We investigated
the extent to which various allelic GCAP positions
are
critical in determining
the enzymatic,
structural,
and immunological
properties
of GCAP phenotypes.
Three homozygous
GCAP phenotypes
[JEG3, BeWo, and wildtype (wt) GCAP] were analyzed
and compared
with a
“core” GCAP mutant that contains the seven amino acid
substitutions
that are consistently
different between PLAP
and GCAP but are common
to the three known allelic
GCAP
genotypes.
Although
some substitutions
could
in
influence
the electrophoretic
behavior
PLAP.
The
selective
of the phenotypes,
the kinetic properties
the immunoreactivity
detected with a panel
antibodies
(MAbs) to
immunoreactivity
of
the
PLAP/
GCAP-discnminating
MAb C2 was critically dependent
on
the nature of the allelic residues
133 and 361 in GCAP.
Residue 133 was also importantfor
the general stability of
the molecule
because
BeWo and wt GCAP, which have
Asn1
and Val1,
respectively,
instead
of Met1,
showed a consistently
reduced heat stability compared
to
core GCAP and JEG3. Because
the core GCAP mutant
consistently
shows the characteristics
of wt GCAP, its use
as an antigen should allow the generation
of monoclonal
antibodies
to GCAP
that
will not cross-react
with
and whose immunoreactivity
will only marginally
enced by allelic GCAP variation.
Additional
.
Keyphrases:
monoclonal
allelic polymorphism
antibodies
.
PLAP
be influ-
genetic
variation
isoenzymes
Human
alkaline
phosphatases
(ALPs)
are encoded
by
a gene family
composed
offour
loci [for review,
see (1)].
Whereas
the three tissue-specific
ALP
(TSAP)
genes,
placental
(PLAP),
germ-cell
(GCAP),
and intestinal
ALPs,
are composed
ofli
exons and occupy
less than 5.0
kb of DNA,
the single
tissue-nonspecific
ALP (TNAP)
1 La Jolla
Cancer
Research
Foundation,
Cancer Research
Center, 10901 North Torrey Pines Road, La Jolla, CA 92037.
2 Department
of Nephrology-Hypertension,
University
of Ant-
werp,
Antwerp, Belgium.
Author for correspondence.
4 Nonstandard
abbreviations:
TSAP, tissue-specific
alkaline
3
ALP,
phosphatase;
alkaline
TNAP,
phosphatase;
tissue-nonspe-
cific alkaline
phosphatase;
PLAP, placental
alkaline phosphatase;
GCAP, germ-cell
alkaline
phosphatase;
wt, wild type; MAb, monoclonal antibody;
DEA diethanolamine;
and pNPP, p-nitrophenyl
phosphate.
Received
May
13, 1992;
accepted
August
3, 1992.
of
Milan”3
The
the allelic differences
did not affect
of GCAP. However,
they did affect
and conformation
of the variants as
of 1 8 epitope-mapped
monoclonal
and Immunoreactivity
contains
an additional,
differentially
spliced
exon
5’ region
and significantly
larger
introns
that
40-50
kb of DNA.
The TSAP
genes
are colocalized
in the long
arm of chromosome
2, but the
TNAP
gene
resides
at the end of the short
arm
of
chromosome
1.
The PLAP gene is subject
to a high degree
of polymorphism.
Three common
alleles
(Plo, pF’, and P1’) account
the
occupy
for over 90% of the PLAP
phenotypes
(2). However,
more than 20 rare allozymes
have been described,
often
only in heterozygous
combinations
because
of their low
allelic
frequency
(3). The two
most
common
PLAP
phenotypes,
S (slow) and F (fast), differ only in an Arg#{176}
to p2o9
substitution
(4), but additional
amino
acid
replacements
have been identified
for the I (intermediate) variant
(5).
Although
GCAP
is encoded
by a different
gene from
PLAP,
the primary
structure
of GCAP
shows
98%
sequence
identity
with
PLAP
(6, 7). However,
when
various
GCAP
samples
were
analyzed
with
respect
to
their
immunoreactivity
with
a number
of monoclonal
antibodies
raised
against
PLAP,
some of the antibodies
reacted
with
high,
intermediate,
or low affinity
(8).
From
these findings,
the existence
of a GCAP
polymorphism
with
up to nine
allelic
GCAP
variants
was
anticipated
(8-10).
Determination
of the sequence
of
GCAP,
derived
from JEG3
choriocarcinoma
cells
(11)
and BeWo
cells (12), confirmed
the existence
of allelic
variation
in the GCAP
gene.
Using
a series
of site-directed
PLAP
mutants,
we
recently
showed
that
individual
amino
acid substitutions in the PLAP
isoenzyme
had a considerable
effect
on the immunoreactivity
and conformation
ofthe
resulting mutants,
as detected
by a panel
of 18 monoclonal
antibodies
(MAbs)
to PLAP
(4). These
results
emphasized the importance
of characterizing
in great
detail
the reactivity
of those
MAbs
used
clinically
for the
serological
evaluation
of ALP
isoenzymes.
PLAP
and
GCAP
are useful
tumor
markers
in the management
of
patients
with
adenocarcinoma
of the ovary
and seminoma
of the testis
(13-1 7). The
existence
of allelic
GCAP
differences
can influence
the accuracy
of the
immunochemical
detection
of GCAP
phenotypes,
their
electrophoretic
identification,
and the molecular
stability of the GCAP
allotypes.
