Identification of the cysteine in the steroid-binding site of human corticosteroid binding globulin by site-directed mutagenesis and site-specific chemical modification

J. Stermd Bwchem. Molec Bwl Vol 48, No. 1, pp. 139-144, 1994 Copyright © 1994 Elsewer Science Ltd Printed in Great Britam. All rights reserved 0960-0760/94 $6 00 + 0 00

Pergamon

0960-0760(93)E0003-P

Identification of the Cysteine in the Steroid-binding Site of H u m a n Corticosteroid Binding Globulin by Site-directed Mutagenesis and Site-specific Chemical Modification J a y a s r i G h o s e - D a s t i d a r , 1 R e z a G r e e n 2 a n d J. B. A l e x a n d e r

R o s s 1.

Departments of XBiochemistry and 2Physiology and Biophysics, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, N Y 10029-6574, U.S.A. Binding o f corticosteroids by h u m a n corticosteroid binding globulin (hCBG) is thought to involve interaction with one o f its two cysteine residues (Cys60 and Cys228). To identify which of the two cysteine residues mediates steroid binding, we have produced m u t a n t hCBGs containing serine or alanine in place o f Cys228 by site-directed mutagenesis. Alteration of Cys22s to serine or alanine does not change the steroid binding affinity o f hCBG, demonstrating that Cys228 is not involved in the binding interaction. This finding strongly suggests that Cys60 is the functionally important cysteine. By modifying the wild-type and m u t a n t hCBGs with the sulfhydryl-specific reagents N - e t h y l m a l e i m i d e , iodoacetamide, and sodium tetrathionate, we have demonstrated that Cys60 is present at the steroid binding site, and that it m a y be directly involved in steroid binding. This result also identifies Cys60 as the accessible cysteine reported in previous studies.

J. Steroid Biochem. Molec. Biol., Vol. 48, No. 1, pp. 139-144, 1994

INTRODUCTION Human corticosteroid binding globulin (hCBG) is a serum glycoprotein that binds cortisol and progesterone with high affinity. Apart from maintaining a balance of bound and free steroids in the circulation, hCBG may be involved in release of cortisol at the site of inflammation, following cleavage of hCBG by neutrophil-derived elastase [1]. We set out to investigate the structure-function relationships within CBG, and to understand the basis for steroid specificity. Affinity labeling studies have suggested that one cysteine residue is present within the steroid binding site of hCBG [2]. The mature polypeptide chain of hCBG contains two cysteine residues, Cys6oand Cys22s [3], and these residues do not form an intramolecular disulfide bond [2]. It is not yet resolved whether the free sulfhydryl (SH) group of the residue associated with the steroid binding site is involved in the steroid-protein interaction or in maintaining the structural integrity of the steroid binding *Correspondence to J. B A. Ross Received 15 July 1993, accepted 5 Oct 1993 139

site. Comparison of the cDNA sequences of human, rat, and rabbit CBG revealed that Cysz2g is conserved among these species [3-5]. Moreover this residue is present within a conserved peptide sequence of 40 amino acids [6]. This observation offers a preliminary indication that Cys228 is the residue present in the steroid binding site. To identify which of the two cysteine residues is associated with the steroid binding site of hCBG, we created two mutant hCBGs, in which the C y s x z g is replaced by serine or alanine (designated as hCBG-Ser22s and hCBG-Ala22s, respectively). Production of these two mutant proteins served several purposes. Observation of a change in the steroid binding properties of these mutant proteins would provide strong evidence that Cyszzs is involved in steroid binding. If, however, the mutant proteins containing only Cys60 are capable of binding steroid, these recombinant proteins would comprise excellent models to address the role of this residue using chemical modification with SH-specific reagents. Here we report expression of mutant hCBGs in insect cells, using the baculovirus system in which we have previously shown high levels of expression

