AbstractHuman interleukin-5 is a homodimer: each subunit contains two cysteine residues that form two inter-subunit disulfide bonds. The topology of the disulfides in recombinant human interleukin-5 produced in Escherichia coli was studied by proteolytic digestion and peptide mapping. Disulfide linked peptides containing cysteine 42 linked to cysteine 84 were isolated. This indicated that cysteine 42 and 84 of one subunit were linked in an antiparallel manner to cysteines 84 and 42 of the other subunit

Davies, J.Gwynfor

Proudfoot, Amanda E.I.

Turcatti, Gerardo

Wingfield, Paul T.

Elsevier - Publisher Connector 

Human interleukin-5 expressed in Escherichia coli: assignment of the disulfide bridges of the purified unglycosylated protein 

Volume 2~D', number 1.61=64 FEB$ 0974"; 
,~:' I¢,~1 Federation or Ettrop~an I~i~hcm|~al ~o~:l¢ll~,~ 0014~79)/~1,:$1.~0 
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blay 1991 
Human interleukin-5 expressed in Escherichia colt: assignment of the 
disulfide bridges of the purified unglycosylated protein 
• 2 Amanda E,I, Proudfoot I, J. Gwynfor Dav ies ,  Gerardo Turcatti I and Paul T. Wingfieid -a 
tGlaxo Institute for Mol¢cmdar Biology $,d,. Raute tics Acacias 4tl, 1221 Geneva 24, Swilzerh#=d, :Dt~parletncnt de Bitwhh~ff¢ 
Mc~dital¢, Universitd de Gtnbv¢. 9 avemt¢ de Champe/, 1211 Gtntv¢~ 4. Swlt:erlmul and ~ Pral¢lt! bL~prtssimt Lahoratary, Nutiomd 
h~slltut¢~ oj' II¢¢tlth, l~¢thcxda, MD 20892. USA 
Received 14 March 1991; revised version received 28 Marcia 1991 
Human interh:ukin.5 is a homed=met: each subunit contains two c.vs|~ine residues that form two inter-subunit disulfide bonds. The topology of 
the di~ttl rides in recombinant human inlcrleukin.5 prod=iced in F,~¢hcrlrhio ¢oli wa~ studied by proteolytic digestion and ~ptide mapping. Disulfide 
linked peptides containing cysteine 42 linked to cysteine 84 w¢r,a isolated. This indicated thai cysteines 42 and 84 of one sabunit were linked in 
an ant=parallel manner to cysteines M4 and 42 of the other subunit. 
lnterleukin.'.~; Recombinant DNA technology; Disulfide bond', Protcotyti¢ di~estion; Pep==de mapping: Homodimeric subunit structure 
1. INTRODUCTION 
Natural IL-5 has been purified from murlne T-cells 
and shown to be a disulfide-linked homed=met [1], 
Both murine and human IL-5 have been cloned [2,31 
and the latter expressed in several eucaryotic systems 
[4,5]. Human IL-5 has also beeta produced in E. colt [6]. 
The various recombinant IL-bs have all been shown to 
be disulfide-linked homodimers. The dimer structure 
appears to be essential for biological activity as the 
monomer is inactive [5,7]. 
Natural IL-5 is glycosylated [1] as is recombinant 
IL-5 expressed in eucaryotic systems. Glycosylation 
neither affects dimer formation or biological activity 
as unglycosylated IL-5 produced in E. con is both 
homodimeric and biologically active [6]. 
Human IL-5 expressed in a eucaryotic system was 
shown to contain two inter-subunit disulfide bonds in 
an ant=parallel arrangement [7]. In this report, we have 
studied the disulfide linkage pattern of human IL-5 pro- 
duced in E. colt. The protein expressed in bacteria is 
produced in an aggregated state. To prepare biological- 
ly active protein, the protein is first extracted and 
purified in a denatured state in which the cysteine 
residues are in the reduced form. This is followed by 
protein renaturation and reo×idation [6]. In protein ex- 
posed to a denaturation/renaturation cycle, it was con- 
sidered most important to establish the disulfide bond 
topology and to confirm that the protein did not con- 
tain mismatched isulfides. 
Correspondence address: A.E.I. Proudfoot, Glaxo l~stitute for 
Molecular Biology S.A., Route des Acacias 46, 1221 Geneva 24, 
Switzerland. 
2. EXPERIMENTAL 
2. I, Protein purification 
