From the Departments of Pediatrics and
¶ Pathology, University of Michigan, Ann Arbor, Michigan
48109-0656
Received for publication, October 11, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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There is currently limited data available
pertaining to the global characterization of the cell surface
proteome. We have implemented a strategy for the comprehensive
profiling and identification of surface membrane proteins. This
strategy has been applied to cancer cells, including the SH-SY5Y
neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon
adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell)
cell lines and ovarian tumor cells. Surface membrane proteins of
viable, intact cells were subjected to biotinylation then
affinity-captured and purified on monomeric avidin columns. The
biotinylated proteins were eluted from the monomeric avidin columns as
intact proteins and were subsequently separated by two-dimensional
PAGE, transferred to polyvinylidene difluoride membranes, and
visualized by hybridization with streptavidin-horseradish peroxidase.
Highly reproducible, but distinct, two-dimensional patterns consisting
of several hundred biotinylated proteins were obtained for the
different cell populations analyzed. Identification of a subset of
biotinylated proteins among the different cell populations analyzed
using matrix-assisted laser desorption ionization and tandem mass
spectrometry uncovered proteins with a restricted expression pattern in
some cell line(s), such as CD87 and the activin receptor type IIB. We
also identified more widely expressed proteins, such as CD98, and a
sushi repeat-containing protein, a member of the selectin family.
Remarkably, a set of proteins identified as chaperone proteins were
found to be highly abundant on the cell surface, including GRP78,
GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase.
Comprehensive profiling of the cell surface proteome provides an
effective approach for the identification of commonly occurring
proteins as well as proteins with restricted expression patterns in
this compartment.
The surface membrane is a cellular compartment of substantial
interest. Comprehensive profiling of proteins expressed on the cell
surface could provide a better understanding of the manner in which the
cell surface proteome is regulated and how it responds to a variety of
intracellular and extracellular signals. This compartment is also rich
in therapeutic targets. For example, the discoveries that the gene for
a growth factor receptor (HER2) is amplified in
breast tumors and its protein product is overexpressed at the cell
surface have led to an effective form of therapy for breast cancer
utilizing an antibody that targets HER2 (1). Also, elucidation of the
role of growth factor receptors expressed on the cell surface in
signaling and in uncontrolled cell proliferation, as in the case of
epidermal growth factor receptor, has led to the development of new
anticancer therapies that target specific components of the epidermal
growth factor receptor signal transduction pathway. Selective compounds
have been developed that target the extracellular ligand binding region
of epidermal growth factor receptor (2). Thus, the development of an
effective strategy for the comprehensive analysis of surface membrane
proteins would have important implications. In cancer, cell surface
proteins that are restricted in their expression to specific cancer(s) or that undergo restricted modifications could be utilized for antibody-based therapy, as in the case of HER2 or for vaccine development or other forms of immunotherapy. Signaling pathways regulated by surface membrane proteins or receptors could also be
targeted for a drug-based therapy.
There is currently a paucity of data pertaining to the comprehensive
analysis of surface membrane proteins due, in part, to a lack of
effective strategies to profile the proteome of surface membranes.
Problems associated with the profiling of this compartment stem from
the limited abundance of surface membrane proteins and the difficulty
in resolving and identifying them. Protein tagging technologies have
been available for a long time and have been utilized in a variety of
applications, yet surprisingly, very few studies have attempted to
incorporate protein tagging as part of strategies to enhance
sensitivity of procedures for quantitative protein analysis, such as
two-dimensional gels. For example, protein radio-iodination has been
utilized for many years in different types of protein studies, yet few
publications have emerged that were based on the analysis of
radioiodinated proteins in complex mixtures, when compared with the
vast literature that exists for protein analysis and detection by
silver staining. Novel approaches to improve the detection of proteins
by post-harvest alkylation and subsequent radioactive labeling with
either [3H]iodoacetamide or 125I have been
described and are promising (3).
The high affinity and specificity of avidin-biotin interactions have
been exploited for diverse applications in immunology, histochemistry,
in situ hybridization, affinity chromatography, and many
other areas (4-8). Biotinylation reagents provide the "tag" that
transforms poorly detectable molecules into probes, which can be
recognized by a labeled detection reagent. Once tagged with biotin, a
molecule of interest such as an antibody or receptor ligand can be used
to probe cells, tissues, or proteins immobilized on blots or arrays.
The tagged molecule is detected with a labeled avidin conjugate.
Although the binding of biotin to native avidin or streptavidin is
essentially irreversible, modified avidins can bind biotinylated probes
reversibly, making them valuable reagents for isolation and
purification of biotinylated molecules from complex mixtures. We
have implemented a biotinylation-based strategy for targeting surface
membrane-derived proteins, to allow capture of these proteins, thereby
providing substantial enrichment, and increased sensitivity through the
use of avidin labels for detection. There have been prior studies that
have combined protein biotinylation with two-dimensional gels (9). In
these studies, protein identification was largely based on the use of
antibodies to specific proteins. However, very recently, a global
surface protein biotinylation strategy, coupled with the use of mass
spectrometry, was applied to Helicobacter pylori, leading to
the identification of 18 proteins (10). We here report on the global
profiling of proteins on the surface of a variety of cancer cell types, which has uncovered an abundance of proteins with chaperone function.
Materials--
All cell culture reagents, including RPMI 1640 (containing L-glutamine) and Dulbecco's modified Eagle's
medium (DMEM,1 containing
L-glutamine, sodium pyruvate, and pyridoxine
hydrochloride), Dulbecco's phosphate-buffered saline
(D-PBS), fetal calf serum, and penicillin/streptomycin were
obtained from Invitrogen (Carlsbad, CA). The ImmunoPure Immobilized
Monomeric Avidin and EZ-Link sulfo-NHS-LC-biotin were obtained from
Pierce (Rockford, IL). D-Biotin was obtained from US
Biologicals (Cleveland, OH). The two-well chamber slides were from
Nalge-Nunc (Napierville, IL). The rabbit anti- Cell Culture--
Established human cancer cell lines cultured
as adherent monolayers (SH-SY5Y neuroblastoma cells, A549 lung
adenocarcinoma cells, and LoVo colon adenocarcinoma cells) were
propagated at 37 °C in a 6% CO2-humidified incubator in
DMEM supplemented with 10% fetal calf serum, 100 units/ml penicillin,
and 100 units/ml streptomycin. The cells were passaged weekly upon
reaching confluence. The non-adherent (Sup-B15 acute lymphoblastic
leukemia-B cell) human Leukemia cell line was grown at 37 °C in a
6% CO2-humidified incubator in RPMI 1640 supplemented with
10% fetal calf serum, 100 units/ml penicillin, and 100 units/ml
streptomycin. The cells were passaged weekly. Freshly isolated ovarian
tumor cells were obtained from ascites fluid by centrifugation
(1000 × g, 10 min at room temp), followed by washing
with DMEM (without added serum or protein), then purified on a 3-ml
layer of Ficoll-Paque (400 × g for 40 min at
20 °C). The tumor cell-containing band was harvested from the
Ficoll-Paque and washed three times in D-PBS
(without added serum or protein).
