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J. Biol. Chem., Vol. 277, Issue 39, 36363-36372, September 27, 2002
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From the Departments of
Received for publication, March 25, 2002, and in revised form, June 13, 2002
The Limulus Factor C (FC), a
multidomain glycoprotein that binds bacterial endotoxin with high
affinity, belongs to the serine protease family of the complement and
blood coagulation cascade. Here, we provide compelling evidence for the
importance of modular arrangement and relevance of the proline-rich
region (PRR) and N-glycosylation to the secretion and
function of FC. We propose that PRR could be a universal conformational
domain that regulates protein folding and targeting. FCs lacking PRR
preceding the serine protease domain, were localized intracellularly.
Misfolded conformers of the intracellular FCs were more susceptible to
trypsin digestion. Glycosylation inhibition studies indicate that the
presence but not the exact structure of the N-glycans
affects the secretion of FC, although the complexity of glycosylation
may influence its endotoxin-induced proteolytic cleavage with resultant
enzymatic activity. Disruption of specific N-glycan sites
at positions 740, 767, and 912, downstream of the PRR, at or near the
serine protease domain, blocks its secretion. Co-expressed molecular
chaperones like canine calnexin associates with glycosylated FCs to
increase its solubility and secretion level but did not alter their
expression profiles. Our results clearly demonstrate that the folding
and secretion of a multidomain serine protease like FC are determined by its modular domain arrangement and site-specific
N-glycans. The secreted FCs containing the N-terminal
portion of FC are able to detect lipopolysaccharide with high
sensitivity. We also identified the lectin-like and sushi 4 domains to
contribute to the binding of lipopolysaccharide.
The availability of engineered serine proteases allows the study
of their activation, substrate specificity, and regulation of various
physiological processes. As the initiator of the coagulation cascade of
limulus amoebocyte lysate
(LAL),1 Factor C (FC) is a
multidomain serine protease, which is activated by a trace amount of
its ligand, endotoxin (lipopolysaccharide; LPS) (1). LAL is widely
employed to detect the presence of LPS in various industrial and
clinical products (2). Analysis of the amino acid sequence indicates
that FC is a complex modular protein, comprising 11 domains (see Ref. 3
and Fig. 1A). Many earlier attempts to express FC in
heterologous hosts have faced limited success (4-7). The native signal
peptide of FC directs this molecule to intracellular large granules of
the limulus amoebocytes, and it is unable to secrete the protein out of
heterologous expression hosts. Instead, it may serve as a membrane
anchor and invariably result in membrane-bound recombinant protein.
Thus, an alternative signal peptide is needed to obtain secreted FC for
investigation of its domain-function relationship. Using a cross-host
secretory signal (8), which leads to the secretion of various
recombinant proteins in both prokaryotic and eukaryotic systems, we
have recently produced full-length and biologically functional domains
of FC that binds LPS (9, 10).
The LPS binding and enzymatic activity of Factor C are functionally
distinct and are localized in the N-terminal sushi (ES) and C-terminal
serine protease domains (SP), respectively (10, 11). However, the
importance of interdomain interactions for expression and biological
activity of FC is hitherto unknown. Of particular interest is the
proline-rich region (PRR) of the FC protein. Proline residues
contribute significantly to the conformation and rigidity of the
protein structure, and PRR serves as a molecular hinge in a number of
proteins (12-14), affecting protein-protein interaction and
protein-ligand binding. In this study, we focus on the potential
involvement of PRR in FC secretion and function. By systematic design
of FC deletion homologues with and without PRR, we are able to examine
their expression profile, post-translational modifications,
localization, and LPS binding activity.
N-Linked glycans play pivotal roles in protein folding,
oligomerization, quality control, sorting, and transport (15). These sugar chains are extensively involved in protein conformation and
function (16). There are six potential N-glycosylation sites in FC at positions 523, 534, 624, 740, 767, and 912 (Fig. 1) (17). However, the functions of these glycans has not been examined. We used
glycosylation inhibition and site-directed mutagenesis to test the role
of N-glycans on expression and secretion of FC and to map
the most critical N-glycans. Further assessment on the
involvement of N-glycans in the secretion pathway was
performed by co-expression of FC with various molecular chaperones,
which are proteins that assist in the correct noncovalent assembly of other polypeptide-containing structures (18).
We report for the first time that expression and secretion of such a
multidomain serine protease requires the proper modular arrangement of
the various domains, and PRR is essential for proper folding of the
downstream SP domain. In addition, we demonstrated the importance of
site-specific N-glycosylation in FC secretion and provided
evidence that N-glycans interact with chaperones and hence
manifest their effect on FC secretion. Interestingly, despite having
proper protein folding, the secreted FC exhibited LPS binding
but lacked enzymatic activity. This loss of LPS-induced enzymatic
activity can be attributed to different complexity in glycosylation.
Unless otherwise stated, all reagents were of molecular biology
grade, purchased from Sigma.
Construction of Expression Plasmids
Drosophila expression vector, pAc5.1/V5-HisA
(Invitrogen) was used for fusion expression. The native signal peptide
sequence of full-length (FL) FC cDNA was replaced by a novel
secretion signal to direct the fusion protein out of the
Drosophila cells. Seven expression plasmids were
constructed, with various combinations of FC domains inserted
downstream of secretory signal and upstream of V5 and His6
fusion tag: FL, FL( Site-directed mutagenesis of the N-glycan sites of FC was
performed using the QuikChangeTM site-directed mutagenesis
kit (Stratagene). Mutations were verified by sequencing.
Transient and Stable Transfections
Plasmids were isolated using CsCl-ethidium bromide gradient
ultracentrifugation. Drosophila S2 cells were maintained at
25 °C in DESTM medium supplemented with 10% fetal
bovine serum (CLONTECH). Calcium phosphate-mediated
transient transfection was performed according to the manufacturer's
recommendation (Invitrogen). For stable transfection, pCoHYGRO
selection plasmid was cotransfected with Factor C expression construct,
selected with 350 µg/ml hygromycin for 4-6 weeks, and adapted to
DESTM serum-free medium.
