Site-specific mutagenesis of the human interleukin-1β gene: Structure-function analysis of the cysteine residues

Vol. 150, No. 3, 1988

BIOCHEMICAL

AND BiOPHYSlCAL

RESEARCH COMMUNICATIONS Pages 1106-1114

February 15, 1988

SITE-SPECIFICMUTAGENESIS OF THEHUllANINTERLEUKIN-1 B GENE: STRUCTURE-FUNCTION ANALYSISOF THECYSTEINE RESIDUES Takashi Kamogashira,Yoshihiro Masui, YasukazuOhmoto, Tohru Hirato, Kenji Nagamura, Keiko llizuno, Yoeng-NanHong, Yoshikazu Kikumoto, Satoru Nakai and Yoshikatsu Hirai Laboratories of Cellular Technology, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno,Kawauchi-cho, Tokushiaa 771-01, Japan Received December 17, 1987

SUMARY: Humaninterleukin-18 (IL-181 has two cysteines located at amino acid residues 8 and 71 of the mature protein consisting 153 amino acids. To clarify the role of these characteristic cysteine residues in IL-lB, at first, an expression plasmid for site-specific mutagenesishas been constructed by inserting the ori and intergenic region of phagefl into the IL-18 expression vector. The plasnid can be used not only for isolation of the modified IL-1B gene but for expression of the mutant protein in Escherichia coli. Using this plasmid, each of the cysteine codons in IL-1B gene was changed tozne or alanine codon, or deleted. The modified IL-l#? showed that the two cysteine residues in 11-1s are not essential for biological activity but not to be eliminated for the maintenance of the functional structure of 11-l/3. 0 1988Academic Press,Inc.

Interleukin-l(IL-1)

was first

defined as a lymphocyte activating

factor

(LAP)

produced by monocytes-macrophages(1) and it has becomeevident that IL-1 activities IL-1 plays a central

are produced by several other cell types (21. activation

of physiological

responses to injury and infection

cloning studies have shownthat IL-l activities molecules

(11-1~~ and IL-lb)

precursor

(4-71.

constructed histiocytic

We also have isolated

clones of IL-18

from mRNAexpressed in human peripheral lyaphoma U937 cells

1106

to

harboring human IL-18 plasmid

Al though it has been reported that

the disulfide

0006-291x/88$1.50

has been purified

gene shows that two cysteines are

both cysteine residues in recombinant human IL-lfl bridge (9-11) and therfore

cDNA from libraries

blood monocytes and human

(8) and mature IL-lb

located at amino acid residues 8 and 71 (4).

Copyright 8 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

17.5 kD

each of which is produced by cleavage of a 31 kD

Nucleotide sequence analysis of IL-18

disulfide

Recent cDNA

can be mediated by 2 distinct

homogeneity from recombinant Rscherichia coli --- (E.coli) (9).

(2,3).

role in the

( rIL-1B)

are not involved in a

bridge was not essential for

Vol. 150, No. 3, 1988

BIOCHEMICAL

exerting the biological activity functional structure of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of IL-lb,

the role of the cysteine residues in the

IL-18 by using site-specific

mutagenesishas not been well

evaluated. In this report,

we describe the construction of the versatile

specific mutagenesisof IL-1s. intergenic

region of phage fl

The plasmid was constructed by inserting the ori and into the IL-18

and purified

Site-specific

expression vector.

mutagenesisprocedures were used to modify the each of the cysteine codons.

plasmid for site-

The modified IL-l/3

IL-lb

gene by changing or deleting

proteins were produced in E.coli

to homogeneity and then assayed for bioactivity.

influence of each cysteine residue on the biological activity

In this way, the of IL-l/3

was studied.

