Analysis o majo in acellula p o eins o Aspe gillus umiga us
by MALDI mass spec ome y: Iden ifica ion and cha ac e isa ion
o an elonga ion ac o 1B p o ein wi h glu a hione ans e ase ac i i y
q
S ephen Ca be y
1
, Clai e M. Ne ille
1
, Ke in A. Ka anagh, Sean Doyle
*
Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional Uni e si y o I eland Maynoo h, Co. Kilda e, I eland
Recei ed 13 Janua y 2006
Abs ac
Aspe gillus umiga us is a ecognised human pa hogen, especially in immunocomp omised indi iduals. The a ailabili y o he anno-
a ed A. umiga us genome sequence will significan ly accele a e ou unde s anding o his o ganism. Howe e , limi ed in o ma ion is
a ailable wi h espec o he A. umiga us p o eome. He e, bo h a di ec p o eomic app oach (2D-PAGE and MALDI-MS) and a
sub-p o eomic s a egy in ol ing ini ial glu a hione affini y ch oma og aphy ha e been deployed o iden i y 54 p o eins om A. umig-
a us p ima ily in ol ed in ene gy me abolism and p o ein biosyn hesis. Fu he mo e, wo no el euka yo ic elonga ion ac o p o eins
(eEF1Bc), e med El A and B ha e been iden ified and phylogene ically confi med o belong o he eEF1Bcclass o GST-like p o eins.
One o hese p o eins (El A) has been pu ified o homogenei y, iden ified as a monome ic enzyme (molecula mass = 20 kDa; pI= 5.9
and 6.5), and ound o exhibi glu a hione ans e ase ac i i y specific ac i i ies (mean ± s anda d de ia ion, n= 3) o 3.13 ± 0.27 and
3.43 ± 1.0 lmol/min/mg, using CDNB and e hac ynic acid, espec i ely. O e all, hese da a highligh he impo ance o new app oaches
o dissec he p o eome o , and elucida e no el unc ions wi hin, A. umiga us.
2006 Else ie Inc. All igh s ese ed.
Keywo ds: Aspe gillosis; P o eomics; 2D-PAGE; Oxida i e s ess; P o ein syn hesis; GST; Glu a hione
The field o ungal p o eomics is ga he ing pace as a
esul o he simul aneous occu ence o ungal genome
sequence a ailabili y and ad ances in high sensi i i y p o-
ein mass spec ome y. Indeed, he ecen publica ion o
a wo-dimensional p o ein e e ence map o , and he anal-
ysis o s ess-induced changes in he p o eome o , Schizo-
saccha omyces pombe clea ly illus a es his poin [1,2].
P o eomic analysis o he filamen ous ungus, T ichode ma
ha zianum, has also yielded insigh s in o he mi ochond ial
p o eome o his mycopa asi ic o ganism [3].
Many Aspe gillus species ha e me i ed a en ion om a
bio echnological s andpoin , as a consequence o a sec e -
ed p o eome ich in indus ially use ul enzymes [4]. Un il
ecen ly, howe e , li le in o ma ion was a ailable wi h
espec o ei he he echnical equi emen s o p o ein
ex ac ion o he p o eome con en o mos Aspe gillus
species. Howe e , dissec ion o he sec e ed p o eome
(sec e ome) o Aspe gillus fla us has demons a ed he
abili y o his o ganism o adap o al e ed condi ions
such as he p esence o a ious ca bon sou ces (e.g., u in)
[5].
Wi h espec o he human pa hogenic ungus, A. umig-
a us, B uneau e al. adop ed a p o eomic app oach o iden-
i y glycosylphospha idylinosi ol-ancho ed p o eins which
a e in ol ed in he o ganisa ion o he ungal cell wall
[6]. Mo e ecen ly, we ha e unde aken he iden ifica ion
o non ibosomal pep ide syn he ases in A. umiga us using
a combined p o eomic and molecula app oach [7]. Along
wi h Candida albicans,A. umiga us is a significan oppo -
unis ic pa hogen o immunocomp omised indi iduals,
0006-291X/$ - see on ma e 2006 Else ie Inc. All igh s ese ed.
doi:10.1016/j.bb c.2006.01.078
q
Abb e ia ions: CDNB, 1-chlo o-2,4-dini obenzene; 2D-PAGE,
wo-dimensional elec opho esis.
*
Co esponding au ho . Fax: +353 1 7083845.
E-mail add ess: [email p o ec ed] (S. Doyle).
1
These au ho s con ibu ed equally o his wo k.
www.else ie .com/loca e/ybb c
Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104
BBRC
ARTICLE IN PRESS
indeed app oxima ely 5% o all hospi al based nosocomial
dea hs a e due o Aspe gillus in ec ions [8].
The ecen comple ion o he sequencing o he A. umig-
a us genome and he a ailabili y o he in silico anno a ed
genome a h p://www.cad e.man.ac.uk [9] should enable
de ailed molecula dissec ion o he biology o his o gan-
ism. Mo eo e , bo h unanno a ed and anno a ed genome
da a can be in e oga ed o u he ou knowledge o he
A. umiga us p o eome and he o ganismal esponse o
al e ed en i onmen al condi ions.