Biotinylation of Membrane Proteins--
Adherent monolayers of
cultured cells (SH-SY5Y, A549, and LoVo) grown in 75-cm2
tissue culture dishes were washed three times with D-PBS
(without added serum or protein). 10 ml of DMEM (without added serum or protein) containing 0.5 mg/ml EZ-Link sulfo-NHS-LC-biotin was added,
and the cells were incubated at 37 °C for 10 min. The biotinylation reaction was terminated by addition of Tris-HCl (pH 7.5) to a final
concentration of 50 mM. Following biotinylation, the cells were washed in PBS and harvested by scraping the cell monolayers in PBS
containing 2% Nonidet P-40. The cells were further disrupted by brief
sonication. Sup-B15 acute lymphoblastic leukemia-B cells were harvested
by centrifugation (1000 × g, 10 min at room temp) and
washed three times with RPMI 1640 (without added serum or protein).
Sup-B15 cells and ovarian tumor cells were suspended at 2.5 × 107 cells/ml in RPMI 1640 and DMEM, respectively (without
added serum or protein), containing 0.5 mg/ml EZ-Link
sulfo-NHS-LC-biotin and incubated at 37 °C for 10 min. The
biotinylation reaction was terminated by addition of Tris-HCl (pH 7.5)
to a final concentration of 50 mM. Following biotinylation,
the cells were washed in PBS, pelleted by centrifugation, and
solubilized in PBS containing 2% Nonidet P-40. The cells were further
disrupted by brief sonication.
Purification of Biotinylated Membrane Proteins--
Solubilized
biotinylated membrane proteins from the various cell populations were
purified on ImmunoPure immobilized monomeric avidin columns (Pierce),
with modifications of the protocol supplied by the manufacturer.
Briefly, 2.5-ml columns of immobilized monomeric avidin were prepared
and extensively washed with PBS. The columns were washed with 2 mM D-biotin in PBS to block any non-reversible biotin binding sites on the column. The loosely bound biotin was removed from the reversible biotin binding sites by washing with 12 ml
of 0.1 M glycine (pH 2.8), and the columns were then
extensively washed with PBS. The disrupted cells with membrane protein
biotinylation were again subjected to sonication, after which the
solubilization solution was clarified by centrifugation (14,000 rpm for
20 min at 4 °C). The solubilization solution was passed through the
immobilized monomeric avidin columns three times, after which the
column was again extensively washed with PBS containing 1% Nonidet
P-40. The bound biotinylated proteins were eluted from the column with 5 mM D-biotin in PBS containing 1% Nonidet
P-40. Fractions containing eluted protein were concentrated on
Centricon YM-3 columns (Millipore).
Two-dimensional PAGE and Western Blotting--
Biotinylated
proteins in the tumor cells and in the cell lines were analyzed by
two-dimensional PAGE (11). Briefly, proteins were solubilized with
lysis buffer, containing 8 M urea, 2% pH 3.5-10 carrier
ampholytes, 2% In-gel Enzymatic Digestion and Mass Spectrometry--
Additional
two-dimensional gels containing proteins eluted from avidin columns
were silver-stained by successive incubations in 0.02% sodium
thiosulfate for 2 min, 0.1% silver nitrate for 40 min, and 0.014%
formaldehyde plus 2% sodium carbonate. The proteins of interest were
excised from the two-dimensional gels and destained for 5 min in 15 mM potassium ferricyanide and 50 mM sodium
thiosulfate as described previously (14). Following three washes with
water, the gel pieces were dehydrated in 100% acetonitrile for 5 min
and dried for 30 min in a vacuum centrifuge. Digestion was performed by
addition of 100 ng of trypsin (Promega) in 200 mM ammonium
bicarbonate. Following enzymatic digestion for 18 h at 37 °C,
the peptides were extracted twice with 50 µl of 60% acetonitrile/1%
trifluoroacetic acid. After removal of acetonitrile in a vacuum
centrifuge, the peptides were concentrated by using pipette tips (C18,
Millipore, Bedford, MA).
Peptide mixtures were analyzed using either a PerSeptive Biosystems
(Framingham, MA) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) Voyager-DE mass spectrometer, operated in
delayed extraction mode, or by nanoflow capillary liquid chromatography coupled with electrospray quadrupole time of flight tandem mass spectrometry (ESI Q-TOF MS/MS) in the Q-TOF micro (MicroMass, Manchester, UK). The peptide mixtures were analyzed using a saturated solution of Immunofluorescence--
The cell lines and tumor cells were
grown in two-well chamber slides for 48 h and then fixed in 2%
formaldehyde, freshly prepared from paraformaldehyde. The fixed cells
were washed briefly in D-PBS. Aldehyde groups resulting
from fixation were quenched in 50 mM L-lysine
(in D-PBS), after which the fixed monolayers were washed
three times in D-PBS (containing 1 mg/ml BSA). Each chamber was incubated with 0.5 ml of the appropriate primary antibody diluted
as indicated in D-PBS (containing 2 mg/ml BSA) for 1 h at room temp. The cells were washed in D-PBS (containing 2 mg/ml BSA). After which they were incubated in 0.5 ml of
D-PBS (containing 2 mg/ml BSA) containing either 10 µg/ml
highly cross-adsorbed Alexa-488-conjugated goat anti-rabbit IgG, 20 µg/ml Alexa-488-conjugated donkey anti-goat IgG, or 20 µg/ml highly
cross-adsorbed Alexa-488-conjugated goat anti-mouse IgG. The stained
monolayers were washed three times in D-PBS (containing 1 mg/ml BSA) and three times in D-PBS, after which a glass
coverslip was mounted on the monolayers in GEL/MOUNT (Biomeda Corp.,
Foster City, CA). Fluorescence images were visualized through a Zeiss
510LSM Confocal Laser Scanning microscope.
DNA Microarray Analysis--
Total RNA was isolated using TRIzol
reagent (Invitrogen), which was followed by clean-up on an RNeasy spin
column (Qiagen), then the total RNA was used to generate cRNA probes.
Preparation of cRNA, hybridization, and scanning of the HuGeneFL arrays
were performed as previously described (65). Briefly, 5 µg of total RNA was converted into double-stranded cDNA by reverse
transcription using a cDNA synthesis kit (Superscript Choice
System, Invitrogen) with an oligo(dT)24 primer containing a
T7 RNA polymerase promoter site added 3' of the poly-T (Genset, La
Jolla, CA). Following second-strand synthesis, labeled cRNA was
generated from the cDNA sample by an in vitro
transcription reaction supplemented with biotin-11-CTP and
biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified on
RNeasy spin columns (Qiagen). 15 µg of each cRNA was fragmented at
94 °C for 35 min in fragmentation buffer (40 mM Tris
acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate). 15 µg of fragmented cRNA was used
to prepare 300 µl of hybridization mixture (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20) containing
0.1 mg/ml herring sperm DNA (Promega, Madison WI), 500 µg/ml
acetylated BSA (Invitrogen), and control cRNAs for comparison of
hybridization efficiency between arrays. Prior to hybridization, the
mixtures were heated to 94 °C for 5 min, equilibrated at 45 °C
for 5 min, and then clarified by centrifugation (16,000 × g) at room temperature for 5 min. Aliquots of each sample
(10 µg of fragmented cRNA in 200 µl of hybridization mixture) were
hybridized to HuGeneFL arrays at 45 °C for 16 h in a rotisserie
oven set at 60 rpm. The arrays were then washed with non-stringent wash
buffer (6× SSPE) at 25 °C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20)
at 50 °C, and stained with streptavidin-R-phycoerythrin. The arrays
were washed again with 6× SSPE, stained with biotinylated anti-streptavidin IgG, followed by a second staining with
streptavidin-phycoerythrin, and a third washing with 6× SSPE. The
arrays were scanned using the GeneArray scanner (Affymetrix). Data
analysis was performed using GeneChip 4.0 software.
