From the Wistar Institute, Philadelphia, Pennsylvania 19104
Received for publication, September 27, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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The GA733-2 antigen is a cell surface
glycoprotein highly expressed on most human gastrointestinal carcinoma
and at a lower level on most normal epithelia. It is an unusual
cell-cell adhesion protein that does not exhibit any obvious
relationship to the four known classes of adhesion molecules. In this
study, the disulfide-bonding pattern of the GA733-2 antigen was
determined using matrix-assisted laser desorption/ionization
mass spectrometry and N-terminal sequencing of purified tryptic
peptides treated with
2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially
reduced and alkylated. Numbering GA733-2 cysteines sequentially from
the N terminus, the first three disulfide linkages are
Cys1-Cys4,
Cys2-Cys6, and
Cys3-Cys5, which is a novel pattern for a
cysteine-rich domain instead of the expected epidermal growth
factor-like disulfide structure. The next three disulfide linkages are
Cys7-Cys8,
Cys9-Cys10, and
Cys11-Cys12, consistent with the recently
determined disulfide pattern of the thyroglobulin type 1A domain of
insulin-like growth factor-binding proteins 1 and 6. Analysis of
glycosylation sites showed that GA733-2 antigen contained
N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential
N-linked glycosylation sites, Asn175 was not
glycosylated, whereas Asn88 was completely glycosylated,
and Asn51 was partially glycosylated. These data show that
the extracellular domain of the GA733-2 antigen consists of three
distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733
type 3 motif).
The GA733-2 antigen is a transmembrane glycoprotein that migrates
on SDS-PAGE1 as a 40-kDa
protein. It is present in the majority of human epithelia (1-5) and is
highly expressed in gastrointestinal carcinomas (6-9). Moreover,
recent studies correlate high GA733-2 expression with malignant tumor
development in other types of cancer lesions, raising intriguing
questions about the role of this antigen in tumor progression (for
review see Ref. 10).
The GA733-2 antigen was originally defined by the CO17-1A monoclonal
antibody (mAb) and GA733 mAb (11, 12). Because a randomized phase II
trial with mAb CO17-1A in colorectal carcinoma patients has
demonstrated a significant decrease in recurrence and mortality of
mAb-treated patients versus control patients (13, 14),
GA733-2 antigen has attracted substantial attention as a target for
immunotherapy for treating human carcinomas. More recently, it has been
reported that a recombinant adenovirus expressing the full-length
GA733-2 antigen has the capability to induce humoral, cellular, and
protective immunity to the antigen resulting in significant and
specific inhibition of the colorectal carcinoma cell growth in mice,
which implies that this recombinant adenovirus may have potential as a
vaccine for colorectal carcinoma patients (15).
The GA733-2 antigen has been shown to function as a calcium-independent
homophilic cell adhesion molecule that does not exhibit any apparent
homology to the four known cell adhesion molecule superfamilies (16).
Although the functional consequence of GA733 antigen-mediated adhesion
on tumorigenesis is unclear, emerging evidence correlates increased
GA733-2 antigen expression with increased cell proliferation and a less
differentiated cell phenotype as demonstrated in cervical
intraepithelial neoplasia (17).
The GA733-2 gene encodes for a 314-amino acid polypeptide that includes
a 23-amino acid signal sequence, a 242-amino acid extracellular domain,
a 23-amino acid transmembrane domain, and a 26-amino acid cytoplasmic
domain (18). The extracellular domain of GA733-2 antigen (GA733-2EC)
has three potential N-linked glycosylation sites and an
N-terminal cysteine-rich region containing 12 cysteines. It has been
proposed that this N-terminal portion is comprised of an epidermal
growth factor (EGF)-like motif followed by a thyroglobulin type 1A
motif based on relatively weak homology (19, 20). However, structural
evidence to support this hypothesis has not yet been reported. In
addition, analysis of glycosylation sites on the GA733-2 antigen is of
interest because changes in glycoprotein glycosylation typically
accompany malignant transformation (for review see Ref. 21). For
example, a recent study showed that modulation of the glycosylation of
CD44 regulated cell adhesion during tumor growth and metastasis (22).
Two of the three possible N-glycosylation sites in GA733-2EC
are located in the N-terminal cysteine-rich region, which appears to
contain the epitope for the CO17-1A mAb used in the clinical trial
described above (10). Hence, it is likely that
N-glycosylation of GA733-2 antigen may play roles in cell
adhesion and/or tumor targeting. A better understanding of the special
features of the GA733-2 antigen may provide insights into the
function(s) of this antigen and should facilitate the rational design
of mutants for structure-function studies.
In this study, we determined the linkages of the six disulfide bonds
and the sites of N-linked glycosylation in GA733-2EC using
MALDI-MS and N-terminal sequencing of purified peptides. The data show
that the cysteine-rich region has two distinct domains with six
cysteines forming three disulfides in each domain. Surprisingly, the
first cysteine-rich domain does not belong to the EGF-like domain
family as previously predicted (10, 19, 20). Instead, it has a unique
disulfide linkage pattern. In contrast, assignment of disulfides in the
second cysteine-rich domain is in agreement with the disulfide pattern
of a thyroglobulin type 1A motif, recently determined in insulin-like
growth factor-binding protein-1 and 6 (23), and consistent with its
apparent sequence homology (20, 24). Of the three potential
N-glycosylation sites, Asn51 was partially
glycosylated, Asn88 was completely glycosylated, and
Asn175 did not contain any carbohydrate moieties.