MATERIALS ANDHETHODS Enzymes Restriction enzymes were obtained from Takara Shuzo Co. and New England Biolabs. E.coli DNA polymerase (Klenow fragment), T4 DNA Iigase and T4 polynucleotide kinase were obtained from Takara Shuzo Co. Bacterial strains and DNAs E.coli strain HV 1304 (A (lac- roAB), t&, EpsL(s trep * 1, endA, sbcBl5, hspR4, A (d- e)306 :: TnlO(tet’ P : traD36, I?proAB,lacI q 2 8#15 1)wa.s used throughout this study fortransformation and express ion. The helper phage Ml3 K07 was obtained from Takara Shuzo Co. The plasmid Bluescribe H13(-) was purchased from Vector Cloning Systems. PIN-RI-ompA3 (12) was generously provided by Dr. H. Inouye (Robert WoodJohnsonMedical School at Rutgers, University of Medicine and Dentistry of New Jersey). The synthetic oligonuclotides were synthesized using a DNAsynthesizer (Applied Biosystems). Construction of fl. IL-18 1ppT The IL-18 expression plasmid for site-specific mutagenesiswasconstructed as diagrammed in Fig. 1. The construction of ptrpIL-l/3 have been described previously (9). It contained the coding region of the mature IL-l,9 under the control of m promotor. We further constructed the plasmid ptrpll-1BlppT by inserting the BamHI-Sal1fragment of PIN-III-ompA3 instead of the &HI-Sal1 fragment of ptrpIL-1BT Thevector was inserted the intergenic region of Bluescribe H13(-1 vector at the pvUII restriction site of ptrpIL-1BlppT. The resulting plasmid fl. IL-1BlppT contained the intergenic region of the phage fl (13) was used for isolation of singIe-stranded DNA (ssDNA)for site-specific mutagenesis and for expression of IL-18 protein. Site-specific mutagenesis(14) The mutagenic oligonucleotides (200 pm011shown in Table I were 5’phosphorylated with 4.5 unit of T4 polynucleotide kinase in a 30 iu 1 reaction containing 1 mHdithiothreitol (DTT) and 1 m! spermidine. After 1 hr at 37C, the reaction was stopped by heating at 65C for 10 min. The ssDNAtemplate from fl.IL-1BlppT (0.5 pm011was mixed with the phosphorylated oligonucleotide (10 pmol) and 1 ~1 of 10 x hybridization buffer (100 mHTris-HCl, 50 mHMgClz , pH 7.5) in a total volume of 10 ~1. The mixture washeated to 9Oc for 5 min and allowed to gradually cool to room temperature over 1 hr. To the 10 ~1 annealing mixture wasadded 1 .ul of 100 mMDTT, 3 yl of 2.5 mti dNTPs, 1 ~1 of 10 mHof ATP, 1 ,~l of 10 x hybridization buffer, 3 units of T4 DNA ligase, 2.5 units of Klenow DNA polymerase I and distilled water to 20 ~1. This reaction was incubated at 15’c for 18 hr. A sampleof the ligation reaction was used for transformation of competent E. g&i HV 1304 cells. Colonies containing mutated plasmids were identified by hybridization with the 5’-SzP-labeled oligonucleotide. Plasmid DNAfrom positive colonies was prepared by the miniprep. procedure (151 and used for a second round of transformation and screening. Plasaid DNAfrom positive secondary transformants was 1107

