RESEARCH

Proteome Analysis of Aspergillus fumigatus Total MembraneProteins Identifies Proteins Associated with the Glycoconjugatesand Cell Wall Biosynthesis Using 2D LC-MS/MS

Haomiao Ouyang • Yuanming Luo •

Lei Zhang • Yanjie Li • Cheng Jin

� Springer Science+Business Media, LLC 2009

Abstract We attempted to identify membrane proteins

associated with the glycoconjugates and cell wall biosyn-

thesis in the total membrane preparations of Aspergillus

fumigatus. The total membrane preparations were first run

on 1D gels, and then the stained gels were cut and submitted

to in-gel digestion followed by 2D LC-MS/MS and database

search. A total of 530 proteins were identified with at least

two peptides detected with MS/MS spectra. Seventeen

integral membrane proteins were involved in N-, O-glyco-

sylation or GPI anchor biosynthesis. Nine membrane pro-

teins were involved in cell wall biosynthesis. Eight proteins

were identified as enzymes involved in sphingolipid syn-

thesis. In addition, the proteins involved in cell wall and

ergosterol biosynthesis can potentially be used as antifungal

drug targets. Our method, for the first time, clearly provided

a global view of the membrane proteins associated with

glycoconjugates and cell wall biosynthesis in the total

membrane proteome of A. fumigatus.

Keywords Aspergillus fumigatus �Cell wall biosynthesis � Glycoconjugate �Membrane protein � Mass spectrometry

Introduction

Aspergillus fumigatus is the most common mold pathogen

of humans and causes both invasive disease in immuno-

compromised patients and allergic disease in patients with

atopic immune systems [1–4]. Though human diseases

caused by this organism are substantial, its basic biology is

to date mostly obscure. In A. fumigatus, a large number of

proteins that play important roles in cell survival and

invasion are located in plasma membrane, endoplasmic

reticulum (ER), Golgi membrane, and other cytoplasmic

organelles. Glycosylation begins in the rough ER [5], as a

co- or post-translational event, and proceeds as the glyco-

proteins migrate through the Golgi to their final destination.

The elaboration of the carbohydrate groups depends on the

expression and subcellular localization of the specific

enzymes required for their biosynthesis and on the final

destination and rate of transport in individual glycoproteins

through the various compartments. Enzymes responsible

for cell wall organization and cell morphogenesis, such

as chitin synthase, b(1,3)-glucan synthase, a(1,3)-glucan

synthase, b(1,3)-glucanosyltransferases, mannosyltransfe-

rases, and chitinase are integral membrane proteins [6, 7].

Sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1)

regulating sphingolipid metabolism and apoptosis is loca-

ted mainly in the endoplasmic reticulum [8].

Since such a large number of proteins associated with

glycoconjugates and cell wall biosynthesis are located in

cytoplasmic organelles as well as plasma membrane,

investigation of the structure and function of these proteins

in A. fumigatus proves to be increasingly significant. With

the recent accomplishment of genomic sequencing of

A. fumigatus [9], identification of membrane proteins on a

large scale becomes practical. To date, mass spectrometry

(MS)-based methods for the identification of proteins have

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12033-009-9224-2) contains supplementarymaterial, which is available to authorized users.

