[30] Production of human fibroblast interferon in the presence of the glycosylation inhibitor tunicamycin

212

INDUCTION

AND PRODUCTION

OF INTERFERONS

[30]

As was shown previously, human type II interferon displayed signifi-, cant heterogeneity on Con A-Sepharose (Fig. 1A). 6 However, type II interferon produced in the presence of tunicamycin (2/zg/ml) did not bind to Con A-Sepharose (Fig. 1B) indicating that neither carbohydrate recognition nor hydrophobic interactions took place, Type II interferon was produced also in the presence of tunicamycin at a lower concentration (0.05 /zg/ml) and was chromatographed on Con A-Sepharose according to the same protocol. Although almost all interferon activity appeared in the breakthrough fraction, a small amount required t~-MM for elution, indicating that the interferon was still partially glycosylated. The absence of a fraction that requires ethylene glycol for elution suggests that hydrophobicity of the type II interferon molecule is dependent upon unaltered glycosylation. Thus it appears that inhibition of glycosylation of human type II interferon not only abolishes its ability to bind to an immobilized lectin, but also alters its conformation to an extent that its hydrophobic binding sites are no longer available or accessible.

[30] P r o d u c t i o n o f H u m a n F i b r o b l a s t I n t e r f e r o n i n t h e Presence of the Glycosylation Inhibitor Tunicamycin B y Y. K. YIP and JAN

VILCEK

Tunicamycin is a glycosylation inhibitor that prevents the assembly of N-linked glycoprotein. 1 It has been shown that tunicamycin inhibits the synthesis of core saccharides by blocking the transfer of GlcNAc- 1-P from UDP-GlcNAc to the dolichol monophosphate. 2 A survey of the literature shows that the strategy of tunicamycin treatment of cell cultures varies among investigators, It is likely that the optimal condition, such as concentration of tunicamycin and duration of treatment will depend on the type of cell used. The procedure to be described in this section deals specifically with the use of tunicamycin in the production of unglycosylated interferon induced by polyinosinate-polycytidylate [poly(I).poly(C)] in cultures of human foreskin fibroblasts. A similar procedure with 2-deoxy-o-glucose or o-glucosamine as inhibitor has been reported by Havell et al. 3 Production of interferon in FS-4 cells by the superinduction procedure has 1 C. J. W a e c h t e r and W. J. L e n n a r z , Annu. Rev. Biochem. 45, 95 (1976). 2 j. S. T k a c z and J. O. L a m p e n , Biochem. Biophys. Res. Commun. 65, 248 (1975). a E. A. Havell, J. Vil~ek, E. Falcoff, and B. B e r m a n , Virology 63, 475 (1975).

METHODS IN ENZYMOLOGY, VOL. 78

Copyright © 1981by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181978-7

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IF

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been described by Havell and Vil~ek. 4 Only the steps involving tunicamycin treatment of the cell culture will be described here. Tunicamycin is a complex molecule consisting of one residue of uridine and two residues of N-acetyl-o-glucosamine, with the amino hydrogen in one of these residues substituted by a long-chain fatty acid. Four major components have been identified in tunicamycin preparations that differ in fatty acid chain length; the molecular weight of each component has been determined to be 970, 984, 998, and 1012, respectively? A more recent study demonstrated that tunicamycin can be resolved into two major and eight minor components by high-performance liquid chromatography (HPLC). Moreover, one of the two major components, while completely blocking incorporation of mannose into protein, has been found to inhibit protein synthesis by 50% in cultures of chick embryo leg tendon fibroblasts. ~ This finding is noteworthy, since it has been generally reported that tunicamycin does not affect protein synthesis. Before this information had become available to us, we observed that tunicamycin suppresses the interferon yield in human foreskin fibroblasts. This result was surprising since it was shown that the carbohydrate moiety is not essential for the biological activity of interferons. 7 We then examined the effect of tunicamycin on the cellular uptake and incorporation of isotopically labeled amino acid and carbohydrate by cells superinduced for interferon production. Effects of treatment with 2-deoxy-D-glucose and D-glucosamine were also examined for comparison. As illustrated in Fig. I, tunicamycin inhibits the uptake of [3H]leucine by the treated cell cultures, the extent of inhibition increasing with the concentration of tunicamycin. When the percentage of incorporation of this amino acid into trichloroacetic acid-precipitable counts was calculated on the basis of uptake by the cells, it was found to be similar in the control and tunicamycin-treated cells, except in cultures treated with l0 ttg of tunicamycin per milliliter (Fig. 1). Thus it seems that the apparent inhibition of protein synthesis by tunicamycin is due mainly to the inhibition of amino acid uptake rather than to a direct effect on biosynthesis of the polypeptide chain. A concentration of 5 mM 2-deoxy-o-glucose showed similar effects as tunicamycin on the uptake and incorporation of [aH]leucine, whereas o-glucosamine at the same concentration showed no inhibitory effect on either of these events. 4 E. A. Havell and J. Vil~ek, Antimicrob. Agents Chemother. 2, 476 (1972). 5 A. Takatsuki, K. Kawamura, M. Okina, Y. Kodama, T. lto, and G. Tamura, Agric. Biol. Chem. 41, 2307 (1977). 6 W. C. Mahoney and D. Duksin, J. Biol. Chem. 254, 6572 (1979). 7 S. Bose, D. Gurari-Rotman, U. T. Ruegg, L. Corley, and C. B. Anfinsen, J. Biol. Chem. 251, 1659 (1976).