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140

of bioactive recombinant hCBG [7]. Contrary to our expectation, comparison of the steroid binding properties and steroid specificities of the wild-type and mutant hCBGs showed that Cys228 is not essential for steroid binding. Using SH-specific reagents with the single cysteine-containing mutant proteins, we instead have identified Cys60 as being present in the steroid binding site. MATERIALS AND M E T H O D S

Site-directed mutagenesis Oligonucleotide directed mutagenesis was carried out in a two step polymerase chain reaction (PCR) as described by Landt et al. [8] and as outlined in Fig. 1. Mutagenic oligonucleotides were designed to change Cys228 to serine or alanine. Two synthetic oligonucleotides were synthesized, corresponding to bp 771-797 of the cDNA in the antisense direction: Oligo 3: 5' C T G CAC CAG C T G G C T G G G GAG CTC TGA 3' Oligo 4: 5' C T G CAC CAG C T G GGC G G G GAG CTC T G A 3' Oligos 3 and 4 contain a codon for serine and alanine in place of cysteine, respectively. The base changes within these codons are underlined. The 5' universal primer (Oligo A) was: 5' GCC AGC AGA CAG ATC AAC 3' (bp 531-548 of the sense strand of the cDNA). The 3' universal primer (Oligo B) was: 5' T C G T G T CCC GGC TCA G T G 3' (bp 871-888 of the cDNA in the antisense direction). The 5' and 3' universal primers flank the Sal 1 and Bsp E1 restriction sites, respectively, enabling the final PCR fragments to be cloned into hCBG cDNA at those sites. PCR was carried out in a DNA thermal cycler (Perkin Elmer Cetus). A first round of PCR was performed in 100/~1 1 x Taq polymerase buffer containing 2 # M oligo A, 2 # M mutagenic oligonucleotide (oligo 3 or 4), 2 ng of pGEM1-CBG DNA [7], 50 # M dNTP mixture, and 10 U of Taq DNA polymerase (Promega). 30 cycles of amplification were carried out, each consisting of 60s at 94°C, 60s at 55°C for oligo 3 or 58°C for oligo 4, and 45s at 72°C. The resulting fragments were gel purified [9]. The second PCR reaction was carried out as described for the first PCR, but in this case the entire intermediate fragment served as the 5' primer and oligo B was used as a 3' primer, the annealing temperature was also changed to 45°C. The purified PCR products of this reaction were digested with Sal 1 and Bsp El, and the products were gel purified and cloned into pGEM1-CBG which has been cleaved with Sal 1 and Bsp El. The cloning protocol was as described previously [9]. The presence of the desired mutations was confirmed by sequencing both strands of the mutagenized insert (Sal 1 to Bsp El). The complete hCBG cDNA sequences were then cloned in the baculovirus derived vector pVL1393 to generate pVL1393CBG-Ser228 and pVL1393CBG-Ala228; these

were then used to produce recombinant baculovirus as described previously [10, 7].

Cell culture Spodoptera frugiperda ( s f g) cells were maintained in Grace's insect cell culture medium supplemented with T C yeastolate, lactalbumin hydrolysate (each at 3.33 gin/l, Difco) and 10% fetal bovine serum (Gibco). This medium is known as T N M F H - 1 0 % FBS. For serum-free medium culture, Grace's medium containing TC yeastolate and lactalbumin hydrolysate was supplemented with 1% ITS supplement containing insulin, transferrin, selenius acid, and bovine serum albumin (BSA) (Collaborative Research). Alternatively, sf900 medium (Gibco BRL) was used. S f 9 cells were always maintained at 27°C. Infection and metabolic labeling To synthesize wild-type and mutant hCBGs in vivo, S f 9 cells were infected with the corresponding recombinant viruses at a multiplicity of infection of 10-25 infectious units per cell. Radiolabeling, immunoprecipitation, and SDS-PAGE were performed as described previously [7].

Steroid binding A binding assay employing [3H]cortisol (40-50 Ci/ mmol, New England Nuclear) was carried out as described previously [11]. Prior to assay, cell lysate samples were diluted with 10 mM Tris-HCl (pH 8.0), and medium samples were concentrated and dialyzed against the same buffer.