Profiling the Cell Surface Proteome by Protein
Biotinylation--
The approach we have implemented for the
comprehensive profiling and identification of surface membrane proteins
involves the selective biotinylation of the surface
proteins of intact cells. Following separation by two-dimensional PAGE
and transfer to PVDF membranes, the biotinylated proteins are
visualized by hybridization with a streptavidin/horseradish peroxidase
complex. Alternatively, biotinylated proteins are captured on an
affinity column, followed by their separation and identification. We
have applied this approach to the analysis of surface membranes of cancer cells and compared biotinylation patterns obtained for different
cell lineages.
Fig. 1 displays the biotinylated proteins
in whole cell lysates of A549 lung adenocarcinoma cells following cell
surface labeling. It is evident that the pattern of visualized
biotinylated proteins (Fig. 1B) is quite rich in separated
proteins that are not visualized in silver-stained two-dimensional gels
of the same whole cell lysates (Fig. 1A). Many of the
resolved biotinylated proteins form trains of spots, as expected
for proteins that undergo numerous post-translational modifications
(e.g. glycosylation, phosphorylation, and sulfation).
Although some proteins could be matched in their location between the
silver-stained (Fig. 1A) and biotinylated (Fig.
1B) two-dimensional patterns, most biotinylated proteins did
not have a match in the silver-stained two-dimensional pattern of whole
cell lysates. This strongly suggests that we were able to obtain a
selective and enhanced visualization of low abundance proteins by
biotinylation of the surface membrane.
Capture and Purification of Biotinylated Surface Membrane
Proteins--
Because the population of surface membrane proteins that
were biotinylated represented low abundance proteins at the whole cell
level, it was necessary to utilize an enrichment procedure following
biotinylation to allow identification of the biotinylated proteins. To
this end, a column of immobilized monomeric avidin was utilized to bind
biotinylated proteins, with their subsequent elution from the column in
PBS containing 5 mM D-biotin, 1% Nonidet P-40.
Column eluates from different cell populations were each concentrated
~400-fold, and aliquots were resolved by either two-dimensional PAGE
or IPG, followed by silver staining for some aliquots or transfer to
PVDF membranes for others. PVDF membranes were hybridized with
streptavidin/horseradish peroxidase and visualized with the ECL
reagents. Two-dimensional patterns for eluted proteins visualized by
silver staining had remarkable similarity with two-dimensional patterns
of proteins that were transferred to PVDF membranes, then hybridized
with streptavidin/horseradish peroxidase and detected with the ECL
reagent, indicating that most proteins captured on the column
represented biotinylated proteins. Fig. 2
displays an IPG two-dimensional pattern for the A549 lung
adenocarcinoma following column elution and silver staining. It shows
similarity with the pattern for the same cell population obtained
following a carrier ampolyte separation of a whole cell lysate
of biotinylated A549 cells in the common pH separation region (Fig. 1).
Moreover, patterns resolved by carrier ampolyte or IPG
overlapped substantially for the same pH separation range (Figs.
1B and 2). Furthermore, Fig. 3
shows that the pattern of biotinylated proteins from ovarian tumor
cells that were visualized by hybridization was highly similar to the
pattern obtained from silver-stained gels of the same aliquot of eluted
protein.
Identification of Biotinylated Surface Membrane
Proteins--
We sought to identify the biotinylated proteins we
considered to be abundant, based on their intensity in images of
two-dimensional blots that were hybridized with
streptavidin/horseradish peroxidase. These proteins also occurred
as relatively abundant proteins in two-dimensional silver stain
patterns of proteins eluted from monomeric avidin columns. Preparative
quantities of biotinylated surface membrane proteins from ~3 × 108 cells were prepared for each of the cell lines
analyzed. Following solubilization, the biotinylated proteins were
captured and purified on monomeric avidin columns. The eluted proteins
were resolved by two-dimensional PAGE, after which the gels were
stained with a mass spectrometry-compatible silver stain. One unstained
gel from each preparation of silver-stained gels for protein
identification was transferred to a PVDF membrane and hybridized with
streptavidin/horseradish peroxidase complex to visualize the proteins
that were biotinylated. In comparison to databases of proteins
previously identified in our laboratory, we noted that some of the
visualized biotinylated proteins actually represent proteins that were
previously identified in whole cell lysates. However, considerable
enrichment was evident in the biotinylated surface protein
patterns (Fig. 4).
We next determined whether patterns obtained from the various cell
populations (e.g. A549 lung adenocarcinoma, papillary
ovarian carcinoma, SH-SY5Y neuroblastoma, LoVo colon carcinoma, and
acute lymphoblastic leukemia-B cell type) were sufficiently distinctive to identify surface proteins with restricted expression patterns. Although it was apparent that some proteins were expressed in all cell
types, some of the visualized biotinylated proteins were detected in
some cell types but not others (Table I).
To verify that proteins considered to be similar in different cell
types based on pattern matching were indeed similar, we excised spots that were evident in two or more cell types from gels prepared from
each of the cell types. These proteins were subjected individually to
mass spectrometric identification, which confirmed that they had the
same identity (Table I). For example, the sushi-repeat-containing protein was identified in the A549 lung adenocarcinoma cell line (Fig.
2), and its presence was confirmed in both the LoVo colon carcinoma and
SH-SY5Y neuroblastoma cell lines, as predicted based on spot matching.
The sushi-repeats are characteristic motifs in members of the selectin
family of cell membrane proteins with functional roles in cell adhesion
(15, 16). Additionally, we identified the Lutheran blood group
glycoprotein in both the A549 and in the SH-SY5Y cell lines (Fig. 2).
