Download Amino-terminal Sequences of the Major Tryptic Peptides Obtained

[CANCER RESEARCH 38, 2199-2208,
0008-5472/78/0038-OOOOS02.00
August 1978]
Amino-terminal Sequences of the Major Tryptic Peptides Obtained from
Carcinoembryonic Antigen by Digestion with Trypsin in the Presence
of Triton X-1001
John E. Shively,2 Michael J. Kessler,3 and Charles W. Todd
Division of Immunology, City of Hope National Medical Center, Duarte, California 91010
ABSTRACT
Sequence studies of Carcinoembryonic antigen (CEA)
were initiated to obtain direct chemical evidence regard
ing the structure of CEA for comparison with that of CEA
cross-reacting antigens. To obtain, purify, and perform
amino acid sequencing on mg amounts of CEA (a glycoprotein with a molecular weight of 180,000, composed of
60% carbohydrate by weight), we needed to develop new
approaches and refine existing techniques. This report
describes the procedures developed during the course of
this study and presents initial results. Trypsin digestion in
the presence of 0.25% Triton X-100 produced seven major
glycopeptide fragments that were separated and purified
by high-pressure liquid chromatography on ion-exchange
resins followed by Sephadex gel chromatography. NH,terminal sequences were determined on 1- to 2-mg
amounts of the glycopeptides by high-pressure liquid
chromatography for the analysis of phenylthiohydantoin
derivatives of amino acids. Four peptides gave sequences
through 20 cycles of Edman degradation, one gave se
quences through 13 cycles, and two gave sequences
through 10 cycles. Three of these peptides also gave
sequences of up to 8 to 13 cycles for a minor component.
The abrupt halt in Edman degradation for each peptide
was interpreted as the failure to sequence beyond a
carbohydrate substitution on the polypeptide chain. Since
each sequence obtained was unique, the results substan
tiate the claim that the polypeptide chain of CEA is a
definite chemical entity and that the microheterogeneity
resides in the carbohydrate portion of the molecule.
INTRODUCTION
CEA4 is a glycoprotein
(26) with a molecular weight of
approximately 180,000 (41), composed of about 60% car
bohydrate and 40% protein by weight (46). Extensive stud
ies on CEA have revealed details of its physical structure by
molecular sizing techniques and by electron microscopy
(41), its carbohydrate structure by periodate oxidation (8,
21) and by methylation-linkage analysis (7, 21), and its NH21 Supported in part by National Cancer Institute Grants CA16434 and
CA19163 from the National Large Bowel Cancer Program.
2 To whom requests for reprints should be addressed, at Division of
Immunology, City of Hope National Medical Center, 1500 East Duarte Road
Duarte, Calif. 91010.
3 Present address: S. U. N. Y. at Buffalo, Department of Cell and Molecular
Biology, Gary Hall, Buffalo, N. Y. 14214.
"The abbreviations used are: CEA, Carcinoembryonic antigen- SOS
sodium dodecyl sulfate: PTH, phenylthiohydantoin.
Received January 23, 1978; accepted April 20, 1978.
AUGUST 1978
terminal amino acid sequence (4, 45) by Edman degrada
tion. In addition these studies and others (22, 31, 52, 53)
support the current view that the antigenic determinants
recognized in the radioimmunoassay for CEA reside in the
protein portion of the molecule. Since CEA was reportedly
shown to be heterogeneous in terms of charge and molecu
lar weight polydispersity (3, 5, 17, 23, 42), a structural
feature attributable to its extensive carbohydrate microhet
erogeneity, it is reasonable to assume that the antigenic
determinants should reside in an invariant portion of the
molecule, namely, the polypeptide chain.
CEA was first described as a tumor-associated antigen by
Gold and Freedman (18, 19) because it was detected in
tumors of the digestive system and in fetal gastrointestinal
tissue. Subsequently, several investigators (9, 20, 22, 30, 50)
have isolated CEA cross-reacting antigens from normal
adult tissues. Recent evidence (33, 34, 48) has shown that
many of these substances differ in chemical composition
and molecular size from CEA. These findings have demon
strated a real need to characterize chemically the CEAreactive molecules from different sources.
Significant progress in the structural studies of CEA and
CEA cross-reacting antigens has been made in this labora
tory. CEA isolated from normal adult colon washings (14)
has been shown to be structurally identical to that isolated
from liver métastasesof colon adenocarcinoma (40), but
significant differences were found in the lower-molecularweight CEA cross-reacting antigens isolated from normal
(16) and malignant tissues (25).
Structural studies on the protein portion of CEA and CEArelated antigens are necessary to establish the basis for
their observed antigenic differences. The results of such
studies may lead to the development of a more specific test
for CEA in malignant and other diseased tissues that show
elevated CEA levels. Protein sequence studies on CEA are
hampered by unusual technical problems since CEA con
tains over 600 amino acids and contains over 50% carbo
hydrate. The carbohydrate, all of which is linked to protein
through /V-acetylglucosamine to asparagine linkages, is
difficult to remove by chemical cleavage (7, 8, 52). Specific
cleavage of peptide bonds by cyanogen bromide treatment
has been unsuccessful due to the lack of methionine in
CEA and the occurrence of hydrolytic cleavage, which
occurs at low pH (presumably aspartylproline bonds). The
use of the cyanylation method of cleaving cysteine peptide
bonds by the method of Jacobson ef al. (24) formed A/
blocked peptides that could not be sequenced (12, 28).
