Proc. Natl. Acad. Sci. USA Vol. 76, No. 8, pp. 3717-3721, August 1979
Biochemistry
Human leukocyte interferon: Relationship between molecular structure and species specificity [antiviral activity/affinity chromatography/poly(I)/poly(U)J
M. N. THANG*, D. C. THANG*, M. K. CHELBI ALIX*, B. ROBERT-GALLIOTt, M. J. COMMOY-CHEVALIERt, AND C. CHANYt *Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France; and tlnstitut National de la Sante et de la Recherche M6dicale,
U43, H6pital Saint-Vincent-de-Paul, 74 boulevard Denfert-Rochereau, 75014, Paris, France
Communicated by Irwin C. Gunsalus, May 11, 1979
Human leukocyte interferon can be separated ABSTRACT into two classes of subspecies by polynucleotide-agarose affinity chromatography; 30-40% of the molecular species have the polynucleotide-binding property and 60-70% lack affinity for the polynucleotide ligand. When analyzed on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the former class of interferon has a slower mobility corresponding to the migration of a polypeptide of 21,000 daltons, while the latter class has a faster mobility corresponding to a polypeptide of 13,500-15,000 daltons. By analogy to the behavior of other interferons and a class of nucleotidyl transferases on the polynucleotide-agarose chromatography, we suggest that the human leukocyte interferon having the polynucleotide-binding site is in a possibly "native" conformation and the loss of affinity for polynucleotide results from a degradative alteration of the native molecules. Moreover, the alteration of interferon is accompanied by an increase in heterospecific activity on bovine cells. It is suggested that the polypeptide domain responsible for species specificity may be closely related to the polynucleotide binding area. The modified interferon molecule, however, still conserves its antiviral activity. The simplicity and the high capacity of polynucleotide-agarose chromatography make this a powerful technique for the purification of interferon. The easy separation of these two classes of human leukocyte interferon makes the purification procedures more rational and will facilitate the preparation of both subspecies to a high degree of molecular
homogeneity. Human leukocyte interferon (Hu Le IF) contains two subpopulations of active molecules, which are differentiated by their molecular sizes, charge properties, and even biological activities (1-6). Such heterogeneity, resulting from the presence of molecular species having different physicochemical properties, complicates the purification of leukocyte interferon. In particular, the strategy for purification has been mostly orientated by the recovery of as much initial activity as possible, necessitating copurification of the two subpopulations. We have recently described an affinity chromatography with blue dextran or polynucleotide as ligand for proteins interacting with nucleic acids (7). Such chromatography not only allows the separation of proteins having a polynucleotide-binding site from other classes of proteins but also can be used to separate one native enzyme from a degraded enzyme that has lost the polynucleotide attachment site (7). The methodology was also applied to the characterization and purification of both viral and immune interferons (8-11). We postulate that the polynucleotide-binding property, observed with mouse types I and II and human interferons, could be a basic characteristic of this The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
antiviral protein. This property also could reveal a structural homology between the mouse types I and II interferons (10). In this paper, we show that Hu Le IF can be separated into two classes of molecules according to their affinity or lack of affinity for the polynucleotide ligand. These two classes correspond to the two subpopulations distinguished by their apparent molecular weights. MATERIALS AND METHODS Chemicals were obtained from the following sources: polyribonucleotides and polynucleotide-agaroses, Choay Laboratories (Paris); blue dextran CNBr-activated Sepharose 4B, Pharmacia; polyacrylamide gel electrophoresis products: International Laboratory, B. V. Lekkerkerk, Holland. Affinity Chromatography. Polynucleotide-agarose column: agarose beads with poly(I) or poly(U) linked covalently were packed into a sterile polystyrene syringe. The column was extensively washed with 1 M NaCl, then equilibrated with 50 mM Tris-HCI, pH 7.5. Blue dextran Sepharose column: blue dextran was covalently bound to CNBr-activated Sepharose by the technique of Ryan and Vestling (12). The blue dextran Sepharose used in this study had approximately 12 mg of blue dextran per g of dry Sepharose. It was extensively washed with 3 M KCI, then equilibrated with 50 mM Tris-HCl buffer, pH 7.5. The gel was packed into a polystyrene column. To both types of column, the interferon preparations diluted 1:3 with 10 mM Tris-HCI, pH 7.5, were applied in phosphatebuffered saline. The column was then washed with 5-10 bed volumes of a 10 mM Tris-HCI buffer (pH 7.5) before elution with a gradient of NaCl (0-1.0 M) or 1 M NaCl in the same Tris buffer. Cells and Viruses. For all experiments human foreskin fibroblasts (F7000 cells, Flow Laboratories, McLean, VA) were used. They were propagated in Eagle's basal medium supplemented with 10% heat-inactivated fetal calf serum. Bovine MDBK cells and mouse L929 cells were routinely propagated in the laboratory in Eagle's minimal essential medium plus 10% newborn calf serum. For maintenance, the serum concentration was reduced to 5%. Vesicular stomatitis virus, Indiana strain, was maintained in the laboratory in 929 cells. The virus titer is expressed in plaque-forming units/0.5 ml. Interferon Production and Purification. Hu Le IF was produced by challenge of leukocytes with Sendai virus, following a techique described by Falcoff et al. (13) and modified Abbreviations: Hu Le IF, human leukocyte interferon; NaDodSO4, sodium dodecyl sulfate.,
3717
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Biochemistry: Thang et al.