214

INDUCTION AND PRODUCTION OF INTERFERONS

2-deoxy- D-Glucose, 5mM

120

U ptoke

Incorporation

[30]

D-Glucosamine, 5raM Uptake

I ncorporotion

I00

8oi 6ol 4(3

FI fi

zo O

Tunicomycin,/~.g/ml:r-lO.O2 I~0,2 Uptake

o~

120

Leu

Man

80 llll 4O 20

Ill,

[] I.O • I0 Incorporation

GrcN

Leu

Man

GIcN

i~A i:.,

ti n.

FIG. 1. Effect of glycosylation inhibitors on the incorporation of isotopically labeled amino acid and carbohydrate by human FS-4 foreskin fibroblasts, superinduced for interferon production. Cells grown in 24-well tissue culture plates from Falcon Plastics were used in the experiment 12 days after seeding. Medium containing either 5/zCi of [aH]leucine, 10 /zCi of [aH]mannose, or 10/zCi [3H]glucosamine per milliliter of MEM supplemented with 5% (v/v) heat-inactivated fetal bovine serum was used as production medium at 1 ml per well. After incubation at 340 for 24 hr in a humidified CO2 incubator, the cell cultures were washed three times with 1 ml of Hanks' balanced salt solution, then were lysed by adding to each well 1 ml of 1% (w/v) sodium dodecyl sulfate (SDS) in water. Cellular uptake of the labeled compounds was measured with 50-/zl aliquots of the SDS lysates, and incorporation was measured with 100-/,d aliquots of the SDS lysates precipitated in 1 ml of a 10% (w/v) trichioroacetic acid solution. Percentage of incorporation was calculated after correcting for a change in precursor uptake in each group of cultures.

In contrast, all three glycosylation inhibitors showed little or no effect on the uptake of mannose or glucosamine (Fig. 1). At 5 mM 2-deoxy-Dglucose and o-glucosamine appear to have inhibitory effects comparable to those of tunicamycin at 1-10/zg/ml. It should be noted here that at concentrations of glycosylation inhibitors employed for production of

[30]

TUNICAMYCIN INHIBITION OF FIBROBLAST

IF

215

unglycosylated interferon, some residual carbohydrate incorporation was always present.

Reagents Distilled water adjusted to approximately pH 9 with 1 M NaOH Eagle's minimal essential medium (MEM), twofold concentrated (2 ×), prepared from a 10× stock solution (Grand Island Biological Company), buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (12 mM), N-trishydroxymethyl glycine (26 mM), and sodium bicarbonate (2.2 g/liter) Tunicamycin obtained from Dr. E. Sulkowski of the Roswell Park Institute, Buffalo, New York, or from Dr. J. Douros of the National Cancer Institute, Bethesda, Maryland, and used without purification

Procedures Preparation of Tunicamycin Stock Solution. Tunicamycin to be made up at 100 ~g/ml is first dissolved in one-half the final volume of distilled water adjusted to pH 9. Dimethyl sulfoxide s and 1 M NH4OH 9 have also been used as solvents for preparing the stock solution of tunicamycin. After gentle mixing on a Vortex mixer to assure complete dissolution of tunicamycin, an equal volume of 2 × MEM is added. A slightly turbid solution may be obtained. Sterilization of the tunicamycin solution can be accomplished by filtration through a Millex filter, 0.45/~m (Millipore Corp.) The stock solution may be aliquoted and stored at - 2 0 ° for several months. Production of Unglycosylated Fibroblast Interferon. The following procedure is to be integrated into the superinduction schedule described by Havell and Vil~ek. 4 Tunicamycin in the range of 0.1 to 10 ~g/ml may be used for treatment of cell cultures. It is convenient to add the desired amount of tunicamycin in a volume equivalent to one-tenth that of the culture medium. Dilution of tunicamycin from the stock solution should be made in MEM. Tunicamycin is added to the induction medium at the same time as actinomycin D, i.e., 4 h r after the addition of poly(I).poly(C). Thus, the cell cultures are treated with tunicamycin for 2 hr during the induction period. After removal of the induction medium and washing of the cell culture at 6 hr after induction, production medium containing 0.01% (w/v) of human serum albumin is added to the cultures with the same amount of tunicamycin as used in the induction medium. The culture medium is collected after a 24-hr incubation at 34° in a hus M. A. H. Surani, Cell 18, 217 (1979). 9 T. J. Housley, F. N. Rowland, P. W. Ledger, J. Kaplan, and M. L. Tanzer, J. Biol. Chem. 255, 121 (1980).