Materials--
Immobilized tosylphenylalanine
chloromethylketone-trypsin F7m columns were purchased from MoBiTec
(Marco Island, FL). Trypsin (sequencing grade) was purchased from
Promega (Madison, WI). N-Glycosidase A was obtained from
Roche Molecular Biochemicals. Tris-(2-carboxyethyl)-phosphine (TCEP)
and 2- (2'-nitrophenylsulfonyl)-3-methyl-3-bromoindolenine (BNPS-skatole) were obtained from Pierce. Pyroglutamate aminopeptidase was obtained from Panvera (Madison, WI). Reagents for PAGE were obtained from Bio-Rad. All other reagents were either high performance liquid chromatography (HPLC) grade or the highest quality analytical reagent grades available.
Purification of GA733-2EC--
Recombinant GA733-2EC was
produced in Hi Five insect cells using the baculovirus system as
previously described (25, 26). The secreted GA733-2EC was purified from
the culture supernatant using a GA733 mAb-Sepharose affinity column.
The column was washed with PBS (10 mM sodium phosphate, 130 mM NaCl, pH 6.0), and proteins were eluted with 50 mM glycine-HCl, pH 2.5. The pH of the peak fractions was
immediately raised to approximately pH 6 using 2 M
Tris-HCl, pH 6.0, followed by dialysis against PBS. Protein samples
were concentrated to 7-8 mg/ml using Centriprep and Centricon concentrators with a 10-kDa molecular mass cut-off membrane
(Amicon, Beverly, MA). The concentrated protein solution was then
injected into two TSK columns G3000 SWXL and G2000
SWXL (Toso-Haas, Japan) connected in series and separated
at 0.8 ml/min using PBS, pH 6.0. The quality of purification was
monitored by SDS-PAGE and Western blot analysis using the GA733 mAb.
The concentration of GA733-2EC was determined by
A280 in a 1-cm pathlength cell, using a molar
absorption coefficient ( Protease Digestion--
For disulfide assignments, a
tosylphenylalanine chloromethylketone-trypsin F7m column was used for
initial fragmentation of GA733-2EC after equilibrating the column with
10 ml of 50 mM sodium phosphate, 3 M urea, pH
6.5, at 22 °C. Purified GA733-2EC (400 µg/134 µl) was treated
with urea (final concentration, 3 M) and loaded onto the
trypsin column. The protein solution percolated through the column by
gravity over the period of ~1 h, and the eluate was reloaded a total
of six times followed by overnight incubation in the column without
flow at 22 °C. The column was then centrifuged at 2,000 rpm for
5 s to collect the digested peptide. Residual peptides were
removed with two successive elutions using 200-µl aliquots of 50 mM sodium phosphate, 3 M urea, pH 6.5, followed
by centrifuging at 2,000 rpm for 5 s.
Mild Trypsin Digestion in Solution for Carbohydrate
Analysis--
GA733-2EC was digested with trypsin in solution under
physiological conditions at a ratio of 1:500 (w/w) enzyme to substrate at 37 °C for 1 h. The reaction was stopped by adding
phenylmethylsulfonyl fluoride (final concentration, 0.15 mM). The sample was denatured and reduced by adding 7 M urea and 10 mM TCEP (final concentration) followed by incubation at 37 °C for 30 min. The fragments were separated by HPLC gel filtration using two TSK columns G3000
SWXL and G2000 SWXL connected in series with
0.6 ml/min flow rate in 7 M urea, 20 mM
Tris-Cl, 1 mM TCEP, pH 7.0. Collected fractions were
immediately dialyzed against 10 mM sodium phosphate, pH
7.0, to remove urea and TCEP.
Reverse Phase HPLC Separation of Peptides--
Peptides were
separated by reverse phase (RP)-HPLC on a ZORBAX 300SB-C18 column
(2.1-mm inner diameter × 150 mm, Hewlett Packard Co.) using a
System Gold HPLC (Beckman, Fullerton, CA) at a flow rate of 0.2 ml/min.
Various gradient conditions were used as described in figure legends
with solvent A (0.1% trifluoroacetic acid in water) and solvent B
(0.085% trifluoroacetic acid in 95% acetonitrile). Where required,
tryptic digests were reduced prior to RP-HPLC by adding 20 mM TCEP in 100 mM ammonium bicarbonate, pH 8.0. The mixture was incubated at 37 °C for 1 h, and 2%
trifluoroacetic acid (final concentration) was then added prior to
injection onto the HPLC column.
Deglycosylation and BNPS-skatole Cleavage--
The purified
tryptic fragment containing glycosylation (T1) was treated with
N-glycosidase A. The peptide solution (3 µg/250 µl in 50 mM sodium acetate, pH 5.0) was mixed with 14 µl of
N-glycosidase A (0.1 µg/µl stock solution). The mixture
was then incubated overnight at 37 °C followed by RP-HPLC to
separate deglycosylated peptide. Deglycosylated T1-N (1.4 µg) in the
presence of 5 M guanidine-HCl was cleaved with BNPS-skatole
essentially as previously described (28).
Partial Reduction with TCEP and Cleavage of N-terminal
Pyroglutamic Acid--
TCEP partial reduction of complexes with
multiple disulfides was performed as previously described (29). The
purified peptide complex (100 pmol/70 µl in 0.1% trifluoroacetic
acid) was mixed with the same volume of 50 mM TCEP in 50 mM citrate, pH 3.5, and incubated for 3 min at 22 °C.