The Lutheran glycoprotein, a membrane protein belonging to the
immunoglobulin superfamily, has been shown to act as a specific
receptor for laminin in cell-matrix interaction (17). Furthermore, the
4F2 heavy chain antigen (also known as CD98) was identified in SH-SY5Y,
A549, LoVo, and the Sup-B15 leukemia cell lines. In vitro
binding studies have shown that the 4F2 heavy chain (CD98) interacts
specifically with integrin
We have also identified biotinylated surface membrane proteins that
were considered to be abundant but whose corresponding spots were found
in only one or two of the cell types examined. Among these were a
protein tyrosine phosphatase receptor type R, found in a spot that
occurred only in A549 cells (21), and the urokinase plasminogen
activator receptor 2 (CD87), found in a spot that occurred only in LoVo
cells. It has been suggested that CD87, a
glycosylphosphatidylinositol-anchored membrane receptor, is involved in
both plasminogen activation and cellular adhesion (22). Additionally, a
spot, found in both ovarian tumor cells and in Sup-B15 acute
lymphocytic leukemia-B cells but not in the other cells examined, was
identified as the activin receptor type IIB. Activin is well known for
its inhibitory regulation of cell proliferation, enhancement of
apoptosis, and suppression of cancer formation and progression.
Aberration in the activin signaling pathway has been considered a
possible cause of malignant transformation in various tissues,
including breast cancer, prostate cancer, and leukemia (23).
Importantly, we also found that the spot identified as corresponding to
Ephrin type B receptor 4, a member of a large family of receptor
tyrosine kinases, was detectable in the LoVo cell line, but not in
other cell lines examined. The Ephrin type B receptor 4 and its ligand
Ephrin B2 appear to provide critical guidance cues at points of
cell-cell contact in cardiovascular development. In adult settings of
neo-angiogenesis, such as in tumors, it appears that the endothelium of
a subset of new vessels strongly expresses the Ephrin B2 ligand (24),
whereas the tumor cells express both the ligand and the receptor (25,
26). Interestingly, it has been demonstrated that the Ephrin type B
receptor 4 is up-regulated in colon tumors when compared with normal
colon tissue from the same patient (26). As such, the Ephrin type B
receptor 4 may provide angiogenic signaling for the recruitment of new blood vessels to support tumor growth. To confirm the restricted expression of the Ephrin receptor, we examined whether we could localize this protein in the plasma membrane of both the LoVo and A549
cell lines by indirect immunofluorescence microscopy. We found that the
Ephrin receptor was present on the surface of LoVo but not on the
surface of A549 (Fig. 5), in confirmation of the results that we obtained by analysis of biotinylated
proteins.
Identification of Chaperone Proteins at the Cell
Surface--
Remarkably, we also found that a relatively large set of
proteins with chaperone function, including heat-shock proteins, was
highly abundant on the cell surface (Fig.
6). These proteins include GRP78, GRP75,
HSP70, HSP71, HSP60, HSP54, HSP27, and protein disulfide isomerase.
Protein disulfide isomerase is an endoplasmic reticulum protein that
catalyzes protein-folding reactions and increases the rate at which
proteins attain their final folded conformation. GRP78 (BiP) is an
endoplasmic reticulum (ER) chaperone whose function is generally
thought to be limited to the structural maturation of nascent
polypeptides and helps to prevent protein-folding intermediates from
aggregating and stabilize energetically unfavorable conformations of
polypeptides to minimize irreversible protein misfolding (27). We
found, by analysis of biotinylation patterns, that GRP78 was expressed
on the surface of all cell types analyzed in this study. To confirm the
ubiquitous expression of GRP78, we examined whether we could localize
this protein to the plasma membrane of the SH-SY5Y, LoVo, and A549 cell
lines and the ovarian tumor cells by indirect immunofluorescence
microscopy. We found that GRP78 was present on the surface of all four
cell lines examined (Fig. 7), in
confirmation of the results that we obtained by analysis of
biotinylated proteins.
HSP70 was originally thought to be ubiquitously expressed as a
cytoplasmic protein whose function was to capture folding intermediates to prevent protein misfolding and aggregation and to facilitate proper
refolding (28-30). We found by analysis of biotinylation patterns that
HSP70 was expressed on the surface of all cell types analyzed, which we
confirmed by indirect immunofluorescence microscopy with LoVo cells
(Fig. 8).
Correlations with RNA Levels--
Comparison of the biotinylation
patterns between the various cell types analyzed revealed that some
biotinylated protein spots exhibited a lineage-restricted expression.
Importantly, however, migration of biotinylated protein may vary
(both by pI and molecular weight) between cell lines, dependent upon
various post-translational modifications (e.g.
glycosylation, phosphorylation, and sulfation). To determine the cell
line expression of genes corresponding to the biotinylated proteins
that were identified by mass spectrometry, high density oligonucleotide
microarrays were utilized to generate gene expression profiles for all
cell types examined. Although not all identified proteins were
represented on our microarrays, in some cases the mRNA expression
data was concordant with the restricted expression of the corresponding
protein (Fig. 9). For example, the
sushi-repeat-containing protein was found to be present in the A549,
LoVo, and SH-SY5Y cell populations, but not in the ovarian or the ALL-B
cells, by both mRNA expression and surface membrane profiling.
Moreover, the multidrug resistance protein (P-glycoprotein) was found
to be present in the SH-SY5Y and LoVo cell lines, but absent in the
A549 cell line, the ovarian tumor cells, and the ALL-B leukemia cells
by both mRNA and surface membrane profiling. In contrast,
however, the mRNA encoding flotilin 2 was expressed in all
cell types examined, but the protein spot of interest was not
visualized in the ovarian and the ALL-B cells (Fig. 9). This suggests
that the identified isoform of flotilin 2 was expressed on the cell
surface in a lineage-restricted fashion.
The approach that we have developed consists of the
biotinylation of plasma membrane proteins of freshly isolated
cells, primary cultures or cell lines, followed by their comprehensive
profiling and identification. Previously, most protein-related
biotinylation applications have dealt with the isolation/enrichment or
assay of individual proteins or a small number of related proteins. A
basic component in a biotin-avidin-based application is the moiety to
be targeted. In the case of proteins, biotinylation is done usually via
the Several findings have emerged from our studies. We have identified both
glucose-regulated proteins (GRPs) and heat-shock proteins as relatively
highly abundant proteins on the cell surface of a wide variety of cell
types. These proteins were originally identified as being either
cytoplasmic (28, 30) or endoplasmic reticulum (36) proteins. However,
it has recently been demonstrated that HSP70 does occur on the surface
of a number of cell types (37-48). However, our ability to
comprehensively profile cell surface proteins has uncovered the high
abundance of heat-shock proteins compared with other cell surface
proteins. It is interesting to note that recent data have implicated
the endoplasmic reticulum (ER) as a membrane source. Fusion of the ER
with the macrophage plasmalemma, underneath phagocytic (49) cups, has
been found to be a source of membrane for phagosome formation in
macrophages (50).
The functionality of heat-shock proteins at the cell surface has begun
to be elucidated, with recent work focusing on heat-shock protein-receptor interactions. Heat-shock proteins derived from tumor
cells are recognized by T cells with either T cell receptor, It is likely that proteins that are routinely found in the endoplasmic
reticulum lumen follow the classic secretory pathway out of the cell.