H. Ouyang � Y. Luo (&) � L. Zhang � Y. Li � C. Jin (&)

State Key Laboratory of Microbial Resources, Institute

of Microbiology, Chinese Academy of Sciences,

A3 Datun Road, Chaoyang District, Beijing 100101, China

e-mail: luoym@im.ac.cn

C. Jin

e-mail: jinc@sun.im.ac.cn

Mol Biotechnol

DOI 10.1007/s12033-009-9224-2

been become a standard platform in proteomics. The most

popular MS-based strategies rely on proteolytic digestion

of proteins into peptides before introduction into the mass

spectrometer. Digestion of proteins into similar sized

peptides helps to overcome the solubility and handling

problems associated with ionization in the mass spec-

trometer. For complex mixtures of proteins which are not

sufficiently resolved in 1DE, a multidimensional separation

may be necessary in that components not separated in the

first dimension are separated in the second. The 2DE is the

most common multidimensional separation technique used

to separate complex mixtures. However, it has obvious

shortcomings such as limited dynamic range, inability to

detect membrane proteins and extremely basic or extre-

mely acidic proteins under standard conditions. Though

Kniemeyer et al. [10] used an optimized 2DE protocol to

improve the 2DE resolution and map quality of A. fumig-

atus, all of the proteins detected were basically cytoplasmic

proteins with low molecular weights. To increase the sol-

ubility of GPI-anchored membrane proteins in 2DE, Bru-

neau et al. [7] reported an applicable method to separate

GPI-anchored membrane proteins, in which the GPI-

anchored proteins were first released from membrane

preparation in a soluble form by an endogenous GPI-

phospholipase C and then separated by 2DE. Though

available for GPI-anchored proteins, this method still faces

challenges in separating the membrane proteins with

transmembrane segments and high MWs. To detect as

many membrane proteins potentially related to the syn-

thesis of glycoprotein, glycolipid, and cell wall in the

whole membrane system comprising plasma membrane

and cytoplasmic organelles, we used the currently devel-

oped multidimensional protein identification technology

(MudPIT) [11, 12] to directly analyze the total membrane

preparations collected by ultracentrifugation. In addition,

cell wall biosynthetic pathways have been recognized for a

long time as essential and unique specific drug targets and

the recent clinical launch of caspofungin, a b(1,3)-glucan

synthase inhibitor, has confirmed that the development of

new drugs can indeed be based on inhibition of cell-wall

biosynthetic enzymes [13]. In the light of the importance of

the biosynthetic pathways of cell wall and ergosterol in

antifungal drug design, we also briefly discussed the pro-

teins that may be used as drug targets.

Materials and Methods

Fungal Culture and Membrane Preparation

A. fumigatus, strain YJ407, was inoculated in complete

medium (0.1% yeast extract, 0.2% peptone, 1% glucose,

0.15% casein, and 2% of salt solution (v/v) containing 2.6%

KCl, 2.6% MgSO4 � 7H2O, 7.6% KH2PO4, 5% of trace-

element solution (v/v), pH 7.0) and grown at 37 �C for 24 h

in a 15-L fermentor (Bioflo410) with stirring (200 rpm) and

fresh air (1 L air min-1). A liter of trace-element solution

contains 40 mg NaB4O7 � 10H2O, 400 mg CuSO4 � 5H2O,

800 mg FeSO4 � 2H2O, 800 mg MnSO4 � 2H2O, 800 mg

Na2MO4 � 2H2O, and 8 g ZnSO4 � 7H2O.

The membrane preparation was performed according to

the method modified by Fontaine et al. [14]. Briefly, the

mycelium was collected by filtration under vacuum,

washed with water, and then ground in liquid nitrogen. The

power was resuspended in disruption buffer solution

(200 mmol/L Tris–HCl, 20 mmol/L EDTA, pH 8.0,

1 mmol/L phenylmethylsulfonyl fluoride buffer) at 4 �C in

presence of glass beads (0.5–0.75 mm diameter). Cell wall

was removed by centrifugation at 10,000g for 10 min at

4 �C. Total membrane pellets were then collected by

ultracentrifugation at 150,000g for 60 min at 4 �C. The

membrane pellets were resuspended in the disruption buf-

fer, homogenized, and then centrifuged at 150,000g for

60 min at 4 �C. The resuspension was repeated three times

to remove the contaminants from nonmembrane compo-

nents. The pellets were finally stored at -80 �C for future

use.

Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis and In-Gel Digestion

Two hundred micrograms of the total membrane prepara-

tion was dissolved in 29 loading buffer containing 4%

sodium dodecyl sulfate (SDS), 20% glycerol, 10%

2-mercaptoethanol, 0.004% Bromophenol Blue, and

0.125 mol/L Tris–HCl, pH 6.8, boiled for 5 min, and

subsequently run on a 12% SDS polyacrylamide gel

without sample loading comb. The gels were silver stained

as previously described [15]. The whole gel was cut for

each run and divided into 27 sections. When cutting the

gels, we have a strategy that the individual gel pieces with

similar staining strength or visually unstained were com-

bined together respectively, preventing interference of

identification of low abundance proteins from high abun-

dance proteins, as shown in Fig. 1. The stained gel pieces

were destained and submitted to in-gel digestion as previ-

ously described [15].

2D LC-MS/MS Analysis

The digested peptide mixtures for each sample were

desalted with Hypersep c18 SPE cartridge (Thermo Sci-

entific, Bellefonte, PA, USA). The cartridge was condi-

tioned before use by 5 9 1 mL of 0.1% TFA in 50%

acetonitrile and then by 5 9 1 mL of 0.1% TFA. The

peptide mixtures to be desalted were dissolved in 100 lL

Mol Biotechnol

of 0.1% TFA and passed through the conditioned cartridge.

