Glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis.

RESEARCH ARTICLE

Glycosylation of liver acute-phase proteins in pancreatic

cancer and chronic pancreatitis

Ariadna Sarrats1, Radka Saldova2, Eva Pla1, Esther Fort3, David J. Harvey4, Weston B. Struwe2,Rafael de Llorens1, Pauline M. Rudd2 and Rosa Peracaula1�

1 Unitat de Bioquımica i Biologia Molecular, Departament de Biologia, Universitat de Girona, Campus de Montilivi,Girona, Spain

2 Dublin-Oxford Glycobiology Laboratory, NIBRT, Conway Institute, University College Dublin, Belfield, Dublin,Ireland

3 Unitat de Digestiu, Hospital Universitari Dr Josep Trueta, Girona, Spain4 Glycobiology Institute, Department of Biochemistry, Oxford University, Oxford, UK

Received: July 10, 2009

Revised: October 6, 2009

Accepted: December 18, 2009

Purpose: Glycosylation of acute-phase proteins (APP), which is partially regulated by cyto-

kines, may be distinct in disease and provide useful tumour markers. Thus, we have

examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic

pancreatitis (CP) and control patients.

Experimental design: Using a specific anti-sialyl Lewis X antibody and N-glycan sequencing,

we have determined glycosylation changes on a-1-acid glycoprotein (AGP), haptoglobin

(HPT), fetuin (FET), a-1-antitrypsin (AT) and transferrin (TRF).

Results: Increased levels of sialyl Lewis X (SLex) were detected on AGP in advanced PaC and

CP and on HPT, FET, AT and TRF in CP. An increase in N-glycan branching was detected on

AGP and HPT in the advanced stage of PaC and CP and on FET and TRF in the CP. A core

fucosylated structure was increased on AGP and HPT only in the advanced PaC patients.

Conclusions and clinical relevance: Changes in APP SLex and branching are probably asso-

ciated with an inflammatory response because they were detected in both advanced PaC and

CP patients and these conditions give rise to inflammation. On the contrary, the increase in

APP core fucosylation could be cancer associated and the presence of this glycoform may give

an advantage to the tumour.

Keywords:

Acute-phase proteins / Core fucose / Liver / Pancreatic cancer / Sialyl Lewis X

1 Introduction

Pancreatic cancer (PaC) is the fourth leading overall cause of

cancer death in the United States and it has the lowest

5-year survival rate (about 5%) [1]. This poor survival is

mostly because of late diagnosis, frequently after metastasis.

Early detection of PaC is currently not available because

existing biomarkers for this disease are inadequate [2]. For

example, the use of CA19-9, the most common tumour

marker for PaC, is restricted by its false-positive results

Abbreviations: 2-AB, 2-aminobenzamide; AGP, a-1-acid glyco-

protein; APP, acute-phase proteins; AT, a-1-antitrypsin; ConA,

concanavalin A; CP, chronic pancreatitis; FET, fetuin; FUT,

fucosyltransferase; HC, healthy control; HCC, hepatocellular

carcinoma; HPT, haptoglobin; IL, interleukin; NP, normal phase;

PaC, pancreatic cancer; Q, quadrupole; RT, room temperature;

SLex, sialyl Lewis X; TBST, 0.1% Tween in TBS; TRF, transferrin;

WAX, weak anion exchange

�Additional corresponding author: Dr. Rosa Peracaula

E-mail address: [email protected]

Correspondence: Professor Pauline M. Rudd, Dublin-Oxford

Glycobiology Laboratory, NIBRT, Conway Institute, University

College Dublin, Belfield, Dublin 4, Ireland

E-mail: [email protected]

Fax: 1353-17166950

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com

Proteomics Clin. Appl. 2010, 4, 1–17 1DOI 10.1002/prca.200900150

[3, 4]. This marker is unable to differentiate PaC from

benign pancreaticobiliary disorders such as chronic

pancreatitis (CP). Thus, a great need exists for new sensitive

and specific biomarkers for PaC.