Human IL-5 expressed in E, colt was purified as previously describ- 
er1 [6] The reduced protein exhibited, a single band of M, = 13 000 on 
SDS.PAGE, The protein was, however, heterogeneous witl~ respect to 
pl, probably due to specific deamidations (A. Proudfoot, unpub- 
lished observations), As this heterogeneity caused multiplication of 
peaks during HPLC peptide mapping (see below), separation of tl~e 
charge isomers was carried out, Tire protein (10 rag) was applied to a 
Mono Q l-iR 10110 anion-excllange column equilibrated with 10 mM 
Tris.HCI, pH 8,0. The protein was eluted with a gradient of 0-0.1 M 
NaCI and tl~e column fractions analysed by =spice=tie focusing using 
a Phasl system (Pharmacia), 
2,2. Tryptic digestion 
Porcine trypsin (Sigma) was treated with TPCK to remove any 
residual chymotryptie activity [8]. hlL-5 (800#1 of 0.21 mg/ml in 10 
mM Tris.HCI, pH 8,0) was mixed with 1,33 ml of 8M urea, 0.123 ml 
1 M Bis.Tris, pH 6.0 (final conditions: 5 M urea and pH 6,0). Trypsin 
was added (20 ml of 1 mg/ml in water) followed by incubation at 
37°C for 2 h. The reaction was monitored by HPLC using a C-18 
reverse phase (4 × 250 ram) column (Maehery-Nagel), After complete 
digestion of the protein° i ml of the reaction mixture was applied to 
a Mono Q HR 10/I0 column equilibrated in20 mM Tris-HCl, pH 7.4. 
A gradient of 0-0.18 M Na2SO.~ was applied. The major eluted pea k 
was further purified on an Uttrapore C.3 RP (4.6 x 75 ram) column 
(Beckman). The column was washed for 10 rain with 0.1070 (w/v) TFA 
and a 15 ml gradient of 0-50070 acetonitrile was applied. The flow rate 
was 1 rnl/min. All separations were monitored at 214 nm and were 
carried out using a Beckman Gold HPLC system. 
2.3, Peptic digestion 
Separations were made on an Aquapore C-8 RP (2,1 x 220 mini col- 
umn (Applied Biosystems) connected to a HPI090 HPLC system 
(Hewlett Packard), Detection was at 214 nm. hlL-5 (1 ml of 0.14 
nlg/ml in 3.5°70 v/v formic acid, pH 1,8) was digested with 2070 w/w 
pepsin (Boehringer) at 25°C for 2 h. A second addition of 1% wlw 
pepsin was added with incubation for a further 3 h, Digestion was 
stopped by lyophilization the digest reconstituted with 0,1% FFA 
(w/v) and then fractionated by reverse phase HPLC. 
Published by Elsevier Scietrce Publishers B. V. 61 
Volume 293, number FEBS LETTERS May 1991 
Peptidex ¢ont~irttn$ disulfide bonds were Identified by t:ompari~oa 
or peplldt; mmp~, of o~idlited arm reduced protein. Dilte~t~ were re. 
du~:ed wl|h 2 mM dithiolllrellol In 6 M Auanldlne.FICI, SO mM Tri~, 
HCI, pH 7.8. The el,tlon position or Ir~ptides ¢ontalnlnlt disulfide 
linkages ~hlfted after eduction, gep~rttted i~ulfld~=.eontalnlnl! I~P. 
tide~ were reehromatoltraphed ellhttr directly or arter reduction, 
Purified pepttde~ were subjected to N.termlnal ~equenetnlb 
2.4, IV.Term#tat sequrnelnlt 
N.Termlaal mino acid sequence analyst~ was ¢arrletl out on.  
Model 477A pulse.liquM sequen*:er (&pplied Biosyslem~); gample~t 
were loaded on tilterJ, preey~;led with polybrene, PTH.amino acid 
derivativeswere analysed on.line by a Model 120 PTH.analyser tap. 
plied Biosystems). 
3, RESULTS 
3.1. Protein characterization 
Recombinant hlL-5 exhibi,'.s charge hetero,zeneity; 
this is true for the glycosylated protein [5], the 
unglycosylated E. coil.derived protein [6] and the 
eucaryotic-derived protein expressed in the presence of 
tunicamycin [9]. Hence, the charge heterogeneity s not 
solely due to carbohydrate, deamidation of  Asn and 
Gin residues being a likely cause in both eucaryotic and 
procaryotic-derived proteins, 
We attempted to resolve the charge isomers using 
anion,exchange chromatography at pH 8,0, Three main 
peaks were obtained (Fig. 1). Isoelectric focusing (Fig. 
1, insert) indicated that although the peak functions 
were not homogeneous, the band patterns were 