Chemical modification with SH-specific reagents S f 9 cell-derived medium containing wild-type or mutant hCBGs was concentrated several fold and dialyzed against 10mM Tris-HCl (pH8.0), using Centricon filter apparatus (Amicon). The final samples contained nanomolar concentrations of hCBG. 320 #1 of each sample were treated with 50 mM dithiothreitol (DTT) (final concentration) for 1 h at room temperature. The reduced samples were then treated with 200mM N-ethylmaleimide [12], 100mm iodoacetamide [13] or 200 mm sodium tetrathionate [14] at room temperature. Excess reagents were removed by passing the samples through a spin column of Sephadex G25 equilibrated in 10 mM Tris-HC1, pH 7.4. Finally, [3H]cortisol binding was measured in the eluate fraction. RESULTS

In vivo expression of mutant hCBG To investigate the role of cysteme residues in steroid binding by hCBG, the codon for Cys228 in hCBG cDNA was changed by site-directed mutagenesis to encode serine or alanine. The resulting hCBG-Ser~28 and hCBG-Ala228 sequences were then expressed in S f 9 insect cells, using recombinant baculoviruses as vectors. To monitor the expression of mutant hCBGs

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and the kinetics of h C B G secretion, a pulse-chase labeling experiment was performed. T h e infected cells were metabolically labeled with [35S]methionine. Intra- and extracellular h C B G were isolated by immunoprecipitation with antihCBG antibody and were resolved by S D S - P A G E [Fig. 2(a)]. Both mutant h C B G s were expressed as efficiently as wild-type hCBG. These mutant proteins exhibit an identical distribution of molecular forms, indicating that they undergo similar posttranslational modifications as does wild-type hCBG. Furthermore, the kinetics of secretion of these mutant proteins are similar to those of wild-type h C B G [Fig. 2(b)]. After 2 h of chase, ~ 4 0 % of pulse-labeled hCBG--Ser22s and ~ 5 0 % of hCBG-Ala22s are secreted.

Biological activity of the mutant hCBGs T o determine whether the replacement of Cys2~ with serine or alanine results in an alteration of steroid binding properties, Scatchard analysis [15] was used to compare the [ 3H]cortisol binding affinities of wild-type SBMB 48/I--G

and mutant hCBGs. Within experimental error, all of these species exhibit an identical Kd for cortisol (1-2 x 10 -9 M at p H 8.0, 4°C). These findings strongly suggest that the SH moiety of Cyse2s does not play a direct role in steroid binding.

Effect of SH-specific reagents on the steroid binding activities of wild-type and mutant hCBGs T h e production of biologically active mutant h C B G s containing only one cysteine (Cys,0) allowed us to investigate the role of this residue in steroid binding. For this purpose, the mutant and wild-type proteins were modified with SH-specific reagents, and the effects on steroid binding were measured. First, serumfree medium derived from infected S f 9 cells was concentrated several fold and buffer exchanged so that the final p H was 8.0. Samples were then treated with 200 m M N-ethylmaleimide ( N E M ) for 30 min, with or without prior treatment with 50 m M D T T . In the case of D T T pretreatment, D T T was removed before exposure to N E M . After N E M treatment, excess

142

Jayasri Ghose-Dastidar et al.

(a) Cys228

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60 TiME (min)

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Fig. 2. T i m e course of synthesis and secretion of wild-type and m u t a n t (Cys~zs---, Ser2zs; Cys2zs--~ Alazzs) hCBGs. Sf9 cells infected with recombinant baculovirus encoding wild-type or m u t a n t hCBGs were pulse-labeled for I h with [3SS]methionine (100 p C i / m l ) , and chased in the presence of 10 m M unlabeled methionine for the indicated times (0.5, 1, and 2 h). Cells and m e d i a were harvested at each t i m e and subjected to i m m u n o precipitation with anti-hCBG antibodies. (a) S D S - P A G E resolution of i m m u n o p r e c i p i t a t e s of the cell lysates (inside) and m e d i u m samples (outside). (b) Quantitation by densitometry.