Liver plays an important role in the acute-phase response

that is one of the key processes of the innate and natural

immunity in response to tissue injury, infection and

inflammation. Cytokines produced during inflammatory

processes, including cancer, are the main stimulators for the

synthesis of liver-positive acute-phase proteins (APP) the

concentration of which in plasma increases [5]. At the same

time, the production of a number of other liver proteins is

reduced (negative APP). Some major plasma APP are

glycosylated, and include a-1-acid-glycoprotein (AGP),

haptoglobin (HPT), a-1-anti-chymotrypsin, a-1-antitrypsin

(AT) and fibrinogen (positive APP); fetuin (FET) and

transferrin (TRF) (negative APP).

Altered glycosylation is a common feature of tumour

cells and it can be reflected in their glycocalyx and secreted

glycoproteins. Generally, N-glycans are more branched and

more sialylated in tumours [6]. In addition, the Lewis

structures sialyl Lewis X (SLex) and sialyl Lewis A (SLea),

which carry an outer-arm fucose linked to N-acetyl-

glucosamine and a sialic acid linked to a-2,3 to galactose, are

overexpressed in carcinomas and have been related to

increase the invasive and metastatic tumour potential [7]. In

particular, Lewis X and related antigens, such as SLex, have

been found to be expressed in PaC tissues at higher rates

than inflamed pancreas (CP) while they were barely detected

in healthy pancreatic tissues [8–10]. PaC sera, but not sera

from healthy subjects, inhibit E-selectin binding of

pancreatic tumour cells [11], suggesting an overexpression

of the sialylated Lewis antigens SLex and SLea in the PaC

sera glycoproteins, which act as E-selectin ligands. Taken

together, these data suggest that serum-specific proteins

secreted by the tumour, which carry SLex or aberrant glycans

moieties, could be used as PaC tumour markers. In fact,

using glycoprotein microarrays with multi-lectin detection

techniques, an increase in both fucosylation and sialylation

of some serum glycoproteins has been described for PaC

patients compared with healthy controls and pancreatitis

patients [12, 13].

Pancreatic adenocarcinoma is a tumour with a high

inflammatory component [14] to which the liver responds by

producing APP. In addition, factors released by tumour

cells, such as cytokines, into the local microenvironment can

have tumour-promoting effects. These include favouring the

proliferation and survival of malignant cells, promoting

angiogenesis and metastasis [15] and modulating the cellu-

lar glycosylation machinery. Proinflammatory cytokines,

such as interleukin-1b (IL-1b), upregulate the biosynthesis

of SLex in hepatoma cells [16], and IL-6 and IL-8 increase the

levels of SLex and 6-sulfo-SLex on bronchial mucins from

cystic fibrosis patients [17]. Pancreatic adenocarcinoma cell

lines and tissues produce proinflammatory cytokines

[18, 19]. These released cytokines could alter the glycosyla-

tion of the tumour neighbouring cells and their secreted

glycoproteins. Given the close proximity of pancreas and

liver, the liver-secreted acute-phase-response glycoproteins

could also alter their glycosylation in pancreatic adeno-

carcinoma. Therefore, not only tumour-secreted glycans but

also tumour host-response glycans could provide useful

biomarkers.

Our aim was to identify glycosylation changes on the

major APP (AGP, HPT, AT, FET and TRF) produced by the

liver in the serum of PaC patients compared with healthy

controls and CP patients. For that we used two approaches.

One based on the use of specific antibodies against glycan

epitopes, as they are known to be highly more specific than

lectins, and if changes in their reactivity in the different

groups of patients could be detected, they would provide

more feasible approaches to develop PaC biomarkers. In

particular, we have used antibodies against SLex to highlight

proteins with altered expression of this cancer-related

epitope. As there are not available antibodies against all

possible N-glycan modifications in tumours, it is necessary

to determine the complete N-glycan structures of these

potential altered glycoproteins in PaC. N-glycan sequencing

is a well-established, direct strategy to evaluate glycan

alterations [20]. Thus, in our second approach, we have used

N-glycan sequencing, including HPLC, exoglycosidase

digestion analysis and MS, in order to identify other specific

glycosylation changes in different stages of PaC patients

compared with healthy controls and CP patients.