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

subjected to nucleotide sequenceanalysis to confirm the presence of the mutation. DNAsequencing of plasmid ssDNA template was according to the chain termination procedure of Sanger e al. (16). Preparation of rIL-Ifi analogue proteins Induction of the 9 prosotor was obtained after tryptophan starvation of E.coli harboring mutant plasmid. After growth in M9 mediumcontaining 0.4 % glucose and 1.0 % casaminoacid for 8 hr at 37C 1 ml portion of the culture was pelleted by centrifugation at 12,000 rpm for 5 min Ht 4c. Extracts for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were madefrom the cell pellets by vigorous vortexing and heating at 1OOC in Laemmlisample buffer (17). E.coli lysates for bioassay were prepared from the cell pellets followed the procedure of preparation of plasmid DNAby the Triton X 100 lysis method (18). Briefly, the cells were suspendedin 450 ~1 of a solution of 50 mM Tris.HCl and 25 % sucrose, pH 8, by vortexing and then placed on ice. Then 50 ,ol of a 10 mg/ml of solution of lysozyme (Sigma) dissolved in 25 m! Tris.HCl, pH 8, was addedand the mixture was incubated on ice for 10 min. Next 50 ,u 1 of 0.25 mll EDTA, pll 8, was added, followed by 450 ,~l of 0.3 % Triton X 100, 50 MMTris.HCl and 25 ml EDTA, pH 8. Each tube was shaken immediately after addition of the Triton solution and then placed at room temperature. After 10 min, the lysates were centrifuged at 12,000 rpm for 10 min at 4C. Supernatants of cell extract were used for biological assay and enzymeimmunoassay. Analysis of rIL-l/3 analogue proteins SDS-PAGE was performed essentially as described by Laemmli (17) using 15 % polyacrylamide gels. Immunoblot analysis and immunostainingof rIL-l/3 protein were performed by a modification of the procedure of Towbin -et al. (19) by using monoclonal antibodies. 11-1s activity was determined its ability to inhibit the Biological assay growth of humanmelanoma cells A375.Sl (20,211. This property of IL-18 is referred to as its growth inhibitory factor (GIF) activity. One GIF unit is defined as the reciprocal of the sample dilution needed to bring about a 50 % cytostasis or cytolysis in 4 days old cultures. Enzymeimmunoassay IL-1B and analogue proteins were quantitated by ELISA using affinity-purified anti-human IL-18 Fab’-peroxidase conjugate as described (22). Purification of rIL-l/3 analogue proteins Analogue proteins, which were produced from the mutants in E.coli, were purified to homogeneity by a combination of cation exchangeHPLCand gel filtration HPLCas described previously (9).

RESULTS ANDDISCUSSION The construction

of the fl.IL-IBlppT

expression plasmid for IL-lb, laboratories

(9).

plasmid ptrpIL-1alppT

plasmid is shown in Fig.

ptrpIL-la,

Because of stability

was previously of the mRNAof

constructed

conventional vector The intergenic

(231,

wasconstructed by inserting the Ban~llI-SaJIfragment of PIN-El

instead of the NHI-SaJI fI.IL-1BlppT

fragment of

for site-specific

ptrpIL-1 B 1ppT with the correct orientation.

ptrpIL-18

restriction

stranded plasmid, fl.IL-1BlppT

and the

site of

In this case, the ssDNAobtained was

The fl intergenic region which is the N$I-&I

vector contains the fI origin of replication,

termination

mutagenesisof IL-l/.? was made.

region of phage fl was inserted at the bD

the coding strand.

The in our

E coli lipoprotein -.-

-0mpAQ(12) which contains a part of coding region and transcription region of lipoprotein

1.

fragment from Bl3

packaging and excretion.

was converted into ssDNAby super-infection 1108

Doublewith a

Vol. 150, No. 3, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EcoRI

Pvu II BamHI+Sal

BamHI I

Sal I

900bp BamHI-Sal I fragment

4.7kb BamHI-Sal I fragment I

I

T4 DNA 1igase NdeI EcoRI

ptrpIL-lf3tppT

4 Ndef-Klenow 4 PvuI-Mung

bean

Nde~Pvul

547bp NdeI-PvuI

P&II

fragment

II

pvull T4 DNA ligase EcoRI

fl*IL-l$lppT

NaeI Fisure 1. Constructionof expressionplasmidfl.IL-1/3lppT. Details of the procedure are describedin HATRRIALS ANDMETHODS. In the resulting plasmidfl. IL-1 @lppT, sequence sources: lL-lfl codingregion, m ; transcription termination region of ~gene, : the intergenic region of phagefl gene, m . Other abbreviations: ampicillin resistancemarker,A’: &J promotor-operator,trp”O .

helper phage Ml3 K07 in E.coli functions,

F+ strain

this plasmid replicated

NV 1304.

