1 Department of Medical Microbiology and Immunology, University of Göteborg, S-413 46 Göteborg, 2 Department of Structural Biology, Pharmacia & UpJohn, S-112 87 Stockholm and 3 Department of Molecular Biology, Astra Hässle AB, S-431 83 Mölndal, Sweden
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: adjuvant/ADP-ribosylation/CTA1/fusion protein/protein engineering
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CT is also one of the most potent orally active adjuvants in experimental use (Elson and Dertzbaugh, 1994; Lycke, 1996
). Unfortunately, the strong inherent toxicity of CT precludes its general use as an adjuvant in human vaccine preparations. Therefore, an important issue has been whether the toxic and adjuvant properties of CT can be separated. Since the apparent biological functions of target cell recognition and binding and enzymatic activity are separated into distinct subunits, we have previously explored the possibility of dissociating the enzymatically active A1 component from the CTB targeting structure and instead added a moiety for selective targeting of CTA1 to B lymphocytes (Agren et al., 1997
). We examined the toxicity and adjuvant properties of the enzymatically active CTA1 protein devoid of the CTB component and instead genetically linked to a dimer of an Ig-binding domain (D) of Staphylococcus aureus protein A (Ljungberg et al., 1993
). In this construct, CTA1 lacked all detectable toxicity whereas the adjuvant capacity of CT was retained (Agren et al., 1997
). The production of CTA1 as a fusion to DD resulted in a molecule with several unique and distinct properties apart from B cell targeting, such as (i) ease of purification by Ig affinity chromatography (Nilsson et al., 1987
) and (ii) improved solubility in aqueous solutions. In this fusion protein we could clearly establish a close link between adjuvant activity and ADP-ribosylating activity (Agren et al., 1997
).
A particular problem with mucosal vaccine administration is to find adjuvants with sufficient water solubility to be incorporated into vaccines together with protein antigens and still maintain effective uptake across the mucosal barrier. It is important to identify strategies to develop such adjuvants. Protein engineering of adjuvant active molecules, such as the CT system, represents one such promising approach. In this work, we have further developed the concept of protein engineering to examine structurefunction relationships by a rational approach to hydrophobicity engineering using algorithms for sequencestructure compatibility and solvation energy calculations.
Surface-exposed hydrophobic amino acids with a large relative contribution to CTA1 hydrophobicity were exchanged for hydrophilic counterparts modeled for the best structural fit.
Our data show that mutant CTA1-DD fusion proteins with increased solubility, stability, maintained in vitro enzymatic activity and in vivo adjuvant activity can be constructed by a rational approach to modify surface-exposed hydrophobicity while maintaining functional integrity. However, recent studies using mutant holotoxins with reduced or lacking enzymatic activity have shown adjuvant effects of these mutants comparable to that of the intact holotoxin. This suggests that CTA1 may engage in activities that are not strictly dependent on ADP-ribosylation, but apparently still important for adjuvanticity (Douce et al., 1997; Yamamoto et al., 1997
; Giuliani et al., 1998
). One such possibility is the known interaction of CT with intracellular ADP-ribosylating factors (ARFs) and perhaps other GTP-binding proteins (Moss et al., 1994
; Sofer and Futerman, 1996
). Knowledge about the exact mechanism for interaction between CTA1 and these molecules is currently lacking. Therefore, it is important to evaluate carefully mutations engineered with the purpose of increasing solubility to ensure that biological function, such as the adjuvant effect, is not compromised (Donaldson and Klausner, 1994
; De Magistris et al., 1998
). Thus, seemingly conservative changes in amino acid sequence in a surface-exposed region of CTA1 may impinge on the biological function, albeit that the alteration is distant from the enzymatically active cleft. Further, CTA1 is thought to act in vivo by covalently modifying intracellular GTP-binding proteins, among them Gs
. This results in an increase in cAMP caused by the activation of adenylate cyclase. Recent findings (Agren et al., 1998
) suggest that CTA1-DD may act via a cAMP-independent signaling pathway possibly by the activation of G proteins other than Gs
. Mutants with retained enzymatic activity in vitro, but markedly reduced adjuvant activity were obtained in this work and these may serve as valuable tools in attempts to dissect the intracellular interactions underlying the adjuvant function of CTA1-DD.