2 Materials and methods

2.1 Serum samples

Serum samples were obtained from five healthy controls

(three females and two males; age range 44–72), nine PaC

patients (six females and three males; age range 52–85, one

each in stages I, II, III and IVA and five in stage IVB) and

three CP patients (all males, age range 38–53) from the

Hospital Universitari Dr J. Trueta (Girona, Spain) following

the standard operating procedures of its Ethics Committee.

Patients were diagnosed by biopsy or image examination by

the Digestive and Pathology Units.

2.2 HSA and IgG depletion and concentration of

serum samples

Serum samples were subjected to HSA and IgG depletion

according to the procedure described by Wang et al. [21],

with modifications. Briefly, 600mL of protein G sepharose

fast flow (GE Healthcare, Uppsala, Sweden) was added to

Spin-X centrifuge tube (Costar, Corning, NY, USA) and

washed three times with MilliQ water. Polyclonal rabbit

anti-human albumin antibody (Dako, Glostrup, Denmark)

was added to the spin tube and incubated with protein G at

2 A. Sarrats et al. Proteomics Clin. Appl. 2010, 4, 1–17


room temperature (RT) for 30 min with rotation, then

washed three times with PBS for removal of unbound

proteins. Thirty microlitre of human serum was diluted 1:10

in PBS, sonicated for 5 min and then incubated in a spin

tube rotating for 30 min at RT. Afterwards, the spin tube

was spun down for 2 min and the flow through was collec-

ted. The spin tube was washed three times with PBS and the

flow-through fractions were pooled (final volume of

approximately 1.8 mL).

To concentrate and desalt the samples, they were

placed on a MicroSep 3K tube (Pall, Ann Arbor, MI, USA)

and centrifuged at 6000� g for 1 h. After that the MicroSep

tube was washed four times with 2 mL of MilliQ

water and centrifuged until the sample volume was

about 300 mL. Protein concentration was determined

by the Bradford protein assay using BSA as standard

(Quick Start Bradford protein assay, BioRad, Hercules, CA,

USA).

2.3 SLex immunodetection in depleted serum

samples

After HSA and IgG depletion and concentration of serum

samples, 40mg of total protein was electrophoresed on a

10% polyacrylamide gel and transferred onto a PVDF

membrane (Millipore, Bedford, MA, USA). The membrane

was then washed with 0.1% Tween in TBS (TBST, 10 mM

Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) for 15 min

and blocked with 1% BSA in TBST for 1 h at RT. After

blocking, it was washed with TBST for 5 min and incubated

with a mouse monoclonal anti-sialyl Lewis X antibody

(Clone KM93, Calbiochem, Darmstadt, Germany) diluted

1/50 in incubation buffer (0.5% BSA in TBST) for 2 h at RT.

The membrane was then washed three times for 5 min with

TBST and subsequently incubated with a goat polyclonal

horseradish peroxidase-conjugated anti-mouse IgG1IgM1

IgA antibody F(ab)2 fragments (Abcam, Cambridge, UK)

diluted 1/40 000 in incubation buffer for 1 h at RT. After

washing out the secondary antibody five times as described

above, the membrane was incubated for 5 min with the

horseradish peroxidase substrate solution West Dura (Pierce

Biotechnology, Rockford, IL, USA). Chemi luminescence

was visualized using the imaging system Fluorochem SP

(AlphaInnotech, San Leandro, CA, USA) under non-satur-

ating conditions.

2.4 2-DE

Two different 2-DE procedures were performed depending

on the type of strip (GE Healthcare) used in the first

dimension.

(i) For the identification of glycoproteins carrying

increased SLex, isoelectric focusing was performed on

immobiline dry strips pH 3–10, 7 cm. Strips were subjected

to active in-gel rehydration (12 h, 50 V, 201C) with 60 or

100 mg of depleted serum dissolved in 200 mL of rehydration

buffer (8 M urea, 0.5% Triton X-100, 65 mM DTT, 1%

pharmalyte 3–10 (GE Healthcare), traces of bromophenol

blue). After rehydration, a water-damp electrode wick was

placed between the strip and each of the electrodes. An extra

50 mM DTT wick was placed at 1 cm from the negative

electrode. Isoelectric focusing was performed as follows:

0–500 V in 30 min, 500 V for 1 h, from 500–1000 V for

30 min, 1000 V for 1 h, from 1000–2000 V for 1.5 h, 2000 V

for 1 h, 2000–4000 V for 1.5 h, 4000 V until 14 250 V h.

