Note: Descriptions are shown in the official language in which they were submitted.
METHOD OE' MAPPING POLYPEPTIDE
LIGAND BINDIMG REGIONS
Field of the Invention
This invention relates to a rapid method of
mapping polypeptide ligand binding regions. More
particularly, this invention is directed to a method
utilizing partial enzymatic digestion of polypeptides and
subsequent separation and hybridizing/labeling of the
product polypeptide partial hydrolysates to locate the
approximate position of regions in the primary structure of
the polypeptide that exhibit selective affinity for other
chemical species.
Backqround and Summary of the Invention
Knowledge of~the mechanisms and the sites of
molecular interactions of polypeptides and proteins
(hereinafter referred to genera]ly as "polypeptides") with
biologically active chemical species, includiny peptides,
polypeptides, ribonucleic acids (RNA), deoxyribonucleic
acids (DNA), DNA/RNA analogs, carbohydrates, phospolipids
and other chemical species of biological significance can
provide insight into the biolQgical activities of such
chemical species. Such knowledge can be an invaluable
resource for medical research and development, including
; particularly, drug design. Examples of common molecular
interactions of biological significance are the high
; affinities and the resulting selective bonding of
antibodies to their respective target antigens, cellular
receptors to their target chemical species, and enzymes to
; their target substrates.
Antibodies are proteins produced by vertebrates
as a defense against infection. Each antibody contains a
unique binding site that exhibits selective affinity for at
least a portion of the molecular structure of the antigen
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that induced its production. Those unique molecular
structures on an antigen that are affinity~targeted by the
antigen binding site on an antibody are referred to as
antigenic determinants or epitopes. Most antigens present
a multiplicity of antigenic determinants on their surfaces.
In the study of antibody-antigen interaction, it is often
important to determine exactly what region (epitope) of the
antigen structure is interacting with (binding to) a
particular antibody and what region on the antibody
structure is interacting with the antigen.
Traditional approaches to antibody epitope
mapping all require amino acid sequence information at some
stage of the characterization protocol. Protein
footprinting relies on the ability of the antibody to
protect the antigenic site from random proteolysis or
chemical modification, but the protected peptide must
invariably be sequenced to identify the epitope. Limited
antigen digestion followed by immunoblotting may also
identify a cross-reactive fragment, but again the fragment
must be sequenced to locate its position in the parent
antigen. While comparison of antibody affinity for a
series of nearly homologous proteins can often correlate
differences in specificity with known amino acid
substitutions, such closely related, previously sequenced
protein families are not common, and considerable effort is
required to generate them artificially by mutagenesis of a
cloned antigen. Alternatively, competition between antigen
and synthetic or natural peptides for binding to the
antibody can often reveal the targeted epitope, but again
the peptide must be sequenced to determine its location in
the antigen's primary structure. In a few cases, epitopes
have been assigned without sequence information to an
enzyme' 5 active site based on the antibody's ability to
inhibit catalytic activity, but even in that case caution
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must be exercised to ensure that the antibody does not
inhibit enzyme activity by a noncompetitive mechanism.
Due to the expense and delay involved in
conventional methods of epitope mapping, there exists a
strong need for a less cumbersome method of epitope
mapping. More generally, there is a need for a means for
mapping regions of polypeptides primary structure that
exhibit selective affinity for certain chemical species.
Accordingly, it is one object of this invention
to provide a method for locating sites or regions (in
polypeptide primary structure) of associative interaction
between a polypeptide and a ligand comprising a second
chemical species.
It is another, more particular object of this
invention, to provide a rapid method for identifying the
location of antibody binding antigenic determinants in the
primary structure of polypeptides.
It is still another object of the invention to
provide a method for mapping regions of the primary
structure of an exoprotease digestible polypeptide which
exhibit sel~ctive affinity to a chemical species (ligand)
by partially digesting the polypeptide with an exoprotease
and determining the molecular weight of the lowest
molecular weight species still exhibiting said selective
affinity.
In still another embodiment of this invention
there is provided a kit for mapping regions or epitopes in
the primary skructure of a polypeptide.
