glutathione-insulin transhydrogenase

Biochimica et Biophysica Acta, 788 (1984) 189-192

189

Elsevier

BBA 31936

P R O D U C T I O N AND CHARACTERIZATION OF A M O N O C L O N A L ANTIBODY T O RAT LIVER T H I O L : P R O T E I N - D I S U L F I D E OXIDOREDUCTASE/GLUTATHIONE-INSULIN TRANSHYDROGENASE R I C H A R D A. R O T H * and M A R Y L. MESIROW

Cell Biology Laboratory and Department of Medicine, Mount Zion Hospital and Medical Center, P.O. Box 7921, San Francisco, CA 94120 and Department of Physiology, University of California, San Francisco, CA 94143 (U.S.A.) (Received January 26th, 1984)

Key words: Monoclonal antibody," Glutathione-insulin transhydrogenase," Protein-disulfide reductase (glutathione)

Rat liver thiol:protein-disulfide oxidoreductase/glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) was purified and found to give two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. A monoclonal antibody was produced against this enzyme preparation and found to remove all the insulin degrading activity of purified preparations of the enzyme. This monoclonal antibody was also found to react with the two different forms of the enzyme observed on gel electrophoresis. These results suggest that glutathione-insulin transhydrogenase can exist in more than one state.

Introduction Glutathione-insulin transhydrogenase, also called thiol : protein-disulfide oxidoreductase (EC 1.8.4.2), was originally identified and purified on the basis of its ability to interchange the disulfide bonds of insulin and thereby inactivate the hormone [1]. Protein disulfide-isomerase, also known as disulfide interchange enzyme (EC 5.3.4.1), was identified and purified on the basis of its ability to promote the reactivation of incorrectly disulfide-bonded ribonuclease [2]. Since the discovery of these two enzymes, the question has been debated as to whether the two activities are mediated by a single enzyme of broad specificity or by distinct enzyme species with overlapping specificities [3,4]. Comparisons of the physiochemical properties of the two enzymes isolated in different laboratories first indicated that the two molecules were * Present address: Department of Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

similar [7]. Subsequent work with highly purified preparations of glutathione-insulin transhydrogenase indicated that this enzyme had both the ability to catalyze the formation of protein disulfide bonds and the cleavage of insulin disulfide bonds [8,9]. However, Hillson and Freedman [10] reported that the two enzymatic activities could be separated by chromatography of the enzyme on thiopropyl-sepharose. Finally, it was recently reported [11] that a polyclonal antibody reacted identically with glutathione-insulin transhydrogenase and protein-disulfide isomerase. Interpretation of these results is complicated, however, by other findings that indicate that glutathione-insulin transhydrogenase is heterogeneous. Studies of Drazic and Cottrell [12], Ohba et al. [13] and Ansorge et al. [14] have all indicated multiple forms of the enzyme in rat-liver cells. In addition, highly purified preparations of glutathione-insulin transhydrogenase from mouse liver and mouse lymphocytes exhibit two bands [9] on the highly sensitive two-dimensional gel electrophoresis systems of O'Farrell [15]. Thus, to ex-

190 amine further whether various forms of glutathione-insulin transhydrogenase exist, we prepared and characterized a monoclonal antibody to ratliver glutathione-insulin transhydrogenase. Materials and Methods

Production of rnonoclonal antibodies Glutathione-insulin transhydrogenase was purified from rat livers by the procedure of Carmichael et al. [16] as described previously [9,17]. Female B a l b / c mice (6-8 weeks old; Simonsen Laboratories, Gilroy, CA) were injected in the peritoneum with 20/~g of purified enzyme emulsified in complete Freund's adjuvant. Mice were periodically boosted over the next few months by the same procedure. 3 days prior to fusion, mice were injected with 20/~g of soluble enzyme in the tail vein. For production of hybridomas, mice were killed by cervical dislocation and the spleens were removed and teased apart to yield a lymphocyte cell suspension. Then 6 - 1 0 7 lymphocytes and 6. 1 0 7 myeloma cells (FO) were fused by the addition of 50% poly(ethylene glycol) 4000 G F (Merck, Darmstadt, F.R.G.) as detailed by De St. Groth and Scheidegger [18]. When the hybrid cells were semi-confluent (10-20 days following fusion), their supernatants were removed and tested for antibodies to glutathione-insulin transhydrogenase by the plate-binding assay described below. Hybridomas making the desired antibodies were cloned by limiting dilution [19] and grown up in culture. The monoclonal antibodies from the supernatants of the cells were purified on protein A-Sepharose (Pharmacia, Piscataway, N J) [19] and their heavy and light chains were identified by the use of specific antisera (Miles, Elkhart, IN) in Ouchterlony two-dimensional immunodiffusion.