However, a number of the proteins that we have identified on the cell
surface, including the majority of heat-shock proteins, do not encode
transmembrane domains or, for that matter, signal sequences (which
target the nascent polypeptide into the secretory pathway) within their
genomic structure. Although it is unclear as to how these proteins were
targeted to the cell surface, it is unlikely that they utilized the
classic secretory pathway (ER to Golgi to plasma membrane) for their
targeting. It has been demonstrated that several proteins, including
basic fibroblast growth factor, interleukin-1 Biotinylation provides an effective tool for the detection and
purification of proteins. However, to retain both biological activity
and ligand-binding properties and to facilitate the identification of
the labeled proteins by mass spectrometry, it is necessary to perform
biotinylation reactions that do not extensively biotinylate proteins.
Furthermore, although complete solubilization of the labeled cells is
desirable, the best reagents for solubilization may interfere with the
ability to capture and purify the biotinylated proteins on a monomeric
avidin column. We have optimized the biotinylation approach for the
analysis of surface membranes by comparing patterns obtained with
different protocols for the solubilization of proteins and for the
capture and purification of the biotinylated proteins. Our methodology
for the identification of surface membrane proteins is based upon the
successful biotinylation and subsequent purification of the
biotinylated proteins on monomeric avidin columns for enrichment purposes. Knowing that the intact protein was biotinylated allows us to
ascertain that the identified protein was present in the membrane
compartment. Such information, which would be lacking if we subjected
the complex mixture of proteins to proteolytic digestion (as is the
case in a MudPIT analysis (64)) provides evidence for us too
properly identify the "tagged" protein. However, it is likely that
additional biotinylated proteins have failed to be adequately
solubilized for their effective capture and identification. Thus,
further refinements of our present strategy, such as by partial
digestion of biotinylated proteins that fail to be solubilized with our
present mixture, may further increase the repertoire of biotinylated
proteins detected on the cell surface.
The approach we have implemented has wide applicability to many
different types of cells, bacteria, or subcellular fractions. A recent
study has been reported using a similar biotinylation approach for the
identification of surface membrane proteins isolated from
Helicobacter pylori (10). Some 82 biotinylated proteins were
resolved by two-dimensional PAGE, of which 18 proteins were identified
by comparison to proteome data and by peptide mass fingerprinting. The
comprehensive profiling of cell surface proteins provides an effective
approach for the identification of novel targets for diagnostics and
therapeutics for a wide range of diseases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EphB4 and the goat
anti-GRP78-N antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). The mouse anti-HSP70 antibody was purchased from
Sigma (St. Louis, MO). Ficoll-Paque Plus, streptavidin-biotinylated horseradish peroxidase complex, and HRP-conjugated goat anti-rabbit IgG
and the ECL (Enhanced Chemiluminescence) kits were obtained from
Amersham Biosciences (Arlington Heights, IL). The biotinylated anti-streptavidin IgG was obtained from Vector Laboratories
(Burlingame, CA). Streptavidin R-phycoerythrin, Alexa 488 highly
cross-adsorbed goat anti-rabbit IgG, and Alexa 488 donkey anti-goat IgG
were obtained from Molecular Probes (Eugene, OR). Immobilon-P PVDF (polyvinylidene difluoride) membranes were purchased from
Millipore Corp. (Bedford, MA). Acrylamide used in the one-dimensional
electrophoresis, urea, ammonium persulfate, and piperazine diacrylamide
were all purchased from Bio-Rad (Rockville Centre, NY). Acrylamide used in the two-dimensional electrophoresis was purchased from Serva (Crescent Chemical, Hauppauge, NY), and carrier ampholytes (both pH
4-8 and pH 3.5-10) and Nonidet P-40 were purchased from
Gallard/Schlessinger (Carle Place, NY). All other reagents and
chemicals were obtained from either Fisher or Sigma and were of the
highest purity available.
-mercaptoethanol, 2% Nonidet P-40, and 10 mM phenylmethylsulfonyl fluoride. Isoelectric focusing was
carried out using either pH 4-8 carrier ampholyte-based tube gels for
13,200 V-h at room temperature or using immobilized 4-10 pH gradient
(IPG)-based strips (12). One-dimensional gels were loaded onto a
cassette containing the second-dimensional gel, after equilibration in
second-dimensional sample buffer (125 mM Tris (pH 6.8),
containing 10% glycerol, 2% SDS, 1% dithiothreitol, and bromphenol
blue). Separation in the second dimension was performed by
electrophoresis in 7-14% polyacrylamide gradient SDS gels, and the
samples were electrophoresed until the dye front reached the opposite
end of the gel. Some gels were silver-stained and digitized for pattern
analysis as previously described (13). For some other gels, the
resolved proteins were transferred to an Immobilon-P PVDF membrane.
Unstained membranes were prepared for hybridization by incubation with
blocking buffer (consisting of Tris-buffered saline (TBS) containing
1.8% nonfat dry milk and 0.1% Tween 20) for 2 h, then washed and
incubated with a horseradish peroxidase-conjugated biotin-streptavidin
complex (at a 1:400 dilution) for 40 min at room temperature. The
membranes were washed five times with TBS containing 0.1% Tween 20, once in TBS, briefly incubated in ECL, and exposed to XAR-5 x-ray film.
Patterns visualized were directly compared with comparable gel
silver-stain patterns.
-cyano-4-hydroxycinnamic acid (Sigma) in acetonitrile containing 1% trifluoroacetic acid (0.5 µl of sample:0.5 µl of matrix). Peptides were selected in the mass range of 800-4000 Da.
Spectra were calibrated using calibration mixture 2 of the Sequazyme
peptide mass standards kit (PerSeptive Biosystems). MALDI-TOF MS gave a
peptide mass fingerprint for each spot based on the molecular mass of
trypsin-digested products. We compared the resulting masses with known
trypsin digest protein sequence databases (SwissProt or NCBInr)
using the MS-Fit data base search engine developed by the University of
California at San Francisco (available at
prospector.ucsf.edu/ucsfhtml3.2/msfit.htm). ESI MS/MS tandem spectra
were recorded in the automated MS to MS/MS switching mode, with an
m/z-dependent set of collision offset values. Singly to triply charged ions were selected and fragmented, with argon used as the collision gas. The acquired spectra were processed and searched against a non-redundant SwissProt protein sequence data base using the ProteinLynx global server
(available at www.micromass.co.uk).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Visualization of surface biotinylation
patterns in A549 lung adenocarcinoma cells. A,
two-dimensional PAGE analysis of cellular proteins from the A549 cell
line. Solubilized proteins from A549 lung adenocarcinoma cells were
resolved by two-dimensional PAGE using carrier ampholytes (pI 4-8) in
the first dimension. The proteins were visualized by silver staining,
as described under "Experimental Procedures." B,
detection of biotinylated surface proteins from the A549 cell line.
Intact A549 lung adenocarcinoma cells were subjected to surface
biotinylation, as described under "Experimental Procedures."
Solubilized proteins from the biotinylated cells were resolved by
two-dimensional PAGE using carrier ampholytes (pI 4-8) in the first
dimension, then transferred to PVDF membranes. The biotinylated
proteins were visualized by hybridization with streptavidin-HRP
complex. Arrows point to biotinylated proteins that were
identified by mass spectrometry. Interestingly, the biotinylated
proteins (B) are not visualized in the silver stain image of
the same lysate (A).
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Fig. 2.