Fo ins ance, a g ea e unde s anding o he ole played
by glu a hione ans e ases (GSTs) in media ing esis ance
o oxida i e s ess and an i ungal d ugs o xenobio ics is
equi ed and we ha e ecen ly iden ified, cha ac e ised,
and cloned h ee GST genes om A. umiga us (gs A-C)
[10]. Howe e , GST-like domains ha e also been de ec ed
in dis inc p o eins such as U e2p, Mak16, and EF1Bc(a
subuni o he EF1B complex) [11]. Fu he mo e, al hough
GST ac i i y has ecen ly been asc ibed o he elonga ion
ac o p o ein complex EF1B isola ed om ice and ypa-
no hione S- ans e ase ac i i y de ec ed in EF1B complex
om Leishmania majo , no de ec ion o GST ac i i y in
na i e EF1Bchas been possible [12]. In an a emp o pu i-
y he GST p o eins (Gs A-C) om A. umiga us, glu a hi-
one (GSH)–Sepha ose affini y ch oma og aphy was
employed. Al hough Gs A, B o C we e unde ec able, a
numbe o o he p o eins we e de ec ed, one o which
was iden ified as a euka yo ic elonga ion ac o 1Bc. This
manusc ip ep esen s one o he fi s de ailed desc ip ions
o a p o eomic app oach o iden i y in acellula p o eins
in A. umiga us and also he use o a sub-p o eomic
app oach, in ol ing affini y ch oma og aphy and MALDI
mass spec ome y o iden i y new biochemical unc ions in
ungal species.
Ma e ials and me hods
P o ein ex ac ion. Aspe gillus umiga us (ATCC 26933) was cul u ed
in Sabou aud’s media (500 ml cul u es), a 37 C wi h shaking a 200 pm.
Mycelia we e ha es ed, fil e ed unde p essu e, and washed wi h PBS and
esuspended in lysis buffe (100 mM T is–HCl, 50 mM NaCl, 20 mM
EDTA, 10% ( / ) glyce ol, 30 mM DTT, 1 mM PMSF, and 1 lg/ml
peps a in A, pH 7.5; 5 ml o lysis buffe pe g am o mycelia). Lysis was
accomplished by g inding in liquid N
2
ollowed by b ie sonica ion.
Mycelial lysa es we e cen i uged (10,000g; 30 min) o emo e cell deb is
and he subsequen supe na an analysed by 2D-PAGE ollowing TCA/
ace one p ecipi a ion [13].
Two-dimensional elec opho esis. P o ein sepa a ion by 2D-PAGE was
as ollows: ex ac s con aining 250 lg o p o ein we e esuspended in 8 M
u ea, 2 M hiou ea, 4% (w/ ) CHAPS, 1% ( / ) T i on X-100, 10 mM
T is–HC1, 65 mM di hio h ei ol (DTT), and 0.8% pH 4–7 ca ie amph-
oly es, and loaded on o Immobiline D y s ips (IPG s ip; Ame sham) in
he pH ange 4–7. Gels unde wen ac i e ehyd a ion a 50 V o 10 h,
ollowed by a u he 10 h ocusing wi h a o al o 26250 V applied. Fol-
lowing IEF, gels we e equilib a ed in educing buffe (50 mM T is–HCl,
6 M u ea, 2% (w/ ) SDS, 30% ( / ) glyce ol, and 2% (w/ ) DTT, pH 6.8)
o 20 min ollowed by equilib a ion in alkyla ion buffe (50 mM T is–
HCl, 6 M u ea, 2% (w/ ) SDS, 30% ( / ) glyce ol, and 2.5% (w/ ) iodo-
ace amide, pH 6.8) o a u he 20 min. The IPG s ips we e placed on
homogeneous 12% SDS–PAGE gels and elec opho esed o 20 h a 100 V
using he P o eanXi-II Cell (Bio-Rad Labo a o ies). Resul ing gels we e
s ained wi h Coomassie b illian blue R and scanned using a Typhoon
T io Va iable Mode Image (Ame sham Biosciences (Eu ope) GmbH,
F eibu g, Ge many).
Ch oma og aphic p ocedu es. Aspe gillus umiga us cell lysa es we e
cen i uged as abo e and he supe na an collec ed while he pelle was
disca ded. Ammonium sulpha e solu ion (100% (w/ )) was hen added o
he supe na an o yield 65% ammonium sulpha e (65% solu ion) which
was s i ed a 4 C o e nigh . This suspension was hen cen i uged, he
supe na an emo ed, and he pelle esuspended in a minimum olume o
lysis buffe . Resuspended ma e ial was ex ensi ely dialysed agains 2 ·50
olumes o 25 mM T is–HCl, 50 mM NaCl, pH 7.4 (buffe A), and he
dialysa e was cen i uged a 10,000g o 10 min and hen fil e ed (0.45 lM)
p io o ch oma og aphy. A GSH–Sepha ose column (3 ml olume) was
equilib a ed wi h buffe A and ollowing dialysa e applica ion and wash-
ing, was elu ed wi h 50 mM T is–HCl, 15 mM GSH, pH 9.2 (buffe B).
F ac ions (0.5 ml) we e collec ed. SDS–PAGE analysis o nea o TCA
p ecipi a ed ac ions was ca ied ou as p e iously desc ibed [13]. GSH–
Sepha ose column ac ions we e subsequen ly pooled (350 lg/10 ml),
applied o a Q-Sepha ose esin (1.5 ·8 cm; flow a e: 1 ml/min), and
0.5 ml ac ions collec ed ollowing elu ion wi h a 25 ml linea g adien o
0–0.5 M NaCl in 25 mM T is–HCl, pH 8.0. Peak ac ions con aining
we e iden ified by SDS–PAGE and enzyma ic ac i i y analysis (glu a hi-
one conjuga ion o 1-chlo o-2,4-dini obenzene (CDNB) and e hac ynic
acid) [14]. Na i e molecula mass de e mina ion was unde aken by size
exclusion ch oma og aphy using an A
¨KTA Pu ifie 100 sys em (Ame -
sham Biosciences, UK) whe eby a Supe ose 12 column (10 ·300 mm) was
equilib a ed in PBS a a flow a e o 0.4 ml/min. The concen a ed
ma e ial om Q-Sepha ose was loaded on he column and 0.5 ml ac ions
collec ed.