The cartridge was washed three times with 1 mL of 0.1%

TFA and the peptides were eluted twice with 1 mL of 0.1%

TFA in 50% acetonitrile followed by evaporation to dry-

ness in a Speed-Vac. The desalted peptides were dissolved

in 20 lL of 0.1% formic acid and loaded on a strong cation

exchange column (BioBasic SCX, 0.32 mm 9 10 cm,

ThermoHypersil, Allentown, PA) via the autosampler, and

then step-eluted with a 10-step NH4Cl gradient (0, 10, 30,

50, 80, 100, 150, 180, 200, and 400 mM in 5% acetonitrile)

from the strong cation exchange column onto alternating

reversed phase columns (BioBasic C18, 300 A, 5 lm sil-

ica, 180 lm 9 10 cm, ThermoHypersil, Allentown, PA)

for the second dimensional separation. The flow rate for the

reversed phase separation was maintained at 100 lL/min

before splitting and at 1.0 lL/min after the flow split. The

gradient was started at 5% acetonitrile in 0.1% formic acid

for 20 min, then ramped to 50% acetonitrile within 80 min,

and then changed to 95% acetonitrile for 20 min, and

finally changed to 5% acetonitrile for an additional 20 min.

The resolved peptides were subjected to MS/MS analysis

with LCQ Deca XP Plus ion-trap mass spectrometer

(ThermoFinnigan, San Jose, CA) equipped with a nano-

spray source. The MS/MS data were acquired in the data-

dependent scan mode including four scan events: one full-

range MS scan and three MS/MS scans on the three most

intense precursors. The mass spectra were measured with

an overall mass/charge (m/z) range of 400–2000.

Database Searching

Each sample analyzed by 2D LC-MS/MS generates 10

peak list files, and 270 peak list files were created from 27

sections. All MS/MS spectra were searched using Thermo

Finnigan Bioworks 3.1 against the protein sequence data-

base of A. fumigatus downloaded from NCBI: http://www.

ncbi.nlm.nih.gov/ containing 21,395 protein entries, with a

static modification of ?57.0215 Da on cysteine residue and

a differential modification of 15.9994 on methionine. The

precursor ion mass tolerance was 1.4 Da, and the fragment

ion mass tolerance was 1.5 Da. We used the following

three steps to perform the data processing. First, we used

SEQUEST criteria below to perform an initial filtration:

DeltaCn C 0.1; Rsp C 1; Xcorr C 1.9 for singly charged

fragments; Xcorr C 2.2 for doubly charged; Xcorr C 3.75

for triply charged. Second, we used the AMASSv1.17.0.17

developed by Sun and co-workers [16, 17] (available at

http://www.proteomics-cams.com) to further filter the SE-

QUEST results based on three parameters: MatchPct C 60,

Cont C 40, and Rscore \ 2.6. Proteins with two or more

spectra approved by AMASS were accepted as positive

identifications. If a protein has multiple isoforms or has

multiple entries in the databases, we only specify the major

form of the protein unless a specific peptide points to a

region of the protein, which exists only in one of the

isoforms. Finally, reverse database searching was used

to estimate the false positive rate. The false positive

rate = peptide number in reverse database/peptide number

in forward database 9 100%.

Results

Proteins Located in Plasma Membrane

and Cytoplasmic Organelles

The results reported here are from merged data of 27

fractions, leading to the identification of 794 proteins from

the total membrane preparations of A. fumigatus. Reverse

database search revealed that the false positive rate

was 3.51% for positive peptides filtered by combined

SEQUEST/AMASS parameters. Of the 794 proteins, 530

were identified with at least two different peptides detected

with MS/MS spectra (Supplementary Table 1) and were

completely discussed. We assigned the subcellular loca-

tions of the 530 proteins according to the ‘‘GO_compo-

nent’’ in NCBI, and for those whose subcellular locations

are not labeled in NCBI, a WoLF PSORT software (freely

Fig. 1 SDS-PAGE of total membrane preparations of A. fumigatus.The left lane indicates the low Mr protein markers, and the right lane

indicates the 27 sections of A. fumigatus cut and divided based on

similar staining intensity

Mol Biotechnol

available at wolfpsort.org) was used to predict the sub-

cellular localization. Due to use of ultracentrifugation at

150,000g, the cytoplasmic organelles were collected as

well as plasma membrane pellets and therefore cytosolic

and excellular proteins located in cytoplasmic organelles

were identified along with the plasma membrane proteins.