For the second dimension, the strips were first equili-

brated for 15 min in equilibration buffer (6 M urea, 30%

v/v glycerol, 2% SDS, 50 mM Tris pH 8.8) containing

65 mM DTT and afterwards for 15 min in equilibration

buffer containing 135 mM iodoacetamide. The strips were

then placed on top of 10% polyacrylamide gels and sealed

with a 0.5% agarose solution containing traces of bromo-

phenol blue in running buffer (192 mM glycine, 25 mM

Tris-HCl pH 8.5, 0.1% SDS). The second dimension was

performed in a Miniprotean III unit (BioRad) under the

following conditions: 10 mA per gel and an increase of

2.5 mA per gel every 15 min.

(ii) For isolation of the APP to perform N-glycan

sequencing, immobiline dry strips pH 3–7 non-linear, 24 cm

were used. Strips were subjected to in-gel rehydration with

10 mL of serum dissolved in 450mL of rehydration buffer 2

(8 M urea; 0.5% CHAPS; 0.2% pharmalyte 3–10; 0.2% DTT,

trace of bromophenol blue) overnight at RT. Isoelectric

focusing was performed in Ettan IPGphor II IEF system

(GE Healthcare) as follows: 3500–75 000 V h, 8000 V for

10 min, 8000 V for 1 h.

For the second dimension, the strips were first equili-

brated for 15 min in equilibration buffer (50 mM Tris-HCl

pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, traces of

bromophenol blue) containing 1% w/v DTT and afterwards

for 15 min in equilibration buffer containing 2.5% w/v

iodoacetamide. The strips were then placed on top of 10%

polyacrylamide gels and sealed with a 1% agarose solution

in running buffer. The second dimension was performed in

a Protean Plus Dodeca Cell (BioRad) under the following

conditions: 1 W per gel overnight at 151C.

Following electrophoresis, proteins separated in the 2-DE

gels were either transferred to a PVDF membrane and

subjected to SLex immunodetection (after using procedure i)

or were Coomassie blue stained (after using both the

procedures i and ii).

2.5 High throughput release and processing of

N-glycans from 2-DE gel spots

Selected 2-DE gel spots were excised from the gel, cut into

1 mm3 gel pieces, transferred to a filter plate (protein

precipitation plate, Whatman, Maidstone, Kent, UK) and

stored at �201C. N-glycans were released from 2-DE gel

Proteomics Clin. Appl. 2010, 4, 1–17 3


pieces and labelled according to the in-gel block method for

human serum described earlier [20], with modifications.

Briefly, gel pieces were treated with protein N-glycosidase F

to release the N-linked glycans. Half of the extracted

N-glycans were fluorescently labelled with 2-aminobenza-

mide (2-AB) by reductive amination (LudgerTag 2-AB

labelling kit, Ludger, Culham, Oxfordshire, UK). The excess

2-AB was removed in this case with Whatman 3MM chro-

matography paper. The 2-AB-labelled N-glycans were then

analyzed by normal-phase HPLC (NP-HPLC) and weak

anion exchange (WAX)-HPLC. The unlabelled N-glycans

were later analyzed by ESI-MS/MS and MALDI-TOF MS.