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INDUCTION AND PRODUCTION OF INTERFERONS

[30]

midified CO~ incubator. It may be stored at 4° for several weeks without significant loss of interferon activity. Isolation of the Unglycosylated Interferon. A convenient method for isolation of the unglycosylated interferon is by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure described here follows the Weber-Osborn method. ~° However, it can be modified for other S D S - P A G E systems simply by equilibrating the sample solution to the appropriate buffer conditions. We have obtained comparable results with the Laemmli gel system. ~ The culture medium is equilibrated to pH 7.2 in 20 mM sodium phosphate by exhaustive dialysis at 4 °. This sample solution, without transfer from the dialysis bag, will then be concentrated by packing it in Aquacide II powder (Calbiochem) and allowing it to stand at 4 ° until the sample volume has been reduced to approximately one-tenth that of the original. For SDS treatment, the concentrated interferon sample solution is adjusted to 1% (w/v) in SDS by adding an appropriate volume of a 10% SDS solution; the mixture is then incubated at 37 ° for 1 hr. Alternatively, SDS treatment of the interferon sample may be carried out by dialysis in the gel buffer for several hours at room temperature. Sodium dodecyl sulfate-treated interferon samples in a volume up to 200/zl may be applied to cylindrical gels 6 × 100 mm in size. For molecular weight calibration, protein markers in the range of 14,300 to 71,500 daltons (BDH Chemicals Ltd) are electrophoresed in a separate gel. Electrophoresis in a 10% polyacrylamide gel is carried out according to the procedure described by Weber and Osborn. ~° Upon removal from the glass tubes, the gels are cut at the center of the bromophenol blue (tracking dye) band. Gels containing interferon samples are to be frozen immediately at - 7 0 ° for at least 30 min before further processing. The molecular weight calibration gel is fixed with a 12% trichloroacetic acid solution for 1 hr and then stained with Coomassie Blue. Mobilities of interferon activity and the molecular weight markers are measured as percentage of the distance traveled by the tracking dye. A calibration curve is constructed by plotting log molecular weight versus relative mobility. The length of the frozen sample gel is measured before cutting it with a gel slicer with cutting edges spaced 1 mm apart. The average length of each gel slice is calculated by dividing the frozen gel length into the number of gel slices obtained.

~0 K. Weber and M. Osborn, in "The Proteins" (H. Neurath and R. L. Hill, eds.), 3rd ed., Vol. 1, pp. 179-223. Academic Press New York, 1975. 11 U. K. Laemmli, Nature (London) 227, 680 (1970).

[30]

TUNICAMYCIN

INHIBITION

OF FIBROBLAST

IF

217

EFFECT OF GLYCOSYLATION INH1BITORS ON YIELD OF HUMAN F1BROBLAST INTERFERON

Inhibitor None Tunicamycin 0.2 txg/ml 2.0 p,g/ml 10.0 p,g/ml 2-Deoxy-o-glucose, 5 mM o-Glucosamine, 5 mM

Interferon (units/ml)

Percent inhibition

20,480

--

20,480 14,000 6,230 1,920 2,680

0 30 70 90 87

Each gel slice is incubated in 1 ml of MEM supplemented with 5% (v/v) of heat-inactivated fetal bovine serum. Incubation is carried out overnight at room temperature to ensure complete equilibration of interferon activity between the gel slice and the medium. Interferon activity is assayed by inhibition of vesicular stomatitis virus using the microtiter method 4 with human GM-258 cells trisomic for chromosome 21.12 Comments

The result of a typical experiment showing the effect of glycosylation inhibitors on interferon production is summarized in the table. Suppression of interferon yield increased with the concentration of tunicamycin. A similar phenomenon was observed with 2-deoxy-o-glucose and oglucosamine/Because of the low yield of interferon from cultures treated with the inhibitor, addition of extraneous protein to the production medium is recommended to promote interferon production; purified human serum albumin added to a final concentration of 0.01% (w/v) has been found to be sufficient for this purpose. Addition of higher concentrations of extraneous protein will hinder subsequent processing, since the lowtiter culture fluid will have to be concentrated to facilitate isolation of the unglycosylated interferon by electrophoresis in SDS-polyacrylamide gels. Although S D S - P A G E is convenient for isolation of the altered interferon species, a serious drawback of this procedure is the substantial loss, up to 95%, of interferon activity due to SDS treatment (before electrophoresis). For elution of interferon from the polyacrylamide gel, it is essential to incubate only one gel slice per 1 ml of elution buffer. This will enhance the resolution of the two closely migrating interferon species, and it will also reduce toxicity caused in the assay cell cultures by SDS. Using this 12 y . H. T a n and A. E. Greene, J. Gen. Virol. 32, 153 (1976).