The subcellular locations of the 530 proteins were pre-

dicted, and the subcelluar location distribution was shown

in Fig. 2.

As shown in Fig. 2, major the proteins are located in

ribosome, mitochondrion, nucleus, and plasma membrane,

accounting for 15.8, 30.4, 15.3, and 16.6%, respectively.

The others are located in the cytoplasmic organelles such

as endoplasmic reticulium (ER), golgi, vesicle, vacuole,

and proteasome complex. For the mitochondrial proteins

identified in this study, based on the annotation in NCBI,

27 are located in mitochondrial inner membrane, 7 in

mitochondrial outer membrane, 9 in mitochondrial matrix,

20 in mitochondrial ribosome, and 1 in mitochondrial inner

membrane space (Supplementary Table 2). It is necessary

to point out though 20 proteins identified in this study were

predicted as cytosolic proteins and 3 as excellular ones

by using the WoLF PSORT software (Supplementary

Table 1), these cytosolic and excellular proteins are not

caused by the contamination of nonmembrane components

for the total membrane pellets were strictly prepared

and washed three times with the disruption buffer (see

‘‘Materials and Methods’’ section). After three washes, the

supernatant was determined by SDS-PAGE to evaluate

washing efficiency and no obvious protein bands were

observed on the silver-stained gel, showing that the total

membrane isolation was reasonably successful. Actually, a

large number of cytosolic and excellular proteins were

identified due to their binding to membrane or transiently

bound to membrane, and their subcellular locations are

much dependent on their different functions, and therefore

multiple localization of the same protein is actually

reasonable [18]. As listed in Supplementary Table 3, 12

cytosolic proteins and 2 excellular proteins have been

reported to be associated with cytoplasmic organelles.

These extracellular and cytosolic proteins were collected

by ultracentrifugation in forms of cytoplasmic organelles

such as ER/golgi transport vesicles [19], sorting vacuoles

[20], degradation vacuoles [21], and storage vacuoles [22].

For example, Hsp70 chaperone (HscA) (gi|70983346) was

predicted as cytosolic protein in this study and was

reported to be associated with peroxisomes and involved in

import of proteins into organelles [23]. Glyceraldehyde

3-phosphate dehydrogenase (gi|70985278), as a typical

cytosolic protein identified in this study, is involved in the

vesicle transport from the ER to golgi [24].

One of the obvious advantages of 2D LC-MS/MS over

2DE is reflected in the identification of proteins with

extreme pIs and high MWs. Of the 530 identified proteins,

29 are with pIs \ 5 and 36 with pIs [ 11, accounting

for 5.5% and 6.8%, respectively, and 45 proteins are

with MWs [ 100 kD (Supplementary Table 1), accounting

for 8.5%, the largest being alpha-1,3-glucan synthase

(gi|70985813) with a MW of 274.4 kD, from which three

unique peptides were identified. However in the previous

0 5 10 15 20 25 30 35

ribosome

cytosol

mitochondrion

nucleus

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ER

vacuole

plasma membrane

cytoskeleton complex

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lipid particle

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phenylalanine-tRNA ligase complex

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Sub

cellu

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loca

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Percent (%)

Fig. 2 Percentage of proteins

located in plasma membrane

and cytoplasmic organelles

Mol Biotechnol

study, the proteome of A. fumigatus separated by 2DE were

major the cytoplasmic proteins with pIs 5.27–9.7 and

MWs \ 90 kD [10].

In this study, it is the proteins with high MWs that play

great roles in the synthesis of glycoconjugates and cell

wall. For example, five proteins with MWs [ 100 kD,

including chitin synthase G (gi|146322408, 102.4 kD),

chitin synthase E (gi|71001992, 206.6KD), a-1,3-glucan

synthase (gi|70985813, 274.4 kD), b-1,3-beta-glucan syn-

thase catalytic subunit FksP (gi|70992539, 219.8 kD), and

chitin synthase A (gi|70988943, 107.4 kD) are involved in

cell wall organization and biosynthesis. These proteins,

except chitin synthase A, are GPI-anchored membrane

proteins. Whereas in the previous report by Bruneau

et al. [7], the MWs of the identified GPI proteins are

below 80 kD. In addition, another two proteins with high

MWs, including GPI-anchor biosynthetic protein (Mcd4)

(gi|70982111, 114.4 kD) and protein mannosyltransferase 1

(gi|71000555, 107.1 kD), are identified to participate in

GPI anchor biosynthesis and O-glycosylation, respectively.

