From the
In a prior study, we found that the processed form of human
interleukin-1
Interleukin-1
The
lysine to glutamic acid mutations, matIL-1
The sequence between amino
acid residues 95 and 104 contains several hydrophobic amino acids
interrupted by a stretch of three glutamic acid residues at positions
99-101 (Fig. 1). To assess the effect of the three glutamic
acid residues on IL-1 secretion, a
Similar to the F162D protein,
the decreased secretion level of the lysine mutants by P388D1 cells may
be a result of an inappropriate conformation. To determine if the
lysine mutants exhibit a major conformational change relative to the
wild-type mature protein, the K-E
The results presented in this study are consistent with the
hypothesis that IL-1 secretion is a tightly regulated process that
requires the processing of the protein and at least two steps that are
differentially sensitive to sequence variations and are differentially
expressed in P388D1 and Jurkat cells. This conclusion is based upon the
assumption that the mechanism of IL-1 secretion in the two cell types
is fundamentally the same, a view that is supported by the following
common features of IL-1 secretion in the two cell lines: 1) mature IL-1
is secreted to a greater extent than the precursor; 2) the secretion of
mature IL-1 is enhanced by calcium ionophores; 3) precursor domains
truncated to amino acid 95 impair secretion; and 4) the secretion of
improperly folded proteins such as the F162D mutant are reduced
relative to the wild-type mature protein.
Using truncated forms of
the IL-1
Although
the sequence of amino acids between positions 105 and 116 has no affect
on the secretion of the protein (), their presence does
reduce the ability of the protein to bind to the IL-1 receptor (13).
Therefore, the truncated protein may be in a slightly different
conformation than the mature protein (as measured by differences in
biological activity), but sequences required for secretion of the
In contrast to the
results with P388D1 cells, the mutations in charged amino acids in
mature IL-1
In the simplest of models, the
secretion of IL-1 would require only a single step, namely,
translocation of the mature protein across the plasma membrane. A
single-step model would be inconsistent with the observed differences
in IL-1 secretion between P388D1 and Jurkat cells. Thus, there must be
at least two steps in the secretion of mature IL-1
A model for secretion may include the partial or
complete refolding of the mature IL-1 in order to account for the
reduced secretory potential of some of the mutants in P388D1 cells.
Does the mature protein translocate across membranes in a folded state?
Pugsley (31) has shown that Escherichia coli pullulanase may be translocated across the bacterial outer
membrane in a form close to the final conformation of the protein. In a
related example, Creighton et al.(32) have recently
reported that the extrinsic 23-kDa protein of photosystem II may be
translocated across the chloroplast thylakoid membrane in a tightly
folded form. Given these examples, it is possible that IL-1 proteins
may cross the membrane in a folded form. The observation that
improperly folded IL-1 proteins (for example,
P388D1 or Jurkat cells were transfected with the indicated
IL-1
P388D1 or Jurkat cells were transfected with
the indicated mature IL-1
Each of the mature IL-1
P388D1 or Jurkat cells were transfected
with the indicated IL-1 constructs, and the level of secretion was
measured after a 30-min incubation with ionomycin. Each value is the
mean ± S.D. of results from three independent experiments.
(mature IL-1
) is secreted to a significantly
greater extent than the precursor form of the protein, indicating that
the precursor domain acts in some manner to reduce the secretory
potential of the protein. In view of this observation, we sought to
define the sequence(s) in the IL-1
precursor that limit the
secretion of the protein as well as the sequences in the mature protein
that promote secretion. The P388D1 murine macrophage cell line and the
Jurkat human T-cell line were transiently transfected with cDNA
expression vectors encoding truncated forms of human precursor
IL-1
proteins, lacking either the first 76, 94, 99, or 104 amino
acids. The removal of increasing numbers of precursor amino acid
residues resulted in a graded increase in the secretion of the
truncated precursor IL-1
proteins from both cell lines. The
minimal region of the precursor sequence required to inhibit the
optimal secretion of IL-1
occurs between amino acids 100 and 104
for P388D1 cells and 95-99 for Jurkat cells. Deletion of the
amino acids within these regions increased the secretion level of the
truncated proteins to that of mature IL-1
. Mutagenesis of the
mature IL-1
sequence revealed that a region of basic amino acids
may play an important role in the optimal secretion of mature IL-1
in P388D1 cells, but not in Jurkat cells. Based on the differences in
the structural requirements for IL-1
secretion in P388D1 and
Jurkat cell lines, it is likely that the secretion of IL-1
may be
subject to multiple levels of regulation that are differentially
operative in different cell types.