MALDI mass spec ome y. Mass spec ome y was ca ied ou
using an E an MALDI-ToF mass spec ome e (Ame sham Biosciences
(Eu ope) GmbH, F eibu g, Ge many). P o ein samples o pep ide
mass de e mina ion we e excised om 2D-PAGE gels using an au o-
ma ed spo cu e (Ame sham Biosciences (Eu ope) GmbH, F eibu g,
Ge many), diges ed wi h ypsin and deposi ed (1 ll) wi h 1 lla-cyano-
4-hyd oxycinnaminic acid (4-HCCA; 5 mg/200 ll o 50% ( / )
ace oni ile in 0.1% ( / ) aqueous ifluo oace ic acid) on o mass
spec ome y slides, and allowed o d y p io o delayed ex ac ion,
eflec on ToF analysis a 20 kV [10]. In e nal calib an s, Angio ensin
III (Sigma–Ald ich) and ACTH agmen 18–39 (Sigma–Ald ich), we e
used o calib a e all spec a. P o ein iden ifica ion was ca ied ou
ei he by m/zda a in e oga ion o (i) he NCBI n da abase a ailable
as pa o he mass spec ome e E alua ion So wa e Ve sion 2.01 o
(ii) a local FASTA e sion o he anno a ed A. umiga us genome
a ailable a h p://www.cad e.man.ac.uk. The anno a ion sys em
employed on his websi e (i.e., A uNg whe e N and g define he
ch omosome and gene numbe , espec i ely) and desc ibed by Mabey
e al. [9] has been adop ed in his manusc ip . Tandem mass spec-
ome y was pe o med by comme cial a angemen (Taplin Biological
Mass Spec ome y Facili y, Ha a d Medical School, MA, USA) using
an LCQ DECAXP Plus ion- ap mass spec ome e (The moFinnigan,
San Jose, CA).
Enzyme ac i i y de e mina ion. GST assays we e ca ied ou as
desc ibed by Habdous e al. [14]. B iefly, assays we e ca ied ou in a o al
olume o 200 ll including 20 ll o p o ein sample wi h CDNB/e hac ynic
acid, GSH in 0.1 M po assium phospha e (pH 5.8) a final concen a ions
o 0.5/0.2 and 2.5 mM, espec i ely. Reac ions we e ini ia ed by he
addi ion o he p o ein sample and a blank was c ea ed by he addi ion o
20 ll o 0.1 M po assium phospha e (pH 5.8) ins ead o he p o ein
samples. The inc ease in abso bance was measu ed a 340 nm (co e-
sponding o he enzyma ic conjuga ion o CDNB/e hac ynic acid o GSH
by GST).
Phylogene ic analysis. Sequence alignmen s and boo s ap neighbou
joining phylogene ic ees we e gene a ed using he Clus al X 1.81 p og am
a ailable a p:// p-igbmc.u-s asbg. /pub/Clus alX/, using he p og am
de aul pa ame e s. A boo s apping alue o 1000 was used, wi h boo -
s apping alues no ed a ee b anch poin s. T ees we e isualised using
T ee iew (h p:// axonomy.zoology.gla.ac.uk/ od/ ee iew.h ml).
S. Ca be y e al. / Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104 1097
ARTICLE IN PRESS
Resul s
Aspe gillus umiga us p o eome analysis
A o al o 180 spo s we e excised om 2D-PAGE gels
(Fig. 1A) which esul ed in he iden ifica ion o 50 dis inc
p o eins (in 65 indi idual spo s), by MALDI-MS (Table 1).
Significan expec a ion alues we e no achie ed o he
emaining 115 spo s o gi e posi i e iden ifica ion. The pe -
cen age sequence co e age ob ained anged om 9.4%
(pe oxisome biogenesis ac o ) o 50.5% (enolase) and
obse ed molecula masses anged om 75 kDa o less
han 25 kDa. Se e al p o eins exhibi ed diffe en obse ed
molecula mass o he heo e ical mass sugges ing he
occu ence o pos - ansla ional modifica ion.
The majo i y o p o eins iden ified appea o be in ol ed
in ene gy p oduc ion, wi h p o eins cha ac e is ic o he
glycoly ic pa hway, ci ic acid pa hway, pen ose phospha e
pa hway, a y acid me abolism, and oxida i e phospho y-
la ion clea ly e iden (Fig. 1B and Table 1). O he classes
o p o eins iden ified include s uc u al p o eins, signalling
p o eins, hea shock and hea shock chape one p o eins,
ansc ip ion ac o s, and wo hypo he ical p o eins.