In the present study it was found that glycopeptides
capable of sequence analysis could be obtained from CEA
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J. E. Shively et al.
by trypsin digestion in the presence of 0.25% Triton X-100
and subsequent separation by ion-exchange high-pressure
liquid chromatography.
In some cases the resulting glycopeptides could be sequenced through 20 degradation cy
cles before the analysis came to an abrupt halt, presumably
due to the presence of a carbohydrate chain linked to an
asparagine residue. Although this report constitutes only a
start on the amino acid sequence analysis of CEA, it shows
for the first time that CEA can be broken up into isolatale
glycopeptide fragments that are amenable to NH2-terminal
amino acid sequencing. These results support the conclu
sion, suggested originally by the finding of a single, uniform
NH,-terminal amino acid sequence for CEA (45), that indeed
the polypeptide portion of CEA is a definite chemical entity.
The authors intend to use the techniques developed here to
answer specific questions about the structural
features
responsible for the antigenic differences observed among
the various CEA and CEA cross-reacting entities.
MATERIALS
AND METHODS
Preparation of CEA. Isolation and purification of CEA
was carried out as previously described (6) and was mea
sured by double-antibody
radioimmune
assay (13) with a
"Co volume marker (15). In addition, contaminating
traces
of mucopolysaccharides
were removed by concanavalin A
Sepharose affinity chromatography
as previously described
(36); recovery of CEA from this step was 78%. CEA from 1
tumor source was used throughout these studies.
Neuraminidase
Digestion. Vibrio cholerae neuraminidase digestion was performed according to Coligan and
Todd (8). A control sample was run in which CEA was
omitted from the reaction. No material capable of sequence
analysis was found in the control sample.
Reduction and Alkylation. CEA (210 mg) treated with
neuraminidase and dissolved in 30 ml of 9.0 M urea buffered
with 0.1 M Tris-HCI, pH 8.3, and purged with N2was reduced
with 0.3 mmol of dithiothreitol
for 1 hr at room temperature
and alkylated with 0.43 mmol of iodoacetamide
(0.30 mCi
of [14C]iodoacetamide
diluted to a final specific activity of
0.70 mCi/mmol) for 1 hr at room temperature in the dark.
The sample was dialyzed exhaustively against water, lyophilized, and dried over P2O5.
Trypsin Digestion. CEA (218 mg) treated with neuramini
dase, reduced, alkylated, and dissolved in 15 ml of 0.25%
aqueous Triton X-100 (Sigma Chemical Co., St. Louis, Mo.)
was adjusted to pH 8.1 with a dilute ammonia solution ana
digested with 2.0 mg of solid u-l-tosylamido-2-phenylethyl
chloromethyl
ketone-treated
trypsin
(Worthington
Bio
chemical Corp., Freehold, N. J.) for 12 hr at 37°.pH 8.1 was
maintained by further addition of dilute ammonia as neces
sary. The reaction mixture was lyophilized and dried over
P205 (yield, 254 mg). A sample of a,-acid glycoprotein was
obtained from Dr. Yu-Lee Hao, American Red Cross Blood
Research Laboratory,
Bethesda, Md., and was used for
model studies of trypsin digestion in the presence of Triton
X-100.
Amino Acid Analysis. Amino acid analysis was performed
by a modification
of the method of Liu and Chang (29).
Samples of 100 to 200 ^g were hydrolyzed under vacuum in
2200
heavy-walled ignition tubes at 110°for 48 hr in duplicate
with 0.5 ml of 3 N p-toluenesulfonic
acid containing 0.2% 3(2-aminoethyl)indole.
The hydrolyzed samples were ana
lyzed on a Beckman 121H amino acid analyzer (Beckman
Instruments, Inc., Palo Alto, Calif.) The basic amino acids
and amino sugars were eluted from a 20- x 0.9-cm column
of PA35 (Beckman) with sodium citrate buffer, pH 5.26 (0.4
N sodium). The acidic and neutral amino acids were eluted
from a 56- x 0.9-cm column of AA15 (Beckman) with sodium
citrate buffers of pH's 3.10 (0.16 N sodium), 3.60 (0.02 N
sodium), and 4.20 (0.20 N sodium).
Carbohydrate Analysis. Carbohydrate analysis was per
formed on the trimethysilyl
derivatives of sugar methyl
glycosides (37). Sialic acid was determined by the previ
ously described method and by the method of Warren (51).
SDS/Gel Electrophoresis. SDS/gel electrophoresis was
performed on either 6 or 12% polyacrylamide
gels accord
ing to the method of Swank and Munkres (44). Gels were
routinely stained with Coomassie blue or periodic acidSchiff reagent (38). For location of 14C-labeled peptides
with CEA activity, portions of the extracts of 1-mm-thick gel
slices shaken overnight in 0.5 ml of phosphate-buffered
saline (0.075 M sodium phosphate/0.075
M NaCI; pH 7.2)
were counted.
Isoelectric Focusing. Isoelectric focusing was performed
on an LKB 8100 electrofocusing
column of 110-ml capacity
according to the method of Vesterberg and Svensson (49).