and -
(IL-1)
(
)are
produced and secreted by macrophages and neutrophils and stimulate a
broad spectrum of cells involved in immune and inflammatory responses
(1). Although the secreted forms of IL-1
and IL-1
exhibit
molecular masses of approximately 15 kDa, they are initially
synthesized as 31-33-kDa precursors that are subsequently cleaved
to their mature forms prior to secretion. In the case of both
IL-1
and IL-1
, the mature (secreted) protein is contained
within the carboxyl terminus of the precursor. Human IL-1
is
synthesized by macrophages as a precursor of 271 amino acids that is
cleaved at Arg
by the calcium-activated protease,
calpain(2, 3) , to generate the major secreted form of
the protein, Ser
-Ala
. Although only very
low levels of the IL-1
precursor are released from
cells(4, 5, 6) , the precursor does appear to be
biologically active(7) . The precursor form of IL-1
contains 269 amino acids and is processed by the IL-1 converting enzyme
at Asp
to yield Ala
-Ser
(Fig. 1)(8, 9, 10, 11, 12) .
In contrast to the situation with IL-1
, the IL-1
precursor is
biologically inactive and must undergo a conformational change
following cleavage by IL-1 converting enzyme that results in the
generation of a biologically active molecule(7) . In support of
this notion, Hazuda et al.(13) established that
recombinant forms of the IL-1
precursor with progressive deletions
of amino acid residues in the amino-terminal precursor domain exhibit a
progressive change in conformation and a marked increase in biological
activity. For example, removal of the first 76 amino acids of the
precursor results in a conformational changes that is reflected in at
least a 600-fold increase in biological activity. The deletion of an
additional 18 amino acids results in a further change in the
three-dimensional structure of the protein and an additional 1500-fold
elevation in biological activity. A truncated form of the protein
lacking residues 1-104 was only slightly less active than the
mature protein itself.
Figure 1:
Structure
of precursor and mature IL-1. The mature IL-1
begins at
Ser
and is the result of cleavage at Asp
by
the IL-1 converting enzyme (ICE). The positions of each
precursor truncation and point mutations are also
presented.
In a recent study(6) , we reported
that the mature forms of IL-1 and -
are secreted to a
markedly greater extent than the precursor proteins, indicating that
the optimal secretion of IL-1
and -
may also be dependent
upon a change in the conformation of the proteins that enhances their
interaction with one or more components of the secretory pathway. In
support of this hypothesis, we have found that mature IL-1
proteins with conformation-altering deletions at the carboxyl or amino
termini are poorly secreted in transiently transfected P388D1 cells, a
murine macrophage cell line(6) . The results of a number of
studies are consistent with the notion that IL-1 proteins are not
secreted by the normal exocytic
pathway(5, 14, 15, 16, 17) , but
are translocated by a novel mechanism that is present in cell types in
addition to macrophages and neutrophils(6) . Evidence from
biochemical and immunoelectron microscopy studies indicates that newly
synthesized IL-1 proteins accumulate in the cytoplasm, even during
periods of peak
secretion(4, 5, 16, 17) . Although the
secretion of IL-1 is a relatively slow process, the rate of secretion
can be dramatically enhanced by calcium
ionophores(5, 15) , a finding that raises the
possibility that calcium may play a key role in the secretory process.
As a first step toward defining the components of the IL-1 secretory
system, we have conducted a detailed analysis of the structural
features of IL-1
that are required for efficient secretion. The
results of these experiments are consistent with the hypothesis that
IL-1 secretion is a multistep process that is subject to regulation in
a cell-type specific manner.
Cell Culture
The P388D1 murine macrophage cell
line has been used extensively to study the synthesis and secretion of
IL-1(3, 4, 6, 18, 19, 20) .
P388D1 cells were grown in suspension culture, whereas the human Jurkat
T cell line was grown in stationary culture. Each of the cell lines was
maintained in RPMI 1640 media supplemented with 10% fetal bovine serum
and 50 µg/ml gentamicin sulfate. All cell lines used in this study
were routinely checked for mycoplasma infection and were found to be
free of contamination.