Those and other objects are realized in
accordance with this invention which is directed
particularly to a protocol that allows identification of
the approximate position of an epitope or other region in
the primary structure of a protein exhibiting affinity to a
chemical species without requiring the tedious procedure of
sequencing said epitope or region. Generally, the method
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in accordance with this invention comprises the steps of
subjecting the targeted polypeptide to denaturing
conditions to initiate unfolding of the polypeptide and
thereafter digesting the polypeptide with an exoprotease
substantially free of endoprotease activity to provide a
mixture of a first set of polypeptide hydrolysate species
containing the region of the polypeptide primary structure
that exhibits selective affinity to the chemical species
and a second set of polypeptide hydrolysate species that do
not contain said region. By determining the moleculax
weight of the lowest molecular weight polypeptide species
conkaining the chemical species-binding region, one can
locate or map that region relative to one or the other
termini of the parent polypeptide structure. The procedure
can thus be applied to define regions of polypeptide
primary structures that exhibit affinity either for other
polypeptides or for other chemical species, including
biologically active drug substances and other biologically
significant chemical species including, but not limited to,
peptides, DNA, RNA, DNA/RNA analogs, carbohydrates, lipids
and phospholipids.
Additional objects, features, and advantages in
the invention will become apparent to those skilled in the
art upon consideration of the following detailed
description of the preferred embodiments exemplifying the
best mode of carrying out the invention.
Detailecl Description of the Invention
In accordance with one embodiment of this
invention, the location of a region on a polypeptide which
exhibits a selective affinity for a chemical species is
determined by a procedure that includes the partial
digestion of the polypepkide with an exoprotease. To
ensure efficient exoprotease diyestion of the polypeptide,
; 35 the polypeptide is preferably subjected to denaturing
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conditions sufficient to initiate unfolding of the
polypeptide, thus ~nhancing exoprotease access to the
terminal end of the polypeptide. Polypeptide denaturants
well known to those of ordinary skill in the art include
urea, aliphatic amines, guanidine hydrochloride, ionic and
non-ionic surfactants, and heat. Each of these denaturants
or denaturant conditions can function separately or in
combination with other denaturant excipients, for example,
art-recognized disulfide-bond-reactive thiols to initiate
or otherwise enable the unfolding of the target
polypeptide. The potency or stringency of the denaturiny
conditions is dependent on both the denaturant selected and
the concentration of the denaturant. The potency of the
denaturing conditions is optimally selected to be within a
range that can initiate unfolding of the polypeptide,
without significantly inhibiting exoprotease activity.
After the at least partially denatured
polypeptide has been subjected to exoprotease digestion for
a time sufficient for the exoprotease to initiate
digestion, the potency or stringency of denaturing
conditions in the digestion medium can be reduced without a
adverse affect on the digestion process due to refolding of
the polypeptide. One preferred means of reducing the
denaturant stringency in the digestion medium is to carry
out the digestion in an aqueous medium in contact with a
dialysis membrane to allow the denaturants and amino acid
digestion products to diffuse from the medium during the
digestion process. High concentrations of cleaved amino
acid residues can work to inhibit exoprotease activity and
30 their removal from the medium allows polypeptide digestion ~`
to continue at a more or less constant rate.
The exoprotease used to digest the polypeptide
can be selected from any of the exoproteases well known to
those of ordinary skill in the art. The exoprotease must
be substantially free of endoprotease activity and capable
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of cleaving amino acids only from one end, either the
; carboxy-terminus or the amino-terminus, of a polypeptide.
Further, the exoprotease should exhibit its proteolytic
activity in the presence of denaturing conditions,
sufficient to initiate unfolding of the target polypeptide
and it should preferably effect a more or less constant or
consistent rate of digestion.
Carboxypeptidases and aminopeptidases comprise
two classes of exoproteases. Carboxypeptidases cleave
amino acids sequentially from the C-terminus of a
polypeptide, while aminopeptidases cleave amino acids
sequentially from the N-terminus of a polypeptide. In a
preferred embodiment of this invention, the exoprotease
used to di~est the polypeptide being assessed in accordance
with the present invention is selected from the group
consisting of carboxypeptidases and aminopeptidases.