By utilizing

phage encoded

as ssDNA, was packaged and excreted

into the

growth mediumin the sameway as Ml3 vectors and the ssDNAtaRplate obtained was used 1109

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BIOCHEMICAL

I.4 I

2

12

3

3

5

4

4

5

6

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6

7

7

8

8

9

9

10

M

10

14.4

Gel analysis and immunoblot detection of rIL-lb protein andanalogues. Lanel-10 referred to SDS-PAGE carried out on a 15 %polyacryl-amidegel as described in MATERIALS ANDMETHODS. A, Coomassie Blue staining. 8, Immunoblot detection of rIL-1 f9. M, Hr Standardsfrom Pharmacia Pine Chemicals.:lane 1, no plasntid; lane 2, wild type rIL-1 B: lane 3, Ser’ mutant: lane 4, Ala’ mutant; lane 5, Ser” mutant: lane 5, Ala” mutant; lane 7, Ala’-Ser” mutant: lane 8, Alas-Ala” mutant: lane 9, des-Cysamutant: lane 10, des-Cys” mutant.

Figure 2.

in dideoxy sequencing or site-specific

mutagenesis.

On the other hand when E.col i

MV 1304 harboring this plasmid was incubated with the production medium, IL-1 B protein

was expressed and followed to the analysis of biological

assay, enzyme

immunoassay,SDS-PAGE and purification. Site-specific

mutagenesisusing ssDNAtemplate from this IL-lb

plasmid, fl.IL-1/3IppT,

E.coli expression

was used to create the mutants, in which cysteine residues

at the position of 8, 71 or both were substituted with serine or alanine, or deleted as shown in Fig. 2 and Table 2.

Synthetic 1110

oligonucleotide

primers used for each

BIOCHEMICAL

Vol. 150, No. 3, 1988

Table I. Synthetic

oligonucleotide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

primers used to produce mutants of rIL-1B Oligonucleotide

Mutants

primer

Se?’ mutant(substitution

of serine for cysteine 8)

5’ -CTCAAC m ACTCTC-3’

Ala” mutant(substitution

of alanine

for cysteine 8)

5’ - CTCTGAAC E

Ser7’ mutant(substitution

of serine

for cysteine 71)

5’ -CTGTCC TCA GTGTTG-3’

Ala”

of alanine

mutant(substitution

Des-Cyss mutanttdeletion

for cysteine

71)

5’-ACCTGTCC Gee GTGTTGA-3’

5’ -GTTCTCTGAAC*ACTCTCCG-3’

of cysteine 8)

Des-Cys” mutantcdeletion

ACTCTCC-3’

5’ -TGTACCTGTCC*GTGTTGAA-3’

of cysteine 71)

The underlined letters indicate where cysteine codons (TGC) were changed. asterisks indicate where codons were deleted.

mutation

are listed in Table 1.

The selected mutant was identified

The

the base change

or deletion by DNAsequencing. Each mutant clone was induced and first and size of the rIL-18

assayedby SDS-PAGE to monitor the amount

mutant protein in the total crude lysates in E.coli under the

reducing condition (Fig. 2).

Mutant proteins were clearly visible on CoomassieBlue

stained gels (Fig. 2A, lane 4, 5, 6,

7, 8) as a band migrating with an apparent

molecular mass of 17.5 kD as sameas the wild type rIL-1B

Table 2.

IL-la

activity

in the crude extract activity

b

protein (lane 2).

of rIL-1S

mutants

Mutants ’

Specific

Control percent

Wild type rIL-1B

2.1

x 10’

20

Se9 mutant

5.7

x 105

2.7

Ala’ mutant*

2.1

x 107

100

Ser”

mutant*

2.0

x 10’

95

Ala”

mutant*

2.2

x 10’

105

x 10’

110

units/mg

Ala*-Ser”

mutant*

2.3

Alas-Ala”

mutant*

2.1 x 10’

100

Des-Cys’mutant

6.7

x 10’

3.2

Des-Cys“

2.1

x

0.1

mutant

10’

a; Extracts were prepared by resuspending in Triton X 100 lysis buffer as described in HATERIALS AND HETIIDDS. *, Partially purified mutant proteins were used for determination of biological activity. b; Biological activity was determined with the ability of growth inhibition for human melanoma cells A375.Sl. The amount of protein was determined by ELISA. Specific activity was evaluated as GIF unit per mg protein.