To evaluate
these
variables,
we used site-directed
mutagenesis
to construct
a series
of PLAP
and GCAP
mutants.
We then compared
these
mutants
with
those
GCAP
phenotypes
for which
the
sequence
is known.
Although
the presence
of Gly at
position
429 in GCAP
is the key element
determining
the enzymatic
properties
ofGCAP
(18), our results
show
that different
amino
acids at certain
allelic
positions
can
CLINICAL
CHEMISTRY,
Vol. 38, No. 12, 1992
2493
influence
significantly
Materials
PLAP
the
and
Mutants
conformation
affect
antibody
of the GCAP
recognition.
molecule
and
with
tively,
low
a rabbit
Genotypes
The
study
PLAP
(F phenotype)
and GCAP
used
in this
have
been
described
previously
(18) and are referred
to as wild-type
(wt) PLAP
and wt GCAP.
A 2.0-kb
Eco RI-Kpn
I fragment
of the PLAP
cDNA
(6) was used
as the source
of template
DNA
to generate
a series
of
PLAP
mutants.
Site-directed
mutagenesis
experiments
were
performed
according
to Kunkel
(19) by using
the
mutagene
M13 in vitro mutagenesis
kit (Bio-Rad
Laboratories,
Richmond,
CA). The generation
of the single
amino
acid
mutants,
[Gln’5]PLAP,
[Thr67]PLAP,
[PhessIPLAP,
[Ser84]PLAP,
[His’]PLAP,
[Leu]PLAP,
and [G1y429JPLAP,
was described
previously
(4). These
cDNAs
were used as a source
of fragments
to reassemble
the
more
complex
mutants.
[HisZl,
Leu,
G1y429]PLAP
([HLG]PLAP)
was constructed
by ligating
a 568-bp
BamHI-SacI
fragment
containing
the [His’1
mutation
(BamHI-[His’]-SacI),
the
154-bp
Sac
I-[Leu254]-SacII,
and
the
937-bp
SacII-[Gly4]-KpnI
fragments
into pSVT7-PLAP
digested
with BamHI
and
KpnI.
A 1414-bp
BstEll-KpnI
fragment
from the [HLG]
PLAP construct
was
then
isolated
and ligated
with a
276-bp
BamHI-[SerJ-BstEII
fragment
into
either
cut with
BamHI
and KpnI
to create
Leu254,
Gly429]PLAP
([SHLG]PLAP)
or
digested
with BamHI
and KpnI to
SerM,
His241,
Leu2M,
G1y429]PLAP
([QSHLG]PLAP).
Finally,
a 276-bp
Barn
HI-[Ser,
r67,
PhessJBstEll
fragment
was ligated
with
the
1414-bp
BstEII-[His1,
Leu,
Gly4}-KpnI
fragment
into pSVT7-[Gln’5IPLAP
to generate
core GCAP.
The
sequence
of the mutagenesis
primer
used
to generate
[Leu361]PLAP
was as follows:
5’-GGA
GAA GAj
GTG
GGA
GTG GTC-3’
(the underlined
base indicates
the
change).
The wt and mutagenized
PLAP
cDNAs
were
antiserum
concentrations
tants,
core GCAP,
the insolubilized
Methods
and GCAP
coated
and GCAP
MAbs.
Upon
fraction
was measured
total enzyme
concentration
surements
were carried
age concentration
divided
by the
fraction
the
binding
step
increasing
that
expressed
in the
in triplicates
(<20%)
an affinity
values
residual
for
index
the
binding.
calculated
by
to the B/F ratio
relative
Mea-
the
aver-
bound
enzyme
(B) was
of the unbound
(free)
between
5% and 10%,
for those phenotypes
(%) were
to the
wells.
and
were
low
mu-
added
to
the bound
relative
deposited
to obtain
ConsecuPLAP
were
recorded
affinities
B/F ratio
and
out
IgG.
PLAP,
The
to 15-20%
showed
ative
wt
phenotypes
equilibration,
(±SD)
of
concentration
(F), in order
binding.
to mouse
of the
(B/F)
SD
occasionally
and mutants
Finally,
expressing
recorded
for
of the
relthe
for wt PLAP.
Gel Electrophoresis
Starch
described
PLAP
lated
gel electrophoresis
(23). Prior
to
mutants,
by treatment
at pH 8.6 was performed
as
electrophoresis,
the wt PLAP,
and GCAP
phenotypes
with neuraminidase
24 h at 37 #{176}C
to eliminate
any
linked
to differences
in the degree
different
cell
were
desialy(0.33 U/mL)
for
charge
heterogeneity
ofsialylation
between
types.
pSVT7-PLAP
[SerM,
His241,
pSVT7-[Gln’5IPLAP
create
[Gln15,
subcloned
into the vector
pSVT’7 downstream
from the
SV4O early
promoter
(20) and transfected
by the calcium
phosphate
procedure
(21) into Chinese
hamster
ovary
(CHO)
cells.
Transfected
cells
were
selected
as
described
(18)
and,
at confluency,
washed
with
20
mmol/L
Tris-HC1
buffer,
pH 7.5, containing
140 mmolJL
NaC1.