Again, care must be taken to ensure that the polypeptide
and the exoprotease are substantially free of endoprotease
activity. The polypeptide is digested for sufficient
amount of time to provide a mixture of a first set of
polypeptide hydrolysate species containing the chemical
species-affinity-exhibiting-region and a second set of
polypeptide species hydrolysate not containing the
affinity-exhibiting region.
The next step for mapping of the
affinity-exhibiting region of the polypeptide is the
determination of the molecular weight of the lowest
molecular weight species of the polypeptide hydrolysates
;; still capable of exhibiting selective affinity for the
chemical species. One preferred means of making such a
determination includes the step o~ chromatographically
resolving the mixture of the partially digested peptide
species on the basis of polypeptide species molecular
weight. After the polypeptide hydrolysate species have
been chromatographically resolved, they can be fixed in
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their separated state, for example, by transfer to a
nitrocellulose or nylon membrane or by other means well
known to those of ordinary skill in the art. The separated
polypeptide hydrolysate species can then be contacted with
the chemical species to which the original whole
polypeptide exhibited a selective affinity under conditions
which said selective affinity can be exhibited by still
existing or intact affinity-exhibiting regions of the
polypeptide.
A polypeptide may exhibit a selective affinity
for a chemical species due to the conformational shape of
the affinity exhibiting region. These conformational
~; affinity-exhibiting regions consist of discontinuous amino
acid sequences that are in close spatial proximity to one
another in the secondary and tertiary structure of the
protein. When the discontinuous amino acid sequences are
separated by large intervening sequences, a partially
digested polypeptide will likely cease to exhibit affinity
for the chemical species once the first most terminal
discontinuous amino acid sequence of the affinity
exhibiting region is cleaved. Thus, without prior knowledge
that the affinity exhibiting region contains component
amino acid regions separated by large intervening
sequences, the interpretation of the exoprotease map could
be misleading. To avoid this problem, and to verify the
accuracy of the mapping, the polypeptide can be subjected
to two separate mapping procedures, each in line with that
described above, except one mapping procedure is carried
out using an aminopeptidase as the exoprotease, while the
other mapping procedure is carried out using a
carboxypeptidase as the exoprotease. In this manner the
affinity exhibiting region will be mapped in reference to
distance from the C-terminus and from the N-terminus. If
the affinity-exhibiting region is a conformationally
dependent affinity-exhibiting region, (i.e., based on the
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cooperation of discontinuous "partial epitop~s" in-the
primary polypeptide structure~ each mapping procedure will
indicate differing locations for the affinity exhibiting
region.
Compositions for mapping regions of the primary
structure of a protein which exhibit selective affinity for
a chemical species in accordance with this invention can be
provided in the form of a reagent kit. The kit comprises
; an exoprotease and an endoprotease inhibitor, and
optionally a protein denaturing composition selected to
provide a denaturant stringency sufficient to initiate
protein unfolding but insufficient to denature the
exoprotease. Thus an epitope mapping kit in accordahce
with this invention can include either one or both of a
carboxypeptidase and an aminopeptidase and an endoprotease
inhibitor. Preferably the kit contain~ one or more
additional components such as a denaturant solution of
predetermined stringency (or components for the preparation
thereof), dialysis membrane tubing, and a solution to
quench or stop the digestion reaction.
One advantage of the method of this invention is
that it does not require purification of the targeted
polypeptide containing a selective affinity exhibiting
region. As long as the polypeptide is free of endoprotease
~ 25 activity, the presence of other contaminants should not
j interfere with performance of this method. Thus, the
regions of the primary structure of an exoprotease
digestible polypeptide which exhibit a selective affinity
to a chemical species can be mapped relative to either
terminus of the polypeptide. The method comprises the
steps of partially digesting the polypeptide with an
;; exoprotease, where the exoprotease and the polypeptide are
substantially free of endoprotease activity to provide a
mixture of polypeptide hydrolysate species having a common
undigested terminus and di~ferent molecular weights. The
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polypeptide hydrolysate species are then
chromatographically separated on the basis of the species
molecular weight. The separated polypeptide species are
then contacted with the subject chemical species under
conditions in which selective affinity can still be
exhibited by still existing or intact affinity exhibiting
regions of the polypeptide. By determining the molecular
weight of the polypeptide hydrolysate species exhibiting
selective affinity for the chemical species, the location
of the affinity exhibiting region on the polypeptide can be
readily determined.