1111

This

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BlOCHEMiCAL

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Table 3. IL-18 activity of the purified rIL-18 and its mutantproteins Polypeptide

Relative biological activity * (%I

Isoelectric point b

Wild type rIL-18

180

6.9

Ala’ mutant

204

6.9

Ala” mutant

187

6.9

Ser’r mutant

174

6.9

Alas-Ala”’ mutant

237

6.9

a; Biological activity wasdeterminedwith the ability of growth inhibition for human melanoma cells A3KSl. b: Isoelectric point wasdeterminedby analytical isoelectric focusing.

The

band was absent in cells not containing the IL-I /3 expression plasrnid (lane 1).

of the 17.5 kD protein bands was confirmed by imrunoblotting with an IL-lB-

identity

specific monoclonal antibody (Fig. 2B).

The low level expression was observed in

Ser 8, des-Cys 8 and des-Cys 71 mutants (lane 3, 9, 10).

Whenplasmids of three

mutants were introduced into an E.coli strain which was deficient the high level expressions were observed (data not shown). that low level expression may be due to instability

in the la protease,

Therfore, we reasoned

of these IL-l fi mutants as a

result of the presence of endogeneous proteases in E.coli. To evaluate the influence of each cysteine residue on the biological activity IL-1B

mutants, protein extracts

inhibition

of

from induced cells were tested by the ability

of

for growth of humanmelanomaA375.51 cells (21) and evaluated as unit.

The amountof IL-Ifi

was determined by ELISA using Fab’ fragment of anti-human IL-l@

antibodies conjugated to peroxidase (22).

Specific activity of IL-1B

mutant was comparedto the

clone as the percent residual bioactivity.

As shownin Table 2, the

unit per mgprotein and then biological activity wild type IL-18 substitutions retained full

was expressed as GIP

of serine or alanine for cysteine at either position biological activity

except Ser 8 mutant.

8, 71 or both

Mutant proteins were purified

to homogeneity to confirm the assay results obtained with crude extract. of the protein was confirmed by SDS-PAGE,isoelectric

The puri ty

focusing, analytical

HPLC,

aminoacid composition and amino acid sequenceanalysis of the amino terminal region. Only alanine was detected as an amino terminal amino acid in all purified As is obvious from Table 3, the substitutions 1112

of either

proteins.

or both cysteine residues

BIOCHEMICAL

Vol. 150, No. 3, 1988

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with serine or alanine showeda little

higher biological activity

protein.

may be the result of a slight

stability

The sharply higher activity of the mutant proteins.

activity

activity.

may reflect

its affinity

increase in the

However, we found that the substitution

for Cys 8 and the deletion of cysteine, their biological

than that of IL-18

especially

of serine

Cys 71, caused the decrease in

It is possible that the sharp decrease in biological

an alteration

for the receptor.

in the conformation of the protein which reduces It is also possible that these mutant proteins are

unstable, although equivalent levels of these mutant proteins were produced in E.coli strains

that did or did not express the la protease (data not shown).

determined the biological

activity

of IL-18

Wealso

mutant proteins with LAF assay using

thymus cells of a mouseof BALB/c strain (24).

There was a very close correlation

betweenGIF assay and LAF assay (data not shown). From the results mentioned above, it was concluded that the cysteine residues in IL-18

are not essential

for biological

activity,

but the amino acid at those

positions can not be deleted if function is to be maintained. The site-specific

mutagenesis using our versatile

useful in determining the tertiary crystallographic

structure of the

IL-IB

expression vector will

be

protein as well as X-ray

analysis. Using this vector, we plan to construct a series of rIL-l/3

analogues to study the structure-function

relationships.

ACKNOWLEDGEBENTS The authors wish to thank Professor K. I. Miura, The University of Tokyo, for his encouragementand helpful discussion. Wealso thank Dr. il. Seidmanfor his critical reading of the manuscript, Hiss M. Sakaguchi for her excellent technical assistance.

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