2.6 Simultaneous oligosaccharide sequencing by

exoglycosidase digestions

All enzymes were purchased from Prozyme (San Leandro,

CA, USA). The 2-AB-labelled glycans were digested in 10mL of

50 mM sodium acetate buffer, pH 5.5 for 18 h at 371C, (except

in the case of jack bean a-mannosidase where the buffer was

100 mM sodium acetate, 2 mM Zn21, pH 5.0) using arrays of

the following enzymes at the indicated concentrations:

Arthrobacter ureafaciens sialidase (EC 3.2.1.18), 1 U/mL;

Streptococcus pneumoniae sialidase (EC 3.2.1.18), 1 U/mL;

bovine testes b-galactosidase (EC 3.2.1.23), 1 U/mL;

S. pneumoniae b-galactosidase (EC 3.2.1.23), 0.1 U/mL; bovine

kidney a-fucosidase (EC 3.2.1.51), 1 U/mL; S. pneumoniae b-N-

acetylglucosaminidase, recombinant in Escherichia coli (EC

3.2.1.30), 8 U/mL; jack bean b-N-acetylglucosaminidase (EC

3.2.1.30), 10 U/mL; jack bean a-mannosidase (EC 3.2.1.24),

50 U/mL; almond meal a-fucosidase (EC 3.2.1.111), 3 mU/

mL; Xanthomonus sp. a-fucosidase (EC 3.2.1.51.), 0.1 U/mL.

After incubation, enzymes were removed by filtration through

a protein-binding EZ filters (Millipore) [22]. N-glycans were

then analyzed by NP-HPLC or WAX-HPLC.

2.7 N-glycan analysis by WAX-HPLC and NP-HPLC

WAX-HPLC was performed using a Vydac 301VHP575

7.5� 50-mm column (Anachem, Luton, Bedfordshire, UK)

on a 2695 Alliance separations module with a 474 fluores-

cence detector (Waters, Elstree Hertfordshire, UK). Solvent

A was 0.5 M formic acid adjusted to pH 9.0 with ammonium

solution, and solvent B was 10% v/v methanol in water.

Gradient conditions were as follows: a linear gradient of

0–5% A over 12 min at a flow rate of 1 mL/min, followed by

5–21% A over 13 min and then 21–50% A over 25 min,

80–100% A over 5 min, and then 5 min at 100% A. Samples

were injected in water. A FET N-glycan standard was used

for calibration [22].

All HPLC units were equipped with Waters temperature

control modules and Waters 2475 fluorescence detectors set

with excitation and emission wavelengths of 330 and

420 nm, respectively [20].

NP-HPLC was performed using a TSKgel amide-80 5-mm

(250� 4.6 mm) (Anachem) column on a Waters 2795 Alli-

ance HT separations module. Solvent A was 50 mM formic

acid adjusted to pH 4.4 with ammonia solution and solvent

B was ACN. The column temperature was set to 301C.

Conditions used were as follows: 60 min method – a linear

gradient of 35–47% solvent A over 48 min at a flow rate of

0.8 mL/min, followed by 1 min at 47–100% A and 4 min at

100% A, returning to 35% A over 1 min and then finishing

with 35% A for 6 min [20]. Samples were injected in 65%

ACN. The system was calibrated using an external standard

of hydrolyzed and 2-AB-labelled glucose oligomers to create

a dextran ladder, as described previously [22].

2.8 N-glycan analysis by MS

2.8.1 ESI-MS and MS/MS

One microlitre of each sample was cleaned by allowing it to

sit for approximately 1 h on the surface of a Nafion 117

membrane (Aldrich, Poole, Dorset, UK) [23] that was float-

ing on water. The samples were then transferred to a small

Eppendorf tube to which was added 6mL of a 1:1 (v/v)

mixture of water:methanol containing 0.2 M ammonium

phosphate and the entire solution was then infused through

Proxeon (Proxeon Biosystems, Odense, Denmark) borosili-

cate capillaries into a Waters (Waters MS Technologies,

Manchester, UK) tandem quadrupole TOF (Q-TOF) mass

spectrometer at a source temperature of 1201C for acquisi-

tion of electrospray (ESI) spectra. Both negative ion MS and

MS/MS (argon collisions) spectra of glycan ions were

recorded. Data acquisition and processing were conducted

with Waters MassLynx version 4.1. Interpretation of the

negative ion MS/MS spectra was according to published

work [24–27].

2.8.2 MALDI-MS

One microlitre of an aqueous solution, cleaned with the

Nafion membrane as above, was mixed on the stainless steel

MALDI target plate with 0.5 mL of a saturated solution of

2,5-DHB in ACN and allowed to dry under ambient condi-

tions. The sample spot was then recrystallized from ethanol.