In accordance with one preferred embodiment of
this invention, the position of an antibody-binding epitope
on a polypeptide antigen is determined. The polypeptide
antigen is digested with a one or more carboxypeptidases to
provide a mixture of polypeptide hydrolysate species, each
containing the same N terminus but different degrees of
truncation at the C-terminus. After electrophoretic
separation and immunoblotting, the epitope-containing
polypeptide hydrolysate fragments are identified by art-
recognized techniques utilizing labelled antibodies capable
of providing a visual signal of the hydrolysates still
containing the intact epitope. The molecular weight of the
smallest molecular weight N-terminal fragment still capable
of selectively interacting with the antibody is then
identified. The molecular weight of the polyp~ptide
hydrolysate correlates to the number of amino acids
subunits contained in the polypeptide. Thus, the distance
- of the epitope from the N-terminus of the target
polypeptide antigen can be directly ascertained from the
immunoblot without need for sequence information.
In a preferred embodiment, an antibody-binding
epitope on a polypeptide can be located relative to either
terminu~ of the polypeptide by a rapid method that does not
require amino acid sequence determination. The method
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defines the location of the epitope in terms of itsdistance generally in molecular weight units from the N-
terminus or C-terminus of the intact protein. When a
protein is diyested by either an aminopeptidase or a
carboxypeptidase for a length of time insufficient to
complete the digestion, there is produced a series of
polypeptide hydrolysates differing only in length (i.e.,
molecular weight). If a polypeptide is digested with a
carboxypeptidase, all the hydrolysate species in the series
will have the same N-terminus; the hydrolysate species will
differ only in the number of amino acids removed from the
carboxyl end. In at least a portion the polypeptide
hydrolysates generated from the partial digestion reaction,
digestion will have proceeded through the location of the
epitope. These species are no longer be capable of
exhibiting selective affinity to the epitope-targeting
antibody. The polypeptide hydrolysates having the epitope
still intact can exhibit selective affinity to the
antibody. Determination of the smallest polypeptide
hydrolysate (lowest molecular weight) exhibiting selective
affinity to the antibody identifies the location of the
epitope relative to the end terminus of the intact protein.
; Highly folded protein structures are often
inefficiently cleaved by exopeptidases. Therefore, to
obtain a relatively continuous distribution of polypeptide
fragments (partial hydrolysates), it is important to
promote at least some degree of polypeptide unfolding of
; such structures prior to digestion. This can be
accomplished by subjecting the polypeptide to denaturing
conditions effective to initiate unfolding. Protein
~-~ denaturants are well known in the art and can be used alone
or in combination at different concentrations to attain a
predetermined denaturing stringency or potency. One
preferred way of initiating unfolding (denaturing) a
protein is to contact the protein in a denaturing solution
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containing, for examplP, urea, dithiothreitol, and
methylamine. The dithiothreitol is included in the
solution to disrupt intramolecular disulfide bonds that
could otherwise interfere with exoprotease digestion. The
exoprotease is typically added to the resulting solution of
the at least partially unfolded polypeptide. After an
initial one hour digestion under denaturing conditions, the
denaturants can be gradually removed by dialysis without
compromising the ability of the exoprotease to continue
digestion. Thus the exoprotease digestion step can be
conducted in a dialysis tubing. Typically the dialysis
tubing has a low molecular weight pore size ranging from
1000-3000 ~, that will allow denaturants and released amino
acids to diffuse out of the dialysis tubing while retaining
small protein fragments. Reducing the concentration of the
denaturants and the amino acids in the reaction medium
allows the exoprotease to function in a more efficient
manner. The dialysis tubing is usually suspended in the
same buffer used for the denaturing/digestion process, but
without (or lower concentrations of) denaturants. The
reaction is preferably supplemented with fresh enzyme
periodically during the digestion process to ensure a
constant digestion rate.