Assays for antibodies to glutathione-insulin transhydrogenase Antibodies to glutathione-insulin transhydrogenase were detected by three different methods. In the first method, purified rat liver enzyme was diluted to 1 ~tg/ml in phosphate-buffered saline (pH 7.5), and 50 #1 were added to each well of a poly(vinyl chloride) microtitration plate (Dynatech, Alexandria, VA) [19]. After 16 h at 4°C, the unbound enzyme was shaken off and the plates

were washed three times with buffer containing 1% bovine serum albumin. Either supernatant or purified monoclonal antibody was added to the wells and incubated 2 h at 24°C; the unbound antibody was removed and the plates were washed again three times. Finally, 50 /al of 125I-labeled rabbit anti-mouse Ig (approx. 10000 cpm) were added and allowed to react for 2 h at 4°C. The wells were washed again, cut out and counted. In the second method, antibodies to glutathione-insulin transhydrogenase were detected by their ability to bind ~25I-labeled enzyme. 25 ng of 125I-labeled enzyme, radioiodinated as described previously [9], were incubated for 14 h at 4°C with either monoclonal or normal IgG coupled to cyanogen bromide-activated Sepharose 4B [20]. The antibody-coupled Sepharose was pelleted and washed three times, and the bound radioactivity was determined. The third assay was identical to this assay except in that 200 ng of unlabeled enzyme were incubated with the antibody-coupled Sepharose. Then the supernatants were tested for insulin-degrading activity as previously described by the trichloroacetic acid precipitation method [9,17].

Gel electrophoresis studies Samples were analyzed on 10% polyacrylamide sodium dodecyl sulfate (SDS) gel by the procedure of Laemmli [21]. Proteins were visualized by staining the gels with Coomassie brilliant blue. For immunochemical localization of the antigens, gels were applied to nitrocellulose paper and the proteins were electrophoretically transferred by the procedure of Towbin et al. [22]. The nitrocellulose paper was then blocked with 3% bovine serum albumin, reacted with 10 ~tg/ml of the monoclonal antibody for 2 h at 24°C, washed and incubated with 125I-labeled rabbit anti-mouse Ig. After 1 h at 24°C the paper was again washed, then dried and autoradiographed. Molecular weights were calculated using the following standards: phosphorylase b (92 500); bovine serum albumin (66 200); ovalbumin (45 000) and carbonic anhydrase (31 000). Results and Discussion

Glutathione-insulin transhydrogenase was purified from rat liver by the method of Carmichael

191 et al. [16]. Previously, this m e t h o d was f o u n d to yield an e n z y m e p r e p a r a t i o n from m o u s e liver which gave a single b a n d on s o d i u m d o d e c y l sulfate gel electrophoresis [9]. I n c o n t r a s t to the results with m o u s e liver, this p r o c e d u r e y i e l d e d a rat-liver e n z y m e p r e p a r a t i o n which gave two b a n d s on s o d i u m d o d e c y l sulfate gel electrophoresis ( M r 60000 a n d 56000) (Fig. 1). F u r t h e r a t t e m p t s at s e p a r a t i n g the two c o m p o n e n t s b y c h r o m a t o g r a p h y on h y d r o p h o b i c , thiol a n d lectin c o l u m n s were unsuccessful. Different forms of the enzyme from rat liver have also been n o t e d previously [12-14]. This enzyme p r e p a r a t i o n was therefore used to i m m u n i z e B a l b / c mice a n d their l y m p h o c y t e s were fused to the F O m y e l o m a cell line. A f t e r six fusions yielding m o r e t h a n 500 h y b r i d o m a s , two h y b r i d o m a s were identified which p r o d u c e d antib o d i e s that could b i n d to m i c r o t i t e r wells c o a t e d with purified enzyme. These two h y b r i d o m a s were c l o n e d a n d g r o w n up, a n d their a n t i b o d y was isolated on p r o t e i n A - S e p h a r o s e columns. Both