reagents were removed by passing the reaction mixture through a spin column of Sephadex G-25, after which cortisol binding was measured for this sample. N E M treatment in the absence of D T T results in 60 and ~40% inhibition of [3H]cortisol binding by the wild-type hCBG and hCBO--Alaz2s, respectively (Table 1). Pretreatment of these proteins with D T T increased the inhibition to ~ 80 and ~ 90%, respectively, suggesting that there is partial oxidation of the cysteine in the expressed protein, hCBG-Ser22 s shows similar NEM sensitivity (data not shown). In all cases, inactivation occurs within 10min of incubation with NEM. These results suggest that the SH moiety of Cyss0 is either directly involved in steroid interaction or is important for maintaining the structure of a functional steroid binding site. Alternatively, it is possible that the inactivation results from steric hindrance

produced by chemical modification of the SH group at the steroid binding site. The wild-type and mutant proteins were also treated with 100mM iodoacetarnide (IAA) using a similar protocol. This treatment was more effective than N E M in eliminating the cortisol binding activity of wild-type and mutant hCBGs (Table 1), and it does not require pretreatment with D T T . Kinetic analysis showed that IAA inactivation is complete within 30 s of treatment (data not shown). This inactivation occurs at pH 8.0 or above, consistent with modification of a cysteine residue in the steroid binding site. Inactivation by IAA does not require prior reduction of wild-type and mutant proteins. The fact that prior reduction is not required to achieve complete inactivation by IAA, may be explained by the high reactivity of IAA (since

Identification of the Cysteine in hCBG

143

Table 1. Effect of S H reagents on the cortisol-binding activity of wild-type and mutant hCBGs Wild-type Treatment

clam

Residual activity (%)

Ala228 cpm

Ser228

Residual activity (%)

clam

Residual activity (%)

650 37

6

1110 301

27

IAA Control 100 m M NEM Control 200 m M (Pretreat with 50 mM DTT) Control 200 m M NazS40e (pretreat with 50 mM DTT) Control 100 m M

2730 34

1

2347 110

5

4200 1523

36

1794 1012

56

1551 259

17

1012 83

8

9490 198

2

4132 450

10

Cortisol binding activity was assessed by a filter assay using [3H]cortisol [11]. The differences

m cpm of the different samples prior to treatment with a particular SH reagent reflect d~fferences in their respective protein concentrations. Samples pretreated with DTT were passed through a second G-25 spin column to remove excess DTT before subsequent treatment with NEM. Control experiments showed that under the assay conditions (saturating steroid) 10 mM DTT did not significantly inhibit steroid binding. iodine is a good leaving group), what may allow it to react with a partially oxidized S H group. T h e wild-type and mutant h C B G s were also treated with another SH-specific reagent, sodium tetrathionate, in a similar experimental protocol. Within 5 min of treatment with 100 m M sodium tetrathionate, cortisol binding activity is effectively abolished. This result confirms that it is the SH group of Cys60 that is in the steroid binding site (Table 1). DISCUSSION Affinity labeling and chemical modification studies on h C B G , purified from human serum, suggested that one or both of the two cysteine residues in this protein are in the immediate vicinity of the steroid binding site and may be directly involved in steroid-protein interaction [2, 16]. Le Galliard and Dautrevaux [17] reported that one cysteine is more accessible than the other, and that modification of the more accessible SH group with ethyleneimine did not induce any loss of binding activity in hCBG. This suggested that the inaccessible cysteine might be buried within the steroid binding site. Comparison of the c D N A sequences of human, rat, and rabbit C B G reveals that only CyS22s is conserved in evolution [3-5], and this residue is present within a conserved flanking sequence of 40 amino acids [6]. T h e other cysteine residue in h C B G , Cys60, is replaced by a serine in rabbit [5] and by an alanine in rat [4]. Moreover, Cys22s is the only cysteine residue in the rat protein [4]. Thus, it was our expectation that Cys22s would be the inaccessible cysteine which is critical for steroid binding in hCBG.