by Cantell et al. (14). However, leukocytes were collected in a continuous flow centrifuge (Elecrem) and scraped off the blades after centrifugation, instead of from buffy coats. The purification was performed according to Cantell and Hirvonen (15) by successive precipitations in ethanol at increasing pH. The preparations used have a specific activity of 2-5 X 106 reference units/mg protein. Interferon Titration. Hu Le IF was titrated by using human foreskin fibroblasts. For estimation of the interferon titer, 50% inhibition of the cytopathic effect was employed. For more precise determination, the interferon titer was expressed as 90% inhibition of the yield of challenge vesicular stomatitis virus. All interferon titers were expressed in reference units. Interferon Neutralization. Anti-human FS-4 interferon
globulin was kindly provided by J. K. Dunnick. The neutralizing titer, when tested against human fibroblast interferon at 10 reference units/ml, was 1024. Anti-Hu Le IF globulin was a gift of K. Paucker. The neutralizing titer, when tested against Hu Le IF at 8 reference units/ml, was 1280. Sodium Dodecyl Sulfate (NaDodSO4)/Polyacrylamide Gel Electrophoresis. Polyacrylamide slab gels (15%) were used. Gel polymerization and electrophoresis were performed using the Laemmli system (16) except that 2-mercaptoethanol was omitted. Interferon samples were dialyzed against the electrode buffer. Samples were applied to wells distant enough one from the other to avoid any accidental contamination. At the end of electrophoresis, each track was cut off and sliced into 2.5-mm portions. Interferon activity was eluted in culture medium containing 5% fetal bovine serum at 4VC for 18 hr. Protein Determination. The protein contents in dilute concentrations were determined by the micromethod described by Schaffner and Weissmann (17). The proteins were quantitatively precipitated on a nitrocellulose filter, then stained with acidic dye amidoschwarz. After washing, the dye retained on the membrane filter was eluted by 25 mM NaOH in 50% ethanol solution containing 0.05 mM EDTA. The absorbance of the eluate was measured at 630 nm against eluant. The protein contents were calculated from a calibrated curve established with bovine serum albumin as standard. RESULTS Separation of Two Molecular Forms by PolynucleotideAgarose Affinity Chromatography. When a preparation of Hu Le IF was chromatographed on poly(I)-agarose or poly(U)-agarose, the antiviral activity was distributed between wash and eluted fractions. The proportion of these two parts of the activity varied from one batch to another. The profile of a typical preparation is shown in Fig. 1. About two-thirds of the product with interferon activity was found in the flowthrough and wash fractions, while the bound material with about onethird of the initial activity was eluted at around 0.3 M NaCl. A similar distribution was also observed with crude interferon. These results contrast with those obtained with mouse viral or immune interferons (9, 10) under identical conditions; in this case, all the antiviral activity was retained. The lack of binding was not due to a saturation of the column by proteins. Indeed, when the E1 fraction was chromatographed again on a poly(I)-agarose column, the activity was recovered completely in flowthrough and wash fractions. No trace of interferon was retained on the column. Similarly, the retained fraction E2, rechromatographed on a poly(I)-agarose column, had all-its activity bound to the polynucleotide and was eluted-by salt at the same molarity. Hu Le IF preparations are known to contain a minor species of interferon (about 1% of the total activity) antigenically related to human fibroblast interferon (18-20). To ascertain that
Proc. Natl. Acad. Sci. USA 76 (1979)
E-6
C
2-
10~~~~~~~~~~~~~.
13
3 5 7 9 Fraction FIG. 1. Chromatography of Hu Le IF on poly(I)-agarose. A sample of Hu Le IF (9 X 106 units in 3 ml of phosphate-buffered saline) was diluted in 6 ml of 10 mM Tris, pH 7.5. The total volume was loaded on a poly(I)-agarose column (2-ml bed volume) at a flow rate of 5 ml/hr. The column was washed (E1) with 6 ml of Tris buffer into 10 fractions then eluted (E2) by a linear gradient of NaCl formed by mixing 3 ml of 10 mM Tris buffer and 3 ml of 1 M NaCl in the same Tris buffer. The activity was assayed on human fibroblasts. One-third of the initial-activity was found in the flowthrough volume, 25% in the wash, and 38% in the eluted fractions. The antiviral activity on bovine 5
7
1
MDBK cells, assayed simultaneously, is represented by the broken lines.
the two classes of Hu Le IF differentiated by their affinity for a polynucleotide ligand are both of the leukocyte type, comparative studies were performed with antisera prepared with either leukocyte or fibroblast interferon. The results obtained showed, as expected, that both classes of Hu Le IF were completely neutralized by anti-Hu Le IF globulin, whereas the anti-human fibroblast interferon globulin had no effect. Characterization by NaDodSO4/Polyacrylamide Gel Electrophoresis. These two classes of interferon were further analyzed by electrophoresis on polyacrylamide gels in the presence of NaDodSO4. The interferon activity profiles (Fig. 2) revealed that each class was mainly composed of one subpopulation of Hu Le IF with a characteristic apparent molecular weight. In fact, interferon having the polynucleotidebinding site migrated to the position corresponding to a polypeptide of 21,500 daltons (Fig. 2A) and interferon without the polynucleotide-binding property migrated as a polypeptide of lower molecular weight (Fig. 2B). Thus, these two classes of Hu Le IF correspond apparently to the two subspecies Les and Lef (2, 6). However, it is clear that each class contained molecules of intermediate sizes. This explains why the initial sample of Hu Le IF before separation by polynucleotide-agarose chromatography had an antiviral activity profile (Fig. 2C) spread
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Thang et al. A
Chn Tr I Cyt ran
2
1
fl2 '96 I II r2.-F.hi :
-
4
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m
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._W
9
c0.4 20
8
7
~~~B
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x
.t 0.893 i
6
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4
5
6
8
7
5 7 6 Migration distance,
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FIG. 2. Electrophoresis of the two classes of Hu Le IF separated by poly(I) agarose chromatography. NaDodSO4/polyacrylamide (15%) gel electrophoresis was performed as described in Materials and Methods. The Laemmli system (16) was used in this experiment. Aliquots of the peak fraction of the polynucleotide-binding species (A) and of the wash fraction from the poly(I)-agarose column (B) were compared. (C) Initial Hu Le IF material before chromatography. The antiviral activity was assayed comparatively on human fibroblasts (solid lines) and on bovine MDBK cells (broken lines). The activity is expressed as units per fraction of gel. For the polynucleotidebinding species (A), 12,000 units, as measured on both human and MDBK cells, was applied. About 5800 units on human fibroblasts (about 50%o recovery) and 2800 units on bovine cells (about 24% recovery) were recovered. For the unbound interferon species (B), 3000 units on human fibroblasts and 9000 units on MDBK were applied. The recoveries were 600 units on human fibroblasts (20%/) and 1900 units on MDBK (21%). For the initial material (C), about 60,000 units for both cell lines was applied. The recoveries were 8000 units on human fibroblasts ( 14%) and 14,000 units on MDBK (24%). The migration positions of protein markers are indicated by arrows: Chn, chymotrypsinogen (25,000 daltons); Tr I, trypsin inhibitor from soybean (21,500 daltons); and Cyt, cytochrome c (13,500 daltons).