For chaperone co-expression studies, Cellfectin-mediated
(Invitrogen) transfection was performed with DNA mixture
containing 1 µg each of FC and chaperone expression construct.
Control transfections were performed with FC expression construct and
parental pAc5.1/V5-HisA DNA instead of chaperone expression constructs.
Partial Tryptic Digestion of FC
Two µl of trypsin solution (100 ng/µl) in 50 mM
acetic acid was added into 200 µg of FC in 100 µl of 50 mM NH4HCO3. The digestion was
carried out at 4 °C. At time intervals, 10 µl of the reaction mixture was removed and terminated by 4 µl of the 4× SDS sample buffer. Western analysis was performed on the digested samples using
polyclonal rabbit anti-FC antibody.
In Vivo Glycosylation Inhibition and ConA-Sepharose Binding
Assay
Tunicamycin (TM) inhibits N-glycosylation of
glycoproteins by blocking the first step in N-glycan
biosynthesis, whereas Immunoprecipitation and Analysis of FC and Chaperone
Association
S2 cells co-transfected with FC and chaperone expression
plasmids were collected on day 5 post-transfection and washed twice with 1.0 ml of PBS. Cells were resuspended in 500 µl of lysis buffer
(0.1× PBS, 1.0 mM phenylmethylsulfonyl fluoride, 1.0 µM aprotinin), sheared seven times through a 27-gauge
needle, and centrifuged at 14,000 × g for 15 min.
Supernatant containing the cell lysate (L) proteins was retrieved. The
pelleted membranes (P) were solubilized in 500 µl of lysis buffer
containing 1% digitonin for 30 min at 4 °C with occasional
agitation. The samples were clarified at 14,000 × g
for 15 min to separate aggregates from solubilized proteins, which were
used for subsequent immunoprecipitation.
Fifty µl of protein A-Sepharose (Amersham Biosciences) was incubated
with 50 µl of PBS containing 2 µl of anti-V5 antibody for 1 h
at 4 °C. After incubation, 100 µl of solubilized protein samples
was immunoprecipitated at 4 °C overnight on a rotator. The samples
were centrifuged and washed three times with cold PBS. Forty µl of
SDS-PAGE loading buffer was added to each tube and boiled for 5 min
before Western analysis.
Concentration and Partial Purification of FC
Culture medium containing secreted FC was collected
after centrifugation of cell culture at 3000 × g for
10 min at 4 °C. Medium was concentrated 10-fold by ultrafiltration
using a regenerated cellulose membrane cassette (Pellicon XL; 30-kDa
molecular mass cut-off; Millipore Corp.) at 4 °C. Concentrated
protein sample was chromatographed on a Sephadex G-100 (1.5 × 90-cm) column. Fractions containing partially purified FC were pooled
and concentrated using Centriprep-regenerated cellulose membrane
(30-kDa molecular mass cut-off; Amicon YM).
In Vitro Endoglycosidase Digestion and Glycan Differentiation
10 µg each of partially purified secreted FC from
Sf9-BEVS and S2 was denatured in 5% SDS, 10%
For the glycan differentiation assay, FCs from LAL, CAL,
Sf9-BEVS, and S2 were purified by protein A-FC antibody affinity chromatography. Fifty µl of protein A-Sepharose (Amersham
Biosciences) was incubated with 50 µl of PBS containing 10 µl of
anti-FC antibody for 1 h at 4 °C. 100 µg of FC samples were
then added to the protein A-Sepharose complex and incubated at 4 °C
overnight on a rotator. The samples were centrifuged and washed three
times with PBS, and FC was recovered from protein A-Sepharose by
boiling for 10 min in PBS. Lectin binding assay of the
immunoaffinity-purified FCs was carried out using the DIG glycan
differentiation kit (Roche Molecular Biochemicals), according to the
manufacturer's instructions.
Functional Analyses of FCs: Endotoxin Binding Assay
The LPS binding potential of various FC proteins were analyzed
using (a) LPS ELISA-based assay and (b) surface
plasmon resonance (SPR).
ELISA-based Assay for LPS Binding--
This assay was
performed as described by Tan et al. (10) with some
modifications. Coating of a polysorp 96-well plate (NUNC) was achieved
by incubation at 37 °C for 2 h, and blocking was performed
overnight at 25 °C. 10 µg of protein sample was added into each
well and incubated for 16 h at 25 °C. Bound FCs were detected
by sequential incubation with primary rabbit anti-FC antibody (1:500
dilution, 3 h at 37 °C) and goat anti-rabbit secondary antibody
(1:2000 dilution) conjugated to horseradish peroxidase (Dako).
SPR to Determine the Kd of FC for Lipid
A--
Anti-V5 antibody at 20 µg/ml was immobilized to CM5 chip
(Amersham Biosciences) according to the manufacturer's specifications. A low capacity coating for kinetic study was achieved as indicated by
an increase of 1418 resonance units. FC samples were injected at 10 µl/min over the anti-V5 antibody surface, resulting in capture of 5.0 pmol of FC (determined by densitometric scanning using pure ES as
standard and normalized based on their peptide size) from the
conditioned medium. Subsequently, lipid A at five different concentrations was injected at 20 µl/min to examine interaction between FC and lipid A. The affinity constant was calculated using BIAevaluation program (version 3.0.2) and reconfirmed using CLAMP (21).
For regeneration, 100 mM NaOH was used. Values represent the mean of three independent experiments.
PRR Is Critical for the Correct Folding and Secretion of Downstream
SP Domain--
Seven expression plasmids were transfected into S2
cells. Immunoblot analysis shows that all the stable S2 transfectants
expressed FCs, albeit displaying different amounts and expression
profiles (Fig. 1A).