Visualization of purified biotinylated
surface proteins isolated from the A549, LoVo, and SH-SY5Y cell
lines. Surface proteins of the A549, LoVo, and SH-SY5Y cell lines
were biotinylated and purified as described under "Experimental
Procedures." Following solubilization, the proteins were resolved by
two-dimensional PAGE using IPG in the first dimension then visualized
by mass spectrometry-compatible silver staining, as described under
"Experimental Procedures." Arrows point to biotinylated
proteins that were identified by mass spectrometry.
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Fig. 3.
Similarity of ovarian biotinylation patterns
as visualized by hybridization and silver-stained images of the same
monomeric avidin column eluate. Surface proteins of ovarian cells
were biotinylated and purified as described under "Experimental
Procedures." Following solubilization, the proteins were resolved by
two-dimensional PAGE using carrier ampholytes (pI 4-8) in the first
dimension then visualized either by mass spectrometry-compatible silver
staining or hybridization with streptavidin-HRP complex, as described
under "Experimental Procedures." Arrows point to
biotinylated proteins that were identified by mass spectrometry.
Interestingly, the patterns visualized by silver stain and
hybridization appear to be virtually identical.
View larger version (88K):
[in a new window]
Fig. 4.
Comparison of the ovarian biotinylation
patterns to a lung adenocarcinoma master image. Surface proteins
of ovarian cells were biotinylated and purified as described under
"Experimental Procedures." Following solubilization, the proteins
were resolved by two-dimensional PAGE using carrier ampholytes (pI
4-8) in the first dimension then visualized either by hybridization
with streptavidin-HRP complex, as described under "Experimental
Procedures." The image was compared with a silver stained image of a
lung adenocarcinoma, which serves as a data base master image.
Arrows point to biotinylated proteins that were found to be
in common between the two images and that have been identified by mass
spectrometry.
1A (18), and it has been implicated in
cell activation and proliferation in various cancer cells, including
those of colon, breast, and lung (19). Moreover, there is a recent
report demonstrating that a specific antibody targeting the 4F2 heavy
chain antigen inhibited the growth of tumor cells that expressed the
antigen (20).
Identified surface proteins from the various cancer cell types
View larger version (118K):
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Fig. 5.
The Ephrin B4 receptor is expressed in a
restricted pattern between the LoVo and A549 cell lines. LoVo
colon carcinoma and A549 lung adenocarcinoma cells were plated
and grown for 48 h then fixed and stained for immunofluorescence
with Rabbit anti-EphB4 antibodies, as described under "Experimental
Procedures." Identical fields are shown for both the
immunofluorescence and the transmitted light image (differential
interference contrast). The LoVo cell line is shown in A and
B. The A549 cell line is shown in C and
D. Bar, 20 µm.
View larger version (42K):
[in a new window]
Fig. 6.
Identification of HSP71 by Q-TOF mass
spectrometry. The MS/MS spectrum of HSP71 obtained after trypsin
digestion is shown by analysis with ESI-Q-TOF, coupled with nanoflow
capillary high-performance liquid chromatography. The precursor ion
shown in the figure is m/z 825.3824, and
resultant peaks were searched against the non-redundant SwissProt
protein sequence data base using the ProteinLynx global server. A total
of nine tryptic peptides, as shown, matched the heat-shock cognate
71-kDa protein.
View larger version (156K):
[in a new window]
Fig. 7.
GRP78 is expressed on the cell surface in a
ubiquitous pattern. Ovarian, SH-SY5Y, LoVo, and A549 cells were
plated and grown for 48 h then fixed and stained for
immunofluorescence with goat anti-GRP78 antibodies, as described under
"Experimental Procedures." The A549 cell line is shown in
A, the LoVo cell line is shown in B, the SH-SY5Y
cell line is shown in C, and the ovarian cells are shown in
D. Bar, 20 µm.
View larger version (85K):
[in a new window]
Fig. 8.
HSP70 is expressed on the cell surface of
LoVo cells. LoVo colon carcinoma cells were plated and grown for
48 h then fixed and stained for immunofluorescence with mouse
anti-HSP70 antibodies, as described under "Experimental
Procedures." Identical fields are shown for both the
immunofluorescence and the transmitted light image (differential
interference contrast). Anti-HSP70 staining is shown in A
and B. The staining obtained with normal mouse IgG is shown
in C and D. Bar, 20 µm.
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Fig. 9.
Concordance of genomic and proteomic data for
SRPX, ABCB1, and FLOT2 in the various cell types examined.
mRNA expression levels were determined for SRPX, ABCB1, and FLOT2
by analysis of high density oligonucleotide microarray data, as
described under "Experimental Procedures," and are shown in the
graph. The presence of protein expression (P) or absence of
protein expression (A) in the identified protein spot is as
indicated. Interestingly, for SRPX and ABCB1 there was strict
concordance between genomic and proteomic data for all cell types. For
FLOT2, however, it appears by genomic data that FLOT2 is expressed
ubiquitously in all of the cell types, although the protein spot of
interest was absent in the ovarian and ALL-B cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of lysine by using an
N-hydroxysuccinimide (NHS) ester of a biotin analog. To
selectively label only those lysine residues that are extracellular in
orientation, non-membrane permeable biotin reagents need to be utilized
to prevent the entry of biotin into the cell. Sulfo-NHS-LC biotin is
water-soluble; thus it is not permeable across hydrophobic lipid
bilayers and can be utilized for the selective labeling of surface
membrane proteins. Other groups that have utilized sulfo-NHS-biotin for isolation of individual cell surface proteins in intact cells have
found selective biotinylation of plasma membrane proteins (31, 32).
Moreover, this reagent has been used to selectively label surface
membrane proteins on either the apical or basolateral surface of
intact, viable epithelial monolayers, because the sulfo-NHS-biotin reagent is impermeable to tight junctions (33, 34). Importantly, calnexin, a molecular chaperone in the endoplasmic reticulum has been
localized on the cell surface by use of this biotinylation reagent
(35).
,
or
,
(49). It has been shown that heat-shock proteins are
linked to TLRs (toll-like receptors) as, unlike in wild type macrophages, HSP60 failed to activate TLR4-defective macrophages (51).
Moreover, it has been demonstrated that the heat-shock protein
Gp96-TLR2/4 interaction results in activation of NF
B-driven reporter
genes and mitogen- and stress-activated protein kinases (52).
Additionally, HSP70 also activates the IL-1 receptor signaling pathway
(53). Furthermore, it has been shown that a signaling complex of
receptors, comprising heat-shock proteins 70 and 90, chemokine receptor
4 (CXCR4), and growth differentiation factor 5, is formed during immune
system recognition of bacterial lipopolysaccharide (54). Thus, it has
been demonstrated that heat-shock proteins and some receptors interact
at the cell surface to stimulate receptor-mediated functions.