Preparation of IL-1 Expression Vectors
The
wild-type pRc/RSV precursor and mature IL-1 cDNA expression
vectors were constructed as described previously(6) . The
following general strategy was employed to create the additional IL-1
constructs. Using the appropriate primers with a BstXI
restriction site in the 5` primer and a XbaI restriction site
in the 3` primer, each cDNA was amplified by the polymerase chain
reaction. The cDNA fragments were gel-purified, digested with the
appropriate enzymes, and directionally cloned into the pRc/RSV
expression vector. The
1-76 precursor cDNA lacks a sequence
encoding the first 76 amino acids of the precursor IL-1
protein,
whereas the
1-94,
1-99, and
1-104
precursor cDNA constructs lack the sequences encoding the first 94, 99,
or 104 amino acids of the precursor IL-1
protein ().
The mature IL-1
mutant cDNAs (21) (kindly provided by Dr.
Grace Ju, Hoffmann-La Roche) were also amplified by the polymerase
chain reaction and subcloned into the pRc/RSV expression vector.
K-E
and
matIL-1
K-E
, were created by a modified megaprimer
method(22) . Briefly, four primers, A and D possessing a BstXI (A) or XbaI restriction site (D) and two
internal primers (B and C), were used to create each construct. For the
matIL-1
K-E
mutant, the internal primers ((B) 5`-TCG
CTT TTC CAT CTC CTC CTC TGG G-3` and (C) 5`-CTA CAG CTG GAG AGT GTA GAT
CCC-3`) were designed in the opposite orientation, yet only primer B
contains the desired mutation. Using primers A and B in one reaction
and primers C and D in another reaction, PCR was employed using the
conditions previously detailed to generate two small cDNA fragments
that overlap by 50 nucleotides. The cDNA fragments were gel-purified,
and 100 ng of each cDNA fragment was used in another round of PCR. The
cDNA was amplified for 8 cycles in the absence of any primers at an
annealing temperature of 65 °C. Since the two cDNA fragments
overlap by 50 nucleotides, the second round of PCR lets the DNA
fragments anneal and extend to full length. This step greatly enhances
the probability that a positive clone encoding the mutation will be
generated. A 20-µl aliquot of this reaction was used as the DNA
template in a subsequent PCR reaction. The final full-length product
was amplified in a third round of PCR for 25 cycles using primers A and
D. The full-length cDNA fragment was gel-purified, digested with BstXI and XbaI, and directionally cloned into the
pRc/RSV expression vector. The matIL-1
K-E
mutant was
created using the same procedure. However, the internal primers ((B)
5`-TGG GTA ATT CTC GGG ATC TAC ACT CTC-3` and (C) 5`-GAG AGT GTA GAT
CCC GAG AAT TAC CCA-3`) were complementary to each other, and each
encoded the desired mutation. By encoding the mutation in both primers,
each of the resulting cDNA fragments generated possessed the desired
mutation. This approach dramatically decreased the chance of generating
a cDNA without the mutation. The quadruple mutant used the triple
mutant cDNA as the template for the PCR reaction. A listing of the IL-1
constructs used in this study is presented in . All of the
constructs were sequenced to assess the fidelity of the PCR reaction.
Transfections
Cell lines were transiently
transfected by electroporation using a Bio-Rad gene pulser(6) .
Aliquots of log phase cells (1 10
cells) were
centrifuged at 600
g for 10 min, resuspended in 250
µl of RPMI 1640 supplemented with 10% fetal bovine serum and
gentamicin sulfate, and pipetted into 0.4-cm gene pulser cuvettes.
Following addition of 25 µg of the appropriate plasmid, samples
were incubated on ice for 10 min. To account for the potential
production of IL-1 proteins from P388D1 genes, control samples received
pRc/RSV vector with no insert. The electroporated cells were
resuspended in 8 ml of complete medium, aliquoted into a 6-well plate,
and rested for 2 days prior to use.