Alkylation of peptides was performed by adding the TCEP-reduced peptide
solution into the same volume of 1 M iodoacetamide in 100 mM HEPES, 1 mM EDTA, pH 7.5, followed by
incubation for 30 min at 37 °C. The reaction was stopped by adding
1.3% trifluoroacetic acid (final concentration). Pyroglutamate aminopeptidase (1.3 milliunits/6.3 µl) was added to the peptide solution (33 pmol/50 µl in 50 mM sodium phosphate, 1 M guanidine-HCl, 10 mM dithiothreitol, 1 mM EDTA, pH 7.0) and incubated overnight at 60 °C to
remove N-terminal pyroglutamic acid from the purified partially reduced
and alkylated peptides.
N-terminal Sequence Analysis--
Automated Edman degradation
was performed using an Applied Biosystems model 494 protein sequencer
as previously described (30).
Mass Spectrometry--
Molecular mass determination was
performed by MALDI-time of flight mass spectrometry using a Voyager
DE-PRO mass spectrometer (PerSeptive Biosystems, Framingham, MA). The
accelerating voltage was set to 20 kV. Data were acquired either in the
linear or reflector mode of operation. Spectra were externally
calibrated with protein A (44,614 Da), chymotrypsinogen (25,657.1 Da),
ubiquitin (8,567.49 Da), insulin Protein Purification, Signal Removal, and Characterization of
GA733-2EC--
The recombinant extracellular domain of GA733-2
(GA733-2EC) was purified from Hi Five insect cell culture supernatant
by GA733 mAb-Sepharose affinity chromatography followed by HPLC gel
filtration. The purified protein migrated on SDS-PAGE as a diffuse
doublet with apparent masses of ~31 and 33 kDa (Fig.
1). MALDI-MS analysis of purified
GA733-2EC showed two broad peaks with average masses (MH+)
of 28,332 and 29,349 Da. Because the calculated mass of the GA733-2EC
is 27,372 Da after removal of the signal peptide, these data suggest
that the molecule is heterogeneously glycosylated, resulting in two
major populations with average carbohydrate masses of about 960 and
1,977 Da. The broad peak shapes of the doublet in the mass spectrum are
indicative of additional mass heterogeneity of these two major
populations of molecules. N-terminal sequence analysis of GA733-2EC
indicated that the majority of the protein has a blocked N terminus,
although a low level sequence representing about 1% of the protein
amount loaded to the sequencer was detected. The low level sequence
(AAQEEC ... ) indicated a minor alternative cleavage of the signal
peptide after residue 21 of the unprocessed full-length protein.
Subsequent MALDI-MS analysis and pyroglutamate aminopeptidase digestion
of tryptic T2 peptide complex (Tables I
and III) showed that the predominant signal peptide cleavage occurred
after residue 23 and the N terminus of the mature protein is pyroGlu
(see below). Hence the amino acid residues of the mature protein are
numbered starting with this pyroGlu residue (Fig. 2). The GA733-2EC eluted from the HPLC
gel filtration column as a single symmetrical peak between the 44- and
17-kDa protein standards. Rechromatogrphy of the protein at high and
low protein concentrations yielded consistent elution volumes (data not
shown). These data suggest that recombinant GA733-2EC is a globular
monomer with a native-like fold.
Isolation of Disulfide-linked Tryptic Peptide Complexes--
A
representative RP-HPLC chromatogram of a GA733-2EC digest using an
immobilized trypsin column after reduction (Fig.
3A) is compared with the
chromatogram of a replicate aliquot that was not reduced prior to
RP-HPLC (Fig. 3B). All peaks in the nonreduced chromatogram
and most peptides in the reduced chromatograms were analyzed by
MALDI-MS. Peptides that could not be unambiguously identified by mass
analysis were subjected to Edman sequencing (data not shown). Three
major peaks and several minor peaks observed in the nonreduced digest
(T1-T3, T2*, and T3* in Fig. 3B) were not seen in the
reduced digest, indicating that they contained disulfide-linked
peptides. In addition, several new peaks appeared in the reduced
tryptic digest chromatogram that corresponded to cysteine-containing
peptides released from disulfide linkages by reduction. The identities
of these peptides are indicated by inclusive residue numbers of the
GA733-2EC sequence (Fig. 3, A and B). Peaks
T1-T3 from the nonreduced tryptic digest were further analyzed to
determine the disulfide linkages in GA733-2EC. T2* and T3* were
identified as incomplete cleavages of T2 and T3, respectively.
Overview of Disulfide Linkage Determination--
MALDI-MS and
Edman sequencing analyses of the tryptic peptide complexes shown in
Fig. 3B are summarized in Table I. The T1 peptide complex
contains four cysteines in two disulfide-linked polypeptide chains,
Ala84-Arg102 and
Thr104-Arg115. Asn88 is entirely
in a glycosylated form with some heterogeneity of the carbohydrate
moiety, as indicated by the broad and multiple peaks in the RP-HPLC
chromatogram (Fig. 3, A and B). The complex also
exhibited heterogeneous trypsin cleavage, i.e. a complex of
Ala84-Arg102 with either
Arg103-Arg115 or
Thr104-Arg115. The T2 peptide complex contains
six cysteines in two polypeptide chains:
pyroGlu1-Lys10 and
Leu11-Lys38. The T3 complex has two cysteines
in two polypeptide chains, Cys43-Lys47 and
Ala59-Lys83, giving one direct disulfide
assignment of Cys43-Cys76. Fig. 3C
shows a summary of additional processing of the T1 and T2 complexes to
complete disulfide assignments of these complexes. To minimize
complexity in data analysis, T1 was deglycosylated with
N-glycosidase A. BNPS-skatole cleavage of deglycosylated T1
(T1-N in Fig. 3C) was subsequently performed to obtain
peptides containing one disulfide bond each. Partial reduction with
TCEP and alkylation with iodoacetamide were performed for T2, which contained three disulfide bonds. One disulfide bond in T2 was assigned
by Edman sequencing of the T2-R5 peak (Fig. 3C).