LKB carrier ampholytes (1%; LKB Instruments, Inc., Rockville, Md.) were used in the pH ranges of 2.5 to 4, 4 to 6,
and 5 to 8.
Peptide Separation Procedures. Ion-exchange separa
tions were performed on either DC4A cation or DA8X8 anión
exchangers (Durrum Instrument Corp., Palo Alto, Calif.)
with a DuPont Model 830 high-pressure liquid Chromato
graph. Gel filtration was performed on either Sephadex
G-50 or G-100 (Pharmacia Fine Chemicals, Inc., Piscataway,
N. J.). Aliquots were analyzed for 14C-labeled peptides by
liquid scintillation counting on a Beckman LS-330 scintilla
tion counter with an Aquasol (New England Nuclear, Boston,
Mass.) cocktail. Free amino groups were detected by reac
tion with fluorescamine
(Hoffmann-La Roche Inc., Nutley,
N. J.). Portions of 10 /JL\were evaporated to dryness under
N, in 0.6- x 5-cm tubes and mixed with 50 /¿Iof 0.2 M
pyridine and 50 /¿Iof 2 mg fluorescamine
per ml acetone
(w/v); the relative fluorescence was read in a 0.10-ml cuvet
on an Aminco fluorometer (excitation, 364 nm; emission, 458
nm).
Two-Dimensional
Mapping Procedures. Chromatogra
phy in the vertical dimension with butyl alcohol/acetic
acid/
water (4/1/5, v/v) for 24 hr and electrophoresis
in the
horizontal dimension with pyridine/acetic
acid/water (20/2/
378, v/v) for 24 hr at 3000 volts was performed according to
the procedure of Bennett (2).
Sequencing
Procedures.
NH,-termmal
peptide se
quences were performed with a modified dimethyl allylamine program (Beckman 102974) on a Beckman Model 890C
sequencer equipped with a dry ice/acetone trap on its lowvacuum pump. The program modifications were the follow
ing. Benzene/ethyl
acetate (1/1, v/v) was used as S1. N2
delivery through S2 at Step 14 was 80 sec. N2 dry at Step 17
was 80 sec. Steps 38 and 39 were interchanged (new Step
CANCER
RESEARCH
VOL. 38
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Tryptic Peptides from CEA
acrylamide gel electrophoresis, and ion-exchange chroma
tography. The failure of trypsin alone to digest denatured
CEA was attributed to the protection of lysyl and arginyl
peptide bonds by the presence of extensive amounts of
carbohydrate on CEA. Model peptide mapping studies on
a, acid glycoprotein treated, reduced, and alkylated by
neuraminidase established that trypsin digestion was more
effective in the presence of 0.25% Triton X-100. Similarly,
this result was confirmed with CEA. The tryptic map of CEA
(not shown here) reveals the formation of 5 to 10 slowmoving, poorly resolved peptides from CEA. In contrast,
trypsin digestion of CEA performed in the absence of Triton
X-100 gave very few large peptides and no smaller peptides.
The tryptic peptides of CEA were eluted and analyzed for
[14C]cysteine by scintillation counting, for carbohydrate
under N2, and evaporated to dryness on the Buchler Eva content by the phenol/sulfuric acid method of Dubois ef a/.
pomix. The PTH's were extracted 3 times with 25 /¿I
of 1,2- (11), and for NH2-terminal amino acids by the method of
dichloroethane/methanol
(7/3, v/v) according to the Neuhoff (32). The results of these analyses (not shown here)
method of Wittman-Liebold (54), transferred to a 0.4- x 4- revealed that the slower-moving peptides were composed
cm polyethylene microfuge tube (Analytical Aids, Wood- of variable amounts of carbohydrate and cysteine, the
side, Calif.), mixed with 25 ¿¿I
of water, and stored at -20° [14C]carboxyamidomethyl derivative. Each peptide con
until analyzed (the PTH is in the bottom, organic phase). tained several NH2-terminal amino acids. In general the 2This procedure has the following advantages, (a) The incor
dimensional mapping procedure was found inadequate for
poration of an internal standard in the method gives more the separation of large glycopeptides.
accurate quantitation and monitors transfer losses, (o) The
The attempted separation of CEA tryptic peptides on
mild conversion conditions prevent amide hydrolysis of SDS/gels is shown in Chart 1. Untreated CEA migrates as a
glutamine and asparagine derivatives and give higher yields single, diffuse band on 6% gels and as a substantially
of serine and threonine. (c) The water extraction step sharper band on 12% gels. An increase in the separation of
removes traces of impurities that would otherwise contrib
CEA tryptic peptides is apparent in 12 compared to 6% gels.