The cells
were
extracted
(30 mm)
with
a 1:1
mixture
of n-butanol
and 50 mmolfL
acetate
buffer,
pH
5.5, containing
100 mmol/L
NaC1, 20 j.unol/L
ZnCl2,
1
mmoLfL
MgCl2, and 0.5 g/L thimerosal
(Sigma
Chemical
Co., St. Louis, MO). JEG3 cells grown
in the presence
of
2 mmolfL
sodium
butyrate
to maximally
stimulate
the
production
of GCAP
(22) and BeWo
cells
grown
to
confluency
were extracted
in the same way. Upon titrating the pH to 7.5 with a 1.0 molfL Tris solution,
these
extracts
were aliquotted
and stored
at -80 #{176}C.
Relative
Affinity
Measurements
Immunoreactivities
were
measured
Briefly,
16 MAbs to PLAP
and
intestinal
ALP were incubated
2494
CLINICAL
CHEMISTRY,
as
(4).
MAbs to
plates
pre-
described
2 cross-reacting
in microtiter
Vol. 38, No. 12, 1992
Heat Inactivation
The wt PLAP,
PLAP mutants,
and
were diluted
in 1 molfL
diethanolamine
pH 9.8, supplemented
alone or 20 mol/L
with
either
ZnCl2 and 0.5
GCAP
0.5
mmol/L
phenotypes
(DEA)
buffer,
mmol/L
MgCl2,
MgCl2
and
incubated
in a water
bath at 56 #{176}C
or 65 #{176}C.
At fixed
time intervals,
50-pL
samples
were removed
and pipetted into each well
of a microtiter
plate
kept
on ice.
Residual
activities
were
then
measured
in duplicate
upon the simultaneous
addition
to the wells of200
L of
10 mmol/L
p-nitrophenyl
DEA
buffer,
pH 9.8,
MgCl2. These
activities
activity
of the unheated
Enzyme
Activation
phosphate
(pNPP)
in 1 mol/L
supplemented
with
0.5 mmoLTL
were expressed
relative
to the
enzyme.
and Inhibition
Kinetics
Michaelis-Menten
kinetics
of the core GCAP
and
different
GCAP phenotypes
were performed
as recently
described,
with Km and Vm
being
derived
from Lineweaver-Burk
plots (24). Catalytic
rate constants
(k)
were calculated
from Vm
upon determination
of the
total enzyme
concentration,
[El#{176},
in an enzyme
antigen
immunoassay
based on the PLAP
MAbs C2 and H7 (8).
Residual
activities
released
during
the inhibition
of the
wt PLAP,
PLAP
mutants,
and GCAP
phenotypes
by
increasing
concentrations
of L-Leu
(0.05-10
mmol/L)
were
measured
in microtiter
plates
by adding
10
mmol/L
pNPP
in 1 mol/L
DEA buffer,
pH 9.8, supplemented
with 0.5 mmolJL
MgC12.
These
activities
were
expressed relative
to the enzyme
activity
in the absence
of inhibitor.
Position
Amino
wt PLAP
[G]PLAP
[HLGJPLAP
acid
at the
residue
tSHLGIPIAP
15
Glu
Glu
Glu
G)u
38’
II.
II.
II.
U#{149}
b
II.
U.
67
II.
II.
Pro
Pro
68
84
133’
241
254
297’
361
429
Pro
Pro
Pro
Mn
Asn
Met
Met
Met
Met
Met:
Arg
Arg
Met
Met
Arg
Arg
Arg
Arg
Arg
Vat
Vat
Vel
Vat
Vat
Pro
Pro
Pro
Glu
Pro
Giy
rs
position
GCAP
JEG3 OCAP
BeWo OCAP
II.
II.
Arg
Arg
Vat
wi GCAP
lie
Asn
479*
indicated
(OSHLG)PLAP-
Met
Vat
Leu
Pro
Arg
$IYL
GJy
Gly
Pro
Cly
.
Pro
Pro
Fig. 1 . Amino acid sequence differences that define wt PLAP, PLAP mutants, and GCAP genotypes
The shaded residues indicate those substitutions consistently different between wt PLAP and the JEG3, BeWo, and wt GCAP genotypes. An asterisk identities
those positions known to be polymorphic by sequence analysis. Boxed residues indicate actual allelic substitutions
A
Results
and
PLAP Mutants
and GCAP
479,
The
comparisons
genotypes
and from
wt GCAP
wt GCAP
and the
the most (at positions
Enzyme
Electrophoresis
at positions 38, 133, and 297.
JEG3
GCAP
phenotypes
differ
38, 133, 297, 361, and 479).
on Starch
Gel
agreement
with the charge
difference
between
the
F PLAP
phenotype
(Arg for Pro at position
209),
gel electrophoresis
separates
the desialylated
FS
heterozygous
PLAP
phenotype
into three bands,
correspending
to the FF, FS, and SS dimers,
respectively
(Figure
2A). The single
substitution
in PLAP
of Glu429
by Gly429 ([GIPLAP)
causes
an important
electrophoretic
retardation
of the resulting
mutant,
compared
with the S PLAP
phenotype
(Figure
2B). This effect is
primarily
conformational,
because
the S PLAP
phenotype
([2O9,
Glu429]PLAP)
and
[G]PLAP
([Pro2#{176},
GIy429JPLAP)
have
an identical
charge
density.
This
result corroborates
our finding
that the substitution
of
residue
429 in PLAP
is associated
with
an important
conformational
change in the molecule
(4). The electroIn
S and
starch
C
Genotypes
(Figure
1) ofthe three different
sequenced
to date
indicate
the existence
of at least
five allelic
amino
acid positions
(residues 38, 133, 297, 361, and 479).