Interestingly, although subcloned downstream of a potent secretion
signal, not all of the FC proteins were secreted, suggesting that the
localization of FCs might be signal sequence-independent. We observed
that the FL could be detected in the culture medium. However, the
deletion of PRR from FL, yielding FL(
A major difference between secreted and cellular FCs that harbor SP was
that the secreted FCs (FL and PRR·SP) have the PRR domain localized
upstream of SP, without which SP is not secreted into the medium. To
verify the importance of this modular arrangement between PRR and SP,
the PRR was subcloned C-terminal to SP (SP·PRR). The PRR domain in
this reciprocal construct was not able to rescue the intracellular
retention of SP·PRR. Taken together, the PRR region of FC is
important for correct folding of the downstream SP domain and that
intracellularly localized FCs might be misfolded.
To shed more light on the role of PRR in folding of the downstream SP
domain, partial tryptic digestion followed by Western analysis was
carried out on two pairs of FCs: (a) FL and FL(-PRR) and
(b) PRR·SP and SP. Over the observed time, distinct
protein fragments of FL (132, 80, 52, and 38 kDa) and fragments of
PRR·SP (56, 52, and 38 kDa) were clearly detectable by anti-FC
antibody (Fig. 1B), suggesting that they were properly
folded into a series of individual compact domains, which are
relatively trypsin-resistant. It is interesting to note that two of the
tryptic fragments, 80 and 52 kDa, corresponded to LPS-activated
autocleavage of Factor C. Importantly, both PRR-deficient mutants,
FL(
Clearly, PRR is important for the folding and, hence, secretion of the
serine protease. Essentially, PRR can only exert its effect when the
serine protease domain is localized downstream of it.
N-Glycosylation Is Required for Proper Post-translational
Processing--
We also observed a differential dependence of the two
functional domains (viz. LPS-binding and serine protease
domains) on PRR for folding and secretion (Fig. 1). The secretion
competence of LPS-binding ES is unaffected by PRR, which is in contrast
to the SP. A distinguishing feature of ES is that it lacks
N-glycosylation sites compared with SP or other SP-harboring
FCs, which contain multiple N-glycosylation sites. Thus, we
next investigated the significance of N-glycosylation on the
expression and secretion of FC.
The secreted FL protein is glycosylated and is able to bind to
ConA-Sepharose (data not shown). FL proteins from S2 cells treated with
TM show progressively decreasing size based on their mobility (Fig.
2A), suggesting a loss of
N-linked sugar chains. Aglycosylated FL was detected only in
the L and P fractions, which clearly indicates that loss of
N-glycosylation inhibited FL secretion. The absence of
N-glycosylation of FL after TM treatment is also confirmed
by its inability to bind ConA-Sepharose (Fig. 2B). In contrast to the effects of TM on FC secretion, cells treated with DNJ
and DMJ exhibited no difference in expression level or distribution pattern of FCs (data not shown). Thus, the conversion of high mannose
oligosaccharide to complex oligosaccharide is dispensable for protein
secretion.
To determine the critical N-glycan sites, a series of
glycomutations were introduced (Table I).
The position of PRR is such that it partitioned the FL into the
N-terminal LPS-binding and the C-terminal serine protease regions.
Thus, we decided to mutate the N-glycosylation sites
sequentially from each terminal. Single disruption of any one of the
six N-glycan sites has no observable impact on the FC
secretion (Fig. 2C, Series I).
However, the elimination of two sites, particularly N523Q/N534Q
and N767Q/N912Q, impaired the expression of FL (Fig. 2C,
lanes 1 and 5 of Series
II). Although mutation of the three consecutive
N-glycan sites located at the N terminus caused drastic
reduction of the expression of FC, this mutant FL is still secreted
into the culture medium (Fig. 2C, lane
1 of Series III). In contrast and
importantly, the mutation of the three consecutive C-terminal
N-glycan sites located downstream of PRR at/near the SP
domain totally abolished the secretion (Fig. 2C,
lane 2 of Series III). The
removal of all six glycosylation sites (i.e. aglycosylated
FL) is not secretion-competent (Fig. 2C, Series
IV). Thus, in addition to PRR, the
N-glycosylation sites in the SP are also critical for
protein expression and secretion. We next sought to assess the
contribution of N-glycans to secretion competence and also
to determine a possible functional dependence on PRR.
N-Glycans Interact with Molecular Chaperones to Enhance Solubility
and Secretion of FC--
To further examine the involvement of
N-glycans in the secretion pathway, co-expression study of
FC with two molecular chaperones was carried out. cCNX and hCRT were
expressed in S2 cells and their expected protein sizes and cellular
localization were detected with their respective antibodies. The
expression levels of the cCNX and hCRT in S2 were also comparable (Fig.
2D).
The overall expression level of FL, as determined by densitometric
scanning of the immunoblot, increased over time, and peaked between 5 and 7 days (data not shown). cCNX caused a 3-fold increase in the level
of secreted FL, whereas hCRT minimally enhanced FL expression. Next,
the effects of chaperone on the expression level of cellular FCs
lacking PRR, FL( Specific Glycosylation Modification Is Required for FC Enzymatic
Activity--
In addition to its contribution to protein solubility,
N-glycans have been reported to influence protein functions
(16). Since glycosylation is the only post-translational modification known so far in FCs, we attempted to precisely investigate the extent
and type of glycosylation in FC that can alter LPS-induced autocleavage
and its enzymatic activity. Sf9-BEVS FC was previously demonstrated to exhibit LPS-induced cleavage and enzymatic activity (22).
Endoglycosidase H and peptide:N-glycanase F digestion
analyses show the presence of both high mannose and complex glycans on
S2 FC and Sf9-BEVS FC. Differences in their mobility indicate that Sf9-BEVS FC contains 12 kDa of complex N-glycans
and S2 FC harbors 8 kDa of complex N-glycans (Fig.