ute to analysis background levels. The PTH's were sepa
38 was cleavage reaction for 80 sec, and new Step 39 was
N2delivery through S3 for 200 sec). For improvement of the
sensitivity of the analysis of PTH-amino acids by thin-layer
chromatography, 1 mCi of phenyliso[35S]thiocyanate was
added to 16 mmol of phenylisothiocyanate in R1 of the
sequencer (final specific activity, 0.06 mCi/mmol). The
thiazolinone derivatives of the amino acids were converted
to their PTH derivatives by a modification of the procedure
of Laursen (27). The PTH derivative of aminoisobutyric acid
(4.5 nmol) was added to the chlorobutane solution of each
thiazolinone as an internal standard. Samples were evapo
rated to dryness on a Buchler Evapomix in 1.5- x 15-cm
conical tubes fitted with ground-glass joints, mixed with
200 fji\ of 20% aqueous trifluoroacetic acid containing 0.05
mg dithiothreitol per ml, allowed to react 15 min at 55°
rated and quantitated by gas chromatography according to
the method of Pisano and Bronzert (35) on a HewlettPackard 571OA gas Chromatograph and by high-pressure
liquid chromatography on a Waters Associates liquid Chro
matograph (Waters Associates, Inc., Milford, Mass.), both
equipped with an Autolab System IV peak integrator. Excel
lent resolution of PTH derivatives of 17 amino acids was
achieved on C1B^Bondapak (Waters Associates) reverse
phase columns by either Program 1, a linear gradient from
0 to 35% Component B over 30 min, or Program 2, a concave
gradient from 10 to 40% Component B over 30 min. In both
cases the flow rates were 2 ml/min, Component A was 0.01
M^odium acetate/acetonitrile buffer (95/5, v/v; pH 7.6), and
Component B was 100% acetonitrile. These separations are
similar to those reported by Zimmerman ef al. (55) and
Downing and Mann (10). In addition the thin-layer Chro
matographie procedure of Summers ef al. (43) was used
with 2,5-bis-2-(5-ferf-butyl benzoxazolyljthiophene
(Pack
ard Instrument Co., Inc., Downers Grove, III.) as the fluor.
Thin-layer chromatograms were recorded and compared by
UV photography and by autoradiography of the [35S]PTH
derivatives on X-ray film. Reagents, solvents, and sperm
whale apomyoglobin obtained from Beckman were used
throughout this work.
RESULTS
Trypsin Cleavage. Initial treatment of CEA (treated with
neuraminidase, reduced, and alkylated) with trypsin gave
only a few peptides in low yields when it was monitored by
2-dimensional mapping, gel chromatography, SDS/poly-
AUGUST 1978
400
4.0
6.0
Mobility
8.0
10.0
12.0
in cm
Chart 1. SDS/polyacrylamide gel electrophoresis profiles of CEA tryptic
peptides. Samples were run on either 6 or 12% polyacrylamide gels (0.5 x 18
cm). The standards were lysozyme (14,000), 0-lactoglobulin (18,000), car
bonic anhydrase (32,000), ovalbumin (45,000), and bovine serum albumin
monomer (68,000) and dimer (136,000). Arrow, migration of CEA treated
with neuraminidase, reduced, and alkylated: bars, molecular weight ranges
for unfractionated CEA tryptic peptides, as detected by either Coomassie
blue staining or location of [14C]carboxyamidomethyl cysteine. The identi
fication of the peptides (T1A1. etc.) corresponds to a separate experiment
in which the peptides purified by ion-exchange and gel filtration chromatog
raphy were run on separate gels and detected by Coomassie blue staining or
location of ["C]carboxyamidomethyl cysteine.
2201
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J. E. Shively et al.
Results similar to these were obtained with a variety of
peptide detection methods: iodination or fluorescent label
ing before separation; Coomassie blue or periodate-Schifi
staining after separation. Peptide bands were eluted from
gels and analyzed for [14C]carboxyamidomethyl
cysteine
and NIVtermmal
amino acids. A minimum of 10 tryptic
fragments could be estimated from these experiments.
Isoelectric focusing of CEA tryptic peptides is shown in
Chart 2. Although CEA treated with neuraminidase,
re
duced, and alkylated does not give a single sharp peak on
isoelectric focusing, it is clear that the profile of its trypsin
digest is different and reveals at least 10 separate peaks.
The acidic nature of the tryptic peptides was confirmed by
the 2-dimensional
tryptic map and by the results of ionexchange chromatography.
Microanalysis
(<5 mg) of the
peptides obtained from either column or polyacrylamide gel
isoelectric focusing proved difficult (complete removal of
ampholytes and urea from the sample was the major prob
lem).
The most practical preparative method for separation and
purification of the tryptic peptides of CEA was ion exchange
followed by gel permeation chromatography.
Trial separa
tions of the tryptic fragments of sperm whale apomyoglobin on a DC4A cation exchanger by high-pressure liquid
chromatography
and fluorescamine
detection
of eluted
peptides gave 16 peptides in a run time of only 2 hr. How
ever, a similar separation of a, acid glycoprotein
treated
with neuraminidase,
reduced, and alkylated gave a large
breakthrough
peak of unseparated
acidic peptides and
6 well-resolved
basic peptides. Further analysis revealed
that the acidic a, acid glycoprotein
peptides were glycopeptides of relatively high molecular weight, which could
be eventually separated on DA8-X8 anion-exchange
resin.
Since the CEA glycopeptides
were of higher molecular
weight (75,000 dallons) and comprised
larger amounts
of carbohydrate, the separation of the CEA tryptic peptides
was more difficult
to achieve than were those ob
tained from a, acid glycoprotein.