The above results suggest that 2D LC-MS/MS combined

with 1D SDS-PAGE is a powerful tool for identifying the

membrane proteins with extreme pIs and high MWs.

Membrane Proteins Involved in Glycosylation

of Proteins

So far, protein glycosylation in A. fumigatus is less

extensively studied. What we described here about the

functions and subcellular locations were much dependent

on Go_function and Go_component in NCBI and related

reports in yeast and other eukaryotic organisms [25]. As

summarized in Table 1, eleven ER- and four Golgi-bound

membrane proteins and two plasma membrane proteins

were identified to be involved in protein glycosylation

(N-glycosylation, O-glycosylation and GPI-anchoring). A

Dense Alignment Surface (DAS) method (available at

http://www.sbc.su.se/*miklos/DAS/) was used to predict

the transmembrane segments of these membrane proteins.

As shown in Table 1, when the cutoff for DAS score is set

at 2.2, at least one transmembrane segment is found in most

of membrane proteins except dolichol-phosphate manno-

syltransferase (gi|70999724) with no transmembrane

segment detected. It is crucial to point out the cDNA

sequences of four proteins have been cloned by our group,

leading to updated accession numbers (Table 1). Based on

the membrane proteins identified in this study, we were

able to develop glycosylation pathways in A. fumigatus

including N-glycosylation, O-mannosylation, and GPI

biosynthesis (Fig. 3).

N-glycosylation is initiated with the transfer of the

N-glycan precursor to nascent polypeptide, which is cata-

lyzed by oligosaccharyltransterase (OST). Biochemical and

genetic investigations have led to the discovery of nine

protein subunits of the OST in yeast, five of which are

essential for growth. In mammals, seven proteins have

meanwhile been identified as components of the OST.

Although the specific role of the individual subunits is not

well understood, some evidences strongly support the idea

that Stt3 is the catalytic subunit of OST [26]. In our study,

five subunits of the OST were identified and Stt3

(gi|70983446) was a putative catalytic subunit (Table 1 and

Fig. 3). Detailed descriptions about the structures, func-

tions, and localization of the OST in yeast and other

eukaryotic organisms can be seen everywhere [26–28].

Although we did not identify the proteins involved

in N-glycan trimming and processing, the calnexin

(gi|70993400), a protein that is involved in the correct

folding of secretory glycoproteins of mammalian cell [26],

was identified in our study, suggesting the existence of

N-glycan-dependent quality control system of protein

folding in A. fumigatus. Once the N-linked core oligosac-

charide is transferred to the Golgi, it is extended by the

addition of the outer chain. In yeast several proteins have

been shown to participate in the synthesis of this structure,

including Och1p, Mannan polymerase I complex (M-Pol I),

M-Pol II, Mnn2p, Mnn5p, Mnn4p, Mnn6p, and Mnn1p

[29]. M-Pol I and M-Pol II are a(1,6)-mannosyltransferases

and responsible for the elongation of the a(1,6)-linked

backbone of the outer chain. M-Pol I consists of Mnn9p

and Van1p, while M-Pol II is a multiprotein complex

including Mnn8p (Anp1p), Mnn9p, Mnn10p, Mnn11p, and

Hoc1p. In the membrane preparation of A. fumigatus, the

Kre5 (gi|70997373) and Ktr4 (gi|70985124) were identified

as a1,2-mannosyltransferases. However, only one subunit

of M-Pol I and M-Pol II, the Mnn9 (gi|70988857) was

discovered, which is consistent with the shorter N-glycan

observed in this species.

The structure of the O-glycans in peptidogalactomannan

of the cell wall of A. fumigatus has been determined

as Glca1-6Man1-O, Galfb1-6Mana1-6Man1-O, Galfb1-

5Galfb1-6 Mana1-6Man1-O, and Galfb1-5(Galfb1-5)3

Galfb1-6Man-O [30]. The protein O-mannosylation of

fungi is initiated in the ER by the transfer of mannose from

dolichyl-phosphate mannose. This reaction is catalyzed by

a family of protein O-mannosyltransferase (PMTs) [31].