A numbe o p o eins (i.e., ca alase 1, HSP 70 chape one
HscA, alanine amino ans e ase, hiamine biosyn hesis
p o ein Nm 1, 14-3-3 amily p o eins, and u idine diphos-
pha e-glucose-4 epime ase) (Table 1) we e indi idually
p esen in mo e han one spo , a iden ical molecula mass,
which indica es sub le cha ge diffe ences possibly due o
side-chain deamina ion. Mos p o eins exhibi ed molecula
mass and pI alues close o he p edic ed alues,
excep o NAD-dependen o ma e dehyd ogenase pI-
obse ed = 7.7; pI-p edic ed = 8.7. Mo eo e , p o ein
A u5g14680 (unknown unc ion) p esen ed a pI4.7, 4.9,
and 5.2 wi h molecula masses all below he heo e ical
25 kDa alue. Analysis o his sequence by BLAST sea ch
indica ed hypo he ical p o eins in Aspe gillus nidu-
lansXM_656639 and Gibbe ella zeae XM_390618 wi h
simila i y o A u5g14680. The heo e ical and ac ual mass
o hypo he ical p o ein A u2g10030 diffe ed by 7 kDa
and BLAST analysis showed simila i y o hypo he ical
p o eins in A. nidulans XM_658516 and Bo y is cine ea
47
62
100
32.5
25
kDapH47
44%
8%
6%
18%
3%
6%
15%
Ene gy
HSP
Hypo he ical
P o einBiosyn hesis
S uc u al
T ansc ip ion
O he
A
B
Fig. 1. (A) 2D-PAGE sepa a ion o whole p o ein ex ac om A. umiga us. Whole p o ein ex ac s (250 lg) we e subjec ed o isoelec ic ocussing on
Immobiline D y s ips (pH ange 4–7) ollowed by SDS–PAGE and Coomassie b illian blue s aining. (B) Func ional classifica ion o all A. umiga us
p o eins iden ified by MALDI-MS whe eby >60% o p o eins we e in ol ed in ei he ene gy gene a ion o p o ein biosyn hesis. HSP, hea shock p o ein.
1098 S. Ca be y e al. / Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104
ARTICLE IN PRESS
Table 1
A. umiga us p o eins (n= 50) p esen a e cul u e in Sabou aud media ollowing iden ifica ion by 2D-PAGE and MALDI-MS (Fig. 1)
CADRE I.D. Spo No. % Co e age M
(kDa) pIP oposed unc ion
A u1g07480 1C7 26.8 50.16 6.9 Cop opo phy inogen III oxidase
A u1g10130 D6 24.7 48.47 5.8 Adenosylhomocys einase
A u1g10350 F12 35.3 44.74 6.5 Phosphoglyce a e kinase
A u1g10630 1A7 44.4 42.18 5.7 S-Adenosylme hionine syn he ase
A u1g12170 1F7 21.8 48.27 6.7 T ansla ion elonga ion ac o EF-Tu
A u1g13500 E5 23.5 74.81 6.1 T anske olase
A u1g13490 2D4 29.7 33.43 5.3 Spe midine syn he ase
A u2g00720 B7 14.4 51.92 5.6 Aldehyde dehyd ogenase
A u2g03290 2A4 25.7 29.08 4.8 14-3-3 Family p o ein A A
A u2g04230 1F1 10.9 46.80 6.0 Fuma ylace oace a e hyd olase ahA
A u2g07420 B5 22.4 72.46 5.8 Fimb in
A u2g09960 G8 30.9 74.45 5.4 Mi ochond ial HSP70 chape one Ssc
A u2g10030 1E11 22.3 28.25 6.2 Hypo he ical p o ein
A u2g11150 1H3 24.6 52.26 5.3 Sec e o y pa hway gdp dissocia ion inhibi o
A u2g13240 A7 24.5 56.4 5.7 V- ype ATPase subuni B
A u3g00590 2B6 41.0 15.18 5.8 Asp-hemolysin
A u3g01110 E6 17.2 61.41 5.8 GMP syn hase
A u3g02270 A3 27.1 79.9 5.6 Ca alase 1
A u3g02270 H2 27.1 79.9 5.7 Ca alase 1
A u3g02270 F4 23.4 79.89 5.5 Ca alase 1
A u3g05450 H9 36.0 53.0 5.5 Glu ama e ca boxypep idase
A u3g07630 2H3 26.8 19.61 4.7 Dihyd op e oa e syn hase
A u3g08160 1A6 15.0 45.76 5.0 Euka yo ic ansla ion ini ia ion ac o eIF4A
A u3g09290 G7 20.4 57.44 5.6 Phosphoglyce a e mu ase 2,3-biphosphoglyce a e-independan
A u3g11070 A6 31.3 62.98 6.1 Py u a e deca boxylase PdcA
A u3g11690 1D10 43.6 39.77 5.5 F uc ose-biphospha e aldolase class
A u3g11690 1E10 33.1 39.77 5.8 F uc ose-biphospha e aldolase class II
A u4g06620 1H1 43.9 49.35 5.8 Glu ama e/leucine/phenylalanine/ aline dehyd ogenase
A u4g07690 G5 13.3 65.01 6.8 Phospho ibosylaminoimidazole ca boxamide o myl ans e ase/IMP hyd ocyclase
A u4g10200 B8 13.2 71.68 5.4 T ansc ip ion ac o R eF
A u4g11730 1C11 16.3 36.8 6.0 Glyce ol dehyd ogenase GldB
A u4g13170 1F11 45.9 34.96 6.4 Guanine nucleo ide binding p o ein b-subuni
A u4g13170 1G11 36.4 34.96 6.4 Guanine nucleo ide binding p o ein b-subuni
A u5g01030 IH8 43.4 36.12 6.5 Glyce aldehyde-3-phospha e dehyd ogenase Ccg-7
A u5g01030 1E8 39.2 36.12 6.2 Glyce aldehyde-3-phospha e dehyd ogenase Ccg-7
A u5g02470 1A11 29.5 38.3 6.0 Thiamine biosyn hesis p o ein Nm 1
A u5g02470 1B11 29.5 38.3 6.0 Thiamine biosyn hesis p o ein Nm 1
A u5g02470 1C10 19.9 38.3 5.7 Thiamine biosyn hesis p o ein Nm 1
A u5g04170 G1 15.4 80.6 4.9 Molecula chape one HSP1
A u5g09230 1D11 40.1 35.43 6.0 T ansaldolase
A u5g10550 1A5 41.4 55.6 4.8 ATPase syn hase F1 subuni b
A u5g10550 1B5 41.4 55.6 4.8 ATPase syn hase F1 subuni b
A u5g10780 1B8 39.9 40.59 6.1 UDP-glucose 4-epime ase
A u5g10780 1C8 27.8 40.59 5.9 UDP-glucose 4-epime ase
A u5g14680 2D7 39.6 25.42 4.7 Hypo he ical p o ein