Subsequently, pyroglutamate aminopeptidase was used to remove the
N-terminal pyroglutamate from T2-R3, making it possible to carry out
Edman sequencing and complete disulfide assignments of T2. Using this strategy it was possible to establish the disulfide bonds as: Cys4-Cys23,
Cys6-Cys36,
Cys15-Cys25,
Cys43-Lys76,
Cys87-Cys93, and
Cys95-Cys112 (Fig. 2). A more detailed
description of the analyses of peaks T1 and T2 is presented below.
Analysis of T1--
The T1 peptide complex has four cysteines in
disulfide-linked polypeptide chains,
Ala84-Arg102 and
Thr104-Arg115, with heterogeneous masses
indicative of glycosylation. The carbohydrate in T1 were completely
removed by N-glycosidase A, indicating that all the
carbohydrate in this peptide complex was located on Asn88
(Table II). RP-HPLC purified
deglycosylated T1 (T1-N) was used for all subsequent experiments.
Because T1-N has two disulfide bonds with one of these two disulfide
bonds linking the two tryptic peptides, a further cleavage was
necessary to generate peptide complexes containing only one disulfide
bridge each for direct assignment. BNPS-skatole cleavage was carried
out to cleave T1-N at Trp94. Fig.
4B shows that BNPS-skatole
treatment generated multiple products (T1-N-S1 to S7). MALDI-MS and
Edman sequencing analyses were used to characterize all peaks. T1-N-S1
was shown to be Ala84-Trp94. T1-N-S2 and
T1-N-S3 were identified as Cys95-Arg102
disulfide-linked to Thr104-Arg115 and
Arg103-Arg115, respectively (Table II).
Because T1-N-S1 and T1-N-S2 each have only a single disulfide bond, it
was possible to assign the disulfide bonds
Cys87-Cys93 and
Cys95-Cys112 for the T1 complex. T1-N-S1
showed a mass 13.5 Da greater than that expected for
Ala84-Trp94 (Table II), consistent with the
formation of an oxolactone by oxidative halogenation of the C terminus
of the tryptophanyl-cleaved peptide as reported by Rahali and Gueguen
(32).
To characterize T1-N-S4 to S7, peak fractions were analyzed by MALDI-MS
after reduction with TCEP to get more precise molecular masses.
Although T1-N-S4 and T1-N-S6 eluted as separate peaks in RP-HPLC (Fig.
4B), MALDI-MS spectra of these two fractions were identical
within expected error and indicated that both fractions were the
uncleaved T1-N species with a mass increase of 15.7 Da on the
Ala84-Arg102 peptide (Table II). This is
consistent with oxidation of the indole ring of tryptophan in
incomplete BNPS-skatole cleavage of peptides, which results in a 16-Da
mass increase (32). T1-N-S5 and T1-N-S7 were also uncleaved T1-N
molecules with a 49.4-Da mass increase on the
Ala84-Arg102 peptide (Table II), which is most
likely due to oxidation of Trp94 by addition of three
oxygen atoms. The differing elutions of T1-N-S4 and T1-N-S6 on RP-HPLC
and their identical masses suggested that they represented two
different constrained conformations of the oxidized indole ring of
Trp94. To test this hypothesis, fraction T1-N-S4 was
isolated following RP-HPLC, incubated at 37 °C for 30 min, and then
rechromatographed using the same gradient. Indeed, the RP-HPLC
chromatogram showed partial conversion to a later elution peak T1-N-S4"
that correspond to the elution position of T1-N-S6 (Fig.
4C). MALDI-MS analysis confirmed that there was no mass
difference between T1-N-S4' and T1-N-S4" (data not shown).
Analysis of T2--
The disulfide assignments for the T2
complex were obtained by characterization of partially reduced and
alkylated peptides using Edman sequencing. The T2 peptide complex is
comprised of pyroGlu1-Lys10 and
Leu11-Lys38, having one intrapeptide disulfide
bond in Leu11-Lys38 and two inter-peptide
disulfide bonds (Table I). This peptide fraction was subjected to
partial reduction using TCEP followed immediately by alkylation with
iodoacetamide and subsequent separation by RP-HPLC. The use of the
strong reducing agent, TCEP, which reduces disulfides at low pH within
a short incubation time, had been shown to minimize both
thiol-disulfide exchange and disulfide rearrangement in the partially
reduced peptides (29). The RP-HPLC analysis of multiple peaks of T2
following partial reduction and alkylation is shown in Fig.
5 and results from MALDI-MS and Edman sequencing analyses are summarized in Table
III. Peptide T2-R2 is the original
unmodified peptide. The T2-R1 and T2-R4 peptides have been completely
reduced and alkylated, whereas peptides T2-R3 and T2-R5 were only
partially reduced prior to alkylation. T2-R5 is the single peptide,
Leu11-Lys38, with alkylation of
Cys23 and Cys36 and no modification of
Cys15 and Cys25, indicating that
Cys15 and Cys25 form an intrapeptide disulfide
bond. T2-R3 is comprised of two disulfide linked peptides,
pyroGlu1-Lys10 and
Leu11-Lys38, with one alkylated cysteine on
each peptide. Because the N-terminal pyroglutamate prevented Edman
sequencing, it was necessary to remove this group prior to Edman
sequencing to determine which cysteine side chains had been modified.