3719
over a region of the gel corresponding to molecular weights from 13,000 to 25,000 daltons, with two peaks of activity located at 21,000 and 15,000 daltons. Species Specificity. The relationship between molecular size and the polynucleotide-binding property would suggest a correlation between the structure of these species and their activities. In fact, when the antiviral activity was assayed comparatively on human and bovine cells, it was found constantly that the activity of the slow mobility species on human fibroblasts was several times higher than that observed with bovine MDBK cells (broken line in Fig. 2A). In the case of the species of high mobility (Fig. 2B), on the other hand, the ratio of the activities was approximately reversed. In particular, at a position corresponding to the migration of cytochrome c, the activity was almost exclusively towards bovine cells. The relative heterospecific activities were also found with the initial sample (Fig. 2C). A similar comparison could be made with the two populations separated on poly(I)-agarose. However, the differences are sometimes less pronounced because each class of molecules is not wholly homogeneous. Purification. The methodology described here may be useful for the purification of Hu Le IF. In fact, it is more rational to separate these two species and to adapt suitable methods to purify each class of Hu Le IF. Obviously, affinity chromatography is the best technique for this from the point of view both of capacity and specificity. In the case of Hu Le IF studied here, most of the preparations contained mainly one dominant contaminating protein which migrated on NaDodSO4/polyacrylamide gels as a polypeptide of 25,000 daltons, and one minor contaminant, serum albumin. Another polypeptide of 14,000 daltons was found in some preparations. Of course, other polypeptides are present in various but low concentrations according to the batch. On poly(I)-Sepharose chromatography, the human serum albumin bound partially to the polynucleotide while the 25,000-dalton and 14,000-dalton polypeptides passed through. Thus human serum albumin was the major contaminant in the Hu Le IFS (slow) fraction after chromatography and the 25,000-dalton protein was the main contaminant in the Hu Le IFf (fast) fraction. Combination of poly(I)-agarose and anti-human serum globulin-agarose leads to a purification of Hu Le IFS to a specific activity of about 108 units/mg of protein. Behavior on Blue Dextran Sepharose. These classes of Hu Le IF could also be separated by affinity chromatography on blue dextran Sepharose, which mimics a polynucleotide ligand as we have postulated (7). In Fig. 3 we show the behavior of Hu Le IF on such a column. The retained activity was eluted by poly(I) at a concentration of A248 = 2. This suggests that this fraction of Hu Le IF has the polynucleotide-binding site similar to the fraction of Hu Le IF, retained on polynucleotide agarose. Preliminary analysis of this poly(I)-eluted material by electrophoresis on NaDodSO4/polyacrylamide gels showed a population of Hu Le IF mainly composed of a 21,000-dalton species.
DISCUSSION The heterogeneity of Hu Le IF is a major difficulty for purification. In addition, because different molecular species are present in Hu Le IF preparations, studies of the relationship between the structure and the function of this glycoprotein (which possesses multiple biological activities) are problematic. The reasons for the presence of two main subpopulations in Hu Le IF have been investigated by several laboratories. Essentially, different degrees of glucosylation of the polypeptide backbone (21, 22) or proteolytic cleavage (3, 5) have been considered. In this study we show that these two subspecies can
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Biochemistry: Thang et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
C
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0
50
1
-
1
3
5
7 9 1 Fraction
3
5
7
FIG. 3. Chromatography of Hu Le IF on blue dextran agarose. A sample of Hu Le IF (5 X 105 units in 0.350 ml of phosphate-buffered saline) was diluted with 0.8 ml of 10 mM Tris-HCl, pH 7.5. The diluted
sample was loaded on a blue dextran agarose column (0.5 ml bed volume). The column was first washed with 3.5 ml 10 mM Tris, then eluted with 3 ml of poly(I) at A24g = 2.0. The activity was assayed on MDBK cells. The recovery was nearly complete: about 8 X 104 units (16% of input activity) in the flowthrough fraction, 3 X 105 units (60%o) in Tris washing fractions, and 6 X 104 units (12%) in poly(I)-eluted fractions.
be recognized by their different binding property to a polynucleotide. By analogy to mouse interferons, it is tempting to equate the polynucleotide-binding molecules with a native (or quasinative) form of the Hu Le IF. Consequently, the other class of molecules, devoid of the polynucleotide-binding property, would be considered as a degraded species having lost the polypeptide domain that interacts with nucleic acids. We have previously suggested (10) that an enzymatic or chemical alteration could explain why some mouse immune interferon preparations have an elution profile from poly(I)Sepharose columns shifted towards lower ionic strength. This kind of alteration of mouse viral interferon, which exists also in two molecular species (11), might be present, because we have succeeded in separating these two subpopulations according to their differential affinity to various polynucleotides (unpublished results). More important is the fact that the antiviral activity of Hu Le IF could be dissociated from the property of polynucleotide binding, whereas species specificity seems to be closely related to the presence or absence of the polynucleotide-binding site.