Three putative PMTs (gi|70991332, gi|71000555, and

gi|70983460) were found in the membrane preparation of

A. fumigatus. In addition, two Golgi-located proteins, Kre2

(gi|70997663) and Ktr4 (gi|70985124), were identified as

a1,2-mannosyltransferases in O-glycosylation pathway. In

S. cerevisiae, KRE2 and KTR4 belong to the KRE2/MNT1

mannosyltransferase gene family. All of them are predicted

to be membrane proteins located in Golgi. The first

member of this family (KRE2/MNT1) is identified to

encode for an a1,2-mannosyltransferase involved in

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Mol Biotechnol

synthesis of O-linked oligosaccharide. Further functional

investigation revealed that KRE2 participates in synthesis

of both O- and N-linked oligosaccharides. Kre2p/Mnt1p,

Ktr1p, and Ktr3p, have overlapping roles, and collectively

add most of the second and the third a(1, 2)-linked mannose

residues on O-linked oligosaccharides as well as some of the

a(1, 2)-linked mannoses in the branches of N-linked oligo-

saccharides [32]. Based on the O-glycan structures identi-

fied in A. fumigatus, the Kre2 (gi|70997663) and Ktr4

(gi|70985124) might be involved in the addition of the

second a(1,6)-linked mannose residue, instead of a(1,2)-

linked mannose residue in yeast. No b-Galf transferase was

identified in this study. Also, a genome analysis has failed to

find eukaryotic gene homologous to b-Galf transferase [31].

Thus, it is likely that A. fumigatus has a novel b-Galf

transferase.

Like most other eukaryotes, A. fumigatus harbors a

GPI-anchoring machinery and uses it to attach proteins to

membranes. While a few GPI proteins reside permanently

at the plasma membrane, a majority of them gets further

processed and is integrated into the cell wall by a covalent

attachment to cell wall glucans. The GPI biosynthetic

pathway is necessary for growth and morphogenesis [33].

In S. cerevisiae, the GPI lipids are synthesized in the ER

and added onto proteins by a pathway comprising 12 steps,

carried out by 23 gene products, 19 of which are essential.

Some of the estimated 60 GPI proteins predicted from the

genome sequence serve enzymatic functions required for

the biosynthesis and the continuous shape adaptations of

the cell wall, others seem to be structural elements of

the cell wall and yet others mediate cell adhesion [34]. It

has been shown that A. fumigatus GPI glycan moiety is

mainly a linear pentomannose structure linked to a glu-

cosamine residue: Mana1-3Mana1-2 Mana1-2Mana1-

6Mana1-4GlcN [14]. However, in contrast to yeast, little

is known for the GPI biosynthesis in A. fumigatus. Several

proteins that might be involved in the GPI biosynthesis

were identified in this study. A protein (gi|14632406) was

identified as a putative mannosyltransferase Smp3, an

enzyme that catalyzes the addition of the Man4 and is

essential in yeast and in C. albicans. Two GPI trans-

amidases, Gpi16p and PIG-S/Gpi17p, may catalyze the

transamidation reaction that results in the cleavage of the

polypeptide chain and the concomitant transfer of the GPI

anchor to the newly formed carboxy-terminal amino acid

of the anchored protein [35]. In addition, a plasma mem-

brane-bound protein, GPI-anchor biosynthetic protein

(Mcd4), (gi|70982111) was identified to be involved in

GPI anchor biosynthesis. It is well known that glycocon-

jugate biosynthesis requires activated form of monosac-

charide as sugar donor. In this study, we also identified

membrane proteins that were involved in specific steps of

galactose metabolism, glycosis, and mannose metabolismTa

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Mol Biotechnol

providing the sugar monomers, such as glucose, galactose,

mannose, and fructose for O-mannosylation, N-glycosyla-

tion, and GPI biosynthesis (Fig. 3). The activated forms of

sugar donors are indicated with gray background.