Pyroglutamate aminopeptidase treatment of T2-R3 was performed under
reducing condition using 10 mM dithiothreitol, generating
Glu2-Lys10 (T2-R3-P1) and
Leu11-Lys38 (T2-R3-P2). After this treatment
both peptides were completely reduced, but the single cysteine in each
peptide that was alkylated prior to pyroglutamate aminopeptidase
treatment could be identified by Edman sequencing (Fig. 5C).
When dithiothreitol was omitted from the enzyme reaction buffer, no
cleavage of the N-terminal pyroglutamate occurred. Edman sequencing of
T2-R3-P1 and T2-R3-P2 showed that Cys6 and
Cys36 were alkylated respectively. Because the
Cys15-Cys25 linkage was found in the analysis
of T2-R5, these results indicate that these two peptides are linked by
the disulfide bond Cys4-Cys23 (Table III).
Similarly, the two reduced and alkylated residues, Cys6 and
Cys36 identified in T2-R3, represent the remaining
disulfide bond. Although the peak fraction labeled with an
asterisk in Fig. 5B also showed a molecular mass
indicative of T2 with two alkylated cysteines, the amount of the
species was negligible compared with the major species (T2-R3), and it
was not analyzed further. Therefore, the partial reduction/alkylation
of the T2 peptide complex indicated that three disulfide bonds
assignments are Cys4-Cys23,
Cys6-Cys36, and
Cys15-Cys25.
N-Linked Glycosylation Sites in GA733-2EC--
The
N-linked glycosylation sites in GA733-2EC were determined by
MALDI-MS and N-terminal sequence analyses of partial or complete tryptic digests. As described above, Asn88 was found to be
completely glycosylated during analysis of the T1 peptide complex. Mass
analysis of reduced Ala84-Arg102 peptide
showed that the carbohydrate moieties at this site were heterogeneous
ranging from ~893 to 1,259 Da (Fig.
6A and Table I). Edman
sequencing of T1 showed no signal for Asn88, further
confirming the complete glycosylation at this site (data not shown).
Because peptides containing nonglycosylated Asn88 were not
found in RP-HPLC fractions from trypsin column digestion (Fig. 3,
A and B), Asn88 is completely
glycosylated. The variable masses of the carbohydrate moiety on
Asn88 are consistent with the average mass increase of 960 Da observed for the major GA733-2EC species compared with its sequence
mass.
The other two potential N-glycosylation sites were evaluated
using fragments from mild tryptic digestion of GA733-2EC in
physiological solution. This partial digestion resulted in three
products when separated under reducing conditions on a SDS gel: a
22-kDa band, an 8-kDa band, and a 6-kDa band (Fig. 6B). This
cleavage site is located at Arg57, Arg58 in a
protease-sensitive loop that has been previously reported to lead to
release of an ~6-kDa peptide from the N terminus of the protein (33,
34). These fragments were separated by HPLC gel filtration under
reducing and denaturing conditions (Fig. 6C). MALDI-MS
analysis of the larger fragment (fraction I) showed a mass of 21,953 Da, which is within experimental error of the sum of the calculated
mass of Ala59-Lys242 and the average mass of
the carbohydrate moiety on Asn88 as described above (21,968 Da expected versus 21,953 Da observed). These data indicate
that the partial tryptic 22-kDa fragment cannot contain additional
glycosylation on either Asn175 or Ser/Thr side chains.
The MALDI-MS spectrum of fraction II in Fig. 6C showed two
clusters of masses (Fig. 6D). The masses at 6,232.7 and
6,388.1 Da correspond to pyroGlu1-Arg57 and
pyroGlu1-Arg58, respectively, with no
N-linked or O-linked carbohydrate. The third peak
(6,533.7 Da) in the first cluster apparently represents a small amount
of protein with an alternative N terminus caused by variable cleavage
of the signal peptide that results in an additional two alanines at the
N terminus (see above). The second cluster of peaks shows masses
1,034-1,037 Da higher than the corresponding peaks in the first
cluster, indicating that the smaller mild tryptic fragment is only
partially glycosylated on Asn51. Because MS is not
quantitative, the relative staining intensities of the 6- and 8-kDa
bands, although consistent with the MS signal intensities, are more
reliable indicators that only a small proportion of the protein is
glycosylated at Asn51. Interestingly, the carbohydrate
moiety on this site appears to be less variable in mass than the
carbohydrate groups on Asn88. Finally, the sum of observed
masses for glycosylations on Asn88 (960 Da) and
Asn51 (1,034 Da) is 1,994 Da, which is close to the
1,977-Da difference between calculated mass and observed mass for the
minor higher mass peak in the intact GA733-2EC MALDI-MS spectrum.
The disulfide bonds in recombinant GA733-2 antigen have been
determined by a combination of MALDI-MS, peptide mapping, and N-terminal sequencing. All peptide cleavage, deglycosylation, and
purification steps were carried out below pH 6.5 to prevent disulfide
scrambling. The disulfide-bonding pattern in the GA733-2 antigen is
summarized in Fig. 2. A 200-µl immobilized trypsin column was chosen
for the primary protease treatment in these studies because its highly
concentrated immobilized enzyme on the matrix surface facilitated
proteolysis with no appreciable contamination of reaction solutions
with enzyme degradation products.