(IL-1
), galectin-3, thioredoxin, and HIV-Tat (55-59), are secreted in a non-classic manner, independent of the ER-Golgi pathway. Furthermore, it has been
recently shown that, although exogenously expressed, properly folded
green fluorescent protein is retained in the cytosol, and improperly
folded green fluorescent protein is secreted from the cell cytosol
through a non-classic secretory pathway (60). Although an ATP-binding
cassette (ABC) transporter appears to be involved in the secretion of
IL-1
(61) and a Na+/K+ ion channel has been
implicated in the secretion of basic fibroblast growth factor (62),
apparently galectin-3 is secreted from the cytosol by membrane
blebbing. This last pathway appears capable of post-translational
export of fully folded proteins (63). One possible explanation as to
how the heat-shock proteins reached the cell surface is that they
accompanied misfolded proteins, or peptide fragments out of the cytosol
via a non-classic pathway. Alternatively, they may be actively
transported from their site of synthesis in the cytosol
to help maintain structural integrity among the individual
components of various receptor complexes.
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FOOTNOTES |
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* 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.
§ Present address: Centre d'Immunologie Pierre Fabre, Saint Julien en Genevois 74164, France.
To whom correspondence should be addressed: Dept. of
Pediatrics, University of Michigan, 1150 West Medical Center Dr., Rm. A520 MSRB-1, Ann Arbor, MI 48109-0656. Tel.: 734-763-0917; Fax: 734-647-8148; E-mail: dmisek@umich.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M210455200
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ABBREVIATIONS |
---|
The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; D-PBS, Dulbecco's phosphate-buffered saline; HRP, horseradish peroxidase; PVDF, polyvinylidene difluoride; NHS, N-hydroxysuccinimide; IPG, immobilized 4-10 pH gradient; MALDI-TOF, matrix-assisted laser desorption time of flight; ESI, electrospray ionization; Q-TOF MS/MS, quadrupole time of flight tandem mass spectrometry; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; SSPE, saline/sodium phosphate/EDTA; ER, endoplasmic reticulum; GRP, glucose-regulated protein; HSP, heat-shock protein; TLR, toll-like receptor; IL-1, interleukin-1; ABC, ATP-binding cassette.
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---|
1. |
Slamon, D. J.,
Leyland-Jones, B.,
Shak, S.,
Fuchs, H.,
Paton, V.,
Bajamonde, A.,
Fleming, T.,
Eiermann, W.,
Wolter, J.,
Pegram, M.,
Baselga, J.,
and Norton, L.
(2001)
N. Eng. J. Med.
344,
783-792 |
2. | Raymond, E., Faivre, S., and Armand, J. P. (2000) Drugs 60 Suppl. 1, 15-23[Medline] [Order article via Infotrieve] |
3. | Vuong, G. L., et al.. (2000) Electrophoresis 21, 2594-2605[CrossRef][Medline] [Order article via Infotrieve] |
4. | Wilbur, D. S., Pathare, P. M., Hamlin, D. K., Stayton, P. S., To, R., Klumb, L. A., Buhler, K. R., and Vessella, R. L. (1999) Biomol. Eng. 16, 113-118[CrossRef][Medline] [Order article via Infotrieve] |
5. | Wilchek, M., and Bayer, E. A. (1999) Biomol. Eng. 16, 1-4[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Diamandis, E. P.,
and Christopoulos, T. K.
(1991)
Clin. Chem.
37,
625-636 |
7. | Bayer, E. A., and Wilchek, M. (1990) J. Chromatogr. 27, 3-11 |
8. | Chapman-Smith, A., and Cronan, J. E., Jr. (1999) Trends Biochem. Sci. 24, 359-363[CrossRef][Medline] [Order article via Infotrieve] |
9. | Hewett, P. W. (2001) Int. J. Biochem. Cell Biol. 33, 325-335[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Sabarth, N.,
Lamer, S.,
Zimny-Arndt, U.,
Jungblut, P. R.,
Meyer, T. F.,
and Bumann, D.
(2002)
J. Biol. Chem.
277,
27896-27902 |
11. | Strahler, J. R., Kuick, R., and Hanash, S. M. (1989) in Protein Structure: A Practical Approach (Creighton, T., ed) , pp. 65-92, IRL Press Ltd., Oxford |
12. | Hanash, S. M., Strahler, J. R., Neel, J. V., Hailat, N., Melhem, R., Keim, D., Zhu, X. X., Wagner, D., Gage, D. A., and Watson, J. T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5709-5713[Abstract] |
13. | Kuick, R., Hanash, S. M., Chu, E. H. Y., and Strahler, J. R. (1987) Electrophoresis 8, 199-204 |
14. | Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999) Electrophoresis 20, 601-605[CrossRef][Medline] [Order article via Infotrieve] |
15. | Johnston, G. I., Cook, R. G., and McEver, R. P. (1989) Cell 56, 1033-1044[Medline] [Order article via Infotrieve] |
16. | Bevilacqua, M. P. (1993) Ann. Rev. Immunol. 11, 767-804[CrossRef][Medline] [Order article via Infotrieve] |
17. |
El Nemer, W.,
Gane, P.,
Colin, Y.,
D'Ambrosio, A. M.,
Callebaut, I.,
Cartron, J. P.,
and Van Kim, C. L.
(2001)
J. Biol. Chem.
276,
23757-23762 |
18. |
Zent, R.,
Fenczik, C. A.,
Calderwood, D. A.,
Liu, S.,
Dellos, M.,
and Ginsberg, M. H.
(2000)
J. Biol. Chem.
275,
5059-5064 |
19. |
Rintoul, R. C.,
Buttery, R. C.,
Mackinnon, A. C.,
Wong, W. S.,
Mosher, D.,
Haslett, C.,
and Sethi, T.
(2002)
Mol. Biol. Cell
13,
2841-2852 |
20. |
Papetti, M.,
and Herman, I. M.
(2001)
Am. J. Pathol.
159,
165-178 |
21. | Sap, J. D., Eustachio, P., Givol, D., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6112-6116[Abstract] |
22. | Dear, A. E., and Medcalf, R. L. (1998) Eur. J. Biochem. 252, 185-193[Abstract] |
23. |
Chen, Y. G.,
Lui, H. M.,
Lin, S. L.,
Lee, J. M.,
and Ying, S. Y.
(2002)
Exp. Biol. Med.