;~ One critical feature of this invention is the
elimination of any endopeptidase activity in the digestion
reaction medium. Even small amounts of contami.nating
endopeptidase in the digestion medium will invalidate the
results, because analysis of the data assumes the digestion
is only occurring from one terminus of the polypeptide.
The presence of contaminating endopeptidases will generate
~ smaller peptide fragments containing the targeted
- interactive site than would be yenerated with exopeptidase
alone. The production of smaller epitope-containing
polypeptide hydrolysates will give an anomalous indication
of the distance of the epitope or interactive site from the
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terminus of the protein. One method of eliminating or
minimizing contaminating endopeptidase activity in the
digestion reaction is to include an inhibitor of
endopeptidase activity such as ~2-macroglobulin. ~2-
5 ~acroglobulin is a potent inhibitor of virtually all
endopeptidases.
Digestion of the polypeptide is continued for a
period of time sufficient to generate a mixture of
polypeptide hydrolysate species having a common undigested
10 terminus and different molecular weights. Essentially
there is produced a set of hydrolysate species that still
contain the target region and a set of hydrolysate species
that do not still contain the target region. The reaction
can be terminated by boiling the digestion mixture or by
15 adding a strong denaturant. The resultant mixture of
hydrolysate species are separated by chromatographic means
according to molecular weight. One preferred means of
chromatographically separating the polypeptide hydrolysate
species utilizes electrophoresis through a gel matrix.
20 Typically, sodium dodecyl sulphate (SDS) polyacrylamide yel
electrophoresis is used to separate the hydrolysate
~; species, but other art-recognized chromatographic means can
also be utilized.
t The separated hydrolysate species are preferably
25 fixed in their separated state prior to assessing their
affinity to the subject chemical species. One preferred
~- manner of fixing the hydrolysate species involves blotting
the fragments onto a membrane such as nitrocellulose or a
nylon filter. The re~ulting immunoblots can then be
30 hybridized with the chemical species, ~or example, an
antibody capable of binding to the target epitope to
determine the lowest molecular weight species containing
the targeted reyion (epitope). Because high resolving SDS
polyacrylamide gels can often determine protein molecular
35 weights within 2,000 daltons, the present methodology
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allows mapping of regions exhibiting the selected a~finity
in the primary structure of a polypeptide with the same
degree of resolution.
Standard laboratory techniques can be used to
label or visualize the hydrolysate species still exhibiting
selective affinity to the chemical species. For example,
to visualize which hydrolysate species still contain an
antibody~binding epitope, the antibody that recc~gnizes the
epitope (primary antibody) is first hybridized with the
blot containing the transferred hydrolysate species. After
unbound primary antibody is washed off the blot, a
secondary antibody (an antibody capable of binding to the
primary antibody) is hybridized with the blot, followed by
washing to remove any unbound secondary antibody. The
secondary antibody is selected to have an enzyme conjugated
to it that is capable of reacting with a substrate to alter
the substrate in a manner that can be visualized.
Preferred secondary antibodies include goat anti-mouse or
~ goat anti-rabbit IgG-horseradish peroxidase conjugates.
; 20 Thus addition of the substrate will provide a visual
indication where the primary ant:ibody has bound and thereby
identify which hydrolysate species still contain the target
epitope.
More generally, a variety of methods well known
to those of ordinary skill in the art can be utilized to
~ visualize chemical species that interact with the
-~ immobilized polypeptide hydrolysate species. 'rhey include
- but are not limited to radioisotope labeling, fluorescent
labeling, and conjugating indicator molecules or enzymes
capable of reacting with indicator molecules to the
chemical species.
One common method for labeling a chemical species uses an
avidin-biotin complex. The methods used to synthesize
biotinylated chemical species, are well known in the art.
See, ~or example, E.A. Bayer and M. Wilcheck, The Use o*
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the Avidin-Biotin Complex as a Tool in Molecular Biology,
Methods of Biochemical Analysis, Vol. 26, pp. 5-9.