Taken together, these studies clearly demonstrate that by carefully selecting sites for amino acid replacements in the CTA1-subunit, it is possible to improve substantially the solubility of the CTA1-DD fusion protein and allow the exploitation of these fusion proteins over a wide dose range. Even a modest increase in solubility will have profound effects on the biological function, allowing, for example, the development of novel vaccine adjuvants. In particular, this is much warranted in the mucosal vaccine field, where CTA1-DD has proved to be an effective means to separate adjuvanticity from toxicity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The approach used to select mutations was to modify amino acids that decreased surface hydrophobicity without affecting the enzymatic activity or the structure of CTA1-DD. In order to do this, the structure of CTA1 was initially modeled on the published structure of the highly homologous heat-labile enterotoxin (LT) from Escherichia coli (Sixma et al., 1991). When the structure of CT became available (Zhang et al., 1995
), the selection of amino acids was further validated by the almost identical structures of CT and LT. Mutants selected to increase the solubility of the protein were designed using solvation energy calculations (Eisenberg and McLachlan, 1986
) as implemented in INSIGHT (Biosym/MSI, San Diego, CA) and sequencestructure comparability calculations using MATCHMAKER (Tripos, St Louis, MO). Selected alterations were generally in amino acids at least 10 Å away from the active site in order to minimize the risk of interference with CTA1-DD enzymatic activity. However, two CTA1-DD variants (Ile64Ser and Leu71Ser) contained mutations in hydrophobic residues aligning the putative active site.
Construction of CTA1-DD mutants
A DNA fragment consisting of 582 bp encoding amino acids 1194 of CTA1 (Mekalanos et al., 1983) with flanking HindIII and BamHI sites was cloned into the pUC19 vector to generate CTA1-pUC19 (Agren et al., 1997
). This vector was used as a template for site-directed mutagenesis and subcloning of PCR products prior to sequencing. The different CTA1 mutations were constructed using the following oligonucleotides (nucleotide changes are underlined): Phe31His, 5'-TTG CAT GAT CAT AAA GGT TGA TAT TCA TTT GAG TAC CTC GGT CAT GGT ACT CAC TCT-3'; Ile64Ser, 5'-TTG ACC CAC TAA GTG GGC ACT TCT CAA ACT ACT TGA GGT-3'; Leu71Ser, 5'-AGA ATG GCC AGA CAA TAT AGT TTG ACC CAC TGA GTG GGC ACT-3'; Ala103Ser, 5'-CCC ACC TAG GGC AGA AAC TTC TTG TTC ATC TGG ATG AGG ACT GTA GGA CCC TAA-3'; Phe132Ser, 5'-TAC TCC CAA ATA TAT GGA TGG TAT CGA GTT CAT TCC GGT GTG CTT GAT GAA-3'; Pro185Gln, 5'-TAC TCC CAA ATA TAT GGA TGG TAT CGA GTT CAT TTT GGG-3' (forward) and 5'-ACC CGG GGA TCC CGA TGA TCT TGG AGC ATT CCC ACA ACC CTG CGG TGC ATG ATG-3' (reverse).
To generate the double mutant Phe132Ser/Pro185Gln, a fragment isolated from CTA1Pro185Gln-pUC19 at BspEIBamHI was cloned into CTA1-Phe132Ser-pUC19. Mutated CTA1-fragments were first cloned into CTA1-pUC19 for sequence confirmation followed by cloning into the expression vector pCTA1-DD as described earlier (Agren et al., 1997) at HindIIIBamHI to generate the vectors, pCTA1Phe31His-DD, pCTA1Ile64Ser-DD, pCTA1Leu71Ser-DD, pCTA1Ala103Ser-DD, pCTA1Phe132Ser-DD, pCTA1Pro185Gln-DD and pCTA1Phe132Ser/Pro185Gln-DD, respectively.
Oligonucleotides were purchased from Scandinavian Gene Synthesis (Köping, Sweden). Low-temperature melting agarose (NuSieve GTG; FMC Bioproducts, Rockland, ME) was used for preparative work and multi-purpose agarose (Boehringer Mannheim, Mannheim, Germany) for DNA analysis. PCR amplifications were performed using the DNA Thermal Cycler and Taq DNA polymerase (Perkin-Elmer, Foster City, CA). Restriction enzymes and T4 DNA ligase (Boehringer Mannheim and New England Biolabs) were used as recommended.