A u5g14680 2G7 34.8 25.42 4.9 Hypo he ical p o ein
A u5g14680 2H7 34.8 25.42 5.2 Hypo he ical p o ein
A u6g04740 1E6 26.7 43.88 5.9 Ac in
A u6g04920 1B9 33.0 45.73 7.7 NAD-dependan o ma e dehyd ogenase
A u6g06750 2D3 28.0 30.05 4.7 14-3-3 Family p o ein
A u6g06750 2C3 28.0 30.05 4.8 14-3-3 Family p o ein
A u6g06770 1H2 50.5 47.29 5.6 Enolase
A u6g07720 E7 15.8 66.8 5.8 Phosphoenolpy u a e ca boxykinase
A u6g07770 F3 24.6 55.12 6.2 Alanine amino ans e ase
A u6g07770 C11 18.2 55.12 6.3 Alanine amino ans e ase
A u6g07770 H10 33.1 55.12 5.9 Alanine amino ans e ase
A u6g08050 1C2 31.4 55.78 5.9 6-Phosphoglucona e dehyd ogenase deca boxyla ing
A u6g12930 F2 16.9 85.5 6.4 Aconi a e hyd a ase
A u7g01860 F3 28.7 65 5.5 Hea shock p o ein S i1
A u7g05070 G10 22.5 51.25 5.7 FAD-dependen oxido educ ase
A u7g05720 1G6 9.6 39.1 5.9 Pe oxisome biogenesis ac o
(con inued on nex page)
S. Ca be y e al. / Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104 1099
ARTICLE IN PRESS
CNS01C5R. Conse ed Domain analysis [15] o
A u2g10030 indic ed ha i may possess an RNA Recogni-
ion Mo i (RRM). The phy anol-CoA dioxygenase amily
p o ein, (A u8g00230) which is a pe oxisomal enzyme ca -
alysing he fi s s ep o phy anic acid a-oxida ion, has no
been p e iously iden ified in A. umiga us, howe e i
exhibi ed simila i y wi h hypo he ical p o ein AN9227.2
in A. nidulans. A Conse ed Domain sea ch iden ified bo h
A u8g00230 and AN9227.2 as con aining a phy anol-CoA
dioxygenase domain as well as a COG5285 domain. In
Bu kholde ia ungo um LB400, a p o ein con aining a
COG5285 domain is in ol ed in biosyn hesis o mi omycin
an ibio ics and he polyke ide, umonisin. Dihyd op e oa e
syn hase (A u3g07630) has been associa ed wi h d ug esis-
ance in Saccha omyces ce e isiae and has been shown o
be de i ed om a polycis onic gene also esponsible o
he p oduc ion o wo o he unc ional p o eins [16]. Ca a-
lase 1 (A u3g02270) in ol ed in oxida i e s ess and iden i-
fied he e as h ee sepa a e p o ein spo s has been
implica ed in A. umiga us pa hogenici y [17].
Almos 44% (22/50) o all p o eins iden ified we e
de i ed om in on-con aining genes. Fo ins ance, 3 pep-
ides o m/z2027.99, 2567.11, and 2630.31 we e de ec ed in
py u a e deca boxylase (A u3g11070) which de i e om
h ee dis inc splicing e en s. In addi ion, wo pep ides
de i ed om exon–exon splicing we e de ec ed in each o
se en p o eins (ca alase, glu ama e ca boxypep idase, ac-
uola syn hase subuni B, enolase, sec e o y pa hway disso-
cia ion inhibi o , F1-ATPase (b-subuni ), and ansla ion
elonga ion ac o EF-Tu) (Table 1). A single pep ide, in
each o he 14 emaining p o eins, was iden ified which
esul ed om exon splicing (da a no shown).
Aspe gillus umiga us sub-p o eome analysis
GSH–Sepha ose affini y ch oma og aphy was unde ak-
en in o de o selec i ely de ec and pu i y glu a hione bind-
ing p o eins in A. umiga us. Affini y column elu ion wi h
5 mM GSH (in 50 mM T is–HCl, pH 9.2) did no esul in
he displacemen o any bound p o eins (da a no shown)
and all esul an GSH-binding p o eins we e displaced om
he column in he p esence o 15 mM GSH. O e all, he o al
p o ein yield was 7.4 mg/g mycelia in cell lysa e supe na an
and he yield o GSH-binding p o eins was 5.3 lg/g mycelia
which indica es ha hese p o eins ep esen 0.0007% o
o al soluble p o ein in A. umiga us.
The p esence o h ee p o eins was de ec ed by
SDS–PAGE analysis ollowing GSH–Sepha ose ch oma-
og aphy and a pep ide (SVDVVEEYLQDR) was iden i-
fied by Tandem-MS om he p o ein p esen in band 3
(Figs. 2A and B). Da abase in e oga ion h p://www. i-
g .o g;h p://www.cad e.man.ac.uk wi h his sequence
iden ified a pu a i e ansla ion elonga ion ac o 1Bc ype
p o ein (A u8g00580; El B) wi h 10% sequence co e age
(Fig. 2B). Fu he analysis o GSH-binding p o eins by
2D-PAGE (Fig. 2C) demons a ed ha a leas 10 p o eins
(molecula mass ange: 18–40 kDa) we e p esen ollowing
GSH–Sepha ose affini y ch oma og aphy. All GSH-bind-
ing p o eins we e excised om 2D-PAGE gels and ana-
lysed by MALDI-MS. Fou p o eins we e iden ified
based on compa ison o bo h he NCBI n da abase and
a FASTA e sion o he in silico-anno a ed A. umiga us
genome a ailable a h p://www.cad e.man.ac.uk (Table
2). Specifically, he mos abundan p o ein (Fig. 2C-spo
3 a,b; A u1g17120) was iden ified, based on 46.4%
sequence co e age, as an ansla ion elonga ion ac o
1Bcsubuni . This p o ein, e med El A, appea ed o be
p esen as wo dis inc spo s o iden ical molecula mass
bu diffe en pI alues (5.9 and 6.5, espec i ely) (Fig. 2C).
