Clinical
42:3
Chemistiy
436-439
.
Un1ai4
(1996)
Relation between
number of apolipoprotein(a)
kringle 4 repeats and mobility of isoforms
in agarose gel: basis for a standardized
isoform nomenclature
M.
SANTICA
The
648
size of apolipoprotein(a)
kDa because
of interallelic
kringle
4-encoding
apo(a)
sizes
sodium
dodecyl
method,
followed
tween
the
in the
apo(a)
number
and
in agarose
related
agarose
gel
designate
was
apo(a)
(r
the
mobility
the
isoform
A nomenclature
isoforms
by
the
was
mIu1s:
genomic
phenotype
blotting
genotype
.
#{149}
pulsed-field
allelic
that
encodes
the
of kringle
genomic
DNA
alleles,
of
electrophoresis,
differences.
#{149}
of a 5.5-kb
of kringle
of individuals
tion
fragments
numbers
that
[apo(a)j
circulates
B component
Apo(a)
shares
ogen:
multiple
one
linked
of
as
LDL
comprises
which
copy
[2]. Each
is a highly
in plasma
three
a high
of
glycoprotein
bridge
{Lp(a)]
domains,
identity
plasminogen-like
5 domain,
and
is stabilized
to the
lipoprotein(a)
structural
of sequence
of the
of kringle
kringle
part
distinct
degree
copies
polymorphic
by a disulfide
with
apo(a)
events
forms.
domain
vary
Department
sity of Washington,
2 Department
Texas
of Medicine,
Northwest
Seattle, WA 98103.
of Internal
Southwestern
*
Address
98103-9103.
November
and
author
e-mail
abbreviations:
Dallas,
at:
2121
Laboratories,
Genetics,
TX
75235.
N.
35th
Univer-
University
St.,
Seattle,
accepted
apolipoprotein;
and
SDS,
December
Lp(a),
sodium
WA
27,
and,
of apo
B- 100,
apo(a)
polymorphs
molecular
sulfate.
apo
these
mass
to immunolocalize
1995.
436
in
by
the
they
associated
Little
the
pathogenesis
by
apo(a)
used
their
with
is known
of
size
isoforms
the
lack
and
possible
to
first
to identify
of a
polymorphic
to determine
sensitivity
not
the
mobility
apo(a)
resolution.
compare
to apo
by sodium
data
B), and
a number
from
1 for
of 23 apo(a)
relative
through
to that
S4 (mobility
et al. [8] resolved
sulfate
the
of
as F, B (mobility
Gaubatz
11 for the highest.
a total
Sl
presence
electrophoresis
isoforms
isoforms
dodecyl
the
gel
of the
the
B). Subsequently,
to
are
complicated
polyacrylamide
designated
or identical
than
restric-
studies.
from
slower
assigned
lipoprotein(a);
dodecyl
(PAGE)
than
different
[1].
apo(a)
the various
currently
isoforms
than
been
et al. [7] were
apo(a)
faster
of
[email protected].
apo,
gel electrophoresis;
14, 1995;
Research
Molecular
at Dallas,
to this
Fax 206-685-3279;
polyaciylamide
Received
Medicine
Center
correspondence
Nonstandard
PAGE,
Medical
Lipid
is
by different
Utermann
six
has
between
the methodologies
it
produced
affects
relation
dramatically
Consequently,
bonds
generate
contain
disease
to designate
Additionally,
isoforms
gel
More
[6].
heterogeneity
of the
system
that
thus
concentrations
cerebrovascular
size
pathological
standardized
plasmin-
disulfide
Examination
the
apo(a)
pulsed-field
blotting.
and
flank
between
using
alleles
sizes
Lp(a)
and
how
apo(a)
4 repeats
plasma
Lp(a).
two
different
of kringle
about
and
of
4 domain,
protease
internal
[J]3
each
kringle
an inactive
by three
apo
have
by
genomic
digested
sites
in number
et
different
they
whose
of
arranged
Lackner
sequences;
DNA
performed
of
Increased
containing
the
and
90%
2 domain.
vary
and
because
sequence,
alleles
that
intra-
in size
enzyme
2 repeat
Lp(a).
4 type
2-encoding
site-fractionated
the
and
apo(a)
a restriction
4 repeats
cardiovascular
Apolipoprotein(a)
4 type
with
type
kringle
34
to
4 domains
3 to 40 repeats
for
of apo(a)
polymorphic
distinguish
referred
the
from
is responsible
heterogeneity
apo(a)
numbers
gel electrophoresis
[5] and
for
2 varies
of copies
to
4 repeats.