227,
75-87 |
24. | Gale, N. W., Baluk, P., Pan, L., Kwan, M., Holash, J., DeChiara, T. M., McDonald, D. M., and Yancopoulos, G. D. (2001) Dev. Biol. 230, 151-160[CrossRef][Medline] [Order article via Infotrieve] |
25. | Liu, W., Ahmad, S. A., Jung, Y. D., Reinmuth, N., Fan, F., Bucana, C. D., and Ellis, L. M. (2002) Cancer 94, 934-939[CrossRef][Medline] [Order article via Infotrieve] |
26. | Stephenson, S. A., Slomka, S., Douglas, E. L., Hewitt, P. J., and Hardingham, J. E. (2001) BMC Mol. Biol. 2, 15[CrossRef][Medline] [Order article via Infotrieve] |
27. | Dill, K. A., and Chan, H. S. (1997) Nat. Struct. Biol. 4, 10-19[Medline] [Order article via Infotrieve] |
28. | Ellis, R. J. (1993) Philos. Trans. R. Soc. Lond. B Biol. Sci. 339, 257-261 |
29. | Georgopoulos, C., and Welch, W. J. (1993) Ann. Rev. Cell Biol. 9, 601-634[CrossRef] |
30. | Welch, W. J. (1993) Philos. Trans. R. Soc. Lond. B Biol. Sci. 339, 327-333[Medline] [Order article via Infotrieve] |
31. | Busch, G., Hoder, D., Reutter, W., and Tauber, R. (1989) Eur. J. Cell Biol. 50, 257-262[Medline] [Order article via Infotrieve] |
32. | Hurley, W. L., Finkelstein, E., and Holst, B. D. (1985) J. Immunol. Methods 85, 195-202[CrossRef][Medline] [Order article via Infotrieve] |
33. | Le Bivic, A., Sambuy, Y., Mostov, K., and Rodriguez-Boulan, E. (1990) J. Cell Biol. 110, 1533-1539[Abstract] |
34. | Lisanti, M. P., Le, Bivic, A., Saltiel, A. R., and Rodriguez-Boulan, E. (1990) J. Membr. Biol. 113, 155-167[Medline] [Order article via Infotrieve] |
35. |
Okazaki, Y.,
Ohno, H.,
Ochiai, T.,
and Saito, T.
(2000)
J. Biol. Chem.
275,
35751-35758 |
36. |
Jolly, C.,
and Morimoto, R. I.
(2000)
J. Natl. Cancer Inst.
92,
1564-1572 |
37. | Botzler, C., Issels, R. D., and Multhoff, G. (1996) Cancer Immunol. Immunother. 43, 226-230[CrossRef][Medline] [Order article via Infotrieve] |
38. | Botzler, C., Li, G., Issels, R. D., and Muthoff, G. (1998) Cell Stress Chaperones 3, 6-11[Medline] [Order article via Infotrieve] |
39. | Botzler, C., Schmidt, J., Luz, A., Jennen, L., Issels, R., and Multhoff, G. (1998) Int. J. Cancer 77, 942-948[CrossRef][Medline] [Order article via Infotrieve] |
40. | Multhoff, G., Botzler, C., Wiesnet, M., Muller, E., Meier, T., Wilmanns, W., and Issels, R. D. (1995) Int. J. Cancer 61, 272-279[Medline] [Order article via Infotrieve] |
41. | Multhoff, G., Botzler, C., Jennen, L., Schmidt, J., Ellwart, J., and Issels, R. (1997) J. Immunol. 158, 4341-4350[Abstract] |
42. | Asea, A., Kraeft, S. K., Kurt-Jones, E. A., Stevenson, M. A., Chen, L. B., Finberg, R. W., Koo, G. C., and Calderwood, S. K. (2000) Nat. Med. 6, 435-442[CrossRef][Medline] [Order article via Infotrieve] |
43. | Di Cesare, S., Poccia, F., Mastino, A., and Colizzi, V. (1992) Immunology 76, 341-343[Medline] [Order article via Infotrieve] |
44. | Hantschel, M., Pfister, K., Jordan, A., Scholz, R., Andreesen, R., Schmitz, G., Schmetzer, H., Hiddemann, W., and Multhoff, G. (2000) Cell Stress Chaperones 5, 438-442[Medline] [Order article via Infotrieve] |
45. | Ferrarini, M., Heltai, S., Zocchi, M., and Rugarli, C. (1992) Int. J. Cancer 51, 613-619[Medline] [Order article via Infotrieve] |
46. | Poccia, F., Piselli, P., Vendetti, S., Bach, S., Amendola, A., Placido, R., and Colizzi, V. (1996) Immunology 88, 6-12[Medline] [Order article via Infotrieve] |
47. | Ishiyama, T., Koike, M., Akimoto, Y., Fukuchi, K., Watanabe, K., Yoshida, M., Wakabayashi, Y., and Tsuruoka, N. (1996) Clin. Exp. Immunol. 106, 351-356[Medline] [Order article via Infotrieve] |
48. | Sapozhnikov, A. M., Gusarova, G. A., Ponomarev, E. D., and Telford, W. G. (2002) Cell Prolif. 35, 193-206[CrossRef][Medline] [Order article via Infotrieve] |
49. | Harada, M., Kimura, G., and Nomoto, K. (1998) Biotherapy 10, 229-235[CrossRef][Medline] [Order article via Infotrieve] |
50. | Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H., Steele-Mortimer, O., Paiement, J., Bergeron, J. J., and Desjardins, M. (2002) Cell 110, 119-131[Medline] [Order article via Infotrieve] |
51. |
Ohashi, K.,
Burkart, V.,
Flohe, S.,
and Kolb, H.
(2000)
J. Immunol.
164,
558-561 |
52. |
Vabulas, R. M.,
Braedel, S.,
Hilf, N.,
Singh-Jasuja, H.,
Herter, S.,
Ahmad-Nejad, P.,
Kirschning, C. J., Da,
Costa, C.,
Rammensee, H. G.,
Wagner, H.,
and Schild, H.
(2002)
J. Biol. Chem.
277,
20847-20853 |
53. |
Vabulas, R. M.,
Ahmad-Nejad, P.,
Ghose, S.,
Kirschning, C. J.,
Issels, R. D.,
and Wagner, H.
(2002)
J. Biol. Chem.
277,
15107-15112 |
54. | Triantafilou, M., and Triantafilou, K. (2002) Trends Immunol. 23, 301-304[CrossRef][Medline] [Order article via Infotrieve] |
55. | Chang, H. C., Samaniego, F., Nair, B. C., Buonaguro, L., and Ensoli, B. (1997) AIDS 11, 1421-1431[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Mehul, B.,
and Hughes, R. C.
(1997)
J. Cell Sci.
110,
1169-1178 |
57. | Mignatti, P., Morimoto, T., and Rifkin, D. B. (1992) J. Cell. Physiol. 151, 81-93[Medline] [Order article via Infotrieve] |
58. | Rubartelli, A., and Sitia, R. (1991) Biochem. Soc. Trans. 19, 255-259[Medline] [Order article via Infotrieve] |
59. |
Rubartelli, A.,
Bajetto, A.,
Allavena, G.,
Wollman, E.,
and Sitia, R.
(1992)
J. Biol. Chem.
267,
24161-24164 |
60. |
Tanudji, M.,
Hevi, S.,
and Chuck, S. L.
(2002)
J. Cell Sci.
115,
3849-3857 |
61. |
Andrei, C.,
Dazzi, C.,
Lotti, L.,
and Torrisi, M. R.
(1999)
Mol. Biol. Cell
10,
1463-1475 |
62. |
Florkiewicz, R. Z.,
Anchin, J.,
and Baird, A.
(1998)
J. Biol. Chem.
273,
544-551 |
63. | Hughes, R. C. (1999) Biochim. Biophys. Acta 1473, 172-185[Medline] [Order article via Infotrieve] |
64. | Liu, H., Lin, D., and Yates, J. R. (2002) BioTechniques 32, 898-911[Medline] [Order article via Infotrieve] |