Anion-exchange ch oma og aphy (Fig. 3A) was subse-
quen ly used o pu i y El A o homogenei y ollowed by
assessmen o p o ein pu i y by SDS–PAGE (Fig. 3B).
When he GST ac i i y o pu ified El A was analysed, bo h
CDNB and e hac ynic acid we e ecognised as subs a es
wi h specific ac i i ies (mean ± s anda d de ia ion, n=3)
o 3.13 ± 0.27 and 3.43 ± 1.0 lmol/min/mg, espec i ely.
No ac i i y was de ec ed agains DCNB and nei he was
any glu a hione pe oxidase ac i i y de ec ed in El A (da a
no shown). Mo eo e , he obse ed specific ac i i ies
agains CDNB and e hac ynic acid we e much g ea e han
hose obse ed o ecombinan A. umiga us Gs C
exp essed in Esche ichia coli (0.01 ± 0.0009 lmol/min/mg)
[10]. Size exclusion analysis iden ified he molecula mass
o El A o be 20 kDa, sugges ing i is p esen as a mono-
me wi hin he cell, unlike mos o ms o GST which a e
loca ed in acellula ly in dime ic o m (da a no shown).
These esul s ep esen he fi s demons a ion o GST
ac i i y associa ed wi h an elonga ion ac o 1Bcsubuni
in a ungus. Mo eo e , i also he fi s demons a ion o
GST ac i i y in a na i e elonga ion ac o 1Bcsubuni dis-
associa ed om o he componen s o he EF1B complex.
Aspe gillus umiga us El A and El B we e compa ed o
28 sequences o GST-like p o eins om a ious species,
p e iously analysed by McGold ick e al. [11], in o de o
assess homology (Fig. 4). F om his analysis, h ee dis inc
clades we e obse ed namely EF1c-like, U e2, and
Table 1 (con inued)
CADRE I.D. Spo No. % Co e age M
(kDa) pIP oposed unc ion
A u7g05720 D8 27.0 52.01 5.4 Py u a e dehyd ogenase complex dihyd olipoamide ace yl ans e ase componen
A u8g00230 2F4 22.3 32.65 5.6 Phy anoyl-CoA dioxygenase amily p o ein
A u8g03930 A9 31.3 66.96 5.3 HSP70 chape one HscA
A u8g03930 H8 22.8 66.96 5.4 HSP70 chape one HscA
CADRE I.D., A. umiga us gene anno a ion nomencla u e acco ding o Mabey e al. [9] and Nie man e al. [33].
1100 S. Ca be y e al. / Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104
ARTICLE IN PRESS

↓El A M 1 2 3 4
kDa
47.5
32.5
25
AB
0
10
20
30
40
50
60
70
80
90
0 10152025
mAU
Elu ion olume (ml)
El A
5
Fig. 3. (A) Pu ifica ion o A. umiga us El A by anion-exchange ch oma og aphy o GSH-binding p o eins om A. umiga us. GSH pu ified p o eins we e
applied o a Q-Sepha ose column (14 ml column olume) and elu ed wi h an inc easing sal g adien (0–0.5 M NaCl in 25 mM T is–HCl, pH 8.0). El A
was iden ified in he fi s peak as indica ed. (B) SDS–PAGE analysis o El A pu ified by ion exchange ch oma og aphy (Q-Sepha ose). Lane M, molecula
mass ma ke s; lanes 1–4, Q-Sepha ose column ac ions con aining pu ified El A.
Table 2
GSH-binding p o eins o A. umiga us sepa a ed using 2D-PAGE and iden ified by MALDI mass spec ome y
A u Code Spo No. % Co e age Mol. mass (kDa) pIP oposed unc ion
A u5g06060 1 14.8 18.1 4 Sulphu me abolism egula o SkpA, pu a i e
A u1g03970 2 10 25 3.2 Mi ochond ial ansla ion ini ia ion ac o IF-2, pu a i e
A u1g17120 3a,b 46.4 24.3 5.9 El A; elonga ion ac o 1-c, pu a i e
A u4g09130 4 25.8 24 10 Mannosyl ans e ase, pu a i e
2
1
43 b
pH 3 10 kDa
3 a
100
62
47.5
32.5
25
A
B
C
Fig. 2. (A) SDS–PAGE analysis o GSH-binding p o eins om A. umiga us. (B) Tandem mass spec ome y iden ified pep ide SVDVVEEYLQDR which
was specific o he sequence o A. umiga us El B (CADRE I.D: A u8g00580) (C) 2D-PAGE analysis o GSH-binding p o eins. Isoelec ic ocussing on
Immobiline D y s ips (pH ange 3–10) ollowed by SDS–PAGE and Coomassie b illian blue s aining. P o eins in spo s 1–4 we e iden ified by MALDI-
MS, one o which was El A (CADRE I.D: A u1g17120; Table 2). El B was no de ec able ollowing 2D-analysis.