These
three
GCAP
phenotypes
differ
from the wt PLAP
sequence
at 12
amino
acid positions.
Seven
of these
differences
are
invariant
and common
to all three
GCAP
alleles.
We
have constructed,
through
site-directed
mutagenesis,
PLAP mutants
containing
an increasing
number
(from
one to seven)
of substitutions
designed
to progressively
confer
more GCAP
character
to the resulting
mutants
(Figure
1). The
most
complex
mutant,
[QTFSHLGIPLAP,
contains
the core of seven amino
acid differences
that
distinguish
GCAP
from
PLAP.
This
core GCAP
mutant
represents
a GCAP
genotype
containing
the
minimal
number
of consistent
differences
compared
with wt PLAP
but displaying
a full GCAP
enzymatic
character
(see below)
common
to all known
GCAP
phenotypes.
This core GCAP
differs
from BeWo
GCAP
at position
133, from JEG3
GCAP
at positions
361 and
Sequence
GCAP
B
Discussion
I
2
3
1
2
3
4
5
1
2
3
4
Fig. 2. Starch gel electrophoresis
of PLAP and GCAP phenotypes
(A) Common PLAP phenotypes: F phenotype (lane 1), FS phenotype (lane 2),
and S phenotype (lane 3); (B) increasingly
more complex PLAP mutants:
reference PLAP S phenotype (lane 1), [GIPLAP (lane 2), [HLG)PLAP
(lane 3),
[SHLGJPLAP
(lane 4), and [QSHLGJPLAP (lane 5); (C) diflerent
GCAP
phenotypes: core GCAP (lane 1), JEG3 GCAP (lane 2), wt GCAP (lane 3),
and BeWo OCAP (lane 4)
phoretic
behavior
of the
mutant
enzymes
is
further
modulated
in increasingly
more complex
PLAP mutants
in which three,
four, or five amino
acids are substituted
for the corresponding
GCAP
residues
(Figure
1). Comparison
ofthe electrophoretic
mobility
ofcore
GCAP and
the other
GCAP
phenotypes
confirms
that GCAP
does
not behave
as an electrophoretically
unique
entity.
The
somewhat
higher
anodic
mobility
ofwt GCAP
than that
of the other
GCAP
molecules
(Figure
2C) can be explained
by the substitution
in wt GCAP
of Arg97
for
Leu297.
These
findings
show
that
the electrophoretic
microheterogeneity
of GCAP
in seminoma
does
not
depend
only
on the
carbohydrate
heterogeneity
and
differences
in hydrophobicity,
as was suggested
(25),
because
allelic
amino
acid
substitutions
involving
residues
also contribute
to the electrophoretic
heterogeneity,
analogous
to the situation
with the electrophoretic
PLAP
polymorphism
(26).
charged
Immunoreactivity
of PLAP
Mutants
We recently
used
a panel
of 18 epitope-mapped
conformationally
dependent
MAbs against
PLAP to define
conformational
changes
induced
by each ofthe
10 amino
acid differences
between
PLAP
and GCAP
(4). Measurements
of the relative
affinities
of the same
panel
of
antibodies
for
the
increasingly
complex
mutants
EGIPLAP,
[HLG}PLAP,
[SHLG]PLAP,
and [QSHLG]PLAP
indicate
that the resulting
immunoreactivity
is a
composite
function
of the contributions
of each
single
CLINICAL
CHEMISTRY,
Vol. 38, No. 12, 1992
2495
(G]PLAP
lx
(OSHLG]
1
PLAP
wt GCAP
120
so
i.ii..IihiL
:BIIdIllhIIhLso
F$,s
QJ8U
IiLhIiiIIIIIk
:[I111.1itttii1
:
[JJ]C[JJJ
#{174}jJjjjP[J
g&’
4c;9
Fig. 3. ImmunoreactiVity
18
epitope-mapped
Values represent
of a series
of complex
PLAP
81p4’g
mutants,
the GCAP
relative affinities with respect to wt PLAP. Note differences
amino
acid substitution
(Figure
3). The conformational
effect of combining
different
amino
acid substitutions
in
a single
mutant
is not necessarily
equal
to the sum of
the conformational
contributions
for the individual
substitutions.
For example,
when His’,
Leu,
and Gly4
are individually
substituted
in PLAP,
the affinity
of
antibodies
D10,
Fil,
B2, and GlO is greatly
reduced
from that of wt PLAP
(4). Yet when these three substitutions
are combined
into
a single
mutant
([HLG]PLAP),
the affinity
for D10 and Fli is largely
reconstitoted
(Figure
3). Furthermore,
the immunoreactivity
of
the triple mutant
[HLG]PLAP
for most ofthe antibodies
was
different
from
that
of a triple
mutant
studied
previously,
[SerM, Leu254,
Gly429]PLAP.
The epitope
for MAb 17E3 is almost
entirely
constituted
by 241
(4) The predominant
contribution
of
the His24’
substitution
in [HLG]PLAP,
as well as in
[SHLG]PLAP
and [QSHLGJPLAP,
is apparent
from
the lack of reactivity
of these
mutants
with 17E3 and
from the relatively
high reactivity
with H5. The additional
inclusion
of SerM ([SHLG}PLAP)
and of GIn’5
([QSHLG]PLAP)
in the mutant
causes
further
modulation
ofthe
immunoreactivities
toward
lower affinities
for most of the antibodies
in the panel.