Membrane Proteins Involved in Sphingolipid Metabolic

Pathway

Glycosphingolipids (GSL) are essential components of the

fungal cell membrane. The sphingolipid moieties of GSL

contains a hydrophobic segment (ceramide), which is a

long-chain base (LCB) that is N-acylated with a very long-

chain a-hydroxy fatty acid, linked to various polar head

groups. It has been shown that A. fumigatus expresses both

glucosylceramide and galactosylceramide (GlcCer and

GalCer). The synthesis of GlcCer is essential for normal

development of A. fumigatus [36]. A comparative genome

analysis of the sphingolipid biosynthetic pathway in fungal

species shows that A. fumigatus has most of the sphingolipid

pathway genes found in other fungi, except for the CSG2

and IPT1 genes; the former is involved in the mannosylation

of inositol phosphorylceramide (IPC) to mannose–inositol–

phosphorylceramide and the latter involved in the synthesis

of mannose–(inositol-P)2–ceramide from mannose–inosi-

tol–phosphorylceramide [37]. Seven proteins involved in

specific steps of sphingolipid metabolic pathway are

detailed in Fig. 4 and the peptide fragments, charges, and

scores that are used to identify these proteins are listed in

Supplementary Table 4. Sphingolipid synthesis begins with

the condensation of palmitol-CoA and serine to yield

3-ketodihydrosphingosine, which is reduced to yield

dihydrosphingosine (DHS). The condensation reaction

is catalyzed by serine palmitoyltransferases, LcbAp

(gi|70992537), and Lcb2p (gi|70995600). Then 3-keto-

dihydrosphingosine is reduced by a 3-ketosphingosine

reductase, Tsc10p (gi|146322596) to produce the LCB. The

next step in sphingolipid synthesis, DHS is converted to

PHS. Sur2p (gi|70996596) is required for the hydroxylation

of DHS at C-4. Two fatty acid hydroxylases (gi|70999962

and gi|70996386) may be involved in the hydroxylation of

the very long-chain fatty acid. In addition, one putative S1P

lyase (gi|70993864) was also identified.

Fig. 3 Metabolic pathways of N-glycosylation, O-glycosylation, and

GPI biosynthesis in A. fumigatus. Glycolysis, mannose metabolism,

and galactose metabolism provide sugar monomers including

GlcNAc, Man, galactose, fructose, and Glu for the biosynthesis of

N-glycans, O-glycans, and GPI. The proteins involved are detailed in

specific steps of individual metabolic pathways. The activated donors

of monosaccharides are indicated with gray background. Dolichyl-

phosphate-mannose, which is generated from mannose metabolism, is

used as donor of mannoses in GPI biosynthesis and O-mannosylation

Mol Biotechnol

Membrane Proteins Involved in Cell Wall Biosynthesis

We successfully found nine plasma membrane-bound

proteins involved in cell wall biosynthesis (Fig. 5 and

Supplementary Table 5), four of which were identified as

chitin synthases (CHSs), including CHSA (gi|70988943),

CHSC (gi|70985514), CHSE (gi|71001992), and CHSG

(gi|70998925), suggesting additive, redundant, or syner-

gistic roles of the various CHSs of this multigene family in

chitin deposition. Indeed, it has been shown that the

mutations in CHSE and CHSG result in a growth pheno-

type such as reduction in hyphal growth, periodic swellings

along the length of hyphae and a block on conidiation

partially restored by growth in presence of an osmotic

stabilizer. While a double CHSE/CHSG disruption mutant

is still viable, suggesting an alternate regulation of the

various remaining CHSs [6].

The Fksp (gi|70992539) was identified as a b-1,3-glucan

synthase. This protein might be essential since only one

FKS gene has been found in A. fumigatus. A protein

gi|70985813 was identified as a a-1,3-glucan synthase

responsible for a(1,3)-glucan synthesis. This enzyme

internally splits a b(1,3)-glucan molecule and transfers the

newly generated reducing end to the nonreducing end of

another b(1,3)-glucan molecule. The Gel1p (gi|70988799)

was identified as a GPI-anchored b1,3-glucanosyltransfer-

ase that generates a new b(1,3)-linkage to allow the elon-

gation of b(1,3)-glucan chains [38, 39]. Besides, the Mnn9

(gi|70988857) may be involved in the biosynthesis of

galactomannan of cell wall, as the yeast Mnn9p has been

shown to be responsible for the elongation of the mannan

chain and involved in the septum formation [40].

When talking about the proteins involved in cell wall

biosynthesis, we will have to mention their roles in

Fig. 4 Diagram of sphingolipid

metabolism in A. fumigatus. In

our study, seven proteins with

accession numbers were

identified to be involved in five

sequential reaction steps of

sphingolipid synthesis, which

begins with the condensation of

palmitoyl-CoA and serine

D-MannoseD-Gluocosamine-6Pα−Glucoseβ−GlucoseD-Galactose

Fig. 5 Diagram of cell wall

biosynthetic pathway. D-

Glucosamine-6P generated in

glycosis is transformed to UDP-

GlcNAc in aminosugars

metabolism and used as sugar

donors for chitin biosynthesis.