Analysis of IL-1
Radiolabeling of
cells and immunoprecipitation were performed as described
previously(6) . Briefly, cells were radiolabeled with 200
µCi of [ Secretion
S]methionine (ICN Radiochemicals) in
methionine minus media (Life Technologies, Inc.) supplemented with 0.1%
fetal bovine serum for 6 h at 37 °C. The cells were washed and
incubated in the presence of 1 µM ionomycin (Sigma) for 30
min. Culture supernatants were collected and concentrated to 400 µl
using Amicon Centricon-10 microconcentrators. Cells were lysed with 400
µl of immunoprecipitation buffer (150 mM NaCl, 0.4%
Nonidet P-40, 10 mM EDTA, and 50 mM Tris, pH 8.0),
and 200 µl of each supernatant and lysate sample was used for
immunoprecipitation. The samples were incubated for 1 h with polyclonal
anti-IL-1
antibodies (kindly provided by Drs. Peter Lomedico and
Richard Chizzoniti, Hoffmann-La Roche) at a final concentration of
1:1000. Matrix-bound protein A (20 µl, Repligen) equilibrated in
immunoprecipitation buffer was added to each sample and incubated for
an additional 45 min. Following several washes, 20 µl of 2
Laemmli sample buffer was added to each sample and then the samples
were boiled for 2 min. Twenty µl of distilled water was added to
each sample, and then protein A was removed by centrifugation. The
supernatant was removed from each tube and analyzed by electrophoresis
on a 12.5% polyacrylamide gel. The gels were fixed, incubated in
Autofluor, dried, and subjected to fluorography. In addition, each gel
was scanned on the AMBIS radioanalytic imaging system to determine the
percent secretion(6) . All experiments were done a minimum of
three times.
Preparation of Radiolabeled Recombinant IL-1 Proteins by
in Vitro Translation
Each of the pRc/RSV mature IL-1 cDNA
constructs was digested with BstXI and XbaI,
gel-purified, and subcloned into the pRc/CMV expression vector
(Invitrogen) containing the T7 promoter and verified by restriction
digest analysis. For all in vitro transcription and
translation experiments, 1.5 µg of XbaI-linearized DNA
template was transcribed, and the resulting mRNA was translated in the
presence of [
S]methionine using an in vitro TnT reticulocyte lysate system (Promega). The samples were
incubated at 30 °C for 90 min to allow the TnT reaction to go to
completion. A 2-µl sample of each radiolabeled protein was
precipitated with ice-cold trichloroacetic acid and counted in a
Beckman scintillation counter to determine the amount of radioactivity
incorporated. In addition, 2 µl of the radiolabeled protein was
spotted on a filter and counted to determine the total radioactive
counts per sample. The percent incorporation of
[
S]methionine was determined by the equation:
(cpm of trichloroacetic acid-precipitated sample/cpm of
non-trichloroacetic acid-precipitated sample)
100%.
Proteinase K Assay
Two hundred fmol of each
protein was incubated with or without 10 µg/ml of proteinase K
(Sigma) for 5 or 180 min at 22 °C in a final volume of 20 µl.
The reactions were terminated by the addition of phenylmethanesulfonyl
fluoride (Sigma) (final concentration of 2 mM; 22 °C for 5
min). The samples were supplemented with 20 µl of 2 Laemmli
sample buffer, boiled for 2 min, and subjected to electrophoresis on a
12.5% polyacrylamide gel. The gel was fixed, incubated in Autofluor,
dried, and subjected to fluorography. The dried gel was scanned on the
AMBIS radioanalytic imaging system to determine radioactive counts for
each protein in order to evaluate the amount of degradation, if any, at
each time point. Each recombinant IL-1 protein was analyzed for
proteinase K sensitivity in at least three separate experiments.
Determination of the Precursor IL-1
Previous
experiments demonstrated that precursor IL-1 Region That
Impairs Secretion of the Protein by P388D1 Cells
is poorly secreted
from P388D1 cells, whereas the mature IL-1
is secreted in a rapid
and efficient manner following the addition of the calcium ionophore,
ionomycin(6) . These observations are consistent with the notion
that the precursor sequence may impair secretion by keeping the protein
in a secretion-incompetent conformation or by masking possible
recognition sequences in mature IL-1
. To define the minimal region
of the precursor sequence that inhibits secretion, we initially
examined the secretory potential of three truncated forms of the
precursor IL-1
proteins that were previously determined by Hazuda et al. (13) to undergo progressive conformational changes upon
the deletion of varying lengths of the precursor sequence and, in so
doing, exhibit graded increases in biological activity (13) (see Fig. 1). Expression vectors encoding precursor IL-1
proteins
lacking either the first 76, 94, or 104 amino acids were transiently
transfected into P388D1 cells. The cells were subsequently
radiolabeled, and secretion levels were determined following a 30-min
incubation with ionomycin. As shown in , the removal of
increasing numbers of precursor amino acid residues resulted in a
graded increase in the secretion of the truncated precursor IL-1
proteins. In confirmation of previous experiments, the precursor
protein was secreted to a lesser extent than the mature protein (3% versus 24%). The deletion of the first 76 or 94 amino acids of
the precursor protein produced a small increase in secretion. However,
when 10 additional amino acids were deleted (
1-104),
secretion was increased to 22%. Since this level of secretion is
comparable to that observed with the mature protein, the region of the
IL-1
precursor sequence that inhibits secretion in P388D1 cells is
present between amino acids 95 and 104.