The separation
out
lined in Chart 3 details the purification
of 8 tryptic frag
ments from CEA. The unfractionated
digest gave a poorly
defined separation
on gel permeation
chromatography
alone but, when it was run first on a cation exchanger,
it yielded a breakthrough
peak (T1) and a retained peak
(T2), each of which yielded a breakthrough
and a retained
peak on anión exchanger (T1A, T2A, T1B, and T2B, respec
tively). Each of the 4 peptide mixtures separated by ion
exchange gave 2 well-defined
peaks on subsequent
gel
permeation chromatography.
The gel permeation results
shown in Chart 3 gave lower estimations
of molecular
weights than that obtained by SDS/gel electrophoresis
(Chart 1). However, the large effect of carbohydrate substi
tution in glycopeptides
on either migration in Sephadex
chromatography
or SDS/gel electrophoresis
precludes ac
curate molecular weight determinations.
The method of purification shown in Chart 3 was adopted
for large-scale preparation of CEA tryptic peptides. Table 1
summarizes peptide yields for this procedure. The apparent
low yields for the initial DC4A cation exchange step is
partially due to the conversion of ammonium salts of pep
tides formed during trypsin digestion in ammonia buffer to
their respective free acid derivatives.
Amino Acid and Carbohydrate Analyses. Table 2 pre
sents the amino acid and carbohydrate
analyses for CEA
and its tryptic fragments. In general the high-molecularweight glycopeptides
resemble intact CEA in overall com
position, except Peptides T1B2 and T2B2, which are low in
carbohydrate,
and Peptide T2A2, which was obtained in
such low yield and contaminated
with foreign matter that
further analysis was impossible.
Sequence Results. It was necessary to refine our se
quencing
procedures
to obtain meaningful
quantitative
data on 2- to 3-mg amounts of the CEA tryptic peptides.
Improvements in the sequenator programs, conversion pro
cedure, and PTH identification
detailed in "Materials and
Methods"
made this possible. The high-pressure
liquid
Chromatographie
separation and quantitation of PTH deriv
atives was especially useful in distinguishing
a major from
a minor sequence in several of the peptides that were later
found to be a mixture (in all cases the minor sequence
comprised 30% or less of the mixture). An example of the
resolution and sensitivity achieved with the high-pressure
liquid Chromatographie
system is shown in Chart 4, along
Chart 2. Isoelectric focusing of CEA tryptic pep
tides. Five mg of CEA tryptic digest mixed with ' " llabeled digest and '"l-labeled
CEA treated with
100
SO
neuraminidase,
reduced, and alkylated were ap
plied to a 1- x 40-cm column, and isoelectric
focusing was performed over a pH range of 2.5 to
8.0 for 72 hr.
M
«O
5
«o ú
3.0
IO
50
60
TO
90
100
110
ml Eluted
2202
CANCER
RESEARCH
VOL. 38
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Tryptic Peptides from CEA
A
4.0
2.0
' \
A
Fraction T l
on DA6X8
4.0
â„¢¿
2.0
i
»hole
D.g«.
on OC44
Summary
of isolation
Separation
methodDC4A
4.0
Table 1
and yields of CEA tryptic peptides
(mg)11.24.115.321.618.640.26.4
yield411556585010836
applied27.4
cationexchanger"DA8-X8wholedigest37.2
of
2.0
Froclion T2
on DA8X8
2.0
ÃŒJO
aniónexchanger*Sephadex
T117.6
of
0.5
1.0
40
T216.3
of
-
,BS
Froction
2.0
TIA2
TÕA
°"s«Ph
G5°
1.0
G-50Sephadex
T1A12.6ofT2A19.2
of
2.0
1.0
2.0
G-100mg
1.0
T1B6.9ofT2BPeptideT1T2TotalT1AT1BTotalT2AT2BTotalT1A1T1A2To
of
2.0
1.0
05
IS
20
25
Effluent ml
Chart 3. Separation of CEA tryptic peptides. A, the peptide mixture (27.4
mg) was applied to a 0.21- x 100-cm stainless steel column packed with
DC4A cation exchanger at 60°,with 1100 psi and a flow rate of 1 ml/mm, and
eluted in steps, first with 0.01 M acetic acid and then with pyridinium acetate
(PyrAc) buffer (0.2 M pyridine/4.5 M acetic acid; pH 3.1). B and C the peptide
mixtures T1 (37.2 mg) and T2 (17.6 mg) were separately applied to a 0.21- x
100-cm stainless steel column packed with DA8-X8 aniónexchanger at 40°,
with 3000 psi and a flow rate of 0.5 ml/min, and eluted in steps, first with 0.1
M A/-ethyl morpholine/0.2 M a-picoline/0.1 M pyridine buffer (pH 8.0) and then
with pyridinium acetate buffer (2.0 M pyridine/2.4 M acetic acid; pH 5.0). D to
G, peptide fractions T1A, T2A, T1B, and T2B were applied to either Sephadex
G-50 (Seph G50, fine) or G-100 (Seph G100; 0.60- x 48-cm) columns, with
flow rates of 0.2 ml/min, and eluted with 0.10 M acetic acid. Arrows, elution
volumes of the standards BSA (bovine serum albumin), RNase (ribonuclease), Ala-Val (L-alanyl-L-valine), and CEA (12*l-labeledCEA). In all cases 0.4ml fractions were collected, and two 0.01-ml aliquots were withdrawn for
analysis. One fraction was counted for "C. whereas the other was reacted
with fluorescamine and the relative fluorescence was determined. Bars in
dicate fractions pooled for sequence determination.
with several cycles of an actual sequence. Unidentified
peaks in Chart 4 correspond to UV-absorbing impurities
that do not change from cycle to cycle and that are generally
encountered when sequencing 2 mg or less of protein. The
PTH-amino acids that could not be separated by this
system were identified by either gas or thin-layer chromatography. The use of [35S]PTH-amino acids on polyamide
plates permitted the unambiguous determination of several
PTH-amino acids that are usually obtained in reduced
yields.