INDEXING
for
except
4 type
is highly
subset
number
copy
motif
of kringle
number
on the
is proposed
respective
in a single
types
in the
al. [5] could
12
number
gene
structural
10 different
of kringle
alleles
ALBERS’
triple-loop
are
size
in tandem,
who
migrated
apo(a)
The
between
kringle
the
variation
of apo(a)
contained
of the
present
number
interindividual
electro-
typical
[4, 5]. The
sequences
gel
the
J.
JOHN
There
[2, 3], each
in
be-
in 29 individuals
that
log
distance
0.999).
=
apo(a)
and
evaluated
The
to the
4-encoding
by pulsed-field
isoforms
4 repeats.
Plasma
relation
produce
as a kringle.
electrophoretic
The
kringle
blotting,
gel
gene.
and
HOBBS,2
that
to
of
by a high-resolution
gel
as assessed
genomic
had 48 expressed
linearly
apo(a)
immunoblotting.
of apo(a)
gene,
41 kringle
in the
determined
H.
HELEN
varies
from
187
in the number
sulfate-agarose
isoforms
kringle
were
by
phoresis
and
[apo(a)]
differences
sequences
isoform
MARCOVINA,l,*
lowest
Kamboh
isoforms
11
(SDS)-PAGE
apparent
and
apo(a)
et al. [9] were
by SDS-agarose
able
Clinical
gel
electrophoresis;
with
they
1 identifying
smaller.
the
More
described
designated
larger
recently,
by
size
totally
consistent
in human
with
the
apo(a)
but
no system
4 repeats
in the
protein
identify
in the
apo(a)
The
of
mathematical
in the
this
agarose
gel
by
(b) to replace
nation
for
each
with
search
based
defined
nomenclature
the
apo(a)
of apo(a)
with
and
numbers
a desig-
of kringle
4
polymorphs
among
the
different
The
sizes
of the
of the
3l)2M754I)
19 21 2324
of apo(a)
7
55’)
32 32 3135
153031
4 Number
isoforms
from
human
plasma.
from each individual
was subjected
to electroagarose gel: the plasma proteins were transferred
and immunoblotted
with apo(a)-specific
monocloprepared
as described
in Materials
arid Methods
and at the end of each gel. The apo(a) isoforms
in
a numerical
system (10). The number of kringle 4
by pulsed-field
gel electrophoresis
with the larger allele designated
as
allele as allele 2. The major expressed
allele is indicated
allele land the smaller
in bold type.
re-
and Methods
LeucoPrep
were
embedded
incubated
alternating
gel
(Beckman
with
number
with
Apo(a)
gel
and
were
from
at the
calibrator
is formed
the
variation,
we
and
distance
being
at the
middle
tration
was
measured
The
capture
size.
isoforms
between
of the
isoform
is insensitive
monoclonal
each
and
to
35 in the
1 and
the
Lp(a)
protein
monoclonal
antibody
(a-6)
size
Lp(a)
epitope
observation).
concentration
agarose
gel electrophoresis
with
phoretic
mobility
in plasma
can
to
the
between
A is
migration
calibrator,
apo(a)
is
concen-
relation
dance
heterogeto
between
or were
4 number
the
2. However,
genomic
blotting
example,
allele
kringles
had
a slower
mobility
but
=
was
and
similar
than
in electro-
and
protein
in lane
in mobility
relation
the
relative
1). The
in
+ 3.7;
not
corre-
The
(Fig.
-0.04x
31
varied
isoforms
resulted
allele
the
in agarose
4 repeat.
was
I product
in amounts
change
number
we
in plasma
Among
in the samples
there
therefore,
present
method.
gel (x) was curvilinear
kringle
after
remaining
expressed
The
in the
isoforms
isoform;
not
kringle
with
The
different
For
28 kringles
apo(a)
between
samples.
with
different
two
apo(a)
apo(a)
log of K4 number
in Fig.
had
4 number.
of
(natural
as shown
1). All
kringle
kringle
distance
a-5 (Fig.
of apo(a)
one
been
a broad
antibody
observed
of
had
12 to
were
our
we
from
immunoblotting.
of
4
gel,
immunoblotting
two
migration
a difference
transformation
in the
isoform
the
who
ranging
and
alleles)
limit
kringle
to represent
4 repeats
either
(null
of
in agarose
individuals
a single
the
number
comparison
alleles
isoforms,
sponds
selected
19 exhibited
sensitivity
inversely
1
19
were
monoclonal
exhibited
individuals
the
mobility
and
these
The
assign
is directed
these
the
relative
(kringle
12 of
individuals
that
between
from
for this
only
identified
antibody-based
to apo(a)
selected
below
gel-to-gel
isoform
of the
spot.