simplified relative to the starting material. The starting 
material migrated as a single band on SDS-PAGE with 
Mr= 13000 under reducing conditions and Mr= 27000 
in the absence of reduetant (Fig. 1, insert). SDS-PAGE 
of the fractions separated by anion-exchange 
chromatography indicated single bands migrating at the 
same molecular weight as the starting material (results 
not shown). Fractions from peak A were pooled as in- 
dicated and used for the peptide mapping studies 
described below. 
Separation of the charge isomers was also attempted 
by chromatofocusing but poor separations were obtain- 
ed with low yields, which we attributed to the poor 
solubility of hlL-5 at pH values below 6 (results not 
shown), 
3.2. Assignment of  disulfide bonds: tryptic digestion 
As protein disulfide-sulphydryl exchange reactions 
occur more rapidly under basic conditions [10], we per- 
formed enzymic digestions at pH values below 7.0. 
Tryptic digestions were carried out at pH 6.0 in the 
presence of 5 M urea using porcine trypsin. The urea 
was necessary to maintain solubility of hlL-5 and thus 
ensure efficient digestion. The porcine enzyme has the 
same specificity as the more commonly used bovine en- 
zyme but is more stable against denaturation by 
chaotropic agents (A. Proudfoot, unpublished observa- 
tions) and denaturation by organic solvents [11,12]. 
0 
< 
0.6  
0.4 
0.3 
0 .2  
0,1 
" - I I  | - I I , , , ~  
2 4 6 8 10 12 14 1(I 18 20 
Fraot lona  
0.3 
==,=~ 
0.1~ 
=It 
0.1 
Fig. I, Anion.exchange ci)rontatography of hIL.5, The elution profile 
is from a Mono Q column. Fractions were pooled as Indicated Frac- 
tion A was used for peptide mapping, Insert ~ shows a 
polyacrylamid¢ isolectric focusing gel, (Lane t) pl standards, pH 3-9; 
(lane 2) Pool A; (lane 3) Pool B; (lane 4) Pool C; and (lane 5) column 
starting material, Insert B shows an SDS.PACE Bel, (Lane 1) starting 
materia I ire the presence ofreductant; (lane 2) starting material intile 
absence of reductant; (lane 3) molecular wei$ht markers. 
Anion.exchange chromatography of tryptic digests 
resolved a major peptide that was eluted with 0.8 M 
NazSO# (Fig. 2). This peptide was further purified by 
reverse phase HPLC and then sequenced. Two in-phase 
N-terminal sequences were recorded) corresponding to
<: 
0.8 
0.2 
0.1 
0,4 
,2 
O) 
z 
5 10 15 ¢;0 2~ 30 
ume tmin=) 
Fig. 2. Anion-e×change chromatography of hlL-5 digested with tryp- 
sin. The peak indicated was used for further analysis. 
62 
¥olume 11~3, number i FEBS LETTERS 
! ! I p 
, , ~ f , P , 
~+++ I+ 
tmo~¢ + PlIIII+N 
w ~ Im I I  p 
Fill, ~t. Reverse phase HPLC o1' hlL.$ dil!e~ted with pepsin, Elution 
~rorlle~ from: ta) oxidized protein dillest; and (B) protein dilleS¢ 
pretreatcd with dlthlothreltol, The peak~ (Pi and P2} that d~it'ted 
after treatmer, l with reductant are indicated wilh ttrrow~ in (A), 
residues 84-88 and 38-46 or' the IL-~ sequence (Fig. 4). 
This result indicated inter-subunit linkage betwee'a cys- 
teines-42 and -84. (The cysteines were nc, t derived from 
an intra-molecular linkage as dimeric IL-5 only con- 
tains inter-molecular disulfides [6].) 
3.3. Assignment of disulfide bonds: peptic digestion 
IL-S was digested with pepsin and fractionated by 
reverse phase HPLC. Two peptides changed elution 
position upon reduction (Fig. 3, peptides PI and P2). 
These peptides were rechromatographed before and 
after reduction with dithiothreitol. Before reduction the 
peptides eluted in their original positions (Fig. 3) 
May 1991 
whereas after reduction each peptide generated two new 
peaks (results not +hown). Sequence analysis of the ox. 
idized peptides P! and P2 and their constituent 
disulfide-linked peptide pairs indicated that peptide PI 
contained residues 73-94 disulfide linked i:o residues 
29=43, Peptic cleavages occurred on one polypeptide 