Table 3 summarizes the results obtained for the first 15
cycles of Edman degradations performed on 7 tryptic pep
tides from CEA. Peptides T1A1, T1A2, T1B1, and T2B1 gave
sequences through position 15 (yields were extremely low
past Cycle 15), but T1B2 and T2B2 gave abruptly lower
yields at Cycle 10. Absolute yields of PTH-amino acids (e.g.,
Cycles 1 and 2) were 30 to 50%, based on the rough
molecular weight estimates for each peptide on SDS/gel
electrophoresis (Chart 1). Since the sequenator program
used gave routine yields of 50% for sperm whale apomy-
" Results
were obtained
for Batch
1 of whole
tryptic
digest.
Similar results were obtained for Batches 2 and 3. The percent
age of yield is based on weight.
6 Results were obtained for Batch 1 of T1 or T2. Similar results
were obtained for the second
yield is based on weight.
batch of each. The percentage
of
oglobin, the previous yields are acceptable.
The NH2-terminal amino acid determination of each of the
peptides by the dansyl procedure of Neuhoff (32) gave the
same NH2-termini as that shown in Table 3. In all cases the
tryptic peptides showed varying amounts of e-dansylamino
lysine.
DISCUSSION
Effect of Triton X-100. These experiments demonstrate
for the first time that specific cleavage of CEA into peptide
fragments capable of sequence analysis can be achieved.
In principle the methodology adopted to produce, separate,
and sequence the glycopeptides of CEA can be used on
other high-molecular-weight,
high-carbohydrate-content
glycoproteins. The use of trypsin digestion in the presence
of Triton X-100 gave higher, more uniform yields of tryptic
peptides for CEA. The binding of this non-ionic detergent to
hydrophobic regions of the protein perhaps unfolded the
collapsed structure of CEA treated, reduced, and alkylated
by neuraminidase [see Slayter and Coligan (41) for a discus
sion of the electron microscopy of CEA] enough to allow
better trypsin binding to the polypeptide chain. This ex
planation of enhanced trypsin digestion is based partly on
the known binding of detergents to hydrophobic portions of
proteins and in part on the resulting NH2-terminal se
quences of the tryptic fragments obtained, which are high
in hydrophobic amino acids. Control experiments have
shown no change in trypsin activity on a synthetic substrate
in the presence of 0.25% Triton X-100.
AUGUST 1978
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research.
2203
J. E. Shively et al.
Amino acid"
and carbohydrate
Table 2
h composition
of CEA tryptic peptides
%3.521.70
Tyrosine
Phenylalanine
Tryptophan
Lysine
HistidineArginine
2.76
1.17
2.85
1.893.50
Carboxymethyl
cysteine
Aspartic acid
ThreonineSerineGlutamic
3.16
1.81
2.84
1.134.59
2.61
0
1.57
0.427.68
2.70
2.12
0
1.161.252.49 1.45
1.452.64
1.97
0
2.06
0.676.88
2.51
8.14
4.27
1.613.98
2.69
1.73
2.28
1.583.61
1.59
0
1.49
1.293.93
1.60
1.37
1.39
1.78
3.49
0.53
2.62
0.60
1.13
13.99
14.03
17.97
21.10
15.20
17.67
10.61
14.29
14.14
8.119.689.5210.275.25
4.238.4411.35011.01
11.2311.914.985.545.58
11.0512.355.6107.78
8.9711.979.509.304.81
6.1612.1711.168.036.92
9.5013.045.214.596.27
7.0710.9810.3511.204.47
6.6710.4711.747.0510.54
acidProlineGlycine
5.540
AlanineHalf-cystine
ValineMethionineIsoleucineLeucine%
6.390
5.480
5.820
6.090
5.800
5.970
6.570
5.590
6.250.104.238.2534.69.79.07.6
4.6402.588.8917.56.610.76.9
4.9702.1413.3232.65.57.56.5
6.5604.666.8656.96.89.17.2
5.5509.056.5917.2%
5.0504.6312.7736.33.05.48.0
6.0709.066.801.70.1<1T2B14.69
6.6205.257.0041.05.45.15.4
6.8007.336.3218.81.1<1
recoveredFucoseGalactoseMannose
of amino acid
by weight1.12.22.0
D-GlucosaminerfD-Glucosaminep%
of carbohydrate
Apparent
molecular
" Samples
14.114.640.4T1A15.38
14.415.238.6T1A22.54
13.013.132.5T1B14.39
12.017.135.1T1B2Mol
11.812.528.2T2A23.33
2.83.68.1T2A13.36
recoveredCEAr5.04
wt'
were hydrolyzed
180,000
50.000
40.000
110,000
12,000
in 0.5 ml of 3 N p-toluenesulfonic
acid for 48 hr in duplicate
Beckman 121H amino acid analyzer.