m o n o c l o n a l a n t i b o d i e s were identified as I g G ] with K light chains b y O u c h t e r l o n y t w o - d i m e n sional diffusion analyses. Also, b o t h a n t i b o d i e s a p p e a r e d to b i n d to the same antigenic site on the enzyme, since the b i n d i n g of the two a n t i b o d i e s to e n z y m e c o a t e d wells was not additive. Consequently, only one of these two m o n o c l o n a l antib o d i e s was further characterized. Three techniques were used to e x a m i n e the i n t e r a c t i o n of the m o n o c l o n a l a n t i b o d y with g l u t a t h i o n e - i n s u l i n t r a n s h y d r o g e n a s e . First, the m o n o c l o n a l a n t i b o d y was tested for its a b i l i t y to r e m o v e the i n s u l i n - d e g r a d i n g activity of a purified p r e p a r a t i o n of enzyme. 10/~g of a n t i b o d y c o u p l e d to Sepharose was f o u n d to r e m o v e 50% of the

"0

4-

¢ ~0 '10

2 -

c~ if)

PROTEIN RTAIN

--~

ORIQIN-

0- 4/

I

I

I

"O c

20--

/

0 .Q

40K8eK-

I

1

-ORIG~N

I-- ~

I/

-80K

I0.

£N ~

O"

-4/

I

I

I

10 0

101

10 9

o

-I

C -O

to

tO "O

"~ o) tO

A

S

¢

D

E

F

G

H

Fig. 1. Sodium dodecyl sulfate gel electrophoresis of rat and mouse liver glutathione-insulin transhydrogenase (GIT). After electrophoresis, the gel was divided into two and a portion (slots A and B) was stained for protein and the rest was reacted with monoclonal antibody after transfer to nitrocellulose paper. Slots A and B contained 2 ~tg rat and mouse glutathione-insulin transhydrogenase, respectively. Slots C, D and E contained 2, 0.6 and 0.2 #g rat glutathione-insulin transhydrogenase and slots F, G and H contained 2, 0.6 and 0.2 #g mouse glutathione-insulin transhydrogenase.

0- 4/ 10 3

I 10 4

Antibody added (ng)

Fig. 2. Reaction of monoclonal antibodies with glutathione-insulin transhydrogenase. O, normal IgG; O, monoclonal anti-glutathione-insulin transhydrogenase. (A) Precipitation of insulin degrading activity. (B) Precipitation of 125I-labeled glutathione-insulin transhydrogenase. (C) Binding of monoclonal antibody to glutathione-insulin transhydrogenase adsorbed to microtiter wells.

192 insulin d e g r a d i n g activity of a purified p r e p a r a t i o n of enzyme (approx. 100 ng) a n d 80/xg of a n t i b o d y r e m o v e d 90% of the d e g r a d i n g activity (Fig. 2A). N o r m a l I g G - c o u p l e d Sepharose h a d no effect on the insulin d e g r a d i n g activity. Second, the m o n o clonal a n t i b o d y - c o u p l e d Sepharose was tested for its ability to b i n d 125I-labeled g l u t a t h i o n e - i n s u l i n transhydrogenase. In this assay, 2 /~g of m o n o clonal a n t i b o d y b o u n d approx. 10 ng of the enzyme (Fig. 2B). Again, n o r m a l I g G - c o u p l e d Sepharose had no effect on the 125I-labeled enzyme. Finally, the m o n o c l o n a l a n t i b o d y was tested for its ability to b i n d wells c o a t e d with purified glutathione-insulin transhydrogenase. Again, the m o n o clonal a n t i b o d y was f o u n d to b i n d to the purified enzyme, whereas n o r m a l I g G d i d not (Fig. 2C). T h e m o n o c l o n a l a n t i b o d y was then tested for its ability to b i n d to mouse-liver glutathione-insulin t r a n s h y d r o g e n a s e a n d to the two forms of the rat liver g l u t a t h i o n e - i n s u l i n t r a n s h y d r o g e n a s e observed on s o d i u m d o d e c y l sulfate gel electrophoresis. T h e enzymes were transferred from the gel to nitrocellulose p a p e r a n d reacted with the m o n o clonal a n t i b o d y . The b i n d i n g of the a n t i b o d y was d e t e c t e d b y the use of an 125I-labeled r a b b i t antim o u s e I g G a n d a u t o r a d i o g r a p h y . T h e two m a j o r forms of the rat liver enzyme were labeled with the m o n o c l o n a l a n t i b o d y (Fig. 1) as well as the mouse-liver enzyme. In addition, several m i n o r b a n d s were also o b s e r v e d reacting with the m o n o clonal a n t i b o d y a n d these smaller m o l e c u l a r weight b a n d s might represent p r o t e o l y t i c fragments of the enzyme. These results strongly suggest that b o t h m a j o r b a n d s represent g l u t a t h i o n e - i n s u l i n transhydrogenase. T h e two forms of enzyme o b s e r v e d on gel electrophoresis could result from differences in either a m i n o - a c i d c o m p o s i t i o n or p o s t - t r a n s l a tional processing of the enzyme (i.e., glycosylation, p h o s p h o r y l a t i o n o r proteolysis). If different forms of g l u t a t h i o n e - i n s u l i n t r a n s h y d r o g e n a s e exist, they m a y have different e n z y m a t i c specificities. Separation of the different forms of the enzyme m a y be achieved in the future b y the p r o d u c t i o n of a m o n o c l o n a l a n t i b o d y specific to one form of the enzyme.