gel.
sizes
but
by
35, with
for
antibody
an
9 (unpublished
gel electrophoresis
apo(a)-specific
as a
used
and
alleles,
concluded
A] + 1, where
±
measurement
by a double
that
of
24,
1 and
isoform
The
agarose
seven
samples
normalize
X B)
of apo(a)
using
system
to obtain
formulation
[(34
spectrum
plasma
7.4,
samples
43),
SDS-
their
genotyped
individuals
was
end
To
following
between
immunoassay
at the
plasma
anti-human
of three
isoforms
1, 8, 14,
samples:
evaluated.
taken
[12].
and
the
in the
apo(a)
isoforms
apo(a)
used
in mm
enzyme
by
largest
number
B is the
different
pH
and
as
as estimated
numerical
buffer,
the
relation
by
by immunoblot-
A mixture
the
previously
probe
electrophoresis,
Tris-NaCI
approach,
in apo(a)
analyzed
[5].
an arbitrary
Before
4 type
monoclonal
recognizes
in nmol/L.
establish
repeats
blotting
by a high-resolution
followed
kringle
this
2, and
specifically
Results
To
designated
sequences
in 20 L.
beginning
the distance
sample
with
with
calibrator
designating
[10].
protein
were
apo(a)
with
4 type
detection,
NJ)
apparatus
genomic
calibrators
by using
diluted
of Lp(a)
individuals
isoform
4-encoding
be expressed
to transverse
4-specific
alleles
Thus,
by
England
Ir
and
in
by
in kringle
for
were
(New
subjected
kringle
method
reported
Park,
segments
GeneLine
identified
designated
were
ng
were
of HpaI
apo(a)
electrophoretic
previously
samples
apo(a)
characterized
isoforms
U
CA),
The
of kringle
by comigration
agarose
an
[11].
3-mm
with
blood
Lincoln
and
were
Fullerton,
described
total
plugs
electrophoresis
performed
125
The
100
whole
present
used
present
described
from
Dickinson,
plugs,
2 h with
Instruments,
previously
ting
(Becton
MA).
by a modification
procedure
isolated
agarose
for
a-40,
estimated
electrophoresis
tubes
Beverly,
were
Leukocytes
in
twice
Biolabs,
alleles
gel
et al. [Ii].
using
was
apo(a)
pulsed-field
Lackner
neity
254131
IS IS
A total of 0.5-5
.tL of plasma
phoresis on a horizontal
1.5%
to a nitrocellulose
membrane
nal antibody
a-5. A calibrator
was included at the beginning
the calibrator
are identified
by
an epitope
as
17
groups.
Materials
the
31)2427
13 14 Il
repeats for each apo(a) allele, as determined
of DNA and genomic
blotting
(5, 11), is given,
available
to standardize
12
Fig. 1. Immunoblot
genomic
(c) to make
3i)$37I924
2
Kringle
the
4 repeats
number
#{149}
3,.
.‘\)ktel
AlkIc
isoforms
nomenclatures
kringle
size
evaluate
of
sequence,
its
to
to
needed.
of kringle
total
of
approach
electrophoresis
on the
were
in the
of the
of apo(a)
number
gel
within
[5]. In
is greatly
mobility
arbitrary
isoform
contained
samples
the
and
the
identified
the number
(a)
Apii (a)
Isolorms
34
migration
plasma
threefold:
between
system
the
437
were
blotting
a standardized
was
pulsed-field
DNA,
to
in human
study
relation
determined
repeats
Clearly,
polymorphs
aim
alleles
to relate
gene
3, 1996
the
detected
findings
genomic
was provided
No.
method
sizes of the apo(a)
isoforms
number
of kringle
4 repeats
apo(a)
gel.
23
the
et al.
34 different
and
and
of
[JO]. Their
reported
gel electrophoresis
gene,
kringle
size
Marcovina
plasma
this latter
study
the relative
shown
to correlate
with the
apo(a)
apo(a)
42,
numerically,
a modification
et al. [9],
isoforms
by pulsed-field
apparent
using
Kamboh
apo(a)
isoforms
the
Chemistry
r
a perfect
resolution
3 (Fig.
I product
to allele
a
=
log
linear
0.999),
concorin all
1) with
28
in lane
14
1 product
438
Marcovina
et al.: Kringle
4 repeats
and
electrophoretic
mobility
of apo(a)
isoforms
Isoform
Q) 45
a>
a)
0.