Assignments of the disulfide linkages in the T1
(Ala84-Arg102/Thr104-Arg115)
and T2
(pyroGlu1-Lys10/Leu11-Lys38)
tryptic peptide complexes were not straightforward because they contained four and six cysteines, respectively. In initial experiments, proteolytic cleavages of deglycosylated T1 (T1-N) and T2 were performed
using various proteases including chymotrypsin, pepsin, subtilisin,
elastase, and thermolysin in the presence of 1 M
guanidine-HCl. However, both complexes were fairly resistant to further
proteolysis under all conditions evaluated, and informative cleavages
between neighboring cysteines were not obtained. Partial reduction of T1-N with TCEP followed by alkylation was then used to attempt determination of disulfide bond assignments. The results of these experiments established that Cys87 was disulfide linked to
either Cys93 or Cys95, but disulfide scrambling
was extensive even when reaction times and pH were lowered (data not
shown). Subsequently, BNPS-skatole cleavage provided unambiguous
disulfide assignments for T1-N. Although several competing side
reactions occurred that resulted in modifications of the peptides
without cleavage at the tryptophan, there was no indication of
disulfide rearrangement. Partial reduction with TCEP followed by
alkylation was performed for T2, and no significant disulfide
scrambling occurred with this complex. After removing the N-terminal
pyroglutamate with pyroglutamate aminopeptidase, Edman sequencing of
both alkylated peptides were performed, and the disulfide linkages in
T2 were unambiguously determined.
Prior reports concluded that the extracellular domain of GA733-2
antigen consisted of a N-terminal cysteine-rich region comprised of an
EGF-like domain followed by a thyroglobulin type 1A motif and a
cysteine-free region with no apparent homology to other proteins
outside of the GA733 protein family based on sequence homology and
cysteine spacing (19, 20). The disulfide linkages of GA733-2 antigen
determined experimentally in the present study are compared with those
of other cysteine-rich domains in Fig. 7.
The first domain of the GA733-2 cysteine-rich region is not an EGF-like
motif. Instead, it has a unique disulfide linkage pattern of
Cys1-Cys4, Cys2-Cys6,
and Cys3-Cys5. We propose that this novel
disulfide structure be called a GA733 type 1 motif. The second
GA733-2EC cysteine-rich motif has Cys1-Cys2,
Cys3-Cys4, and
Cys5-Cys6 linkages, which match the disulfide
pattern of the thyroglobulin type 1A motif recently determined for
human insulin-like growth factor-binding proteins 1 and 6 (24). In
contrast, the EGF-like motifs contain six cysteine residues, with a
Cys1-Cys3, Cys2-Cys4,
and Cys5-Cys6 linkage pattern (35, 36). The
tumor necrosis factor receptor also contains motifs with six cysteines.
The generally accepted disulfide pattern of the tumor necrosis factor
receptor cysteine motif is Cys1-Cys2,
Cys3-Cys5, and
Cys4-Cys6 (37), but this pattern is equivalent
to those of the EGF-like and the laminin type EGF-like motifs after a
circular permutation (36). The laminin type EGF-like motifs contain
eight cysteine residues, with a Cys1-Cys3,
Cys2-Cys4, Cys5-Cys6,
and Cys7-Cys8 pattern determined for human EGF
receptor and mouse laminin
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
g/l) of 0.926, calculated from the GA733-2EC sequence according to Pace et al. (27).
chain (3,496.96 Da), and
bradykinin (1,061.24 Da). Intact GA733-2EC and the tryptic 22-kDa
fragment from partial digestion in solution (10 pmol/2 µl) were mixed
1:1 with a saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid
(sinapinic acid, Sigma) in 33% acetonitrile and 0.1% trifluoroacetic
acid. The 6-kDa fragment from partial tryptic digestion of GA733-2EC in solution was analyzed using
-cyano-4-hydoxy cinnamic acid (Sigma) in
33% acetonitrile and 0.1% trifluoroacetic acid as the matrix. Peptides with molecular masses smaller than 5 kDa were directly applied
to the MS sample plate that was precoated with a saturated solution of
nitrocellulose and
-cyano-4-hydoxycinnamic acid (1:4 w/w) in
2-propanol and acetone (1:1 v/v) as previously described (31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SDS-PAGE analysis of purified GA733-2EC.
Recombinant GA733-2EC was separated on 7% Tris-Tricine gels under
nonreducing conditions and stained with Coomassie Blue. Lane
1, crude Hi Five insect cell culture supernatant; lane
2, GA733-2EC after immunoaffinity purification; lane 3,
after HPLC gel filtration. The positions of the standard proteins in
kDa are shown on the left.
MALDI-MS and N-terminal sequence analyses of tryptic peptide complexes
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Fig. 2.
Amino acid sequence of GA733-2EC showing
experimentally determined disulfide linkages and
N-glycosylation sites. Amino acids are numbered
starting with the N-terminal residue of the major species of the mature
protein as determined in this study; the predominant N terminus of
GA733-2EC is pyroglutamate (E*), which represents removal of
a 23-amino acid residue signal peptide. The N terminus of a small
amount of the protein involves cleavage of 21 amino acid residues with
retention of an additional two residues (Ala-Ala); the N terminus of
this minor component is not blocked. Bold lines indicate
disulfide bonds. Glycosylated N-linked consensus sites have
solid underlines, whereas the unoccupied site at
Asn175 has a dashed underline. The major sites
of cleavage by trypsin to produce disulfide-linked peptides used to
define disulfide linkages are indicated by downward
arrows.
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Fig. 3.
Chromatographic identification of
disulfide-linked tryptic peptides and summary of GA733-2EC disulfide
bond determination. A, chromatographic separation of
GA733-2EC tryptic digest (45 µg) after reduction with TCEP on a
ZORBAX 300SB-C18 column as described under "Experimental
Procedures" using the following gradient conditions; 2% B for 5 min;
2-32% B over 75 min; 32-60% B over 35 min. B,
chromatographic separation obtained with 90 µg of GA733-2EC tryptic
digest (not reduced) using the same gradient as in A. Major
peaks, which disappeared following reduction, are indicated by T1-T3.