EXAMPLE ONE
Yeast carboxypeptidase Y and porcine pancreas
carboxypeptidase B (From Calbiochem) were selected because
of their resistance to denaturation by 6 M urea. Also, a
mixture of the two proteases yielded a less interrupted
digestion pattern than either carboxypeptidase alone. ~2-
Macroglobulin immobilized on agarose beads was ~btainedfrom Boehringer-Mannheim. Nitrocellulose paper (0.2 ~m
pore siæe) from Schliecher and Schuell, prestained low-
molecular-weight markers from GIBCO BRL, electrophoresis
reagents from Bio-Rad, and dialysis membranes (molecular
weight cutoff ~2000) from Spectrum were obtained.
Antigen and Antibodies
The 43,000 Da (residues 1-379) cytoplasmic domain
of human erythrocyte band 3 protein (cdb3) was purified to
homogeneity from fresh human blood. The polyclonal cdb3
anti-peptide antibodies, p32-34, p22 23, pl6-17, and pOO-O1
were raised in rabbits against synthetic peptides
corresponding to residues 283-297, 189-203, 142-154, and 1-
15 of the cdb3, respectively. The monoclonal antibodies,
`~ mAb41-43 and mAb36-41 have been mapped to residues 360-379
and 317-359 of cdb3 previously using conventional epitope
mapping techniques.
Carboxypeptidase Digestion
Because even small amounts of contaminating
endoproteases in the digestion mixture could invalidate the
results (vide infra), it became necessary to take special
; precautions to inhibit such contaminants. For this
purpose, both the antigen (1 mg/ml cdb3) and the
carboxypeptidase mixture (1000 units/ml of carboxypeptidase
Y and 100 units/ml of carboxypeptidase B) dissolved in
digestion buffer (50 mM sodium citrate, 10% acetonitrile,
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pH 6.0) were separately incubated for thirty (30) minutes
at room temperatura with 0.1 volume of agarose beads
containing immobilized ~2-macroglobulin, a potent inhibitor
of virtually all endoproteases. After incubation, the
heads were removed by centrifugation and an ali~uot of the
carboxypeptidase mixture (10 units of Y plus 1 unit of B)
was added to 1 ml of the cdb3 solution supplemented with 6
M urea, 10 mM dithiothreitol, and 20 mM methylamine (final
concentration) to promote cdb3 unfolding. The unfolded
cdb3 was allowed to digest for one (1) hour at room
temperature, after which the mixture was transferred to ;-
dialysis tubing suspended in digestion buffer supplemented
with 1 mM dithiothreitol. The digestion mixture in the
tubing was then treated again with ten (10) units of
carboxypeptidase Y plus 1 unit of carboxypeptidase B and
the digestion was continued in the dialysis bag for five
(5) more hours. After this period and again six (6) hours
~ later the dialysis bag was opened and the cont~nts were
; treated with the same amount of the carboxypeptidase
~; 20 mixture. After a total of twenty-four (24) hours
~ digestion, the reaction was terminated by boiling (5
-; minutes) and the polypeptide hyclrolysate species were
separated on a 15% Laemmli polyacrylamide gel and then
transferred to nitrocellulose for three (3) hours at 240
mA. The resulting blots were blocked for fifteen (15)
minutes with 4~ bovine serum albumin in blotting buffer (20
mM NaCl, pH 7.5) and incubated three (3) hours with ascites
fluid containing monoclonal antibody diluted 1:500 in
blotting buffer or with rabbit anti-cdb3 peptide antibody
diluted 1:100 in blotting buffer. The nitrocellulose
sheets were then washed with blotting buffer containing
0.05~ Tween 20 and incubated two (2) hours with goat anti-
mouse or goat anti-rabbit IgG-horseradish peroxidase
conjugate diluted 1:500 in blotting buffer. Blots were
stained with 4-chloronaphthol.