Havell et al. (6) have also obtained different ratios of antiviral activity on human fibroblast versus bovine cells with Hu Le IF species separated by isoelectric focusing. However, these authors considered that the values of the ratios are similar. It has also been mentioned (21) that a subspecies of Hu Le IF obtained after periodate treatment and separated on NaDodSO4/polyacrylamide gel at a position of 13,500 daltons was active on cat cells but practically devoid of activity on human cells. Stewart et al. (21) suggested that the conversion of Hu Le IF to a low molecular weight species by cleavage of carbohydrate from interferon eliminates its activity for homologous cells. We have succeeded in modifying the ratio of antiviral activity on human fibroblast versus bovine cells of the poly(I)-bound Hu Le IF species that has been previously treated by limited proteolysis (unpublished results). This suggests that proteolytic cleavage of the 21,000-dalton species could also be one of the origins for the genesis of the fast mobility species. Gresser et al. (23) reported that Hu Le IF exhibits an antiviral activity on bovine cells much higher than that on human fibroblasts. This might indicate that the preparations used had a higher proportion of the modified species of Hu Le IF. We postulate that interferon, like many hormones and other multifunctional enzymes, has distinct domains assigned for separated functions: antiviral activity, species specificity, immune response, etc. The loss of the polynucleotide-binding area could indicate the alteration of the domain recognized by the cell receptor(s) (24) for species restriction while the "core" molecule remains active for induction of the antiviral process. One question we are asking is whether part of this core polypeptide of interferon could be conserved during evolution. When this article was submitted, we learned that Rubinstein et al. (25) had succeeded in separating the subspecies of Hu Le IF by high performance liquid partition chromatography and in purifying one species of 17,500 daltons. We thank Christine Nicoletta for her valuable technical assistance and the Centre National de Transfusion Sanguine (Orsay, France) for the leukocytes. This work was supported by the Centre National de la Recherche Scientifique (G.R. 18), the Delegation Generale a la Recherche Scientifique et Technique (Convention GBM 78.7.0693), the Institut National de la Sant6 et de la Recherche Medicale (U.43 and CRL 77.04.0741), and the Fondation pour la Recherche Medicale
Frangaise. 1. Fantes, K. H. (1970) in l'Interferon, Colloque No 6 de l'INSERM (INSERM, Paris), pp.181-186. 2. Stewart, W. E., II & Desmyter, J. (1975) Virology 67,68-73. 3. Torma, E. T. & Paucker, K. (1976) J. Biol. Chem. 251, 48104816. 4. Chen, J. K., Jankowski, W. J., O'Malley, J. A., Sulkowski, E. & Carter, W. A. (1976) J. Virol. 19,425-434. 5. Chadha, K. C., Sclair, M., Sulkowski, E. & Carter, W. A. (1978) Biochemistry 17, 196-200. 6. Havell, E. A., Yip, Y. K. & Vilcek, J. (1977) Arch. Virol. 55, 121-129. 7. Drocourt, J. L., Thang, D. C. & Thang, M. N. (1978) Eur. J. Biochem. 82, 355-362. 8. Thang, M. N., de Maeyer-Guignard, J. & de Maeyer, E. (1977) FEBS Lett. 80,365-370. 9. de Maeyer-Guignard, J., Thang, M. N. & de Maeyer, E. (1977) Proc. Natl. Acad. Sci. USA 74,3787-3790. 10. Wietzerbin, J., Stefanos, S., Lucero, M., Falcoff, E., Thang, D. C. & Thang, M. N. (1978) Biochem. Biophys. Res. Commun. 85, 480-489. 11. de Maeyer-Guignard, J., Tovey, M. G., Gresser, I. & de Maeyer, E. (1978) Nature (London) 271, 622-625. 12. Ryan, L. C. & Vestling, C. S. (1974) Arch. Biochem. Biophys. 160, 279-284.
Biochemistry: Thang et al. 13. Falcoff, E., Falcoff, R., Fournier, F. & Chany, C. (1966) Ann. Inst. Pasteur 111, 562-584. 14. Cantell, K., Hirvonen, S., Mogensen, K. E. & Pyhala, L. (1974) in The Production and Use ofInterferon for the Treatment and Prevention of Human Virus Infections, In Vitro, Monograph, ed. Waymouth, C. (Tissue Culture Association, Rockville, MD), Vol. 3, pp. 35-38. 15. Cantell, K. & Hirvonen, S. (1978) J. Gen. Virol. 39,541-543. 16. Laemmli, U. K. (1970) Nature (London) 227,680-685. 17. Schaffner, W. & Weissmann, C. (1973) Anal. Biochem. 56, 502-514. 18. Anfinsen, C. B., Bose, S., Corley, L. & Gurari-Rotman, D. (1974) Proc. Natl. Acad. Sci. USA 71, 3139-3142. 19. Berg, K., Ogburn, C. A. & Paucker, W. A. (1975) J. Immunol. 114,640-644.
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20. Havell, E. A., Berman, B., Ogburn, C. A., Paucker, K. & Vilcek, J. (1975) Proc. Natl. Acad. Sci. USA 72, 2185-2187. 21. Stewart, W. E., II, Lin, L. S., Wiranowska-Stewart, M. & Cantell, K. (1977) Proc. Natl. Acad. Sci. USA 74,4200-4204. 22. Havell, E. A., Yamazaki, S. & Vilcek, J. (1977) J. Biol. Chem. 252, 4425-4427. 23. Gresser, I., Bandu, M. T., Brouty-Boye, D. & Tovey, M. (1974) Nature (London) 251, 543-545. 24. Chany, C., Gregoire, A., Vignal, M., Lemaitre-Montcuit, J., Brown, P., Besanqon, F., Suarez, H. & Cassingena, R. (1973) Proc. Natl. Acad. Sci. USA 70,557-561. 25. Rubinstein, M., Rubinstein, S., Familletti, P. C., Miller, R. S., Waldman, A. A. & Pestka, S. (1979) Proc. Natl. Acad. Sci. USA 76,640-644.