T1 is comprised of two disulfide-linked polypeptide chains, 84-102 and
(103)104-115. T2 consists of two disulfide-linked polypeptide chains,
1-10 and 11-38, and T3 consists of two disulfide-linked polypeptide
chains, 43-47 and 59-83. T2* and T3* indicate incomplete tryptic
cleavages of T2 and T3, respectively. Single peptides that appear as a
result of reduction are indicated in A by their inclusive
residue numbers. C, summary of strategy used to complete
GA733-2EC disulfide bond assignments. T1 was deglycosylated with
N-glycosidase A and further cleaved chemically with
BNPS-skatole. Partial reduction with TCEP and alkylation was performed
for T2 followed by enzymatic deblocking of the N-terminal
pyroglutamate. Each peak is named after the reagent and protease used
as well as with the peak number after RP-HPLC. T, trypsin
(cleavage after K or R); N, N-glycosidase A; S,
BNPS-skatole (cleavage after W); R, partial reduction and
alkylation; P, pyroglutamate aminopeptidase (cleavage after
pyroGlu).
MALDI-MS and N-terminal sequence analyses of deglycosylated and
BNPS-skatole-treated T1
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Fig. 4.
RP-HPLC analysis of BNPS-skatole cleavage of
tryptic and deglycosylated peptide T1-N complex. Peptides were
chromatographed on a ZORBAX 300SB-C18 column as described under
"Experimental Procedures" using the following gradient conditions:
2% B for 5 min (A) or for 15 min (B and
C); 2-10% B over 5 min; 10-30% B over 50 min.
A, T1-N before treatment with BNPS-skatole. B,
T1-N after BNPS-skatole cleavage. Peaks marked with
asterisks were in a BNPS-skatole reaction control (no
protein). The analysis of each peptide peak is summarized in Table II.
C, rechromatogram of T1-N-S4 fraction in B after
incubation at 37 °C for 30 min, showing two peaks. T1-N-S4' and
T1-N-S4" eluted at the same retention times as T1-N-S4 and T1-N-S6 in
B, respectively.
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Fig. 5.
RP-HPLC analysis of partial reduced,
alkylated and pyroglutamate aminopeptidase treated tryptic peptide T2
complex. Peptides were chromatographed on a ZORBAX 300SB-C18
column as described under "Experimental Procedures" using the
following gradient conditions: 2% B for 15 min; 2-10% B over 5 min;
10-35% B over 62 min. A, T2 control before partial
reduction and alkylation. B, T2 after partial reduction with
TCEP and alkylation. The analysis of each peak is summarized in Table
III. The small peak marked with asterisks showed the
molecular mass of T2 with two alkylated cysteines by MALDI-MS, although
the amount of this species was negligible compared with the major
species with two alkylated residues, T2-R3. C, pyroglutamate
aminopeptidase treatment of T2-R3 generating free N-terminal T2-R3-P1
peptide. The peak marked with an arrow was in the
pyroglutamate aminopeptidase control.
MALDI-MS and N-terminal sequence analyses of partially reduced,
alkylated, and pyroglutamate aminopeptidase-treated T2
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Fig. 6.
Characterization of
N-glycosylation sites in GA733-2EC. A,
MALDI-MS spectrum of Ala84-Arg102 after
reducing T1 with TCEP. Mass differences between the observed series of
peaks and the peptide sequence mass (2000.3 Da) are due to
glycosylation on Asn88 (summarized in Table I), which
explains the 960-kDa mass difference between the sequence mass and the
major observed mass of GA733-2EC. B, partial tryptic digests
were separated on 15% Tris-Tricine gels under reducing conditions and
stained with Coomassie Blue. Lane 1, purified GA733-2EC;
lane 2, partial digestion with trypsin in PBS (E: S = 1: 500) at 37 °C for 1 h. The positions of standard proteins in
kDa are shown on the left. The solid and
dashed arrows indicate the apparent 6- and 8-kDa bands,
respectively. C, HPLC gel filtration chromatography of
GA733-2EC after partial tryptic digestion using a TSK G3000
SWXL and a G2000 SWXL column connected in
series under reducing and denaturing conditions. Peaks I and
II were pooled separately and analyzed by MALDI-MS after
dialysis to remove the buffer, reducing reagent, and urea.
D, MALDI-MS spectrum of fractions I and
II from C. The average mass of fragment
I agrees within experimental error with the sum of the sequence
mass of Ala59-Lys242 and the average mass of
glycosylation on Asn88 deduced from A. The
MALDI-MS spectrum of fragment II showing series of peaks
with masses that differ by ~1,034 Da, indicating partial
glycosylation on Asn51.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 domain (36, 38).
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Fig. 7.
Cysteine spacings in cysteine-rich
motifs. Comparison of disulfide bonding patterns of cysteine-rich
motifsis shown. Disulfide linkages and spacings for the GA733-2
cysteine-rich motifs are based on the results of this study. The
generic ranges of spacing for thyroglobulin (Tg) type 1A
motif were derived from alignments of human thyroglobulin
(hThyroglobulin) type 1A motifs (20). Disulfide linkages for
this protein have not been experimentally determined (shown as
dashed lines). However, the linkages are expected to be the
same as for the human insulin-like growth factor-binding proteins 1 and
6 (hIGFBP-1,6) domains. Note that the spacings between
linked cysteines in hthyroglobulin motifs are highly variable whereas
the spacings between adjacent disulfide linked pairs are highly
conserved. Experimentally determined disulfide linkages and cysteine
spacings for human insulin-like growth factor-binding proteins 1 and 6 (23) are shown for comparative purposes. A generic EGF-like
cysteine-rich motif was derived from alignments using the EGF-like
sequences for which three-dimensional structures were available (36).