3A). Thus, it appears that S2
FC is less glycolytically processed. Galanthus nivalis agglutinin treatment reveals the presence of high mannose
glycans in all four FCs. Sambucus nigra agglutinin treatment
yielded positive results for FCs from LAL, CAL, and Sf9-BEVS and
only weakly reacted with S2 FC. Interestingly, reaction with
Maackia amurensis agglutinin yielded the strongest signal
with S2 FC, pointing to a difference in the branching of terminal
sialic acids. S2 FC contain greater sialic acid linked
LPS activation of the FC expressed in the Sf9-baculovirus
expression system was cleaved into two protein fragments of 80 and 52 kDa (Fig. 3C), with resultant enzymatic activity (22). In contrast, attempts at measuring LPS-inducible enzymatic activity of S2
FC showed that it does not autocleave and exhibited no enzymatic activity. The above mentioned host cell-specific complexity of glycosylation of the FCs offers plausible explanations for this lack of
cleavage and enzymatic activity.
Secreted FL and ES·PRR Retained Their LPS Binding
Activities--
Secreted S2 FCs were tested for their LPS binding
efficacy using both ELISA and SPR. Using gel filtration-purified FCs,
significant lipid A binding activity of FL and ES·PRR was observed,
as compared with the partially purified conditioned serum-free medium
from wild-type S2 cells. Both secreted FCs were capable of detecting as
low as 0.05 µg/ml LPS (Fig.
4A). The secreted PRR·SP was
unable to detect LPS, indicating that there is no LPS-binding domain in
this molecule. The Kd values of FL and ES·PRR for binding to E. coli lipid A was determined using SPR. Fig.
4B shows a sensorgram of lipid A binding to FL. The
Kd values were 2.23 × 10 In this study, we have identified two important determinants of
protein secretion of a mosaic serine protease: the PRR and certain
strategically located N-glycans. PRR is necessary for proper
folding of the downstream SP domain. The N-glycans,
particularly those residing downstream of PRR, at or near the SP
domain, contribute to stable interaction with specific chaperone(s) and
consequently increase the solubility of FCs. Although both factors are
involved in different aspects of protein expression, they are equally
critical for the maturation and secretion of the protein (Fig.
5). We also identified two potential
domains that contribute to the LPS binding activity of FC. The absence
of the lectin-like and sushi 4 domain in SP·PRR leads to an apparent
decrease in affinity for LPS by 4-fold compared with FL, indicating
that these two domains could either affect the conformation of the
upstream LPS binding domains or directly contribute to the binding of
LPS. Finally, we provide evidence that the N-glycans are
dispensable for LPS binding but that the differential complexity in
glycosylation is critical for LPS-induced cleavage and enzymatic
activity.
PRR Governs the Folding of Downstream SP Domain--
The
significance of modular arrangement to the structural integrity and
functionality of multidomain proteins such as Factor XII and FC may
provide us with important insights into their regulation, activation,
and substrate specificity. The availability of secreted recombinant
proteases in a heterologous system is usually preferable for ease of
subsequent purification and functional studies. Since the secretion of
a protein relies not only on its signal peptide but also on the
intrinsic amino acid sequence that determines the final tertiary
structure of the mature protein, we examined the secretion of FC
constructs containing different modular domains in their native and
reciprocal positions.
In the middle of the FC molecule is the basic PRR (amino acids
641QNPPVPSYGSVEIKPPSRTNSISRVGSPFLRLPRLPLPLARAAKPPPKPR690),
which contains three Src homology 3 binding core motifs (23), PXXP (underlined; P denotes proline). Src
homology 3 domains affect protein targeting and enzymatic activity and
play a role in signal transduction (24). It is possible that the PRR of Factor C is involved in transducing LPS-induced activation of the
serine protease enzymatic activity as well as contributing to the
LPS-dependent multiactivation of Factor C via
protein-protein interaction (25). In addition, the PRR of Factor C
shares homology with the hinge region of human coagulation Factor XII
(26). Hence, it is possible that the PRR acts as a crucial
conformational motif necessary for the secretion and function of such
serine proteases.
In this study, we constructed and expressed a pair of FCs (FL and
FL(
The differential susceptibility to trypsin clearly
demonstrates the importance of PRR in the correct folding of the
downstream SP region and in directing the secretion of glycosylated FC
(Fig. 1B). Trypsin acts preferentially at the unfolded
regions in proteins, such as the domain boundaries of a mosaic protein.
It is worth noting that on the basis of sequence specificity alone,
there are close to 100 and 50 potential trypsin cleavage sites for FL and PRR·SP, respectively. However, only three or four characteristic bands, representing two sets of relatively protease-resistant fragments, were detected after partial tryptic digestion. Therefore, the stable fragments are attributable to the compactly folded individual domains. The 38-kDa stable fragment observed after digestion
of both FL and PRR·SP could be the properly folded SP domain. On the
contrary, PRR-deficient mutants, FL(
The crystal structure of a catalytic fragment from human C1s, which
shares high similarity to Factor C modular architecture, has been
determined (28). The CCP2 module (sushi domain) of C1s is oriented
perpendicularly to the surface of the SP domain, and the connection is
maintained through a rigid module-domain interface involving
intertwined proline- and tyrosine-rich polypeptide segments (28). Since
the residues maintaining the interface framework are highly conserved
in the sushi-SP family, it is believed that the rigid sushi-SP assembly
is also present in Factor C (29). Consistent with the role of a
module-domain interface, our data show that the absence of PRR changes
the sushi-SP assembly, resulting in misfolding of the deletion
homologues, rendering them nonsecretory.
Many other studies have documented the importance of PRR to the
biological functions of different proteins, and that its removal adversely affects the localization and activity of such proteins (30-32). Thus, rather than being restricted to a small subset
of serine proteases, the PRR could be a universal conformational motif
that regulates protein targeting.