Biochemistry
Human leukocyte interferon: Relationship between molecular structure and species specificity [antiviral activity/affinity chromatography/poly(I)/poly(U)J
M. N. THANG*, D. C. THANG*, M. K. CHELBI ALIX*, B. ROBERT-GALLIOTt, M. J. COMMOY-CHEVALIERt, AND C. CHANYt *Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France; and tlnstitut National de la Sante et de la Recherche M6dicale,
U43, H6pital Saint-Vincent-de-Paul, 74 boulevard Denfert-Rochereau, 75014, Paris, France
Communicated by Irwin C. Gunsalus, May 11, 1979
Human leukocyte interferon can be separated ABSTRACT into two classes of subspecies by polynucleotide-agarose affinity chromatography; 30-40% of the molecular species have the polynucleotide-binding property and 60-70% lack affinity for the polynucleotide ligand. When analyzed on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the former class of interferon has a slower mobility corresponding to the migration of a polypeptide of 21,000 daltons, while the latter class has a faster mobility corresponding to a polypeptide of 13,500-15,000 daltons. By analogy to the behavior of other interferons and a class of nucleotidyl transferases on the polynucleotide-agarose chromatography, we suggest that the human leukocyte interferon having the polynucleotide-binding site is in a possibly "native" conformation and the loss of affinity for polynucleotide results from a degradative alteration of the native molecules. Moreover, the alteration of interferon is accompanied by an increase in heterospecific activity on bovine cells. It is suggested that the polypeptide domain responsible for species specificity may be closely related to the polynucleotide binding area. The modified interferon molecule, however, still conserves its antiviral activity. The simplicity and the high capacity of polynucleotide-agarose chromatography make this a powerful technique for the purification of interferon. The easy separation of these two classes of human leukocyte interferon makes the purification procedures more rational and will facilitate the preparation of both subspecies to a high degree of molecular
homogeneity. Human leukocyte interferon (Hu Le IF) contains two subpopulations of active molecules, which are differentiated by their molecular sizes, charge properties, and even biological activities (1-6). Such heterogeneity, resulting from the presence of molecular species having different physicochemical properties, complicates the purification of leukocyte interferon. In particular, the strategy for purification has been mostly orientated by the recovery of as much initial activity as possible, necessitating copurification of the two subpopulations. We have recently described an affinity chromatography with blue dextran or polynucleotide as ligand for proteins interacting with nucleic acids (7). Such chromatography not only allows the separation of proteins having a polynucleotide-binding site from other classes of proteins but also can be used to separate one native enzyme from a degraded enzyme that has lost the polynucleotide attachment site (7). The methodology was also applied to the characterization and purification of both viral and immune interferons (8-11). We postulate that the polynucleotide-binding property, observed with mouse types I and II and human interferons, could be a basic characteristic of this The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
antiviral protein. This property also could reveal a structural homology between the mouse types I and II interferons (10). In this paper, we show that Hu Le IF can be separated into two classes of molecules according to their affinity or lack of affinity for the polynucleotide ligand. These two classes correspond to the two subpopulations distinguished by their apparent molecular weights. MATERIALS AND METHODS Chemicals were obtained from the following sources: polyribonucleotides and polynucleotide-agaroses, Choay Laboratories (Paris); blue dextran CNBr-activated Sepharose 4B, Pharmacia; polyacrylamide gel electrophoresis products: International Laboratory, B. V. Lekkerkerk, Holland. Affinity Chromatography. Polynucleotide-agarose column: agarose beads with poly(I) or poly(U) linked covalently were packed into a sterile polystyrene syringe. The column was extensively washed with 1 M NaCl, then equilibrated with 50 mM Tris-HCI, pH 7.5. Blue dextran Sepharose column: blue dextran was covalently bound to CNBr-activated Sepharose by the technique of Ryan and Vestling (12). The blue dextran Sepharose used in this study had approximately 12 mg of blue dextran per g of dry Sepharose. It was extensively washed with 3 M KCI, then equilibrated with 50 mM Tris-HCl buffer, pH 7.5. The gel was packed into a polystyrene column. To both types of column, the interferon preparations diluted 1:3 with 10 mM Tris-HCI, pH 7.5, were applied in phosphatebuffered saline. The column was then washed with 5-10 bed volumes of a 10 mM Tris-HCI buffer (pH 7.5) before elution with a gradient of NaCl (0-1.0 M) or 1 M NaCl in the same Tris buffer. Cells and Viruses. For all experiments human foreskin fibroblasts (F7000 cells, Flow Laboratories, McLean, VA) were used. They were propagated in Eagle's basal medium supplemented with 10% heat-inactivated fetal calf serum. Bovine MDBK cells and mouse L929 cells were routinely propagated in the laboratory in Eagle's minimal essential medium plus 10% newborn calf serum. For maintenance, the serum concentration was reduced to 5%. Vesicular stomatitis virus, Indiana strain, was maintained in the laboratory in 929 cells. The virus titer is expressed in plaque-forming units/0.5 ml. Interferon Production and Purification. Hu Le IF was produced by challenge of leukocytes with Sendai virus, following a techique described by Falcoff et al. (13) and modified Abbreviations: Hu Le IF, human leukocyte interferon; NaDodSO4, sodium dodecyl sulfate.,
3717
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Biochemistry: Thang et al.