1-94 mutant was generated
that contains a lysine residue in place of each glutamic acid. These
mutations had no effect on secretion over and above that observed with
the wild-type
1-94 deletion mutant (). Thus,
the inhibitory activity of the 95-104 region is not due to a
direct effect of the three glutamic acid residues at positions
99-101. In view of this result, an additional deletion mutant,
1-99, was generated to determine if the inhibitory region
spanned a more limited set of residues. As shown in , the
1-99 protein was secreted at a higher level than the
1-94 protein, but not to the same extent as the
1-104 protein. This finding indicates that amino acid
residues 100-104 are sufficient for the impairment of IL-1
secretion in P388D1 cells. It is important to note that the truncated
precursor proteins are not processed because the P388D1 cells have
little or no IL-1 converting enzyme activity (6).
(
)Thus the observed differences in secretion are due to
the relative secretory potential of each IL-1 protein and not to
differences in susceptibility to proteolytic processing. Taken
together, these results indicate that the 100-104 region is
sufficient to inhibit the secretion of IL-1
in P388D1 cells,
possibly by sterically hindering the interaction of a secretory signal
sequence with one or more components of the IL-1 secretory pathway.
Alternatively, this stretch of amino acids may prevent the protein from
assuming a secretion-competent conformation.
Determination of the Precursor IL-1
The ability to
secrete the mature forms of IL-1 Region That
Inhibits Secretion of the Protein by Jurkat Cells
and IL-1
is not restricted
to macrophages. For example, HeLa cells as well as the Jurkat and EL4 T
cell lines possess the ability to efficiently secrete the mature forms
of IL-1(6) . When these non-macrophage cell types are
transiently transfected with a cDNA encoding the precursor forms of
IL-1 proteins, there is very little secretion because these cell types
lack the active enzymes that convert the precursor proteins to their
mature forms. To determine if the domain of the precursor sequence that
impairs IL-1
secretion in P388D1 cells also interferes with
secretion in non-macrophage cell types, we evaluated the secretion
potential of the precursor deletion proteins in the Jurkat T cell line.
As was the case with P388D1 cells, Jurkat cells exhibited a very
limited ability to secrete proteins with deletion of the first 76 or 94
amino acids. Likewise, the conversion of glutamic acid residues to
lysine at positions 99-101 did not enhance secretion in Jurkat
cells. In contrast to the findings with P388D1 cells, the
1-99 protein was secreted at levels comparable with the
wild-type mature protein in Jurkat cells (34% versus 40%).
This result indicates that the residues between positions 95 and 99 in
the precursor sequence are sufficient for the inhibition of secretion
in Jurkat cells. Furthermore, the deletion of amino acid residues
100-104 markedly enhanced the secretion of the truncated protein
by Jurkat cells. Indeed, in Jurkat cells, the
1-104 protein
appears to be a better substrate for secretion than the fully
processed, mature IL-1
protein (67% versus 40%). These
results raised the interesting possibility that IL-1 secretion may be
susceptible to different forms of regulation and that such regulatory
mechanisms may be present to varying degrees in different cell types.
Effect of Point Mutations on the Secretion of the Mature
IL-1
In a prior study, we found
that mature IL-1 Protein by P388D1 Cells
proteins with deletions in the first or last 20
amino acids are not efficiently secreted from P388D1 cells(6) .
Such deletions are known to markedly reduce biological
activity(23) . These observations are consistent with the notion
that a specific conformation is required for the optimal secretion and
biologic activity of IL-1. To determine if this is the case, we
examined the secretion potential of mature IL-1
proteins
possessing point mutations (see Fig. 1) that greatly reduce the
ability of the mutant proteins to bind to the type I IL-1 receptor and
trigger biological responses(21) . As shown in I,
none of these point mutants were secreted at wild-type levels. The
secretion of the mutant proteins ranged from 7% to 11%, as compared to
22% for the wild-type mature protein. Although these results are
consistent with a linkage between secretion potential and receptor
binding, we also found that the K204G mutant that retains the wild-type
ability to bind to the IL-1 receptor was not secreted to the same
extent as the wild-type protein (13% versus 22%). This result
indicates that the structural requirements for secretion and receptor
binding may overlap, but they are not coincident.