S. Ca be y e al. / Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104 1101
ARTICLE IN PRESS
MAK16. Bo h El A and El B appea in he EF1c-like clade
o GSTs showing homology o EF1c, bo h clea ly dis inc
om he U e2 and MAK16 clades. El A and EAA47645
(56% simila i y) appea o be o hologues, as do El B
and Q00717 (59% simila i y), confi ming ha hese p o-
eins con ain GST-like domains (Fig. 4).
Discussion
He e we p esen da a on he iden ifica ion o 54 in acel-
lula p o eins om he pa hogenic ungus A. umiga us
using a combined 2D-PAGE/sub-p o eomic/MALDI-MS
app oach. In addi ion, conclusi e e idence is p esen ed
o he fi s ime ha a euka yo ic elonga ion ac o 1Bc
p o ein o molecula mass 20 kDa exhibi s glu a hione
ans e ase ac i i y.
A che and Dye [18] ha e no ed ha p o eomics will
play an impo an ole in u u e in es iga ions in o he
biology and pa hogenici y o A. umiga us. Ye , i is qui e
su p ising ha eliable me hods o he ex ac ion o p o-
eins om filamen ous ungi did no eme ge un il he ea ly
2000s. Since hen, we, and o he s, ha e epo ed essen ially
simila s a egies o mycelial lysis in ol ing igo ous
physical dis up ion (bead-bea ing [19], g inding in liquid
N
2
[7,20], sonica ion o he use o F ench p ess echnology
[21]). Despi e he a ailabili y o efficien app oaches o cell
wall dis up ion, i is only ecen ly ha a s a egy o he
iden ifica ion o in acellula p o eins om A. umiga us
has been o hcoming [22]. Indeed, 28 o he 50 p o eins
iden ified in ou s udy ha e no p e iously been epo ed
in A. umiga us. The pe cen age sequence co e age ange
(9.4–50.5%) ob ained he e o MALDI-MS iden ifica ion
o A. umiga us p o eins is consis en wi h ha ecen ly
epo ed by Medina e al. o he iden ifica ion o u in-
induced sec e ed p o eins om A. fla us (7–43% sequence
co e age) and G inye e al. who mapped mi ochond ial
p o eins om T ichode ma ha izium (8–54% sequence co -
e age) [3,5].
Many o he p o eins iden ified we e shown o be p es-
en in se e al spo s ollowing 2D-PAGE, including
Fig. 4. Phylogene ic analysis o El A and B om Aspe gillus umiga us along wi h 28 p edic ed GST p o eins om a ange o ungal species. I is clea ha
bo h El A and B clus e in he EF1Bc-like g oup o GST-like p o eins. EF1Bc-like p o eins comp ise: EAA47645 (Magnapo he g isea); Q00717
(Aspe gillus nidulans); EAA77260 (Gibbe ella zeae); EAA55279 (Magnapo he g isea); NP_011717 (Saccha omyces ce e isiae); KLLA0D11594G
(Kluy e omyces lac is); KLLA0F26092G (Kluy e omyces lac is); AAA16892 (S. ce e isiae); NP_012842 (S. ce e isiae); YALI0C24420G (Ya owia
lipoly ica); YALI0B12562G (Ya owia lipoly ica); DEHA0D17369G (Deba yomyces hansenii); EAA57903 (A. nidulans); XP_323127 (Neu ospo a c assa);
EAA64302 (A. nidulans); NP_587885 (Schizsaccha omyces pombe). U e 2 p o eins comp ise: KLLA0D19624G (K. lac is); NP_014170 (S. ce e isiae);
CAGL0J07392G (Candida glab a a); DEHA0F08635G (D. hansenii); YALI0C03069G (Ya owia lipoly ica). MAK 16 p o eins comp ise: NP_009377(S.
ce e isiae); NP_586198(Encephali ozoon cuniculi); CAGL0G06248G (C. glab a a); KLLA0A04037G (K. lac is); DEHA0F02112G (D. hansenii);
YALI0C08052G (Ya owia lipoly ica).
1102 S. Ca be y e al. / Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104
ARTICLE IN PRESS
HSP70 chape one HscA, alanine amino ans e ase, hia-
mine biosyn hesis p o ein, UDP-glucose 4 epime ase, ca a-
lase, and glyce aldehyde-3-phospha e dehyd ogenase.
Glyce aldehyde-3-phospha e dehyd ogenase was also iden-
ified om se e al p o ein spo s in Candida albicans [23].
The exp ession o h ee p e iously uniden ified p o eins
in A. umiga us (py u a e deca boxylase, glyce aldehyde-
3-phospha e dehyd ogenase, and 6-phosphoglucona e
dehyd ogenase) has been p e iously epo ed o be up-
egula ed in C. albicans in esponse o he p esence o he
an i ungal azole agen , ke oconazole [23]. In e es ingly,
py u a e deca boxylase, along wi h wo o he p o eins
(HSP90 and enolase), was iden ified as a ungal an igen
exp essed du ing in asi e aspe gillosis [24]. A numbe o
iden ified p o eins ha e been shown o be loca ed in he cell
wall o o he ungi, included HSP90 and HSP70 in C. albi-
cans [25], enolase and glyce aldehyde-3-phospha e dehy-
d ogenase [26]. O he s, such as ca alase, ATP syn hase
(subuni b), and phosphoglyce a e mu ase we e ecen ly
shown o be sec e ed by A. fla us [5]. O e all, i is clea ha
a mo e sys ema ic and ocused app oach is equi ed o
u he elucida e he unc ional a ionale go e ning he
unexpec ed localisa ion, and dynamic na u e, o hese un-
gal p o eins which we e p e iously hough o be p edom-
inan ly o cy oplasmic o igin.