by Cantell et al. (14). However, leukocytes were collected in a continuous flow centrifuge (Elecrem) and scraped off the blades after centrifugation, instead of from buffy coats. The purification was performed according to Cantell and Hirvonen (15) by successive precipitations in ethanol at increasing pH. The preparations used have a specific activity of 2-5 X 106 reference units/mg protein. Interferon Titration. Hu Le IF was titrated by using human foreskin fibroblasts. For estimation of the interferon titer, 50% inhibition of the cytopathic effect was employed. For more precise determination, the interferon titer was expressed as 90% inhibition of the yield of challenge vesicular stomatitis virus. All interferon titers were expressed in reference units. Interferon Neutralization. Anti-human FS-4 interferon
globulin was kindly provided by J. K. Dunnick. The neutralizing titer, when tested against human fibroblast interferon at 10 reference units/ml, was 1024. Anti-Hu Le IF globulin was a gift of K. Paucker. The neutralizing titer, when tested against Hu Le IF at 8 reference units/ml, was 1280. Sodium Dodecyl Sulfate (NaDodSO4)/Polyacrylamide Gel Electrophoresis. Polyacrylamide slab gels (15%) were used. Gel polymerization and electrophoresis were performed using the Laemmli system (16) except that 2-mercaptoethanol was omitted. Interferon samples were dialyzed against the electrode buffer. Samples were applied to wells distant enough one from the other to avoid any accidental contamination. At the end of electrophoresis, each track was cut off and sliced into 2.5-mm portions. Interferon activity was eluted in culture medium containing 5% fetal bovine serum at 4VC for 18 hr. Protein Determination. The protein contents in dilute concentrations were determined by the micromethod described by Schaffner and Weissmann (17). The proteins were quantitatively precipitated on a nitrocellulose filter, then stained with acidic dye amidoschwarz. After washing, the dye retained on the membrane filter was eluted by 25 mM NaOH in 50% ethanol solution containing 0.05 mM EDTA. The absorbance of the eluate was measured at 630 nm against eluant. The protein contents were calculated from a calibrated curve established with bovine serum albumin as standard. RESULTS Separation of Two Molecular Forms by PolynucleotideAgarose Affinity Chromatography. When a preparation of Hu Le IF was chromatographed on poly(I)-agarose or poly(U)-agarose, the antiviral activity was distributed between wash and eluted fractions. The proportion of these two parts of the activity varied from one batch to another. The profile of a typical preparation is shown in Fig. 1. About two-thirds of the product with interferon activity was found in the flowthrough and wash fractions, while the bound material with about onethird of the initial activity was eluted at around 0.3 M NaCl. A similar distribution was also observed with crude interferon. These results contrast with those obtained with mouse viral or immune interferons (9, 10) under identical conditions; in this case, all the antiviral activity was retained. The lack of binding was not due to a saturation of the column by proteins. Indeed, when the E1 fraction was chromatographed again on a poly(I)-agarose column, the activity was recovered completely in flowthrough and wash fractions. No trace of interferon was retained on the column. Similarly, the retained fraction E2, rechromatographed on a poly(I)-agarose column, had all-its activity bound to the polynucleotide and was eluted-by salt at the same molarity. Hu Le IF preparations are known to contain a minor species of interferon (about 1% of the total activity) antigenically related to human fibroblast interferon (18-20). To ascertain that
Proc. Natl. Acad. Sci. USA 76 (1979)
E-6
C
2-
10~~~~~~~~~~~~~.
13
3 5 7 9 Fraction FIG. 1. Chromatography of Hu Le IF on poly(I)-agarose. A sample of Hu Le IF (9 X 106 units in 3 ml of phosphate-buffered saline) was diluted in 6 ml of 10 mM Tris, pH 7.5. The total volume was loaded on a poly(I)-agarose column (2-ml bed volume) at a flow rate of 5 ml/hr. The column was washed (E1) with 6 ml of Tris buffer into 10 fractions then eluted (E2) by a linear gradient of NaCl formed by mixing 3 ml of 10 mM Tris buffer and 3 ml of 1 M NaCl in the same Tris buffer. The activity was assayed on human fibroblasts. One-third of the initial-activity was found in the flowthrough volume, 25% in the wash, and 38% in the eluted fractions. The antiviral activity on bovine 5
7
1
MDBK cells, assayed simultaneously, is represented by the broken lines.
the two classes of Hu Le IF differentiated by their affinity for a polynucleotide ligand are both of the leukocyte type, comparative studies were performed with antisera prepared with either leukocyte or fibroblast interferon. The results obtained showed, as expected, that both classes of Hu Le IF were completely neutralized by anti-Hu Le IF globulin, whereas the anti-human fibroblast interferon globulin had no effect. Characterization by NaDodSO4/Polyacrylamide Gel Electrophoresis. These two classes of interferon were further analyzed by electrophoresis on polyacrylamide gels in the presence of NaDodSO4. The interferon activity profiles (Fig. 2) revealed that each class was mainly composed of one subpopulation of Hu Le IF with a characteristic apparent molecular weight. In fact, interferon having the polynucleotidebinding site migrated to the position corresponding to a polypeptide of 21,500 daltons (Fig. 2A) and interferon without the polynucleotide-binding property migrated as a polypeptide of lower molecular weight (Fig. 2B). Thus, these two classes of Hu Le IF correspond apparently to the two subspecies Les and Lef (2, 6). However, it is clear that each class contained molecules of intermediate sizes. This explains why the initial sample of Hu Le IF before separation by polynucleotide-agarose chromatography had an antiviral activity profile (Fig. 2C) spread
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Thang et al. A
Chn Tr I Cyt ran
2
1
fl2 '96 I II r2.-F.hi :
-
4
C.
m
5
C)
._W
9
c0.4 20
8
7
~~~B
II e
x
.t 0.893 i
6
_1 L
4-
4
5
6
8
7
5 7 6 Migration distance,
cm
FIG. 2. Electrophoresis of the two classes of Hu Le IF separated by poly(I) agarose chromatography. NaDodSO4/polyacrylamide (15%) gel electrophoresis was performed as described in Materials and Methods. The Laemmli system (16) was used in this experiment. Aliquots of the peak fraction of the polynucleotide-binding species (A) and of the wash fraction from the poly(I)-agarose column (B) were compared. (C) Initial Hu Le IF material before chromatography. The antiviral activity was assayed comparatively on human fibroblasts (solid lines) and on bovine MDBK cells (broken lines). The activity is expressed as units per fraction of gel. For the polynucleotidebinding species (A), 12,000 units, as measured on both human and MDBK cells, was applied. About 5800 units on human fibroblasts (about 50%o recovery) and 2800 units on bovine cells (about 24% recovery) were recovered. For the unbound interferon species (B), 3000 units on human fibroblasts and 9000 units on MDBK were applied. The recoveries were 600 units on human fibroblasts (20%/) and 1900 units on MDBK (21%). For the initial material (C), about 60,000 units for both cell lines was applied. The recoveries were 8000 units on human fibroblasts ( 14%) and 14,000 units on MDBK (24%). The migration positions of protein markers are indicated by arrows: Chn, chymotrypsinogen (25,000 daltons); Tr I, trypsin inhibitor from soybean (21,500 daltons); and Cyt, cytochrome c (13,500 daltons).