chain between Thr-28-Leu-29 and Thr,43-Glu-44 and 
on the other chain between Leu-72-lle,73 and 
Phe-94-Leu-gL The peptide P2 contained residues 
73-95 disulfide linked to residues 29-47; Peptide P2 
tt~us contained the same disulfide pairing 
(Cys-42--Cys-84} as PI except peptic cleavage had oc. 
cuffed between Phe,47-GIn-48 instead of 
Thr-43-Glu-44. The results of the tryptic and peptic 
digestion ztudiez .-.re summarized in Fig. 4. 
4, DISCUSSION 
While the manuscript was in preparation, the assign- 
ment of the disulfide linkages of 81ycosylated human 
IL-5 produced in CHO cells was published [7]. These 
authors described the same anti-parallel disulfide 
linkage configuration as we have described here. Thus, 
the same disulfide linkage pattern exists in bacterial 
produced protein that has undergone denatura- 
0.8 -  
0;4 - 
I 
o.2L. 
i 
0~--,. 
0.8 
0.0 
0.4 
0.2 
O.  • 
o 
I I I i i , . . .  
- I  + 
i 
i 
10 20 ~O 
t ime ( rn lns )  
J 
i I 
4_0 ~0 
0,6 
0,4 
0,3 
0,2 
0.1 
Z 
0,8 ~,~ 
0.4 
0.3 
0.2 
0.1 
Fig, 4. Disulfide linkage pattern in hlL-5, The proteolytic cleavage sites are indicated by arrows: t, refers to tryptic cleavage and p refers to peptic 
cleavage. The peptides after the indicated cleavage sites were ident fled by N-terminal sequencing, The large tryptic peptide (residues 38-66) was 
only partially sequenced. The sequence determined (residues 38-48) is underlined, The complete amino acid sequence of hlL-5 is given in [2]. 
63 
Vol:umc 283, number  1 FEBS LETTERS May 1991 
¢i0n/renalurat ion and disulphide bond reduct ion/ox-  
iclation cycles, Based on the I~ilIh molar yields of  the 
disulf ide cont~ininB peptides (> 90%). there is little or 
no mismatched disulfide linked protein in the protein 
fractions analysed. 
Disulf ide.l inked dimers are not uncommon and are 
found in proteins such as immunoglobul ins and mem- 
brane receptors uch as the insulin and transferrin 
receptors. However, cytoplasmic proteins containing 
intermolecular disulf ide-l inked homodimeric 
topologies are not common. Some secreted and 
cytoplasmic proteins have been shown to be 
homodlmers, Most of" these proteins are not disulfide, 
l inked, although glutathione redactase contains a single 
covalent disulfide bridge between cystelne-90 of  each 
monomer [13] and ribonuclease has been shown to exist 
as a disulfide-linked dimer in bovine seminal fluid [141. 
The steroid binding protein uteroglobulin is also 
homodinteric containing two antip:~allel inter. 
molecular disulfide linkages analogous to hIL-5 [15]. 
Mason et al. [16] have proposed a family of hor. 
monal proteins having the disulfide.linked 
homodimcric motif, including transforming rowth 
factor beta, Mullerian inhibiting substance and inhibin. 
Another growth factore, platelet-derived growth factor, 
adopts homo- and heterodimeric configurations, with 
the monomer and dimers having different activities 
[16,17], IL-5 may belong to this structural class of pro. 
tein hormones, 
5, CONCLUSIONS 
Human IL-5 is a homodimer which contains two in- 
ter-molecular disulfide bonds. The arrangement of ~he 
disulfides was determined by peptide mapping. The 
linkages occurred between cysteines-42 and -84 on op- 
posite subunits. This anti-parallel arrangement is
similar to that found in glycosylated mammalian cell 
produced protein. As cysteines-42 and -84 are conserv. 
ed in routine [L.$ [5], the disulfide bond topo~:3gy is 
probably ll~e same as in the human protein. 
Ack,mwl¢~l~¢mcm:t: J ,¢3. D, thanks l i l t  Swis~, National Science Foun. 
dalton for finctn¢i.I support. 
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64 


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Human interleukin-5 expressed in Escherichia coli: assignment of the disulfide bridges of the purified unglycosylated protein

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