Immunoreactivity
of GCAP
Comparisons
MAb
panel
of the
with
the
Phenotypes
pattern
immunoreactivity
core
GCAP
mutant
and
the
of the
three
GCAP
phenotypes
of known
sequence
revealed
major
differences
in affinity
between
the different
GCAP
allelic variants.
First,
in comparison
with [QSHLG]PLAP,
the two additional
substitutions
in core GCAP
([QTFSHLG]PLAP)
further
affected
the reactivity
of 8 out of
18 MAbs
by reducing
their
affinities
fivefold
compared
with
those of PLAP
(Figure
3). Together,
the seven
amino
acid substitutions
that distinguish
core GCAP
from PLAP
shape
a GCAP
phenotype
that is structur2496
CLINICAL
phenotypes,
and the single
[Leu1JPLAP
mutant
with
the panel of
MAbs to PLAP
CHEMISTRY,
Vol. 38, No. 12, 1992
in the scales of the yaxes
ally
different
from
PLAP.
This
conformational
difference
is detected
by MAbs
B2, Gb,
and E6, which
recognize
the F/S allelic
PLAP difference,
as well as by
MAbs
A3, E5, F6, 327,
and 7E8,
which
bind to the
central
antigenic
domain
of the molecule.
The BeWo
GCAP
phenotype
can be regarded
as resulting
from
the additional
substitution
in core GCAP
of
a single
amino
acid, Met’33
for Asn’”
(Figure
1). This
additional
substitution
is important
for two reasons:
first,
the domain
recognized
by the F/S discriminating
antibodies
B2, Gb,
and E6 is reexposed,
and second,
the
reactivity
with C2 drops by a factor of 10 (Figure
3). The
C2 antibody
has
been
described
before
as a reagent
reacting
with PLAP but not with GCAP
(8). We recently
concluded
that this selectivity
is largely
conformationally determined
(4). It is somewhat
surprising
that the
substitution
of seven
GCAP
amino
acids in PLAP
(core
GCAP)
only leads to a twofold
decrease
in the immune
reactivity
for C2, whereas
the additional
substitution
of
the allelic
Met’
generates
the expected
C2 selectivity.
Moreover,
this
substitution
is
operational
only
in
a
context,
because
our previous
analysis
(4) could
not define
any
structural
role for Met’33
when
we
analyzed
the phenotype
in a PLAP
context
by measuring the immune
reactivity
pattern
of[Val’]PLAP.
The
wt GCAP
showed
a more
modest
decrease
in immunereactivity
than the BeWo GCAP,
but the drop was more
general,
in that all antibodies
tested
reacted
to a lower
extent
with wt GCAP than with PLAP.
Although
in wt
GCAP
Met’33
is substituted
for Val’33
(and
not for
Asn’33),
the same
low reactivity
with
C2 is observed,
indicating
conformational
similarities
between
the
BeWo GCAP
and wt GCAP.
The slightly
higher
affinities ofwt GCAP
with some antibodies
are a consequence
ofthe
Arg297 to Leu7
substitution,
known
to positively
GCAP
influence
immune
The JEG3
GCAP
recognition
shows
(4).
a distinctive
immunoreactiv-
ity pattern
compared
with the other GCAP phenotypes.
The affinity
for the group
of antibodies
binding
to the
central
antigenic
domain
of the molecule
(A3, E5, F6,
7E8) is entirely
reconstituted
in JEG3
GCAP
compared
with
wt GCAP.
On the contrary,
the reactivity
measured
with C2 is even five- to tenfold
lower
for JEG3
GCAP
than
for wt and BeWo
GCAP.
Likewise,
lower
reactivities
are found
with
MAb
17E3
for the JEG3
GCAP
than
for the other
GCAPs.
The low reactivity
with C2 cannot
be accounted
for, in this case, by residue
133 since this position
is not substituted
in JEG3 GCAP.
Moreover,
the larger
reduction
in affinity
for this phenotype
in comparison
with
the others
suggests
the
involvement
of the other
two residues
that
are substituted in the JEG3
GCAP:
Val36’ (for Leu361) and Prom
(for Arg’79).
The construction
of a PLAP mutant
carrying the single
Va136’ to Leu36’
substitution
generated
a
mutant
with
a reactivity
pattern
that
largely
corresponded
to that
of JEG3
GCAP
itself
(Figure
3), suggesting
that residue
361 played
an important
conformational
role in JEG3
GCAP.
If one considers
all the
individual
mutations
investigated
previously
(4), apart
from the essential
G1u429 to Gly4
substitution,
the
Va138’ to Leu36’
substitution
is the only single
amino
acid replacement
that could reduce
the immunoreactivity ofC2
by 50%. The low reactivity
ofJEG3
GCAP can
be explained
by this mutation,
which,
when analyzed
in
the GCAP context
ofthe JEG3 GCAP phenotype,
is fully
operational.
These
data clearly
show that the low reacof C2 with
different
GCAP
phenotypes
is very
dependent
on conformation,
and that
several
combinations of substitutions
also influence
reactivity,
which
explains
why different
GCAP
phenotypes
have varying
residual
reactivities
with C2. On the contrary,
the low
reactivity
of GCAP
with
17E3
largely
depends
on the
Arg24’
to His24’ substitution
(4), which is common
to all
tivity
four
GCAP
genotypes.