6 Samples were treated with 1.5 N methanolic
HCI for 24 hr in duplicate
Packard 7620 gas Chromatograph
equipped with a flame ionization
c CEA treated with neuraminidase,
reduced, and alkylated.
d Determined by gas chromatography.
'' Determined by amino acid analysis.
! Molecular
weight values calculated
from migration
on SDS/gels
The potential interference of the carbohydrate chains of
CEA in the trypsin cleavage is great since there are on the
average 80 carbohydrate chains of about 7 residues/chain
present and >600 amino acids/mol (39). With the assump
tion that the carbohydrate substitution on asparagine resi
dues is heterogeneous in terms of degree and extent of
substitution, there is the likelihood that different CEA mol
ecules can be cleaved by trypsin at different arginine or
lysine residues, depending on the degree of interference
caused by carbohydrate chains. The beneficial effect of
Triton X-100 may be in part due to a lessening of the
carbohydrate steric effects.
Although CEA contains sufficient arginine and lysine
residues to produce up to 40 tryptic peptides, only 8 were
isolated. Thus it seems likely that many of these residues
are inaccessible to cleavage by trypsin. Also, possibly,
several small peptides (<10 amino acids) were lost due to
the small amounts of the CEA fractionated and the fractionation procedure adopted. The relatively large-molecularweight (75,000 daltons) glycopeptides obtained contained
variable amounts of carbohydrate. In each case the se
quence was lost before 20 cycles of Edman degradation
could be achieved. Most probably, the sequence was lost
due to the drop in yields, which often occurs when an
2204
8.09.323.9T2B23.48
50,000
16.000
70.000
28,000
at 110°under vacuum and analyzed on a
at 80°and analyzed as trimethylsilyl
derivatives
on a Hewlett-
detector.
shown in Chart 1 should be considered
approximate.
asparagine linked to a carbohydrate chain is encountered
(47). Amino acid analysis of CEA gives an average of 90
asparagine plus aspartic acid residues/mol of CEA and,
since there are 80 carbohydrate chains (all linked to protein
through asparagine) per molecule, the likelihood of en
countering carbohydrate linked to asparagine in sequence
studies on CEA is high. Indeed, 4 of the 7 peptides sequenced encountered asparagine before 20 degradation
cycles. Peptide T2A1 contains the sequence Asn-X-Thr,
which appears to be a common recognition sequence for
carbohydrate linked to asparagine in glycoproteins (1). The
fact that these asparagines were identified in the usual
chlorobutane washes obtained from the sequenator sug
gests that these residues had little or no carbohydrate at
tached, since the presence of hydrophilic carbohydrate
substituents on PTH-asparagine would render these deriva
tives insoluble in chlorobutane.
The finding that Peptides T1A2, T1B1, and T2B2 gave
both a major and a minor sequence demonstrates that
these 3 peptides were not completely pure. However, in no
case did the minor sequence of these 3 peptides corre
spond to the sequence of the other peptides. Evidently, the
minor sequences represent distinct peptides obtained in
lower yield. A tentative reason for the lower yields of these
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research.
Tryptic Peptides
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J. E. Shively et al.
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CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research.
Tryptic Peptides from CEA
03
PTH
Ammo
Acid
Stondards
linear (rom 0%B to 35% B
over 30 mm, 2ml/mm
A<Ph 76, 01MActtoit/CHjCN
95/5 U/»)
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N
GA
02
MP
u
01
LJ
03
Program 2
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A* B same as above
PTH
Ammo
Acid
Standards
o>
o
02
01
CO
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I
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,
»
1
determine if the tryptic peptides could be used to make
antisera against specific portions of the CEA molecule. CEA
has been shown to require the presence of intact disulfide
bridges to retain activity in the radioimmunoassay (21, 53).
We have produced high-liter antiserum against reduced
and alkylated CEA, which cross-reacts with intact CEA in a
radioimmunoassay although it has no activity against trypsinized, reduced, and alkylated CEA. Evidently, the tryptic
fragments obtained from CEA do not possess sufficient
similarity in tertiary structures to reduced and alkylated
CEA to yield significant cross-reactivity in this sensitive test.
Alternatively, these fragments may not be derived from
peptide regions involved in the antigenic sites recognized
by this antiserum. This result was confirmed for the unfractionated and fractionated CEA tryptic peptides. The unfractionated tryptic fragments were injected into rabbits unconjugated and conjugated to either bovine serum albumin or
methylated bovine serum albumin, but in no case was
specific antibody formed against the injected antigens.
Additionally, 500 /.¿g/injection(total, 3 injections) of purified
CEA tryptic peptides gave no detectable antisera as judged
by direct binding against their respective antigens. These
negative results further suggest that tertiary structure is of
paramount importance in determining CEA antigenicity.
Possibly, the antigenic determinants in CEA recognized by
anti-CEA are present in those portions of the molecule,
which are also accessible to cleavage by trypsin. Although
trypsin-cleaved CEA is not antigenically active, specific
cleavage at cysteine residues in CEA does yield antigeni
cally active peptides (12, 28) that are blocked at their amino
termini. Structural studies on these peptides should yield
meaningful information on the nature of the antigenic
determinants of CEA.