N-Glycans Contribute to the Interaction with Chaperones and
Solubility of the Recombinant Proteins--
Whereas the data above
clearly demonstrate that PRR is an important secretory determinant of
FC, PRR also exerts a preferential effect on the serine protease
domain, SP, and not on the LPS-binding ES domain. A distinctive
characteristic is that ES does not harbor any
N-glycosylation sites. Using specific glycosylation
inhibitors and site-directed mutagenesis, we demonstrate that
inhibition of glycosylation blocked the secretion of FL. The
aglycosylated FL became intracellularly localized although their
corresponding PRR was present, thus indicating that
N-glycosylation is also critical for
PRR-dependent secretion (Fig. 5). The importance of
N-glycans for the secretion of FC was recapitulated using
site-directed mutagenesis of the N-glycosylation sites.
Interestingly, the ablation of the three N-glycan sites at
the C terminus at positions Asn740, Asn767, and
Asn912 completely abolished secretion. Coincidentally,
these three sites reside downstream of PRR at or near the SP domain.
The partial dependence of FC secretion on N-linked
oligosaccharide appendices suggests that the individual
N-glycans do not have local effects on protein folding.
However, when the N-glycosylation sites at positions 740, 767, and 912 are modified at the same time, folding and secretion of FC
are compromised although none of the glycans are needed individually.
In view of this observation, the oligosaccharides of FC seem to have a
global effect on the folding/secretion process.
These data suggest that correct folding of the SP domain is the most
critical for secretion of FC; the absence of PRR preceding this domain
or the absence of N-glycans within this domain leads to
misfolding, which impairs FC secretion.
The mechanism by which N-glycans aid in FC secretion is
attributable to its ability to interact with chaperones. Overexpression of chaperones has been proven to improve protein folding and stimulate secretion of certain target proteins (34-37). In this study, the secretion of FL and solubility of the cellular glycosylated FCs (FL(
Functional FC should display both LPS binding activity and
LPS-activated enzymatic activity. However, these two properties were
uncoupled in S2-produced FC. S2 FC binds LPS, but it is not cleaved and
not enzymatically activated. This is in contrast to Sf9-BEVS FC,
which we demonstrated previously to harbor both functions (22). Further
analyses reveal that the loss/gain of enzymatic activity stems from
subtle differences in the complexity of glycosylation of FC. In
agreement with our results, over the last decade, various reports
(38-41) have described that viral infection of Sf9 cells temporally effects protein glycosylation (i.e. the
activation of glycosyltransferase genes takes place under proper
conditions, depending on the virus type, promoter, and medium). Normal
glycosylation characteristics of insect cells were thus altered to
allow complex type oligosaccharide processing to occur. Thus, during
baculoviral infection of Sf9 cells for expression of FC, the
viral infection process affects the glycosylation of FC, consequently
allowing its conformational change upon LPS binding, leading to limited proteolysis and activation of the serine protease enzyme. In contrast, being nonvirally infected, the S2 FC bears differently processed glycans, especially those at/near the catalytic domain. Thus, due to
the nonnative branching of the adjacent N-glycans, the S2 FC
is not proteolytically cleaved, although it possesses LPS binding
properties. This is despite the correct overall protein folding and
successful passage of the S2 FC through the quality control of the ER
and Golgi, which allows its successful secretion. However, since FC is
such a demanding glycoprotein, retaining the LPS binding activity in
these secreted FCs does not satisfy the requirement for their
conformational change induced by LPS binding to yield enzymatic
activity. This reveals another surprising impact of
N-glycans on FC functions. Although differences in
recombinant protein localization (42) and N-glycosylation
heterogeneity (43) have been reported in these two insect cell
expression systems, FC from these two hosts is so far the first
recombinant protein to exhibit such marked functional difference.
Future understanding and manipulations in the post-translational
processing pathway with exoglycosidase/glycotransferases could possibly
resolve this problem faced by FC as well as other serine proteases.
In summary, we confirm that PRR of a serine protease is essential for
the folding and secretion of the downstream catalytic domain. These
results suggest that the PRR serves a simple but essential role in
tethering the structurally distinct but interacting domains of the
protein. In addition, the secretion ability of FCs was impaired in the
absence of N-glycans, especially those residing at or near
the catalytic domain. We also report for the first time that
co-expression of chaperones significantly increases the soluble and
secreted recombinant protein level in S2 cells but has no appreciable
effect on the localization of the protein. These results help to
elucidate the function of different structural domains of Factor C and
how the intrinsic properties of various domains of such a mosaic
protein influence its destination (illustrated in Fig. 5). This general
concept of the modular arrangement may be applicable to other members
of the multidomain protein family, such as blood coagulation Factor
XII, in particular the influence of domain interactions on their cell
sorting, secretion, and function. Finally, PRR is therefore a potential
universal conformational domain that regulates protein folding and
secretion, and it is probably applicable as domain fusion junctions for
engineered chimeric multidomain proteins expressed in eukaryotic
expression systems.
We thank Prof. D. Williams (University of
Toronto) and Prof. D. Llewellyn (University of Wales College of
Medicine) for kindly providing the cDNAs of canine calnexin and
human calreticulin.
*
This work was supported by National Science and Technology
Board Grant NSTB/LS/99/004.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.
§
A postgraduate research scholar of the National University of Singapore.
¶
Both authors contributed equally to this work.
Published, JBC Papers in Press, June 27, 2002, DOI 10.1074/jbc.M202837200
The abbreviations used are:
LAL, Limulus
polyphemus amoebocyte lysate;
FC, Factor C;
cCNX, canine calnexin;
hCRT, human calreticulin;
FL, full-length Factor C;
ES, Eco47III-SalI fragment of Factor C cDNA;
SP, serine protease;
PRR, proline-rich region;
TM, tunicamycin;
DMJ,
Modular Arrangement and Secretion of a Multidomain
Serine Protease
EVIDENCE FOR INVOLVEMENT OF PROLINE-RICH REGION AND
N-GLYCANS IN THE SECRETION PATHWAY*
§¶,
,
Biological Sciences and
** Microbiology, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PRR), ES·PRR, ES, PRR·SP, SP, and SP·PRR.
Fig. 1 depicts the domain structures of these recombinant constructs.