Acknowledgments This w o r k was s u p p o r t e d b y N I H R e s e a r c h G r a n t AM-26918, an N I H Research Career De-

v e l o p m e n t A w a r d (AM-01171) to R.A.R., a n d the Elise Stern H a a s Research F u n d , H a r o l d Brunn Institute, M o u n t Z i o n H o s p i t a l a n d M e d i c a l Center. The assistance of Jennifer Meek in the initial stages of the p r o d u c t i o n of the m o n o c l o n a l a n t i b o d y is gratefully acknowledted. W e also t h a n k Dr. S.F. F a z e k a s de St. G r o t h for the F O cells.

References 1 Tomizawa, H.H. and Halsey, Y.D. (1959) J. Biol. Chem. 234, 307-310 2 Goldberger, R.F., Epstein, C.J. and Anfinsen, C.B. (1963) J. Biol. Chem. 238, 628-635 3 Freedman, R.B. (1979) FEBS Lett. 97, 201-210 4 Varandani, P.T. (1977) in Diabetes, Proceedings of the IX Congress of the International Diabetes Federation (Bajaj, J.s., ed.), pp. 213-223, Excerpta Medica, Amsterdam 5 Freedman, R.B. and Hawkins, H.C. (1977) Biochem. Soc. Trans. 5, 348-357 6 Varandani, P.T. (1978) in Mechanisms of Oxidizing Enzymes (Singer, T.P. and Ondarza, R.N., eds.), pp. 29-42, Elsevier/North-Holland, Amsterdam 7 Ansorge, S., Bohley, P., Kirschke, H., Langner, J., Marquardt, I., Wiederanders, B. and Hanson, H. (1973) FEBS Lett. 37, 238-240 8 Morin, J.E., Carmichael, D.F. and Dixon, J.E. (1978) Arch. Biochem. Biophys. 189, 354-363 9 Roth, R.A. and Koshland, M.E. (1981) Biochemistry 20, 6594-6599 10 Hillson, D.A. and Freedman, R.B. (1980) Biochem. J. 191, 373-388 11 Bjelland, S., Wallevik, K., Kroll, J., Dixon, J.E., Morin, J.E., Freedman, R.B., Lambert, N., Varandani, P.T. and Nafz, M.A. (1983) Biochim. Biophys. Acta 747, 197-199 12 Drazic, M. and Cottrell, R.C. (1977) Biochim. Biophys. Acta 484, 476-485 13 Ohba, H., Harano, T. and Omura, T. (1977) Biochem. Biophys. Res. Commun. 77, 830-836 14 Ansorge, S., Kirchke, H., K6hler, E., Langner, J., R6mer, I. and Weideranders, B. (1977) Acta Biol. Med. Germ. 36, 1713-1721 15 O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021 16 Carmichael, D.F., Morin, J.E. and Dixon, J.E. (1977) J. Biol. Chem. 252, 7163-7167 17 Roth, R.A. (1981) Biochem. Biophys. Res. Commun. 98, 431 438 18 De St. Groth, S.F. and Scheidegger, D. (1980) J. Immunol. Methods 35, 1-21 19 Oi, V.T. and Herzenberg, L.A. (1980) in Selected Methods in Cellular Immunology (Mishell, B.B. and Shiigi, S.M., eds.), pp. 351-372, Freeman, San Francisco 20 March, S.C., Parikh, I. and Cuatrecasas, P. (1974) Anal. Biochem. 60, 149-152 21 Laemmli, U.K. (1970) Nature (London) 227, 680-685 22 Towbin, H., Staehlin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4358