Effect of Point Mutations on the Secretion of the Mature
IL-1
In view of the potentially
important differences in the secretion potential of the precursor
truncations in P388D1 and Jurkat cells, we also tested the secretion of
the mature IL-1 Protein by Jurkat Cells
point mutants in Jurkat cells. In marked contrast
to P388D1 cells, Jurkat cells efficiently secreted most of the mutant
proteins (I). Three of the mutant IL-1
proteins,
K209L, K219S, and E221S, were secreted at levels comparable to
wild-type mature IL-1
(46%, 49%, and 52% versus 41%). In
addition, the biologically active protein, K204G, was also efficiently
secreted from Jurkat cells (49%). The R120A protein was secreted at a
slightly reduced level compared to the mature protein (25% versus 41%). However, as with P388D1 cells, secretion of the F162D mutant
in Jurkat cells was reduced relative to the mature protein (18% versus 41%). Unlike the situation in P388D1 cells, mutations
at residues involved in IL-1 receptor binding, with the exception of
Phe
and possibly Arg
, did not affect
secretion in Jurkat cells. These results provide additional support for
the conclusion that the regulation of IL-1 secretion may vary between
cell types, possibly because one or more regulatory components of the
IL-1 secretory pathway may exhibit cell type-specific expression or
activity.
Determination of the Proteinase K Sensitivity of the
Mature IL-1
Since the secretion of IL-1 may
be conformation-dependent, it was possible that the decrease in
secretion of the mature IL-1 Mutant Proteins
point mutants from P388D1 cells might
be due to conformational changes created by the mutations. Hazuda et al.(13) established that correctly folded mature
IL-1
and -
proteins are highly resistant to proteinase K
digestion whereas the precursor IL-1 proteins or truncated forms of the
precursor proteins are rapidly degraded by proteinase K. To examine the
possibility that the mature IL-1 mutants are not secreted because they
have undergone a substantial change in conformation, we tested the
proteinase K sensitivity of these proteins. Radiolabeled samples of
each mutant were incubated with or without 10 µg/ml proteinase K
for 5 min or 3 h at 22 °C. Although it has been shown that a 5-min
incubation is sufficient for nearly complete digestion of IL-1
precursor proteins, an additional 3-h incubation was used to assess the
possible presence of more subtle conformational changes. As expected,
the precursor IL-1
protein was rapidly degraded (),
whereas the mature IL-1
protein was resistant to proteinase K
degradation. Among the point mutants, only the F162D protein was
degraded by proteinase K, demonstrating that the decrease in secretion
of the F162D mutant in P388D1 and Jurkat cells may be due to a
substantial change in the conformation of the protein. On the basis of
proteinase K sensitivity, it appears that the R120A, K204G, K209L,
K219S, and E221S mutants are not in a dramatically different
conformation than the wild-type mature IL-1
protein. Therefore,
the decreased secretion level exhibited by these proteins in P388D1
cells may be due to the effects of the mutations on the interaction of
these proteins with components of the secretory pathway. Alternatively,
these mutant proteins may possess subtle conformational changes that
are not detectable by prolonged proteinase K treatment.
Effect of Mutations in the Basic Loop Region of Mature
IL-1
Because mutations in
charged amino acids decreased secretion of the mutant mature IL-1 on Secretion by P388D1 Cells
proteins in P388D1 cells, we explored the possibility that the
distribution of charged amino acids within the mature protein may play
a role in IL-1 secretion in these cells. PCR mutagenesis was used to
convert specific lysines to glutamic acid. The lysines chosen for
mutagenesis are located within a highly basic loop of the mature
protein. Based on the three-dimensional structure of mature
IL-1
(24, 25) , this loop is located in proximity to
the residues involved in IL-1 receptor binding (21), creating a
relatively basic face on one side of the protein. A triple mutant,
matIL-1
K-E
, containing Lys to Glu mutations at
positions 208, 209, and 210, and a quadruple mutant, matIL-1
K-E
, containing an additional mutation at lysine 204, were
created. As shown in , secretion of both lysine mutant
proteins by P388D1 cells was decreased by 50% compared to the wild-type
mature protein (11% and 12% compared to 24%). Although these results
indicate that these basic residues contribute to the overall secretory
potential of mature IL-1
, they are not essential since the
secretion was not totally abolished.
and K-E
proteins were analyzed for proteinase K sensitivity. As shown in , these mutant proteins were not susceptible to
degradation by proteinase K. Thus, the decreased secretion of these
mutants is not due to a major change in conformation. The possibility
still exists, however, that these mutant proteins possess minor
conformational changes that affect secretion, but are not detectable by
proteinase K treatment. Nonetheless, it is quite possible that these
lysine residues may directly or indirectly influence the interaction of
mature IL-1 with one or more components of the IL-1 secretory pathway.