Reactivities
of GCAP Phenotypes
Cloned GCAP Alleles
Deduced
from the
The immunoreactivity
patterns
of the three
GCAP
phenotypes
investigated
here
are far from identical
and
provide
a rational
explanation
for the differences
in
immunoreactivity
observed
previously
during
the
screening
ofrandom
GCAP-positive
tumor
extracts
and
serum
samples
(8, 10, 15). GCAP,
like
PLAP,
is a
dimeric
enzyme
that results
from the random
association oftwo
(either
identical
or allelic)
GCAP monomers.
The
genotypes
we studied
could
give
rise
to three
heterozygous
GCAP
phenotypes:
wt
GCAP-JEG3
GCAP,
wt
GCAP-BeWo
GCAP,
and
JEG3-BeWo
GCAP.
These
heterozygous
GCAP
phenotypes
would
have
immunoreactivities
intermediate
between
those
depicted
count
for
the
homozygotes
for the existence
ofsix
and
different
could,
therefore,
ac-
GCAP phenotypes
distinguishable
immunologically
with MAbs
from
the
panel.
Although
additional
GCAP
genotypes
are likely
to be discovered
in the future,
on the basis ofour current
knowledge
of the binding
of epitope-mapped
MAbs,
we
are able to predict
the likely
substitutions.
We recently
reported
that the epitopes
for the MAbs
F11 and 17E3
were almost
entirely
constituted
by Arg#{176} and Arg41,
respectively
(4). Previously,
a type H GCAP
phenotype
(8) was characterized
as being
fully reactive
with
F11
compared
with PLAP (S phenotype).
This indicates
that
the type
II GCAP
probably
has a Pro#{176} to Arg#{176}
substitution.
Similarly,
type VU and VIII GCAP
were
defined
as highly
reactive
with
MAb
17E3 (10). This
points
to the existence
of GCAP phenotypes
that have a
His241 to Arg24’
substitution
and identifies
residue
241
as another
allelic
position.
The
core
GCAP
mutant
constructed
in this study
contains
the minimal
number
of substitutions
that confer
a GCAP
character
to the
molecule.
The MAb reactivity
ofcore
GCAP,
that is, low
reactivity
with 7E8, 17E3,
327, and E6, is compatible
with the definition
oftype
IX GCAP
(10) and thus may
well represent
a naturally
existing
genotype.
Finally,
the reactivity
pattern
of those same
antibodies
indicate
that JEG3
GCAP
is compatible
with
a type
V GCAP
phenotype,
whereas
the BeWo
and wt GCAP
currently
cannot
be correlated
with any of the previously
defined
phenotypes.
Heat Stability
At
ing
of PLAP Mutants
physiological
temperatures
and GCAP
pH, PLAP is extremely
of 65 #{176}C
for 60 mm.
Phenotypes
stable,
resistTo facilitate
an
of the general
stability
of PLAP
mutants
phenotypes,
we conducted
heat stability
tests
at a higher
pH (1 mollL
DEA buffer,
pH 9.8). Heating
enzyme
samples
at 65 #{176}C
for 6 mm in the presence
of 0.5
mmol/L
MgCl2
caused
a moderate
(20%) loss of enzymatic
activity
for wt PLAP,
whereas
the same
treatment
caused
a considerable
reduction
(>60%)
in the
activity
of the PLAP
mutants
and the GCAP
phenotypes
(Figure
4A). These
results
show that the conformational
change
that
occurs
in PLAP
when
Glu429
is
substituted
for Gly4
has a major impact
on the general
stability
of the isoenzyme.
They also show that further
substitutions
can partially
correct
for this
decreased
stability.
However,
because
all PLAP
mutants
and
GCAP
phenotypes
were
not inactivated
to the same
degree,
we also measured
residual
activities
after mactivation
at a less critical
temperature
(56 #{176}C).
Exposure
to 56 #{176}C
for 30 mm (Figure
siB) caused
a comparable
destabilization
for wt PLAP
(80% residual
activity),
but
differences
within
the group
of PLAP
mutants
and
GCAP
phenotypes
were also evident.
The stability
ofthe
PLAP mutants
and GCAP
phenotypes,
expressed
relative
to that of wt PLAP,
is greater
at 56 #{176}C
than
at 65 #{176}C,
even
when
the enzymes
are
exposed
for longer
time intervals.
The addition
of ZnCl2
during
incubation
at elevated
temperatures
could
partially
protect
some
GCAP
phenotypes
from
denaturation (not shown).
Therefore,
to describe
the kinetics
of
heat inactivation
in more detail,
we analyzed
the residual activity
of wt PLAP
time-dependently
in 1 molJL
DEA buffer,
pH 9.8, containing
20 moI/L
ZnCl2 and 0.5
mmol/L
MgCl2,
both at 56 #{176}C
and at 65 #{176}C
(Figure
5A).
