©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-1 Secretion
A POSSIBLE MULTISTEP PROCESS THAT IS REGULATED IN A CELL TYPE-SPECIFIC MANNER (*)

William M. Siders , Steven B. Mizel (§)

From the (1)Department of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In a prior study, we found that the processed form of human interleukin-1 (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.


INTRODUCTION

Interleukin-1 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.


MATERIALS AND METHODS

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.

The lysine to glutamic acid mutations, matIL-1 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 Secretion

Radiolabeling of cells and immunoprecipitation were performed as described previously(6) . Briefly, cells were radiolabeled with 200 µCi of [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.


RESULTS

Determination of the Precursor IL-1 Region That Impairs Secretion of the Protein by P388D1 Cells

Previous experiments demonstrated that precursor IL-1 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.

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 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 Region That Inhibits Secretion of the Protein by Jurkat Cells

The ability to secrete the mature forms of IL-1 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 Protein by P388D1 Cells

In a prior study, we found that mature IL-1 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 Protein by Jurkat Cells

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 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 Mutant Proteins

Since the secretion of IL-1 may be conformation-dependent, it was possible that the decrease in secretion of the mature IL-1 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 on Secretion by P388D1 Cells

Because mutations in charged amino acids decreased secretion of the mutant mature IL-1 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.

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 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 by Jurkat Cells

Since the K204G, K209L, K219S, and E221S mutant IL-1 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.


DISCUSSION

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 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).

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 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.

In contrast to the results with P388D1 cells, the mutations in charged amino acids in mature IL-1 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.

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 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.

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, 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

P388D1 or Jurkat cells were transfected with the indicated IL-1 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

P388D1 or Jurkat cells were transfected with the indicated mature IL-1 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

Each of the mature IL-1 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

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.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI25836 and National Institutes of Health Training Grant AI07401 (to W. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 910-716-4471; Fax: 910-716-9928.

The abbreviations used are: IL, interleukin; PCR, polymerase chain reaction; RSV, Rous sarcoma virus.

U. Kavita and S. B. Mizel, unpublished observations.

W. M. Siders and S. B. Mizel, unpublished observations.


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