Expression and purification of mutant fusion proteins
For the production of mutant fusion proteins, E.coli TG-1 cells transformed with the different expression vectors were grown in 250 ml flasks overnight in 2xYT or LB, with kanamycin (50 µg/ml), at 37°C. After culture, the cells were collected by centrifugation and the fusion proteins, produced as inclusion bodies, were solubilized by treatment with 6 M guanidineHCl. After addition of distilled water to allow refolding, the fusion proteins were purified by affinity chromatography on IgG-Sepharose (Pharmacia) as described (Nilsson et al., 1987) and stored in 0.2 M HOAc at 4°C until use.
Protein analysis
A Mini-Protean system (Bio-Rad, Hercules, CA) was used for SDSPAGE as suggested by the manufacturer. A 10 µg amount of the protein material was dissolved in buffer and analyzed under reducing (5% ß-ME) conditions in 420% SDSPAGE. The gel was stained with Coomassie Brilliant Blue R-250 and the molecular weight was estimated using SDSPAGE Low Range Molecular Weight Standards (Bio-Rad). Protein concentrations were determined by the Bio-Rad DC protein assay (Bio-Rad) according to the manufacturer's instructions.
Analyses of the solubility of mutant fusion proteins were performed as follows. Proteins were diluted to 1 mg/ml in 0.2 M HOAc, pH 3.2. The pH was increased gradually to 7 by the addition of 5 M NaOH. Any precipitate that formed was removed by centrifugation for 10 min at 8000 g and the protein concentration in the supernatant was determined as described above. All experiments were performed at room temperature (22°C).
ADP-ribosyltransferase activity
Determination of enzymatic activity was performed using the NAD:agmatine assay as described earlier (Tsuji et al., 1990; Spangler, 1992
). Briefly, the ADP-ribosyltransferase activity was determined by assaying 10 µg of the different CTA1-DD mutants and assessing the ADP-ribosylagmatine formation through incorporation of [U-14C]adenine. Each sample contained 50 mM sodium phosphate (pH 7.5), 100 µM GTP, 5 mM MgCl2, 100 mM [U-14C]adenine-labeled NAD, 10 mM agmatine, 0.1 mg/ml of ovalbumin and the respective mutant fusion proteins. After 3 h at 30°C, three 50 µl samples were transferred to an AG1-X4 column which was washed four times with 1.25 ml of water. Eluates containing [U-14C]adenine-labeled ADP-ribosylagmatine were collected for determination of radioactivity. The values represent mean counts per minute (c.p.m.) of three experiments with SD <5%.
Analysis of in vivo adjuvant activity
Immunizations of C57Bl/6 female mice, aged 812 weeks (B&K Universal, Sollentuna, Sweden) were performed twice intraperitoneally at 10 day intervals (five mice per group) and the mice were killed 68 days after the final injection. Affinity-purified fusion proteins were used at 10 µg per dose. The probe antigen keyhole limpet hemocyanin (KLH) (Calbiochem, San Diego, CA) was given at 5 µg per dose together with the putative adjuvants.
ELISA analysis
Mice were bled and sera were prepared and stored at 20°C until assayed. Analysis of anti-KLH log10 titers in serum was performed by ELISA as described (Bromander et al., 1996). Briefly, polystyrene 96-well microtiter plates (Nunc) were coated with KLH at 100 µg/ml in PBS. After blocking with 0.1% BSAPBS for 30 min at 37°C, sera were added at a 1:50 dilution and serial threefold dilutions were performed in corresponding subwells. After an overnight incubation at 4°C, bound total antibodies were visualized with HRP-conjugated rabbit anti-mouse Ig antibodies (Dako) at 1:200 dilution followed by o-phenylenediamine (OPD) substrate (1 mg/ml) containing 0.04% H2O2 in citrate buffer (pH 4.5). The reaction was followed in a Titertek Multiscan spectrophotometer (Flow Laboratories, Irvine, UK). The anti-KLH titers were defined as the interpolated value giving rise to an absorbance of 0.4 above the background. The mice were analyzed individually and specific log10 titers were expressed as means ± SD of five mice per group.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Finally, we analyzed the in vivo capacity of the different mutants to augment the immune response to an unrelated antigen (KLH) by mixing the putative adjuvants with KLH followed by intraperitoneal immunization of mice. The Phe132Ser analogue which demonstrated a 50% increase in solubility fully maintained wild-type stability and in vitro and in vivo activity (Table I). However, this was not a general finding since the two most soluble mutants, CTA1Pro185Gln-DD and CTA1Phe132Ser/Pro185Gln-DD, gave lower enhancement of anti-KLH titers as compared with CTA1-DD (Table I
). By contrast, the less soluble CTA1-DD analogues Ala103Ser, Ile64Ser and Leu71Ser all demonstrated good adjuvant activity (Table I
), suggesting that Ala103, Ile64 and Leu71 are not critical for the biological activity of CTA1-DD. By contrast, Phe31His had reduced adjuvant activity, indicating a significant role for Phe31 in this respect.