Experimentally determined disulfide linkages for human tumor necrosis
factor-binding protein (hTNFBP) are also shown (37). The
laminin type EGF-like (LE) motifs have similar disulfide
linkages to those of EGF-like motifs, although they contain eight
cysteines instead of six cysteines. Disulfide linkages of human
epidermal growth factor receptor (hEGFR) and mouse laminin
1 chain (mLaminin
1 domain) were
experimentally determined previously (36, 38).
It has been reported that treatment of colon cancer cells in culture with N-glycosylation inhibitor, tunicamycin, resulted in a reduced migration on SDS-PAGE of GA733-2 antigen that was consistent with the sequence predicted mass, whereas treatment with an O-glycanase showed no effects, suggesting that carbohydrates in GA733-2 are predominantly or exclusively N-linked (33, 34). Our data now confirm that the carbohydrate moieties on the GA733-2 antigen are N-linked with no evidence of O-linked carbohydrate moieties. The extracellular domain of GA733-2 antigen has three potential N-glycosylation sites on asparagine residues 51, 88, and 175 (Fig. 2). Through analysis of T1, we found that Asn88 is completely glycosylated with carbohydrate moieties ranging in size from about 893 to 1,259 Da, which accounts for the average 960-Da mass difference between the calculated mass of the GA733-2EC sequence and the major peak observed using MALDI-MS. Because GA733-2EC has a proteolytic cleavage sensitive site on Arg58 (18), purified GA733-2EC was subjected to mild tryptic digestion to generate the N-terminal 6-8-kDa fragment containing Asn51 and the C-terminal 22-kDa fragment containing Asn88 and Asn175. MALDI-MS analysis showed evidence of partial glycosylation on Asn51 but no glycosylation on Asn175. A prior analysis of GA733-2 antigen from keratinocytes using radioimmunoprecipitation concluded that the N-glycosylation was exclusively on the larger proteolytic fragment (34), which implied that Asn51 did not possess glycosylation. However, it is quite likely that a minor diffuse 8-kDa band on a SDS gel might have been overlooked in this earlier study. The degree of posttranslational modification at Asn51 may be functionally and clinically important because it is close to the CO17-1A mAb epitope (10) and to the proteolysis sensitivity site. Interestingly, different ratios of cleaved/noncleaved species of GA733-2 antigen were observed from different epithelial cell lines (33). Although proteolytic regulation of the function of GA733-2 antigen has not been directly demonstrated, the possible correlation between proteolytic cleavage, glycosylation, and adhesion characteristics of the protein is an intriguing question for future studies.
A schematic model of the GA733-2 antigen based upon the data in this
study is compared with previous models in Fig.
8. In all models, the location of the
protease sensitive site is indicated with an arrow. The early model by
Schon et al. (34) predicted that multiple disulfide bonds
linked the small N-terminal fragment to the larger fragment after
cleavage in the protease sensitive loop. In addition, three
glycosylation sites on the larger fragment were predicted with no
glycosylation of the small fragment. Recently, Balzar et al.
(10) predicted that the cysteine-rich region is comprised of two tandem
EGF-like motifs rather than the previously predicted EGF-like motif
followed by a thyroglobulin type 1A motif (19, 20). In contrast, our
current model (Fig. 8) is based upon the disulfide bond structure and
glycosylation sites established in the present study and delineate at
least three distinct structural domains.
|
In conclusion, we have determined the disulfide bond linkages and
glycosylation sites in the human gastrointestinal carcinoma GA733-2
antigen. The extracellular portion of this protein is comprised of at
least three distinct motifs; an N-terminal six-cysteine motif with a
unique disulfide linkage pattern (GA733 type 1 motif), a six-cysteine
motif with the same disulfide linkage as thyroglobulin type 1A motifs
(GA733 type 2 motif = thyroglobulin type 1A motif), and a larger
cysteine-free motif with no apparent homology to other proteins outside
the GA733 protein family. Among the three potential
N-glycosylation sites, Asn88 and
Asn51 were found to have complete and partial
glycosylation, respectively, whereas no glycosylation was found on
Asn175. There was no evidence of O-linked
glycosylation. Knowledge of the disulfide bonds and locations of the
carbohydrate moieties of GA733-2 antigen provides a basis for further
structural and functional studies of this protein and will aid in
rational design of mutations in this protein.
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ACKNOWLEDGEMENTS |
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We thank David Reim for performing N-terminal sequence analyses and helpful comments on the manuscript, Olivera Kolbas for assistance with RP-HPLC, Kaye Speicher for advice concerning MALDI-MS, and Peter Hembach for assistance in preparation of the figures. We also thank Drs. Gavin Manderson and Ronen Marmorstein for helpful comments on the manuscript. We are grateful to the Wistar Institute Recombinant Protein Production Facility for providing baculovirus-infected cells.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA74294 and CA10815.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Wistar Inst., 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3972; Fax: 215-898-0664; E-mail: speicher@wistar.upenn.edu.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M008839200
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; EC, extracelluar; EGF, epidermal growth factor; MALDI, matrix-assisted laser desorption/ionization; mAb, monoclonal antibody; TCEP, tris-(2-carboxyethyl)-phosphine; BNPS-skatole, 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine; RP, reverse phase; HPLC, high performance liquid chromatography; MS, mass spectrometry; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine.
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