Among the seven constructs, FL, ES·PRR, PRR·SP, and SP·PRR
contain PRR. The remaining three constructs (FL(PRR), ES, and SP)
lack PRR. Only ES, which contains the N-terminal LPS-binding region,
lacks both PRR and N-glycan sites. Details on cloning strategies and secretory signal sequence are available upon request. Canine calnexin (cCNX) (19) and human calreticulin (hCRT) (20) cDNAs were subcloned into pAc5.1/V5-HisA.
-deoxynojirimycin (DNJ) and
-deoxymannojirimycin (DMJ) specifically inhibit -glucosidase and
-mannosidase activities, respectively, thus blocking the conversion
of high mannose oligosaccharides to complex-type oligosaccharides. S2
cells stably expressing FC were exposed to sublethal doses of TM (5 µ g/ml), DNJ (5 mM), and DMJ (2 mM). Proteins
from treated cells were collected at 24 h and subjected to
affinity chromatography on ConA-Sepharose (Amersham Biosciences).
Conditioned medium containing 10 µg/ml secreted FC and cell lysate
protein containing 1 µg/µl cellular FC were incubated with
one-fifth volume of ConA-Sepharose at 25 °C for 1.5 h. The
unbound fraction containing aglycosylated FC was recovered by
centrifugation at 2000 × g for 5 min. The resin-bound
glycosylated FC was dissociated via boiling for 5 min in 3 resin
volumes of SDS-PAGE loading buffer. Both fractions were subjected to
Western analysis.
-mercaptoethanol for 10 min at 100 °C and subjected to
endoglycosidase H and peptide:N-glycanase F (New
England Biolabs) digestion at 37 °C for 16 h, followed by
Western analysis using anti-FC antibody. Control samples were treated
in the same way except in the absence of enzyme.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PRR), is not secreted (Fig.
1A, constructs 1 and 2).
This indicates that PRR is important for the secretion competency of
FCs. We next investigated the relative contribution of PRR to the
functionally distinct LPS-binding and serine protease domains, using
paired truncated FCs (ES·PRR versus ES and PRR·SP
versus SP) containing or lacking the PRR (Fig.
1A). Neither the preservation nor the removal of PRR affects
the secretion competency of the LPS-binding domain, ES (ES·PRR and
ES; Fig. 1A, constructs 3 and
4). Similarly, when PRR was localized upstream of SP
(PRR·SP), as in the native FC, the PRR·SP protein was easily
detectable in the medium. Interestingly, when the PRR was deleted, the
serine protease domain alone (SP) is no longer detectable in the
culture medium (Fig. 1A, constructs 5 and 6). This clearly indicates that PRR is critical for the secretion of the serine protease domain.
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Fig. 1.
Domain structure, targeting, localization,
and folding of FCs. A, domain structure of the
full-length Factor C and the deletion homologues. The recombinant
fusion sites of different domains are indicated by upward
arrows with the amino acid numbering in the original
Carcinoscorpius rotundicauda FC21 (GenBankTM
accession no. S77063). The glycosylation sites are marked with
green closed diamonds. Darkly
shaded area, secretory signal (ss);
CR, cysteine-rich region; horizontally
striped area, epidermal growth factor-like
domain; S1-5, sushi-like domains 1-5; LECTIN,
lectin-like domain; PRR, proline-rich region, in
red; SP, serine protease coding region;
diagonally striped region, V5 epitope;
vertically striped region, His6
tag. Targeting of the seven recombinant FCs was detected by
Western blot. M, 1.0 µg of culture medium containing
secreted proteins; L, 20 µg of cell lysates in PBS
containing cellular proteins were obtained by five cycles of
freeze-thawing of cells, followed by centrifugation at 12,000 × g for 15 min at 4 °C; P, 20 µg of insoluble
pellet obtained at this step was also collected, weighed, and
solubilized in 1× SDS-PAGE sample buffer. Protein concentration was
measured by Bradford assay. The relative distribution of FC proteins in
M, L, and P fractions was revealed
using anti-V5-horseradish peroxidase antibody (Invitrogen). Membranes
were visualized using the Supersignal® West Pico Chemiluminescent
Substrate (Pierce). The amount of FC was quantitated densitometrically
by using ImageMaster (Amersham Biosciences) software. Pure ES protein
(10) was used as standard. Band intensities within the linear range
were measured. B, partial proteolysis of FCs. FCs (FL,
FL(-PRR), PRR·SP, and SP) were partially digested with trypsin and
collected at time points as indicated. Digested samples were analyzed
by Western analysis using polyclonal rabbit anti-FC antibody. Distinct
stable protein fragments of FL (132, 80, 52, and 38 kDa) and PRR·SP
(56, 52, and 38 kDa) were detectable.
PRR) and SP, were rapidly digested by trypsin and were not
detectable by anti-FC antibody after 1 h. Two isoforms of SP, of
~38 kDa, were observed, of which the larger isoform was rapidly
digested by trypsin. Interestingly, the minor smaller isoform increased
slightly over the tested time period. This increase could be generated
from a small subpopulation of the larger isoform, and the smaller
isoform was relatively resistant to trypsin digestion. These
results show differences in protein folding between intracellular
PRR-deficient FCs and secretion-competent FCs. It is envisaged that
lack of PRR upstream of the SP domain tends to result in misfolded FCs,
which expose more accessible trypsin cleavage sites that render them
more susceptible to protease digestion.
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Fig. 2.