T o ask whether Cys2z~ is indeed involved in steroid binding, we used site-directed mutagenesis to create h C B G cDNAs in which the codon for Cys22s encodes serine or alanine. Serine was chosen because it is chemically and sterically similar to cysteine. I f the cysteine SH group is directly involved in hydrogen bonding with some part of the steroid molecule, we predicted that this interaction would be maintained in the serine mutant, which should retain that hydrogen bonding possibility. However, in the alanine mutant, we expected that this kind of interaction should be abolished, since the side chain of alanine has a methyl group that would eliminate the possibility of hydrogen bond formation. T o compare the properties of the wild-type and mutant proteins, we produced these proteins in baculovirus-infected insect cells. T h e rates of synthesis and secretion of the mutant h C B G s are very similar to those of the wild-type protein (Fig. 2). Furthermore, the spectrum of molecular forms (representing differentially glycosylated species) is also identical among these proteins, suggesting that the posttranslational modifications of these mutants are identical in the mutant and wild-type polypeptides. We conclude that these single amino acid substitutions cause no major change in either protein biogenesis or overall conformation. Scatchard analysis of steroid binding data obtained from the filter assay shows that both mutant h C B G s have a high affinity for cortisol, identical with that of wild-type hCBG. On the one hand, if Cys22s is involved in steroid binding, we might predict that hCBG-Ser22g could retain cortisol binding activity because serine is chemically and structurally a conservative mutation. In

144

Jayasrl Ghose-Dastidar et al.