At 65 #{176}C,
the wt PLAP
inactivation
showed
a biphasic
process,
with
the first phase
lasting
approximately
10
assessment
and GCAP
CLINICAL CHEMISTRY,
Vol. 38, No. 12, 1992
2497
100
A
100
65C
A
80
50
60
20
wt PLAP
56 C : 0
wtPLAP
65C:I
I
)
100
I
10
I
20
I
I
30
40
50
60
30
40
50
60
40
50
60
56C
B
.80
B
100
>1
u6O
a
4O
50
20
0
0
Q
Q9
20
C,
[GJPLAP:o
[HLG] PLAP :
[SHLG] PLAP : o
b
0
Fig. 4. Residual enzyme activity of the wt PLAP, PLAP mutants, and
GCAP phenotypes after heat inactivation in 1 mol/L DEA buffer, pH
9.8, containing
0.5 mmol/L MgCI2 at (A) 65 #{176}C
for 6 mm and at (B)
10
[QSHLG]PLAP:u
Cl)
10
I
20
56 #{176}C
for 30 mm
Values expressed
mm;
tion
relative to the activity of the unheated
samples
100
at 56 #{176}C,
a slower
process
was
heat
inactivation
PLAP
mutants
turation
but almost
monophasic
inactivaapparent.
Likewise,
kinetic
analysis
of
of [GIPLAP
and the more
complex
at 56 #{176}C
(Figure
5B) showed
that dena-
occurred
by
way
of a monophasic
mechanism.
The introduction
of additional
mutations
in [GIPLAP
increased
the stability
of the more
complex
mutants.
The heat inactivation
behavior
ofthe
GCAP phenotypes
at 56 #{176}C
(Figure
5C) could
also
be described
as a
monophasic
process.
Core GCAP and JEG3 GCAP had a
general
stability
comparable
with that of the multiply
substituted
PLAP
mutants.
However,
wt GCAP
and
BeWo
GCAP
consistently
showed
lower stability.
This
difference
must
be
related
to
the
single
amino
acid
substitution
of Met’
(core GCAP)
for Asn’
(BeWo
Met’33
for Val’33
(wt GCAP)
substitution
effect on the stability
of wt GCAP.
This
already
described
as being
conformationally critical,
causing a 10-fold loss in immunoreactivity
with
MAb
C2. Therefore,
from the antibody
affinity
studies
and the heat
inactivation
analysis,
we can
attribute
a structural
role to the allelic
amino
acid
residue
at position
133.
The double
Val36’
(for Leu361) and Pro
(for Arg479)
substitution
in core GCAP is silent
in terms
of heat
inactivation
behavior.
Yet, from the pronounced
effect of
the Val36’
to Leu36’
substitution
in wt PLAP
on the
immunoreactivity
of C2, we can attribute
a structural
role to this amino
acid position
that
is evident
only
when
it is combined
with additional
amino
acid substitutions
that confer
the general
GCAP
structure.
GCAP).
The
had a similar
residue
was
Active
Site Properties
of GCAP
Allelic Variants
We showed
recently
that the Glu429 for Gly429 substitution
in wt PLAP
is accompanied
by a small
decrease
2498
CLINICAL
CHEMISTRY,
50
Vol. 38, No. 12, 1992
20
CoreGCAP:
0
JEG3 GCAP :
wtGCAP:
0
10
IBeW0GCAP:
a
io
20
Time
Fig. 5. Kinetics
containing
ofthe
heat inactivation
30
(mm)
in 1 mol/L
DEA buffer,
pH 9.8,
20 moVL
ZnCI2 and 0.5 mmol/L MgCI2 of (A) wt PLAP at
56 #{176}C
and 65 #{176}C,
(B) the different PLAP mutants at 56 #{176}C,
and (C)
the different GCAP phenotypes at 56 #{176}C
Michaelis
constant
(Km) from 0.35 mmol/L
to 0.1
when measured
in 1 mol/L DEA containing
0.5
mmol/L
MgCl2
(18, 24). This substitution
only slightly
affected
the turnover
number
(k)
from 460 s’
(wt
PLAP) to’344 s’ ([Gly429]PLAP).
Additional
substitutions
in wt PLAP
did not further
affect
these
kinetic
parameters.
We have
now confirmed
that
this
result
also holds
for th
core GCAP
mutant
and the three
GCAP
phenotypes
studied;
they
have
very similar
Km
(0.1 mmolJL)
and
(280-300
1)
values.
The substitution
in wt PLAP
of Glu429 for Gly429 accounts
for the
differential
inhibition
ofGCAP
by L-Leu (18), a phenomenon
explained
by steric
hindrance
exerted
by the
Glu4
side chain in PLAP,
but absent
in GCAP,
during
positioning
of the inhibitor
in the active
site of the
enzyme
(24). Our present
comparison
ofthe
inhibition
of
the different
GCAP phenotypes
by increasing
concentrations of L-Leu (Figure
6A) confirms
that
all GCAPs
in
mniol/L
#{149}
A
100
These
mutant
we
phenotype
displaying
the consistent
characteristics
of GCAP,
including
immunoreactivity,
molecular
stability,
and inhibition
properties.
The use ofcore
GCAP
as an antigen
for the production
of monoclonal
antibodies
is likely
to
allow the generation
ofreagents
that will show very low
cross-reactivity
with PLAP
and will enable
us to produce
antibodies
for which
immunoreactivity
is only
constructed
80
40
>1
20
0
0.05
0
0.1
1
10
studies
show
behaves
that
as
the
core
a prominent
GCAP
GCAP
marginally
determined
by allelic
amino
in GCAP.
These
monoclonal
antibodies
valuable
in the specific
determination
serum
of patients.
acid variations
would
prove
of GCAP
in the
100
801-
Supported
by grant CA42595
from the National
Health and by the Veremging
voor Kankerbestrijding,
We thank Elisabeth
Bossi for help with the transfections
60
DNAS.