In an a emp o mo e owa ds a consis en nomencla-
u e o A. umiga us p o eins, he anno a ion desc ibed
by Mabey e al. and employed on he CADRE websi e a
h p://www.cad e.man.ac.uk has been adop ed [9]. The ini-
ial a ailabili y o he unanno a ed genome o he A.
umiga us [h p://www. ig .o g] and subsequen ly ha o
hein silico anno a ed e sion a h p://www.cad e.man.
ac.uk ha e been c i ical o he success o he wo k
p esen ed he e wi h espec o p o ein iden ifica ion. Mo e-
o e , he a ailabili y o he A. nidulans genome sequence
[h p://www.b oad.mi .edu/anno a ion/ ungi/aspe gillus/]
has also been o g ea assis ance in p o ein anno a ion and
iden ifica ion. Howe e , i should be no ed ha p o eomic
da a also se e o alida e he p edic i e bioin o ma ic
ools (e.g., A emis so wa e [27] and Glimme M [28,29])
used, in pa , o p edic in on–exon splice si es in ungal
genes. Consequen ly, a leas 29 pep ides om 22 p o eins
ha e been iden ified as a ising om mRNA o med as a
consequence o in on excision. No ably, 22 o 50 p o eins
iden ified in his wo k de i e om in on-con aining genes
in A. umiga us and ep esen biochemical alida ion o
bioin o ma ic gene p edic ion da a.
The euka yo ic elonga ion ac o 1B complex (eEF1B)
plays a key ole in he elonga ion s ep o p o ein syn hesis
and is comp ised o h ee subuni s e med a,b, and c,in
o de o inc easing molecula mass [30]. The iden ifica ion
o an EF1Bcp o ein (El A) in A. umiga us, which exhibi s
GST ac i i y, significan ly ex ends he ole o S- ans e ase
ac i i y beyond glu a hionyl conjuga ion o xenobio ic
compounds and ad ances expe imen al and bioin o ma ic
e idence o he p esence o GST-like domains in eEF1Bc
subuni s [11,30,31]. In ac , a ecen s udy has demons a -
ed an S- ans e ase ac i i y in he eEF1Bcsubuni o he
pa asi e C i hidia ascicula a, capable o u ilising ypano-
hione (N
1
,N
8
-bis(glu a hionyl)spe midine), bu no glu a-
hione, as a subs a e o CDNB conjuga ion [32].No
glu a hione pe oxidase ac i i y was de ec able in El A
p epa a ions. In e es ingly, Vicke s e al. [12] ha e shown
ha he in ac eEF1B complex is necessa y o pe oxidase
ac i i y ound in he eEF1Bcsubuni o Leishmania majo
eEF1B. Mic oa ay analysis o A. umiga us gene exp es-
sion has e ealed ha El A ( e med elonga ion ac o -1c)
exp ession is up- egula ed wo old wi hin 1 h o a empe -
a u e shi (30–48 C) which sugges s a ole o El A in p o-
ec ing agains hea shock [33].
Fi e clus e s o GST-like p o eins we e p e iously iden-
ified, h ee o which included EF1c, U e2, and MAK16
[11]. Rec ea ing a simila phylogene ic ee e ealed ha
bo h El A and El B om A. umiga us clus e ed and sha ed
homology o GST-like p o eins wi h EF1csimila i y.
These da a sugges ha bo h El A and El B a e eEF1Bc
p o eins con aining GST-like domain and ha his GST-
like domain is conse ed in a ious euka yo es. These
genes could possibly ha e a wide ange o unc ions includ-
ing p o ec ion om oxida i e s ess, con olling ansla ion
in esponse o oxida i e s ess [34] o egula ing p o ein
olding in a chape one like manne [35]. Unlike he high
affini y o El A o GSH-affini y media, he eEF1Bcsub-
uni s/eEF1B complexes iden ified by Kobayashi e al.
and Kamiie e al. did no exhibi affini y o glu a hione-
affini y media, al hough eEF1Bcsubuni s pu ified om
ice and Bombyx mo i we e amenable o pu ifica ion by
GSH-affini y ch oma og aphy [36,37] and GST ac i i y
was p esen in he pu ified EF1B complex p epa a ions.
Iden ifica ion o he comple e enzyma ic and unc ional
epe oi e o El A will ha e o awai eEF1B complex s ud-
ies in A. umiga us.
In summa y, ou findings ep esen one o he fi s
de ailed iden ifica ions o he mos abundan in acellula
p o eins o A. umiga us, iden ifies he impo ance o sub-
p o eomic s udies in ungal biology, as exemplified by he
iden ifica ion and cha ac e isa ion o he eEF1Bcp o ein,
El A and B, and will acili a e u u e s udies in o he
dynamic p o eome o A. umiga us.
Acknowledgmen s
This wo k was unded by he I ish Highe Educa ion
Au ho i y h ough he P og amme o Resea ch in Thi d
Le el Ins i u ions (PRTLI) Scheme-Cycle 3. P elimina y
sequence da a we e ob ained om The Ins i u e o Geno-
mic Resea ch websi e a h p://www. ig .o g. Sequencing
o Aspe gillus umiga us was unded by he Na ional Ins i-
u e o Alle gy and In ec ious Disease U01 AI 48830 o
Da id Denning and William Nie man, he Wellcome
T us , and Fondo de In es icagiones Sani a ias. Mass
spec ome y acili ies we e unded by he I ish Heal h
Resea ch Boa d.
S. Ca be y e al. / Biochemical and Biophysical Resea ch Communica ions 341 (2006) 1096–1104 1103
ARTICLE IN PRESS
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