The success of these preliminary sequence studies holds
considerable hope that CEA and CEA cross-reacting anti
gens may be directly compared in terms of their protein
chemistry. These studies are currently underway.
ACKNOWLEDGMENTS
Time (seconds)
Chart 4. High-pressure liquid Chromatographie separations of PTH deriv
atives. Five to 10 fil of the sample were injected onto a /¿BondapakC„
(0.04x 30-cm) column. Pan A. Program 1 separation of <1 nmol each of 16 PTH
standards, except arginine (R) (6 nmol) and cysteine (C) (3 nmol). Under
these conditions methionine (M) and proline (P) are not separated from
valine (V). and tryptophan (W) is not separated from isoleucine (/) and leucine
(L). The internal standard PTH derivative of aminoisobutyric acid is desig
nated by y. Part B, Program 2 separation of P from M and V. Part C,
Program 2 results for the first 6 cycles of Edman degradation performed
on CEA Tryptic Peptide T2B1. D, aspartic acid; £, glutamic acid; N,
asparagine; S, serine; 7, threonme; G, glycine; A, alanine; H, histidine:
C?,glutamine; Y, tyrosine; F, phenylalanine; K, lysine.
peptides may be that a given population of CEA molecules
possesses a spectrum of specific trypsin cleavage sites that
vary in their accessibility to the enzyme. One interesting
and perhaps anomalous cleavage has occurred in CEA to
produce peptide T1B2 with a NH2-terminal lysine. This
somewhat surprising result suggests an unusual structural
feature such as a cluster of 2 or more basic residues in the
sequence of CEA.
Antigen Studies. An important goal of this work was to
AUGUST 1978
The authors are grateful to David Bills, Jaga Nath Singh Glassman, and
Nancy Buker for their excellent technical help. We thank Jean Warren for
performing all of the radioimmunoassays.
REFERENCES
1. Aubert, J.-P., Biserte, G., and Loucheux-Lefebvre, M.-H. CarbohydratePeptide Linkage in Glycoproteins. Arch. Biochem. Biophys., 775: 410418,1976.
2. Bennett, J. Paper Chromatography and Electrophoresis, Special Proce
dure for Peptide Maps. Methods Enzymol., 11: 330-339, 1967.
3. Chism, S. E.,-Bell. P. M., and Warner, N. L. Heterogeneity of CEA and
CEA-like Preparations Determined by Farr Assays for Lectin Binding. J.
Immunol. Methods, 13: 83-89,1976.
4. Chu, T. M., Bhargava, A. K., and Harvey, S. R. Structure Studies of the
GlycoproteinsAssociatedwith Carcinoembryonic Antigen (CEA). Feder
ation Proc., 33: 1562,1974.
5. Coligan, J. E., Henkart, P. A., Todd, C. W., and Terry, W. D. Heteroge
neity of the Carcinoembryonic Antigen. Immunochemistry, 10: 591-599,
1973.
6. Coligan, J. E . Lautenschleger, J. T., Egan, M. L., and Todd, C. W.
Isolation and Characterization of Carcinoembryonic Antigen. Immuno
chemistry, 9: 377-386, 1972.
7. Coligan, J. E., Pritchard, D. G., Schute, W. C., Jr., and Todd, C. W.
Methylation Analysis of the Carbohydrate Portion of Carcinoembryonic
Antigen. Cancer Res., 36:1915-1917,1976.
8. Coligan, J. E., and Todd, C. W. Structural Studies on Carcinoembryonic
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J. E. Shively et al.
Antigen: Periodate Oxidation. Biochemistry, 14: 805-810, 1975.
9. Darcy, D. A., Turberville, C., and James, R. Immunological Study of
Carcinoembryomc Antigen (CEA) and a Related Glycoprotein. Brit. J.
Cancer. 28: 147-160, 1973.
10. Downing, M. R., and Mann, K. G. High-Pressure Liquid Chromato
graphie Analysis of Amino Acid Phenylthiohydantoins: Comparison with
Other Techniques. Anal. Biochem., 74: 298-319, 1976.
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Colorimetrie Method for Determination of Sugars and Related Sub
stances. Anal. Chem.,28. 350-356, 1956.
12. Egan, M. L., Coligan, J. E., Morris, J. E., Schnute, W. C., Jr.. and Todd,
C. W. Antigenic Determinants on Carcinoembryonic Antigen: Chemical
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Cascinelli (eds.), Proceedings of the Eleventh International Cancer
Congress, Florence. Italy, October 20 to 26, 1974, Vol. 1, pp. 244-248.
Amsterdam: Excerpta Medica, 1975.
'3. Egan, M. L., Lautenschleger, J. T., Coligan, J E., and Todd, C. W.
Radioimmune Assay of Carcinoembryonic Antigen. Immunochemistry,
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CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research.
Amino-terminal Sequences of the Major Tryptic Peptides
Obtained from Carcinoembryonic Antigen by Digestion with
Trypsin in the Presence of Triton X-100
John E. Shively, Michael J. Kessler and Charles W. Todd
Cancer Res 1978;38:2199-2208.
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