3719
over a region of the gel corresponding to molecular weights from 13,000 to 25,000 daltons, with two peaks of activity located at 21,000 and 15,000 daltons. Species Specificity. The relationship between molecular size and the polynucleotide-binding property would suggest a correlation between the structure of these species and their activities. In fact, when the antiviral activity was assayed comparatively on human and bovine cells, it was found constantly that the activity of the slow mobility species on human fibroblasts was several times higher than that observed with bovine MDBK cells (broken line in Fig. 2A). In the case of the species of high mobility (Fig. 2B), on the other hand, the ratio of the activities was approximately reversed. In particular, at a position corresponding to the migration of cytochrome c, the activity was almost exclusively towards bovine cells. The relative heterospecific activities were also found with the initial sample (Fig. 2C). A similar comparison could be made with the two populations separated on poly(I)-agarose. However, the differences are sometimes less pronounced because each class of molecules is not wholly homogeneous. Purification. The methodology described here may be useful for the purification of Hu Le IF. In fact, it is more rational to separate these two species and to adapt suitable methods to purify each class of Hu Le IF. Obviously, affinity chromatography is the best technique for this from the point of view both of capacity and specificity. In the case of Hu Le IF studied here, most of the preparations contained mainly one dominant contaminating protein which migrated on NaDodSO4/polyacrylamide gels as a polypeptide of 25,000 daltons, and one minor contaminant, serum albumin. Another polypeptide of 14,000 daltons was found in some preparations. Of course, other polypeptides are present in various but low concentrations according to the batch. On poly(I)-Sepharose chromatography, the human serum albumin bound partially to the polynucleotide while the 25,000-dalton and 14,000-dalton polypeptides passed through. Thus human serum albumin was the major contaminant in the Hu Le IFS (slow) fraction after chromatography and the 25,000-dalton protein was the main contaminant in the Hu Le IFf (fast) fraction. Combination of poly(I)-agarose and anti-human serum globulin-agarose leads to a purification of Hu Le IFS to a specific activity of about 108 units/mg of protein. Behavior on Blue Dextran Sepharose. These classes of Hu Le IF could also be separated by affinity chromatography on blue dextran Sepharose, which mimics a polynucleotide ligand as we have postulated (7). In Fig. 3 we show the behavior of Hu Le IF on such a column. The retained activity was eluted by poly(I) at a concentration of A248 = 2. This suggests that this fraction of Hu Le IF has the polynucleotide-binding site similar to the fraction of Hu Le IF, retained on polynucleotide agarose. Preliminary analysis of this poly(I)-eluted material by electrophoresis on NaDodSO4/polyacrylamide gels showed a population of Hu Le IF mainly composed of a 21,000-dalton species.
DISCUSSION The heterogeneity of Hu Le IF is a major difficulty for purification. In addition, because different molecular species are present in Hu Le IF preparations, studies of the relationship between the structure and the function of this glycoprotein (which possesses multiple biological activities) are problematic. The reasons for the presence of two main subpopulations in Hu Le IF have been investigated by several laboratories. Essentially, different degrees of glucosylation of the polypeptide backbone (21, 22) or proteolytic cleavage (3, 5) have been considered. In this study we show that these two subspecies can
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Biochemistry: Thang et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
C
.~100 C
0
50
1
-
1
3
5
7 9 1 Fraction
3
5
7
FIG. 3. Chromatography of Hu Le IF on blue dextran agarose. A sample of Hu Le IF (5 X 105 units in 0.350 ml of phosphate-buffered saline) was diluted with 0.8 ml of 10 mM Tris-HCl, pH 7.5. The diluted
sample was loaded on a blue dextran agarose column (0.5 ml bed volume). The column was first washed with 3.5 ml 10 mM Tris, then eluted with 3 ml of poly(I) at A24g = 2.0. The activity was assayed on MDBK cells. The recovery was nearly complete: about 8 X 104 units (16% of input activity) in the flowthrough fraction, 3 X 105 units (60%o) in Tris washing fractions, and 6 X 104 units (12%) in poly(I)-eluted fractions.
be recognized by their different binding property to a polynucleotide. By analogy to mouse interferons, it is tempting to equate the polynucleotide-binding molecules with a native (or quasinative) form of the Hu Le IF. Consequently, the other class of molecules, devoid of the polynucleotide-binding property, would be considered as a degraded species having lost the polypeptide domain that interacts with nucleic acids. We have previously suggested (10) that an enzymatic or chemical alteration could explain why some mouse immune interferon preparations have an elution profile from poly(I)Sepharose columns shifted towards lower ionic strength. This kind of alteration of mouse viral interferon, which exists also in two molecular species (11), might be present, because we have succeeded in separating these two subpopulations according to their differential affinity to various polynucleotides (unpublished results). More important is the fact that the antiviral activity of Hu Le IF could be dissociated from the property of polynucleotide binding, whereas species specificity seems to be closely related to the presence or absence of the polynucleotide-binding site.