218

INDUCTION AND PRODUCTION OF INTERFERONS

[30]

SOS-PAGE r';

i' --

Tunlcomycin, 2/~.g / ml

11

\ FIG. 2. Molecular weight estimation of human fibroblast interferons made in the presence oftunicamycin by sodium dodecyl sulfatepolyacrylamide gel electrophoresis ( S D S PAGE). - . . . . , Interferon made by control superinduced cultures; , interferons made by superinduced cultures in the presence of tunicamycin.

'~o_ x ~ g

\ -- Tunicomycio, I0 ~u-glml

~! i

,', i t

;:¢i

10 20 30 40 50 60 70 Gel length (mm)

procedure, we were able to detect as little as 0.4 reference unit of interferon per gel slice in the GM-258/VSV assay, 13 and quantitative recovery of interferon activity can often be obtained. Size heterogeneity of human fibroblast interferon made in the presence of tunicamycin is shown in Fig. 2. Similar results were obtained by Havell e t a l . 14 with 2-deoxy-D-glucose and o-glucosamine. Two components with estimated molecular weights of 18,000 and 27,000 have been obtained with all three inhibitors; the molecular weight of the latter species is identical to that obtained with control superinduced interferon. As shown in Fig. 2 the proportion of the low molecular weight component increases with the concentration of tunicamycin. It should be noted here that, within the concentration range of the glycosylation inhibitors tested, we have never been able to eliminate the larger molecular weight component (27,000). This observation is in accord with the carbohydrate incorporation results shown in Fig. 1. 13 T. G. Hayes, Y. K. Yip, and J. Vil~ek, Virology 98, 351 (1979). 14 E. A. Havell, S. Yamazaki, and J. Vil~ek, J. Biol. Chem. 253, 4425 (1978).

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TUNICAMYCIN INHIBITION OF FIBROBLAST I F

219

In view of the recent success in the cloning of interferon genes 15-1~ and the prospect of producing interferon by bacterial cultures, it should be ot interest to characterize the biological and physiocochemical properties of the unglycosylated interferons made by cells treated with glycosylation inhibitors. Since prokaryotes lack glycosylating enzymes comparable to animal cells, interferons produced in bacteria are not likely to be glycosylated. The diminished yield of active interferon produced by cultures treated with glycosylation inhibitors may be the result of altered molecular properties of the unglycosylated interferon. Alterations in the folding of the newly synthesized polypeptide chain due to the absence of the carbohydrate moiety may result in decreased stability and/or lower intrinsic specific activity. Such an explanation does not contradict the observation reported by Bose e t al. 7 that no loss of biological activity results from removal of part of the carbohydrate moiety by treatment with glycosidases, since in this case removal of the prosthetic group is presumably from the exterior of already folded molecules. Havell e t al. 3 observed that human fibroblast interferon made in the presence of 2-deoxy-D-glucose and D-glucosamine showed decreased thermal stability and lower affinity for anti-F (anti-/3) interferon serum. Diminished interferon yield in the culture medium due to a reduced secretion rate of the unglycosylated interferon appears to be unlikely, since intracellular levels of interferon activity are also found to be suppressed in cells treated with glycosylation inhibitors.* Some biological properties of the high and low molecular weight components of interferon produced in the presence of tunicamycin, separated by SDS-PAGE, have been examined. Both molecular weight components exhibit little antiviral activity in bovine cells, and their antiviral activity is neutralized by anti-F but not anti-Le (anti-a) interferon sera. Thus in these respects they resemble fibroblast (F or/3) interferon made in the absence of glycosylation inhibitors. Acknowledgments This work was supported by Public Health Service Grants AI-07057 and AI-12948 from the National Institute of Allergy and Infectious Diseases.

is N. Mantei, M. Schwarzstein, M. Streuli, S. Panem, S. Nagata, and C. Weissmann, Gene 10, 1 (1980). 16 T. Taniguchi, S. Ohno, Y. Fujii-Kuriyama and M. Muramatsu, Gene 10, 11 (1980). 17 R. Derynck, J. Content, E. De Clercq, G. Volckaert, J. Tavernier, R. Devos, and W. Fiers, Nature (London) 285, 542 (1980).