Glycosis and galactose

metabolism provide Gal and

Man for galactomannan

biosynthesis, a-glucose for

a-glucans biosynthesis, and

b-glucose for b-glucan

biosynthesis

Mol Biotechnol

antifungal drug design. As fungal cell wall polysaccharides

do not have a counterpart in humans, theoretically, those

proteins involved in cell wall biosynthesis can be used as

ideal antifungal drug targets, as in the case of caspofungin,

a new antifungal that is designed as a b(1,3)-glucan

synthase inhibitor [13]. As shown in Fig. 5, four chitin

synthases are involved in chitin biosynthesis, Mnn

(gi|70998857) is involved in galactomannan biosynthesis,

AGS (gi|70985813) is involved in a1,3 glucan biosynthesis,

and Gel1p (70988799) and Fksp (gi|70992539) are

involved in b-1,3 glucan biosynthesis, the inhibitors of

these enzymes that can disrupt the cell wall biosynthesis in

fungi may be potentially used as drug targets. And to some

extent, development of new antifungals based on inhibition

of cell wall biosynthesis may solve the current problem in

resistance to azole antifungals [41].

In addition, in this study, we successfully found six ER-

bound enzymes (Supplementary Table 6) that are associ-

ated with antifungal drug design based on inhibition of the

formation of cell membrane by blocking the ergosterol

biosynthetic pathway [41], which provides another alter-

native method to overcome the resistance to azole anti-

fungals. As shown in Fig. 6, two sterol c14-reductases

(gi|66847697, gi|66847953) were identified in this study

and have been successfully used as drug target of

amorolfine, an antifungal [42]. As in the case of azoles

directed against C14a-demethylase in the ergosterol path-

way, design of the inhibitors directed against the identi-

fied enzymes, farnesyl-diphosphate farnesyltransferase

(gi|66844434), Erg26 (gi|66853474), Erg28 (gi|66853125),

and Erg3 (gi|66845190), should be a reasonable starting

point for new antifungal development, which will effec-

tively promote the antifungal drug discovery based on

inhibition of ergosterol biosynthesis.

Besides the proteins described above, many other pro-

teins that are localized in ubiquitous cytoplasmic organ-

elles also fulfill extensive functions in A. fumigatus. Since

our current interest is focused on membrane proteins

related to the synthesis of glycoconjugates and cell wall,

the proteins that are putatively involved in cell adhesion,

Fig. 6 Ergosterol biosynthetic pathway. Six enzymes indicated with accession number are identified in this study. The enzymes involved in the

specific steps of ergosterol biosynthesis can potentially be used as drug targets for antifungal drug design

Mol Biotechnol

regulation of autophagy, cell cycling, SNARE interaction

in vesicular transport system, regulation of actin cyto-

skeleton, ubiquitin mediated proteolysis, phosphatidylino-

sitol signaling pathway, MAPK signaling pathway, ERBB

pathway, calcium signaling pathway, and other signaling

pathways are not discussed here.

Discussion

In A. fumigatus, a large number of membrane proteins that

fulfill different functions are located in ubiquitous cyto-

plasmic organelles as well as cell membrane. In order to

get a global view of the membrane proteins involved in

biosynthesis of cell wall and glycoconjugates, the total

membrane pellets were directly subjected to one-dimen-

sional SDS-PAGE followed by 2D LC-MS/MS capable of

high throughput separation and identification. Many of the

membrane proteins involved in protein glycosylation, cell

wall biosynthesis, and glycolipid synthesis were identified

to be localized in endoplasmic reticulum, Golgi apparatus,

and other cytoplasmic organelles as well as plasma mem-

brane, strongly demonstrating the availability and necessity

of this method. Bands from 1D gels were cut according to

invisibly distinguished abundance to achieve the enrich-

ment of low abundance proteins, avoiding the interference

of high abundance proteins. Besides, we combined 1DE

with 2D LC-MS/MS rather than directly subjected the total

membrane preparations to 2D LC-MS/MS. In this way, in-

gel digestion was carried out in 1D gel instead of in-

solution. The major problem in in-solution digestion is the

introduction of denature reagent (8 mol/L urea or 6 mol/L

guanidine hydrochloride) to dissolve the proteins, whereas

the denatured protein solution must be diluted to below

2 mol/L urea or 1 mol/L guanidine hydrochloride prior to

proteolytic digestion to maintain the activity of the trypsin,

leading to loss of proteins caused by dilution precipitation.

Identification of total membrane proteome of A. fumigatus

provides only a starting point for functional analysis. We will

hopefully continue to make progresses in deciphering the

structures and functions of membrane proteins related to

glycoconjugates in A. fumigatus as we have been doing [33].

Acknowledgments This project was supported by the State ‘‘863’’

High-tech Project (2007AA02Z164), the National Basic Research

Program of China (2006CB504400), and the Youth Fund from Institute

of Microbiology, Chinese Academy of Sciences (0654041005).

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