Havell et al. (6) have also obtained different ratios of antiviral activity on human fibroblast versus bovine cells with Hu Le IF species separated by isoelectric focusing. However, these authors considered that the values of the ratios are similar. It has also been mentioned (21) that a subspecies of Hu Le IF obtained after periodate treatment and separated on NaDodSO4/polyacrylamide gel at a position of 13,500 daltons was active on cat cells but practically devoid of activity on human cells. Stewart et al. (21) suggested that the conversion of Hu Le IF to a low molecular weight species by cleavage of carbohydrate from interferon eliminates its activity for homologous cells. We have succeeded in modifying the ratio of antiviral activity on human fibroblast versus bovine cells of the poly(I)-bound Hu Le IF species that has been previously treated by limited proteolysis (unpublished results). This suggests that proteolytic cleavage of the 21,000-dalton species could also be one of the origins for the genesis of the fast mobility species. Gresser et al. (23) reported that Hu Le IF exhibits an antiviral activity on bovine cells much higher than that on human fibroblasts. This might indicate that the preparations used had a higher proportion of the modified species of Hu Le IF. We postulate that interferon, like many hormones and other multifunctional enzymes, has distinct domains assigned for separated functions: antiviral activity, species specificity, immune response, etc. The loss of the polynucleotide-binding area could indicate the alteration of the domain recognized by the cell receptor(s) (24) for species restriction while the "core" molecule remains active for induction of the antiviral process. One question we are asking is whether part of this core polypeptide of interferon could be conserved during evolution. When this article was submitted, we learned that Rubinstein et al. (25) had succeeded in separating the subspecies of Hu Le IF by high performance liquid partition chromatography and in purifying one species of 17,500 daltons. We thank Christine Nicoletta for her valuable technical assistance and the Centre National de Transfusion Sanguine (Orsay, France) for the leukocytes. This work was supported by the Centre National de la Recherche Scientifique (G.R. 18), the Delegation Generale a la Recherche Scientifique et Technique (Convention GBM 78.7.0693), the Institut National de la Sant6 et de la Recherche Medicale (U.43 and CRL 77.04.0741), and the Fondation pour la Recherche Medicale
Frangaise. 1. Fantes, K. H. (1970) in l'Interferon, Colloque No 6 de l'INSERM (INSERM, Paris), pp.181-186. 2. Stewart, W. E., II & Desmyter, J. (1975) Virology 67,68-73. 3. Torma, E. T. & Paucker, K. (1976) J. Biol. Chem. 251, 48104816. 4. Chen, J. K., Jankowski, W. J., O'Malley, J. A., Sulkowski, E. & Carter, W. A. (1976) J. Virol. 19,425-434. 5. Chadha, K. C., Sclair, M., Sulkowski, E. & Carter, W. A. (1978) Biochemistry 17, 196-200. 6. Havell, E. A., Yip, Y. K. & Vilcek, J. (1977) Arch. Virol. 55, 121-129. 7. Drocourt, J. L., Thang, D. C. & Thang, M. N. (1978) Eur. J. Biochem. 82, 355-362. 8. Thang, M. N., de Maeyer-Guignard, J. & de Maeyer, E. (1977) FEBS Lett. 80,365-370. 9. de Maeyer-Guignard, J., Thang, M. N. & de Maeyer, E. (1977) Proc. Natl. Acad. Sci. USA 74,3787-3790. 10. Wietzerbin, J., Stefanos, S., Lucero, M., Falcoff, E., Thang, D. C. & Thang, M. N. (1978) Biochem. Biophys. Res. Commun. 85, 480-489. 11. de Maeyer-Guignard, J., Tovey, M. G., Gresser, I. & de Maeyer, E. (1978) Nature (London) 271, 622-625. 12. Ryan, L. C. & Vestling, C. S. (1974) Arch. Biochem. Biophys. 160, 279-284.
Biochemistry: Thang et al. 13. Falcoff, E., Falcoff, R., Fournier, F. & Chany, C. (1966) Ann. Inst. Pasteur 111, 562-584. 14. Cantell, K., Hirvonen, S., Mogensen, K. E. & Pyhala, L. (1974) in The Production and Use ofInterferon for the Treatment and Prevention of Human Virus Infections, In Vitro, Monograph, ed. Waymouth, C. (Tissue Culture Association, Rockville, MD), Vol. 3, pp. 35-38. 15. Cantell, K. & Hirvonen, S. (1978) J. Gen. Virol. 39,541-543. 16. Laemmli, U. K. (1970) Nature (London) 227,680-685. 17. Schaffner, W. & Weissmann, C. (1973) Anal. Biochem. 56, 502-514. 18. Anfinsen, C. B., Bose, S., Corley, L. & Gurari-Rotman, D. (1974) Proc. Natl. Acad. Sci. USA 71, 3139-3142. 19. Berg, K., Ogburn, C. A. & Paucker, W. A. (1975) J. Immunol. 114,640-644.
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20. Havell, E. A., Berman, B., Ogburn, C. A., Paucker, K. & Vilcek, J. (1975) Proc. Natl. Acad. Sci. USA 72, 2185-2187. 21. Stewart, W. E., II, Lin, L. S., Wiranowska-Stewart, M. & Cantell, K. (1977) Proc. Natl. Acad. Sci. USA 74,4200-4204. 22. Havell, E. A., Yamazaki, S. & Vilcek, J. (1977) J. Biol. Chem. 252, 4425-4427. 23. Gresser, I., Bandu, M. T., Brouty-Boye, D. & Tovey, M. (1974) Nature (London) 251, 543-545. 24. Chany, C., Gregoire, A., Vignal, M., Lemaitre-Montcuit, J., Brown, P., Besanqon, F., Suarez, H. & Cassingena, R. (1973) Proc. Natl. Acad. Sci. USA 70,557-561. 25. Rubinstein, M., Rubinstein, S., Familletti, P. C., Miller, R. S., Waldman, A. A. & Pestka, S. (1979) Proc. Natl. Acad. Sci. USA 76,640-644.