0)
40
References
(GI PLAP:
[HLG] PIAP:
201-
1. Harris H. The human
and what we don’t know.
2. Beckman
G, Beckman
PLAP:
PLAP:
I.
0L
0.05
0.1
1
10
[L-Leu]
(mM)
Fig. 6. Inhibition of the enzymatic activity of (A) the different GCAP
phenotypes
and (B) the different PLAP mutants by increasing L-Leu
concentrations
(0.05-i 0 mmol/L) in comparison with wt PLAP
investigated
efficiencies
are
than
Significance
of Core
inhibited
by L-Leu with 10-fold
higher
wt PLAP.
The [Gly41PLAP
and the
triple
[His’,
Leu4,
Gly429]PLAP
mutant
are inhibited with even slightly
higher
affinities
(Figure
6B). We
have
shown
previously
that
Ser
in GCAP
plays
a
modulating
role on the L-Leu inhibition.
As soon as this
mutation
is superimposed
on top of the other
PLAP
mutations,
L-Leu
inhibition
profiles
are identical
to
those
obtained
for GCAP.
All
the
raised
immunological
MAbs
GCAP
and 130) used in this study
Therefore,
it is not surprising that
GCAP
was generally
recognized
with
lower
affinities
than
PLAP.
However,
it is clear
from
the
present
study
that amino
acids
in allelic
GCAP
positions can strongly
affect the conformation
and immunoreactivity
ofthe
enzyme.
Only two MAbs
(17E3
and C2)
consistently
showed
a low reactivity
with the different
GCAP
phenotypes,
a property
that has been exploited
in
the clinical
evaluation
and quantitation
of PLAP
and
GCAP
in serum
(9, 10). A third
MAb
(H317),
with
properties
comparable
with those of 17E3 and C2, has
also been
described
(27); however,
to date there are no
were
(except
against
reagents
Institutes
of
Belgium.
of mutant
151
PLAP.
that
allow
the
specific
mea-
surement
of GCAP
in the presence
of PLAP.
Yet,
a
correct
assessment
ofthe
GCAP concentration
would be
clinically
valuable
because
in seminoma
(9, 10, 13-15,
25), other testicular
tumors
(14, 15), and ovarian
cancer
(16, 1 7) GCAP
concentrations
increase
in serum
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monoclonal
antibodies.
Prog
Clin Biol Res 1984;166:67-75.
Appendix
The technical
issue
discussed
here was raised
when
this manuscript
was being
reviewed
and merits
consideration.
Reviewer’s
question:
The reactivities
of antibodies
C2
and H7 with the different
isoenzymes
and mutants
are
not uniform,
as shown
in Figure
2 of this paper and in
previous
reports
[Mill#{225}n JL, Stigbrand
T. Eur J Biochem
1983;136:1-7;
Hoylaerts
MF, Mill#{225}nJL. Eur J
Biochem
1991;202:605-16].
The concentration
of antigen [El#{176}
was determined
by use of these
monoclonal
antibodies.
Why can you get a true value
of [E]#{176}?
Authors’
response:
Initially
we used an ELISA
procedure [Hoylaerts
MF, Manes
T, Mill#{225}nJL. Biochem
J
1992;286:23-30]
in which
plates
were
coated
with
a
polyclonal
antiserum
to PLAP.
After
deposition
of the
2500
CLINICAL
CHEMISTRY,
Vol. 38, No. 12, 1992
samples,
we used 500 gfL
ofH 7/C2
for the detection
of
bound
PLAP/GCAP
mutants,
using
H7 preferentially
for the GCAP-related
enzymes
(or mutants).
After
revealing
the bound
monoclonal
antibody
with
biotinylated rabbit
antiserum
to mouse
IgG and Vectastain
ABC reagent,
the absorbance
was read on calibration
curves
constructed
with purified
PLAP.
Concentrations
thus determined
yield only estimates
of [El#{176}.
Because,
in principle,
the
enzyme
concentrations
could be estimated
from their catalytic
activity,
we have
determined
Km and
for each enzyme
mutant.
Km
determinations
are straightforward,
but to circumvent
the problems
encountered
in the ELISA
during
the
determination
of [El#{176},
we adapted
our immunoassay
as
follows:
A limited
amount
of H7 (10 ngfL)
was bound
onto
rabbit
antiserum
to mouse
Ig-coated
plates
and, during
the
incubation
amount
of antibody
was
increasing
concentrations
ofPLAP,
GCAP,
or mutant
enzymes.
The activity
of the
bound
enzymes
was measured
at 405 nm. Plots
of 1/A
(405 nm) vs the dilution
factor,
in the range
of saturating concentrations,
are then linear.
The A (405 nm) at
infinite
concentration
(intersection
with y-axis)
represents
the activity
of fully
saturated
H7 monoclonal
antibody.
At saturation,
independent
of the mutant
studied
or of its affinity,
the absolute
amount
of the
enzyme
is constant.
Therefore,
the differences
in the A
(405 nm) values
at the intersection
reflect
differences
in
When
relating
these
A (405 nm) values
to that
found for the reference
PLAP
(for which
can be
calculated
easily),
it is possible
to calculate
for the
progressively
different
step,
this
saturated
enzyme
mutants.
low
with
Thus,
we
found
that
values
are only marginally
influenced
by the different
substitutions
investigated.
This, we believe,justifies
the
choice of enzyme
concentrations
based
on activity
measurements,
as was done
during
the measurements
of
relative
affinities.