To conclude, we have shown that it is possible to introduce mutations in CTA1-DD which increase water solubility without loss of enzymatic, stability or adjuvant properties. However, none of the mutations resulted in a compound with stronger biological effects than native CTA1-DD. In particular, a direct relationship between solubility and adjuvant activity in vivo was not found following intraperitoneal administration. Nonetheless, it is possible that for other applications such as incorporation into vaccine adjuvant formulations or given by alternative routes, e.g. intranasally or orally, the mutations with increased solubility may provide a stronger adjuvant effect than the original CTA1-DD fusion protein.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two of the initial amino acid alterations of CTA1-DD (Pro185Gln; Phe132Ser) resulted in analogues with increased water solubility at neutral pH and in vitro enzymatic properties similar to those of wild type CTA1-DD. A combined mutant showed further enhanced solubility, demonstrating an additive effect of the two mutations. However, two out of three mutants with significantly improved water solubility produced in this work showed a dramatically reduced capacity to act as adjuvants in vivo. Both of these contained the Pro185Gln alteration, suggesting an important role of Pro185 for the biological function of CTA1-DD. Alteration of Phe31 also resulted in an analogue with dramatically reduced biological activity. Interestingly, the only mutations that resulted in loss of adjuvant activity, Pro185 and Phe31, are both positioned in close vicinity distal to the active site of CTA1 (Figure 1). This suggests a possibility that they comprise part of a structure which is essential for biological activity. Of the different mutants analyzed in this work, only one analogue, Phe132Ser, was more soluble than wild-type CTA1-DD and still retained full stability and enzymatic and adjuvant activity. This finding clearly demonstrates the feasibility of introducing amino acid alterations to enhance solubility properties without affecting the major biological activity of CTA1-DD. Also, our results suggest that the fine specificity of target protein ADP-ribosylation by CTA1-DD may be dependent on interactions via different, yet undefined, regions of the molecule. As demonstrated earlier, CTA1 can ADP-ribosylate a number of different target proteins (Spangler, 1992
; Burnette et al., 1995
; Francis et al., 1995
). The specificity of this activity is poorly understood. However, based on the present findings, it seems feasible that regions of CTA1 outside the active site are involved in regulating this activity by interaction with different intracellular components. Burnette et al. (1995) have previously shown that substitution of His70 resulted in a qualitatively distinct type of activity loss with a markedly reduced ADP-ribosylation of Gs
. Their interpretation of this finding was that His70 constituted part of a structure involved in specific binding of CTA1 to its substrate Gs
. We believe that the specificity of the ADP-ribosylation of cellular target proteins is linked to the adjuvant activity of the CTA1-DD molecule. If so, alterations in parts of CTA1-DD which affect the fine specificity of the ADP-ribosylation by CTA1 will have an impact on adjuvant activity. Our findings point to that possibility, since mutation of Pro185 or Phe31 but not Phe132 influenced the adjuvant activity in such a manner. In addition, other properties, such as biological half-life, cellular uptake, compartmentalization or intracellular enzymatic activity of CTA1-DD, can also affect the adjuvant function of the Pro185Gln or the Phe31His analogue. Nevertheless, to address these issues, our panel of CTA1-DD mutants will be further analyzed with respect to changes in the pattern of target protein ADP-ribosylation using whole cell membranes. This may provide tools for dissecting the biological activities underlying the powerful adjuvant properties of CTA1-DD and may aid in the efforts to construct more efficient and stable adjuvants for use in, e.g., vaccine development.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agren,L., Lowenadler,B. and Lycke,N. (1998). Immunol. Cell Biol., 76, 280.[ISI][Medline]
Bromander,A.K., Ekman,L., Kopf,M., Nedrud,J.G. and Lycke,N.Y. (1996). J. Immunol., 156, 4290.[Abstract]
Burnette,W.N., Kaslow,H.R. and Moss,J. (1995). Adv. Exp. Med. Biol., 2, 1513.