this case, we w o u l d e x p e c t a d e c r e a s e in affinity, since t h e O H o f serine is a w e a k e r n u c l e o p h i l e t h a n t h e S H g r o u p o f cysteine; h o w e v e r , no c h a n g e in affinity was o b s e r v e d . O n the o t h e r h a n d , t h e d e m o n s t r a t i o n t h a t hCBG--Ala22s is e q u a l l y active a r g u e s a g a i n s t Cys228 b e i n g t h e f u n c t i o n a l l y i m p o r t a n t c y s t e i n e since in this m u t a n t t h e h y d r o g e n b o n d i n g a b i l i t y o f r e s i d u e 228 has b e e n e l i m i n a t e d . T h i s l e d us to the c o n c l u s i o n t h a t Cys60, r a t h e r t h a n Cys2zs, is t h e c y s t e i n e r e s i d u e i n v o l v e d in s t e r o i d b i n d i n g . A l t h o u g h the p r e v i o u s i n v e s t i g a t i o n s p o i n t e d to a b u r i e d o r i n a c c e s s i b l e c y s t e i n e r e s i d u e w i t h i n the stero i d b i n d i n g site [16-18], t h e p r e s e n t w o r k p r o v i d e s e v i d e n c e t h a t the accessible c y s t e i n e is f u n c t i o n a l l y i m p o r t a n t . F o r e x a m p l e , w e find t h a t after t r e a t m e n t w i t h N E M , t h e b i n d i n g activities o f b o t h t h e w i l d - t y p e a n d t h e m u t a n t p r o t e i n s are significantly r e d u c e d ( T a b l e 1). T h e i n a c t i v a t i o n o f s t e r o i d b i n d i n g b y I A A , w h i c h has a p H o p t i m u m a r o u n d 8.0, f u r t h e r suggests t h a t an accessible S H g r o u p is i n v o l v e d in s t e r o i d b i n d i n g , since the pK~ o f t h e c y s t e i n e S H g r o u p is a r o u n d 8.0. M o r e o v e r , t h e o b s e r v a t i o n t h a t the kinetics o f this i n a c t i v a t i o n are v e r y r a p i d (30 s for 1 0 0 % i n a c t i v a t i o n ) e l i m i n a t e s h i s t i d i n e as t h e t a r g e t for m o d i f i c a t i o n , since t h e rate o f h i s t i d i n e m o d i f i c a t i o n b y I A A is v e r y slow [13]. T o p r o v i d e f u r t h e r c o n f i r m a t i o n t h a t a c y s t e i n e is t h e r e s i d u e m o d i f i e d in t h e a b o v e e x p e r i m e n t s , we t e s t e d t h e effect o f s o d i u m t e t r a t h i o n ate w h i c h r e v e r s i b l y m o d i f i e s S H g r o u p s [14]. S o d i u m t e t r a t h i o n a t e also c o m p l e t e l y a b o l i s h e d t h e a c t i v i t y o f both wild-type and mutant hCBGs. In conclusion, site-directed mutagensis coupled with specific c h e m i c a l m o d i f i c a t i o n u n a m b i g u o u s l y d e m o n strate t h a t Cys22s is f u n c t i o n a l l y u n i m p o r t a n t . T h e d a t a s u p p o r t b u t do n o t p r o v e the h y p o t h e s i s t h a t the S H g r o u p o f Cys60 is d i r e c t l y i n v o l v e d in t h e s t e r o i d - p r o t e i n i n t e r a c t i o n in h C B G (at a m i n i m u m , this r e s i d u e m u s t be in close p r o x i m i t y to the b o u n d s t e r o i d m o l e c u l e ) . T h e fact t h a t t h e side chains o f Cys60 a n d Cys228 d o n o t f o r m a disulfide b r i d g e [16] suggests t h a t the t w o c y s t e i n e s r e s i d u e s are n o t i m m e d i a t e l y a d j a c e n t in t h e t e r t i a r y s t r u c t u r e . N e v e r t h e l e s s , t h e y c o u l d b e sufficiently close t h a t c h e m i c a l m o d i f i c a t i o n o f one c o u l d sterically p r e c l u d e m o d i f i c a t i o n o f the o t h e r , as o b s e r v e d b y L e G a i l l a r d a n d D a u t r e v a u x [17]. I n a d d i t i o n , t h e fact t h a t a c y s t e i n e f o r m s a c o v a l e n t a d d u c t w i t h 6 f l - b r o m o p r o g e s t e r o n e [3] does n o t n e c e s s a r i l y r e q u i r e t h a t t h e r e s i d u e ts essential for s t e r o i d b i n d i n g . I n s t e a d , it is c o n s i s t e n t w i t h t h e g e n e r a l c o n c l u s i o n t h a t one o f t h e c y s t e i n e s m u s t be a s s o c i a t e d w i t h the s t e r o i d b i n d i n g site [16]. T h e r e f o r e , an i n t e r p r e t a t i o n o f o u r d a t a t h a t is c o n s i s t e n t w i t h t h e g e n e r a l l i t e r a t u r e , is t h a t b o t h Cys228 a n d Cys60 reside in o r n e a r t h e s t e r o i d b i n d i n g site, a n d it is p o s s i b l e t h a t n e i t h e r c y s t e i n e p e r se is r e q m r e d for steroid binding. Further mutagenesis and chemical modification e x p e r i m e n t s are in p r o g r e s s to d e t e r m i n e w h e t h e r t h e S H g r o u p o f Cys60 is d i r e c t l y i n v o l v e d in t h e s t e r o i d p r o t e i n i n t e r a c t i o n or w h e t h e r c h e m i c a l m o d i f i c a t i o n

s i m p l y b l o c k s access to t h e s t e r o i d b i n d i n g site. I n a d d i t i o n , t h e single c y s t e i n e h C B G m u t a n t s p r o v i d e us a basis for h i g h e r r e s o l u t i o n i n v e s t i g a t i o n b y use o f S H - s p e c i f i c p r o b e s in c o n j u n c t i o n w i t h s p e c t r o s c o p i c methods. Acknowl~Igements--The authors thank Dr Gerald Schwartz and

Dr Ronald Kohanskl for suggestions and advice regarchng specific chemical modificauon of cysteine residues, and we thank Dr Arthur Cederbanm and coworkers for sharing their ussue culture facilities This work was supported by NIH Grants GM-38501 (R.G) and GM-39750 (J.B.A.R).

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