Effect of Mutations in Basic Amino Acids on the Secretion
of Mature IL-1
Since the K204G, K209L,
K219S, and E221S mutant IL-1 by Jurkat Cells
proteins are efficiently secreted
from Jurkat cells but not from P388D1 cells, we also examined the
secretion of the K-E
and K-E
mutants in Jurkat
cells. Although the K-E
mutant was secreted as well as the
mature wild-type protein in Jurkat cells (39% versus 48%), the
secretion of the K-E
mutant was markedly reduced (8%) (). As shown in I, single mutations within
charged amino acids do not affect secretion in Jurkat cells to the same
degree as in P388D1 cells. Furthermore, the mutation of three basic
amino acids at positions 208, 209, and 210 did not reduce the secretion
of the mutants from Jurkat cells. However, the lack of secretion of the
K-E
mutant indicates that there is a finite number of basic
amino acid mutations in IL-1
that the Jurkat cells can tolerate
before secretion is reduced. Taken together, these results are
consistent with the hypothesis that basic amino acids play a role in
the secretion of mature IL-1 from P388D1 and Jurkat cells. However, the
dependence on these residues is much more pronounced in P388D1 cells
than in Jurkat cells.
precursor, we have found that the presence of a region of
only 5 amino acids (residues 100-104) restricts the secretion of
the protein by P388D1 cells. The presence of this sequence may maintain
the protein in a secretion-incompetent conformation or mask possible
recognition sequences that are required for optimal secretion. With
regard to the latter possibility, the masking of recognition sequences
might be dependent upon the specific interaction of the precursor
fragment with one or more sites within the mature IL-1. For example,
Kessler and Safrin (26) have shown that Pseudomonas
aeruginosa elastase is inhibited in trans by its
propeptide sequence. This inhibition is dependent on the propeptide
sequence binding to the active enzyme sequence. However, co-expression
of a peptide encoding amino acids 100-116 of the IL-1
precursor in P388D1 cells did not inhibit the secretion of mature
IL-1
.
(
)Although this observation indicates
that the short IL-1 precursor sequence does not function in
trans, it may be that without additional flanking sequences the
100-116 fragment cannot assume an appropriate conformation for
interaction with the mature domain of the IL-1 protein. Clearly,
additional studies are necessary to evaluate this interesting
possibility. In all likelihood, however, the impairment of secretion by
the precursor domain of IL-1 probably occurs through a dual requirement
for a specific conformation and recognition sequence(s).
1-104 protein are accessible for interaction components of
the secretory pathway. In contrast to the situation in P388D1 cells,
the deletion of the region between positions 95 and 99 is sufficient to
promote IL-1
secretion in Jurkat cells. Indeed, the
1-104 IL-1 protein is secreted more efficiently than the
mature wild-type protein (). These findings raise the
possibility that the secretion of IL-1 is not a single-step process,
but rather involves several steps, one or more of which are subject to
regulation by factors that may be expressed in a cell type-specific
manner. Thus, the dissimilarity in the extent of secretion of the
truncated precursor proteins in P388D1 and Jurkat cells may be a
reflection of differences in the nature or activity of specific
regulatory factors in the two cell lines. The observed differences in
the secretion of the point mutants of mature IL-1 by P388D1 and Jurkat
cells (Tables III and V) are also consistent with this hypothesis.
These experiments revealed that substitutions of charged amino acids at
a number of sites in the primary sequence of the mature protein affect
secretion in P388D1 cells, but not Jurkat cells. Mutation of these
residues to uncharged species significantly decreased the secretion of
the mutant proteins by P388D1 cells. Even though the basic amino acids
that affect secretion in P388D1 cells are located at multiple sites in
the primary sequence, they are primarily localized to one face of the
three-dimensional structure of the protein. In this regard, it is of
interest to note that the histidine residues that are concentrated in
the surface-exposed loops and turns of hisactophilin, a protein that
possesses a three-dimensional structure that is similar to that of IL-1
proteins (the
-barrel), may be essential for the interaction of
this protein with actin(27) . Thus, the basic loop structure may
play an important role in the functions of several members of the
-barrel structural family of proteins.
had little or no effect on secretion by Jurkat cells.