De Magistris,M.T., Pizza,M., Douce,G., Ghiara,P., Dougan,G. and Rappuoli,R. (1998). Dev. Biol. Stand., 92, 123.[ISI][Medline]
Domerighini,M., Montecucco,C., Ripka,W.C. and Rappuoli,R. (1991). Mol. Biol., 5, 23.
Donaldson,J.G. and Klausner,R.D. (1994). Curr. Opin. Cell Biol., 6, 527.[ISI][Medline]
Douce,G., Fontana,M., Pizza,M., Rappuoli,R. and Dougan,G. (1997). Infect. Immun., 65, 2821.[Abstract]
Eisenberg,D. and McLachlan,A.D. (1986). Nature, 319, 199.[ISI][Medline]
Elson,C.O. and Dertzbaugh,M.T. (1994). In Ogra,P.L., Mestecky,J., Lamm,M.E., Strober,W., McGhee R. and Bienenstock, J. (eds), Handbook of Mucosal Immunology. San Diego, p. 391.
Francis,M.L., Okazaki,I., Moss,J., Kurosky,A., Pecanha,L.M. and Mond,J.J. (1995). J. Immunol., 154, 4956.
Giuliani,M.M., Del Giudice,G., Giannelli,V., Dougan,G., Douce,G., Rappuoli,R. and Pizza,M. (1998). J. Exp. Med., 187, 1123.
Levine,M.M., Kaper,J.B., Black,R.E. and Clements,M.L. (1983). Microbiol. Rev., 47, 510.[ISI]
Ljungberg,U.K., Jansson,B., Niss,U., Nilsson,R., Sandberg,B.E. and Nilsson,B. (1993). Mol. Immunol., 30, 1279.[ISI][Medline]
Lycke,N. (1996). In Kagnoff,M. and Kiyono,H. (eds), Essentials in Mucosal Immunology. Academic Press, San Diego, p. 563.
Mekalanos,J.J., Swartz,D.J., Pearson,G., Harford,N., Groyne,F. and Wilde,M. (1983). Nature, 306, 551.[ISI][Medline]
Merritt,E.A., Sarfaty,S., van den Akker,F., L'Hoir,C., Martial,J.A. and Hol,W.G.J. (1994). Protein Sci., 3, 166.
Moss,J., Tsai,S.C. and Vaughan,M. (1994). Methods Enzymol., 235, 640.[ISI][Medline]
Nilsson,B., Moks,T., Jansson,B., Abrahamsén,L., Elmblad,A., Holmgren,E., Henrichson,C., Jones,A.T. and Uhlén,M. (1987). Protein Engng, 1, 107.[Abstract]
Sixma,T.K., Pronk,S.E., et al. (1991). Nature 351, 371.[ISI][Medline]
Sixma,T.K., Kalk,K.H., van Zanten,B.A., Dauter,Z., Kingma,J., Witholt,B. and Hol,W.G. (1993). J. Mol. Biol., 230, 890.[ISI][Medline]
Sofer,A. and Futerman,A.H. (1996). J. Neurochem., 67, 2134.[ISI][Medline]
Spangler,B.D. (1992). Microbiol. Rev., 56, 622.[Abstract]
Tsuji,T., Inoue,T., Miyama,A., Okamoto,K., Honda,T. and Miwatani,T. (1990). J. Biol. Chem., 265, 22520.
Yamamoto,S., Takeda,Y., Yamamoto,M., Kurazono,H., Imaoka,K., Yamamoto,M., Fujihashi,K., Noda,M., Kiyono,H. and McGhee,J.R. (1997). J. Exp. Med., 185, 1203.
Zhang,R.G., Scott,D.L., Westbrook,M.L., Nance,S., Spangler,B.D., Shipley,G.G. and Westbrook,E.M. (1995). J. Mol. Biol., 251, 563.[ISI][Medline]
Received November 24, 1997; revised October 21, 1998; accepted October 27, 1998.