N-Glycans are required for proper
FC synthesis and targeting. A, immunoblot of FL from the M,
L, and P after treatment of S2 cells with TM. Western analyses used 10 µg of medium (M), 20 µg of lysate (L), and 20 µg of solubilized pellet (P). B, immunoblot
after ConA-Sepharose binding assay of FL from M and L. Protein samples
were collected from cells treated or not with TM for 48 h.
UB, unbound proteins; B, proteins bound to
ConA-Sepharose. C, immunoblots of FC samples from S2 cells
transfected with FC glycomutants. Cell culture supernatant was prepared
from S2 expressing wild type FL and FL glycomutants. Protein samples
were collected as described in legend of Fig. 1. 1.0 µg of medium was
resolved in a 8% gel by SDS-PAGE. All transfections were done in
parallel. The blot shown is representative of three independent
experiments. Immunoblotting was performed with anti-V5-horseradish
peroxidase antibody. D, expression level of FCs co-expressed
with molecular chaperones. Expression of cCNX (1) and hCRT
(2) in S2 cells is shown. S2 cells transfected with
pAc5.1/V5-HisA vector or chaperone expression constructs were collected
on day 5. Soluble lysate (L) and particulate (P)
fractions were resolved by SDS-PAGE and analyzed using respective
chaperone antibodies. Antibody for cCNX is from Transduction
Laboratories, and antibody for hCRT is from Stressgen. cCNX is detected
in the P fraction, and hCRT is detected in both the L and P fraction.
These expression profiles conform to their original nature, in that
cCNX is integrated into the ER membrane, whereas hCRT are ER luminal
proteins. E, comparison of secreted FL, ES, and cellular
FL( PRR), SP co-expressed with chaperones on day 5 post-transfection. Relative values calculated from the densitometric
data are shown, with the value for the S2 cells transfected with FC
construct alone arbitrarily set as 1. Error bars
represent the S.E. (n = 3). F,
left panel, direct interaction of glycosylated FC
with chaperones. Digitonin-solubilized cell lysates (L) from
S2 cells transfected with vector-pAc5.1/V5-HisA, cCNX alone, FL(PRR) + cCNX and SP + cCNX were precipitated overnight with anti-V5 antibody.
The immunoprecipitates were subjected to SDS-PAGE and probed with
anti-cCNX antibody. Right panel, association of
chaperone and FC under glycosylation-inhibited conditions. S2 cells
co-transfected with cCNX and SP (lanes 2-5) were
pretreated overnight with TM (5 µg/ml, lane 3),
DNJ (5 mM, lane 4), and DMJ (2 mM, lane 5) before transfection and
incubated with these inhibitors after transfection. FCs expressed were
then precipitated with anti-V5 antibody, and the chaperones that
associated with FC were analyzed by SDS-PAGE and immunoblotting.
Glycomutants of Factor C generated by site-directed mutagenesis
PRR) and SP, were examined. Although overexpression
of cCNX and hCRT enhanced the solubility of FL(PRR) and SP to
variable degrees (Fig. 2E), none of these chaperones was
able to rescue the nonsecretion of PRR-deficient FCs: FL(PRR) and SP.
The chaperones did not affect ES expression and secretion. This is not
surprising, since the LPS-binding ES domain is not glycosylated (Fig.
2E). Co-immunoprecipitation experiments showed that cCNX
co-precipitated with FL(PRR) and SP (Fig. 2F,
left panel), but no apparent interaction was
observed with hCRT (data not shown). Since hCRT consistently gives a
small increase in FC expression, it is conceivable that the association
between FC and hCRT is transient. Specific interaction between the
N-glycans of FC and cCNX was further confirmed using
N-glycosylation inhibitors, TM, DNJ, and DMJ. Consistent
with the above observation, treatment with TM and DNJ abolished the
interaction between FC and cCNX, whereas DMJ had no effect (Fig.
2F, right panel). This clearly indicates that the N-glycans on the FC molecules are
responsible for interaction with cCNX. In addition, cCNX probably
recognizes certain structures of FC N-glycans and binds
specifically to the deglucosylated form of FC. Taken together, the
results indicate that N-glycans are important for
interaction with cCNX, which increases the solubility of the FC
protein, and that its observed effect on protein secretion is secondary
to the lack of this interaction. Furthermore, the PRR region, although
critical for the correct conformation of the downstream serine protease
domain, is dispensable for the interaction with these chaperones.
(2-3) to
galactose, which is in contrast to Sf9-BEVS FC, LAL, and CAL.
The latter three FCs contain more sialic acid linked (2-6) to
galactose. In agreement with the endoglycosidase H digestion results,
Datura stramonium agglutinin yielded the weakest signal with
S2 FC, confirming that S2 FC is the most poorly processed among the
four FCs (Fig. 3B). Collectively, we have shown that the
glycan structures of FC expressed in S2 are different from FCs derived
from Sf9, CAL, and LAL. Such differences in glycomodifications can translate into disparity in protein functions.
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Fig. 3.
Comparison of the extent and complexity of
glycosylation in S2 FC and Sf9-BEVS FC. A,
endoglycosidase treatment revealed that Sf9-BEVS FC has more
complex N-glycans. The molecular mass shift after
endoglycosidase H (Endo H) treatment indicates the extent of
high mannose N-glycan chains, whereas the shift after
peptide:N-glycanase F (PNGase F) treatment
indicates total N-glycosylation (including both high mannose
and complex sugar chains). The size difference between
peptide:N-glycanase F-treated FC sample and FC TnT
(transcription-coupled translation) product is attributable to
O-glycosylation. B, glycan differential assay of
S2 FC and Sf9-BEVS FC. Lane 1, FC;
lane 2, FC without N-glycans. The
N-glycans were detached from FC peptide chain by treatment
with peptide:N-glycanase F, and the free glycan chains are
removed by centrifugation through a 30-kDa molecular mass cut-off
Microcon filter (Amicon). G. nivalis agglutinin
(GNA), S. nigra agglutinin
(SNA), M. amurensis agglutinin (MAA),
peanut agglutinin (PNA), and D. stramonium
agglutinin (DSA) have specific affinity for terminal
mannose, sialic acid linked (2-6) to galactose, sialic acid linked
(2-3) to galactose, O-glycosidically linked sugar chain,
and Gal-(1-4)GlcNAc in complex and hybrid N-glycans,
respectively. G. nivalis agglutinin treatment
yielded positive signals for all four FCs; S. nigra
agglutinin treatment yielded positive results for FCs from LAL, CAL,
and Sf9-BEVS and only weakly reacted with S2 FC. Reaction with
M. amurensis agglutinin yielded the strongest signal with S2
FC. S. nigra agglutinin did not react with
peptide:N-glycanase F-treated FC samples. D. stramonium agglutinin yielded the weakest signal with S2 FC.