! 35
a)
a)
.E 25
I.-
20
‘‘
0
0.
a>
0
I-
a>
.0
E
10
0
-5
5
10
15
20
25
30
Isoform Number
Apo(a)
between number of apo(a) kringle 4 repeats
in SOS-agarose
gel.
Fig. 2. Relation
relative mobility
kb
35
and their
The apo(a)
isoform
number
was assigned
by using the equation
given in
Materials
and Methods and is indicative
of the relative migration
rate in agarose
gel.
in lane
13 with
contained
one
degradation
kringles.
from
one
apo(a)
None
of the
bands
upon
of
their
samples
agarose
the
log
The
All
number
5, 9, and
exhibited
distance
in
the
extra
+ 3.7;
kringle
the
r
4
gel
previous
was
The
there
number
individuals
whose
pulsed-field
apo(a)
in agarose
number
mathematical
4 repeats
gel.
of
samples
content
rather
of sialic
acid,
drate
upon
that
their
could
relative
be
with
related
concordance
size
[13],
the
the
after
an
gel for
log of the
is given,
with the larger of
electrophoretic
in pathological
conditions
glycosylation
in the
Even
present
if the
study.
one
is affected
will
results
alone
size
approach
is the
maintained
kringle
of the sialic
frequency
nearly
per-
probably
K4 number
and
is observed,
allele
size
same
in diabetic
assume
other
or by
the
3 suggest
The
that
of two
of
these
phenotypes
quite
low.
In those
samples
can
gel
potential
isoforms
in
instances
be reanalyzed
the
misclassiby
an
4 number.
difficulty
differing
of
with
this
by only
one
Fortunately,
general
in which
of
in the
classification
are large.
the
and
electrophoresis
of a credible
isoforms
apo(a)
designated
kringle
agarose
and
mobility
size,
are
ex-
percentage
their
apparent
major
if both
a low
apo(a)
isoforms
the establishment
4, particularly
the
that
because
than
the
resolution
be
be required
is the
whether
phenotypes.
will
apo(a)
by factor(s)
in Fig.
studies
gel.
in which
cannot
mobility
classified
permit
occur,
between
can
nomenclature
as diabetes,
to
Additional
correctly
occur
such
is known
relation
though
are not
fication
apo(a)
differences
isoform
samples
of apo(a)
carbohydrate
of apo(a)
individuals.
data
that
of alteration
nondiabetic
The
suggest
possibility
electrophoretic
Removal
the
allele
in agarose
arbitrary
et al. [8] reported
would
polymorph
carbohy-
removal
for each apo(a)
migration
to determine
size.
system
34
in apo(a)
cluded
total
mobility
28
for differences
nonenzymatic
the
in carbohydrate
of the
27
account
mobility
gel
in agarose
Furthermore,
natural
of apo(a)
providing
protein
in their
positions
treatment.
between
rate
of
isoforms.
Gaubatz
detected
have
number
to determine
mobility
alters
by
we
the
thus
in apo(a)
[8]. However,
isoforms
blotting,
to differences
apo(a)
from
determined
migration
-30-50%
electrophoretic
fect
relative
for
samples
between
in apo(a)
represents
by neuraminidase
genomic
of apo(a)
differences
acid
and
samples,
than
11 apo(a)
previously
it is possible
which
electrophoresis
was
relation
differences
associated
the
the
or nomenclature
of plasma
unknown
designation
Conceivably,
some
this
4 of
use
relation
and
From
kringle
unambiguous
size
electrophoresis
the
kringle
the
apo(a)
gel
established
method
Through
31
4 Number
4 repeats
of kringle
mobility
The
is no standardized
phenotyping.
23
the two alleles provided first.
apo(a)
Discussion
apo(a)
32
Fig. 3. Direct comparison
of apo(a) immunoblots
(top) and genomic
blots (bottom)
on fresh plasma
and DNA from 10 individuals
preselected to have a relatively broad range of apo(a) sizes.
rarely
Currently
30
samples
0.997).
=
27
Kringle
10.
analysis
apo(a)
29
16181822211824262532
two
in lanes
immunoblot
with
-0.04x
=
had
polymorphs.
and
19
electro-
apo(a)
between
observed
DNA
compared
agarose
10 individuals
migration
(7-9)
apo(a)
and
individuals
two
relation
to that
of K4
with
observed
these
relative
identical
(natural
3).
gel electrophoresis
plasma.
and
essentially
from
plasma
we directly
10 exhibited
was
lanes
reflect
fresh
analyses
(Fig.