Indeed, these proteins were secreted at levels comparable to that
achieved with the mature protein. In light of the results with the
Jurkat cells, we favor the view that changes in charged residues do not
affect the intrinsic ability of the proteins to be secreted,
but instead subject the resultant proteins to a quality control
mechanism that is operative in P388D1 cells, but not Jurkat cells.
Perhaps P388D1 cells contain a factor(s) that acts in a manner that is
analogous to that of BiP in the endoplasmic reticulum(29) .
Dorner et al.(30) have reported that the secretion of
an overexpressed form of human tissue plasminogen activator was
enhanced when the synthesis of BiP was reduced following the expression
of an antisense RNA. It is possible that the enhanced ability of Jurkat
cells to secrete IL-1 proteins (relative to P388D1 cells; see Tables
II, III, and V) is due, at least in part, to a reduced level of a
cytoplasmic or membrane-associated factor that acts in a manner that is
analogous to BiP to prevent the secretion of improperly folded
proteins. Although most of the IL-1 mutants are resistant to
proteolytic cleavage by proteinase K, it is still possible that the
mutant proteins are inappropriately folded and thus are recognized by
one or more BiP-like regulatory factors and are, as a consequence,
prevented from being secreted.
that differ in
their sensitivity to amino acid sequence variation. As evidenced by the
lack of secretion of the various point mutants in P388D1 cells, the
step that is more sensitive to sequence variation is
rate-limiting for secretion in these cells. In Jurkat cells, however,
the step that is less sensitive to amino acid sequence
variation appears to be rate-limiting and promotes a greater overall
rate of IL-1
secretion. Hence, Jurkat cells secrete the wild-type
and point mutant proteins to a significantly greater extent than P388D1
cells. Our results indicate that the secretory process in Jurkat cells
is able to reject only IL-1
mutants with substantial sequence or
conformational perturbations, for example, the F162D and the
matIL-1
K-E
mutants. However, the secretory process in
P388D1 cells is clearly able to distinguish more subtle changes. Since
macrophages are a major source of secreted IL-1 in immune and
inflammatory responses(1) , it may be that this cell type has
evolved unique and relatively stringent mechanisms to prevent the
release of altered forms of the IL-1 protein. In view of the T cell
origin of Jurkat cells, such a mechanism is not present or only weakly
active in these cells and thus the release of wild-type and altered
IL-1 proteins can occur at a relatively high rate in comparison to
P388D1 cells.
117-136 and
250-269 (6) and F162D (I)) are poorly
secreted is certainly consistent with the notion that proper folding is
a requirement for efficient secretion. However, it is also conceivable
that a regulated unfolding step is also required for optimal secretion.
Such a step would likely involve a chaperone, since this class of
proteins is known to play a key role in the membrane translocation of a
relatively large number of proteins destined for secretion along the
normal exocytic pathway or into mitochondria(29, 33) . A
chaperone may aid in maintaining the mature IL-1 in an unfolded state
and thus allow it to translocate through a membrane or associate with
components of the IL-1 secretory pathway. Experiments are currently
underway to determine if IL-1 secretion is dependent upon unfolding
and, if so, to characterize the cellular factors that may be involved
in regulating the process.
Table: IL-1 expression vectors and relevant properties
Table: Secretion of truncated precursor IL-1
proteins
expression vectors, and the level of IL-1 secretion was
assessed following a 30-min incubation with ionomycin. Each value is
the mean ± S.D. of the values from three independent
experiments.
Table: Secretion of mature IL-1 mutants by
P388D1 and Jurkat cells
cDNA expression vectors, and the level
of secretion of each protein was subsequently assessed following a
30-min incubation with ionomycin. Each value is the mean ± S.D.
of results from three independent experiments.
Table: Proteinase K sensitivity of mature
IL-1 proteins
cDNAs was subcloned
into the pRc/CMV expression vector. mRNA was transcribed and translated
in the presence of [
S]methionine, and the
resultant radiolabeled proteins were incubated in the presence or
absence (Control) of Proteinase K for 5 min or 3 h. The samples were
subjected to SDS-polyacrylamide gel electrophoresis analysis, and the
amount of the remaining undegraded IL-1 was determined by scanning the
gels on the AMBIS radioanalytic imaging system. The data presented in
this table are representative of three separate experiments.
Table: Secretion of mature IL-1 proteins
with Arg to Glu mutations
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.