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Results
Carboxypeptidases comprise a class of proteases
that cleaves amino acids s~quentially from the C-terminus
of a polypeptide. Polypeptide cdb3 (Mr ~3,000; 379
residues) was digested by the carboxypeptidase mixture into
a series of different molecular weight peptides. When
digestion was extended to thirty-six (36) hours or
terminated prematurely at twelve (12) hours, a
disproportionately large number of low- or high-molecular-
; lo weight hydrolysate species, respectively, was obtained,
suggesting the twenty-four (24) hour digestion period was
roughly optimal for cdb3. The derived peptides were all
found o contain the amino-terminal region of cdb3, as
evidenced by their reactivity with pOO-Ol antibody. When
stained with antibodies previously mapped by more tedious
methods to relatively evenly spaced epitopes along cdb3,
~i the fragmentation mixture yielded a staining pattern that
- differed for the different epitope-specific antibodies.
Thus, m41-43, which recognizes an epitope between resides
360 and 379, stained only the largest molecular weight
~` component of he digestion mixture corresponding to a band
at Mr >35,000, but no fragments of smaller molecular weight.
The staining of p32-34 was terminated at M~ ~33,000, the
staining of p22-23 at Mr 22,000, pl6-17 at Mr -17,000 and
pOO-O1 near the bottom of the gPl. Thus, the digestion
mixture in each lane was immunostained from the molecular
weight position of the intact antigen to the approximate
position of tne Ppitope and no further. By observing where
the staining pattern terminated, a rough estimate of the
3G position of the antibody's epitope in the primary structure
of the antigen was obtained. Importantly, when an impure
mixture of the antigen and other proteins was trPated
similarly, an analogous result was obtained as long as
unwanted proteases did not contaminate the mixture.
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In developing this methodology, a number of
significant obstacles were encountered that required
modifications in the protocol. First, all commercial
carboxypeptidases tested came contaminated with small
amounts of endoproteases that cleaved the antigen
internally. Proteolysis with such enzyme preparations
generated fragments that were predictably stained by the
above antibodies at anomalously low molecular weights. For
example, the anti-peptide IgG, p32-34, stained blots of
cdb3 digested with an unmodified mixture of commercial
carboxypeptidases Y and B discontinuously from the
molecular weight of the intact protein (Mr ~42,000) to
~16,000 Da. In contrast, when the carboxypeptidase mixture
was pretreated with ~2-macroglobulin (a protein that
inhibits all endoproteases without inactivating
` exoproteases), the staining pattern terminated at the
expected molecular weight of 33,000 Da. Thus, the correct
epitope of p32-34, located between residues 283 and 297
(i.e., 33,000 Da from the N-terminus) was correctly
identified only when all endoprotease activity was
eliminated.
A second obstacle arose from the tendency of the
carboxypeptidases to balk at cleaving highly folded
protein. However, if the antigen was first unfolded in 6
urea, 10 mM dithiothreitol, and 20 mM methylamine, a more
continuous/complete digestion pattern was observed.
Therefore, to obtain a relatively continuous distribution
of N-terminal cdb3 fragments, it was necessary to promote
at least some degree of protein unfolding. After the
initial one (1) hour digestion under denaturing conditions,
the denaturants could be gradually removed by dialysis
without comprising the ability of the proteinase mixture to
continue cdb3 digestion. A dialysis membrane with a low-
molecular-weight cutoff (M, ~2000) was necessary to avoid
loss of the smaller protein fragments.
2~7~
-18-
Finally, because intrachain disulfide bonds
interfere with carboxypeptidase digestion, the reaction
mixture was always supplemented with dithiothreitol.
omission of this reagent l~d to appearance of major
discontinuities in the cleavage pattern of cdb3. The
digestion was also carried out mainly in a dialysis bag to
allow the escape of released amino acids that at high
concentrations inhibited the carboxypeptidases.
Discussion
For many experimental applications of antibodies,
; a crude evaluation of the antibody's epitope on its antigen
is sufficient. The present invention provides a relatively
~- simple method for identifying such epitopes in terms of
their distance in molecular weight units from the N-
terminus of the intact antigen. The most obvious
advantages of this protocol are (i~ that no sequence
in~ormation is required, and (ii) that the antigen need not
~- be pure. That is, as long as the antigen is not degraded
by contaminating endoproteases, other polypeptides should
not interfere with the map since they should not be
visualized in the immunoblots.
Although the invention has been described in
detail with reference to certain preferred embodiments,
~; variations and modi~ications exist within the scope and
spirit of the invention as described and defined in the
following claims.
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