7 of
polymorph
fresh
number
using
blot
data
and
several
presumably
individuals,
gel/genomic
alleles,
that
Therefore,
phoresis/immunoblot
Only
bands
10 additional
pulsed-field
different
Furthermore,
extra
products.
obtained
the
30
to two
the
population
only
one
at a different
is
isoform
apo(a)
Clinical
concentration
bands.
to optimize
Note
that
considerably
the
narrower
field
gellgenomic
differ
by only
analysis,
8). Moreover,
the
method
is also
subject
degree
of
of two
in Fig.
in Fig.
not
closely
3 are,
1. Even
samples
4 are
lane
its
those
kringle
bottom,
and
resolution
bands
than
blot
one
the
isoform
to measurement
reproducibility
and
remains
2.
pulsed-
two
alleles
Fig.
3,
be
tein(a).
rigorously
4.
van
Proteins
der
Additional
minor
bands
or aggregated
Fig.
1. To
low
frequency
cover
use
samples
a large
of the
that
a partial
excluded.
fresh
samples
and
the
the
sizes
isoform
sizes,
over
plasma
potential
the
sample
of apo(a)
designation
Addition-
analyzed,
no
of the
handling
5.
and
6.
the
Lackner
C,
extreme
size
Mol
repeat
Beisiegel
JC,
U,
kringle
kringle
Genet
Cohen
ME,
apolipoprotein(a)
major
Hum
are
Kastelein
JJ,
IV repeats
which
in variably
sized
present
1993:2:361-6.
Hobbs
polymorphism
variation
use of
HH.
Molecular
definition
in apolipoprotein(a).
Hum
of
Mol
the
Genet
may
E, Leffert
in
CC,
gene
plasma
J, Lackner
Lin
accounts
for
Iipoprotein(a)
C, Chiesa
greater
G, Hobbs
than
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Boerwinkle
Apolipoprotein(a)
extra
that
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storage
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YY,
ML.
isoforms.
to
cannot
importance
band
the
necessary
period.
degradation
of minor
in
considering
shipment
are
differ
samples
it was
a 5-year
emphasize
proper
and
during
samples
products
in some
of apo(a)
collected
We
degradation
apparent
of the samples
fresh
are observed.
minimize
range
were
bands
represent
are
extreme
thawing
VVhen
that
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ally,
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to
439
3, 1996
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and apolipoproteins.
Washington,
DC: AACC Press, 1994:235-63.
gel electrophoresis
errors
No.
spaced
(see
the
resolved
pulsed-field
42,
in general,
in the
in which
readily
Chemistry
7. Utermann
G, Menzel
Hi, Kraft HG, Duba MC, Kemmler
HG, Seitz C.
Lp(a) glycoprotein
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inheritance
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In conclusion,
ing
bands
in our
constant
had
because
the
SDS-agarose
throughout
the
number
We
recommend
and
establish
spectrum
the
and
different
relation
closely
relative
phenotyping
system.
basis
for
a common
standardized
ing
direct
of
nomenclature,
the
results
mobility
provide
thereby
obtained
gel.
evaluate
and
may
by
JW, Ghanem KI, Guevara ii, Nava ML, Patsch W, Mornsett ID. Polymorphic
forms of human apolipoprotein(a):
inheritance and relationship
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I Lipid Res 1990:31:603-13.
relation
in the
number
approach
is not
8. Gaubatz
we have
carefully
kringle
This
sizes,
a linear
migration
laboratories
migrat-
system
to obtain
between
in their
comparison
two
of apo(a)
K4 number
of kringles
that
between
gel electrophoresis
the entire
to log-transform
between
distance
the
9. Kamboh MI, Ferrell RE, Kottke
morphism
of apolipoprotein(a).
74.
10. Marcovina
SM, Zhang ZH, Gaur VP, Albers ii. Identification
of 34
apolipoprotein(a)
isoforms:
differential
expression
of apolipoprotein(a) alleles between
American
blacks and whites.
Biochem
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1993:191:1192-6.
allowdifferent
researchers.
11.
This
research
Health
We
thank
excellent
gator
was
Program
Tommy
technical
for
the
supported
Project
Hyatt,
the
HL3
Jean
Mernaugh,
assistance.
American
by
Grant
H.H.H.
Heart
National
0086,
and
and
Institutes
ROl
Hal
is an Established
Kennedy
12.
for
Investi-
Association.
13.
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