C, comparison of LPS activation of Sf9-BEVS FC and S2
FC. Incubation of 10 µg of Sf9-BEVS FC and S2 FC with 50 EU of
LPS was carried out at 37 °C for 1 h. The activation of FC from
Sf9-BEVS was readily observable (autocleavage into 80- and
52-kDa bands). S2 FC remained intact after the LPS treatment.
7
M and 9.58 × 107 M for FL
and ES·PRR, respectively. It is interesting to observe that the
absence of the lectin-like and sushi 4 domains leads to a 4-fold
decrease in affinity for LPS.
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Fig. 4.
Functional analysis of FC. A,
ligand binding assays of FC samples. ELISA of 10 µg each of protein
samples: control serum-free medium, FL, ES·PRR, and PRR·SP
partially purified through Sephadex G-100 (void fraction). Three
concentrations of LPS (0, 0.05, and 5 µg/ml) were used.
Error bars represent the S.E. (n = 3). B, a sensorgram depicting the interaction (in response
units (RU)) of captured FL with various concentrations of
lipid A. ES·PRR gave similar response profiles. 100 µl of five
concentrations (5.00, 2.50, 1.25, 0.62, and 0.31 µM) of
lipid A were injected. The affinity constants
were calculated using the BIAevaluation program version 3.0.2 and reconfirmed using CLAMP (21).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
An illustration of PRR and
N-glycans involved in secretion of FC.
Gray string, cysteine-rich region;
orange string, epidermal growth factor-like
domain; green string, sushi-like domains;
black circle, lectin-like domain; red
string, proline-rich region; blue
oval, serine protease native conformer; blue
string, misfolded serine protease; black
oval, N-glycan; round gray
shapes, proteins involved in folding, assembly, and
secretion (e.g. chaperones, foldases, signal peptidase, and
glycosidase). The hypothesis was confirmed by (a) the
removal of PRR from FL, which completely switches its expression
profile and protein conformation and reconfirms PRR to be an important
secretory determinant for glycosylated FC; (b) mutation of
N-glycan sites that impaired the secretion competency of FC;
and (c) co-expressed chaperone (e.g. calnexin),
which associates with glycosylated FL protein and assists the reversal
of this impairment to a certain extent, to enhance its solubility and
secretion. Thus, whereas PRR is crucial for correctly directing folding
and secretion of the downstream glycosylated SP domain, chaperones and
foldases also play a role in enhancing protein folding, to ensure that
the modular arrangement of the glycosylated protein is properly folded
to remain soluble or targeted for secretion and to exhibit full
functionality.
PRR)) with a view to examine the functional significance of PRR.
The FL was efficiently secreted and fully capable of binding LPS. In
contrast, the FL(PRR), which lack the PRR, were expressed intracellularly and were nonfunctional. The expression profile of three
additional FCs, SP, PRR·SP, and SP·PRR, confirmed that PRR is
essential for folding and secretion of the downstream SP domain. The
introduction of PRR C-terminal to ES has no observable effect on ES
expression profile, suggesting that PRR only exerts its influence on
the downstream domains. Thus, PRR itself may act as an intramolecular
chaperone for correct folding of the downstream SP region for its
subsequent transportation through the organelle. In support of our
data, it was reported that the minimal portion of secreted Factor XII,
which is also a multidomain serine protease similar to FC, also
contains PRR (27).
PRR) and SP, failed to give rise
to characteristic patterns of stable products, suggesting that they are
incorrectly folded and are more susceptible to proteolytic digestion.
PRR) and SP) were greatly enhanced by the presence of cCNX. Specific interaction between certain structures of N-glycans
and calnexin was further confirmed by a glycosylation inhibition study. Thus, it is evident that the N-glycans of FC function
through interaction with molecular chaperones to aid in protein folding and subsequent secretion. On the other hand, co-expressed chaperones did not overcome the nonsecretory problem of FL(PRR) and SP, suggesting that their intracellular localization was determined by
their conformation and not the lack of certain molecular chaperones in
the host cell secretion pathway.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Institut de Biologie Animale, Bâtiment
de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland.
To whom correspondence should be addressed: National University
of Singapore, Marine Biotechnology Laboratory, Dept. of Biological Sciences, 14 Science Dr. 4, Singapore 117543, Singapore. Tel.: 65-8742776; Fax: 65-67792486; E-mail: dbsdjl@nus.edu.sg.
ABBREVIATIONS
-deoxymannojirimycin;
DNJ, -deoxynojirimycin;
LPS, lipopolysaccharide;
SPR, surface plasmon resonance;
CAL, C.
rotundicauda amoebocyte lysate;
S2 FC, full-length
Factor C derived from the Drosophila melanogaster S2
expression system;
Sf9-BEVS FC, full-length Factor C derived
from Spodoptera frugiperda, Sf9-baculovirus
expression vector system;
ELISA, enzyme-linked immunosorbent assay;
PBS, phosphate-buffered saline.
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(1997)
Protein Expression Purif.
9,
388-398[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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