Spectra were acquired with a Waters Micro MX MALDI-TOF

mass spectrometer in reflectron mode with an acceleration

voltage of 12 kV. Data acquisition and processing were

conducted with Waters MassLynx version 4.1.

2.9 Identification of the proteins in 2-DE spots by

MS analysis

Proteins contained in the 2-DE spots were in-gel digested

with trypsin (sequencing grade modified, Promega Biotech



Iberica, Madrid, Spain) in the automatic investigator

ProGest robot from Genomic Solutions. Briefly, excised gel

spots were washed with ammonium bicarbonate buffer

(50 mM NH4HCO3) and ACN. Proteins were reduced with

10 mM DTT solution during 30 min and alkylated

with a 55 mM solution of iodoacetamide. After washing with

buffer and ACN, proteins were digested overnight,

at 371C with 0.27 nmol of trypsin. Tryptic peptides were

extracted from the gel matrix with 10% formic acid and

ACN; the extracts were pooled and dried in a vacuum

centrifuge. Proteins were either analyzed in a MALDI-TOF-

TOF (4700 Proteomics Analyzer, Applied Biosystems, IL,

USA) or LC-ESI-Q-TOF (Q-TOF Global, Waters) mass

spectrometer.

For MALDI-MS and MS/MS analyses, the digests

were redissolved in 5mL of 0.1% TFA in 50% ACN.

Typically, a 0.5–1 mL of sample was mixed with the same

volume of a matrix solution (5 mg/mL CHCA; Waters)

in 0.1% TFA in 50% ACN and spotted to the MALDI

plate. MS spectra were acquired in positive reflector mode

(voltage of 20 kV in the source and a laser intensity that

ranged from 5800 to 6200). Typically, 500 shots perspectrum were accumulated. MS/MS spectra were

acquired using collision-induced dissociation with atmo-

spheric air as the collision gas. An MS/MS 1 kV positive

fragmentation mode was used. MS and MS/MS spectra

from the same spot were merged in a single MASCOT

(Matrix Science, London, UK) generic file prior to submis-

sion for database searching.

For on-line LC-ESI-MS/MS analysis (Cap-LC-nano-ESI-

Q-TOF) (CapLC, Waters), the tryptic digested peptide

samples were resuspended in 25mL of 1% formic acid

solution and 4 mL was injected to a reversed-phase capillary

C18 column (75 mm internal diameter and 15 cm length,

PepMap column, LC Packings (Dionex Biogen, Madrid,

Spain)). The eluted peptides were ionized via coated nano-

ES needles (PicoTipTM, New Objective, Woburn, MA, USA).

A capillary voltage of 2000–2800 V was applied together with

a cone voltage of 80 V. The collision energy for collision-

induced dissociation was 20–35 eV, and argon was employed

as the collision gas. Data were generated in PKL file format

and submitted for database searching in the MASCOT

server against a non-redundant Swiss-Prot database. The

search parameters were: human taxonomy, two missed

cleavages allowed, carbamidomethyl of cysteine as a fixed

modification and oxidation of methionine as a variable

modification. The peptide tolerance was 100 ppm and

0.25 Da for MS and MS/MS spectra, respectively. The

significance threshold was set at po0.05.

For the MASCOT generic file searches, only proteins

with scores above significant MASCOT level (456) were

considered as positive hits and protein summary was

selected as the report format. For the PKL files, only

peptides with scores above the significant MASCOT level

were considered for protein identification (428) and peptide

summary was selected as the report format.

3 Results

3.1 HSA and IgG serum depletion yields

Depletion of HSA and IgG from serum samples reduced the

quantity of serum proteins by about 70%. Coomassie

staining of SDS-PAGE gels from the retained and flow-

through fractions showed that only albumin and IgG were

removed (data not shown). To check that other minor serum

proteins were not extracted along with albumin and

removed from the sera, ELISA against ribonuclease 1, which

is one minor serum protein, was performed with the serum

and the corresponding serum-depleted fraction. The recov-

ery yield for ribonuclease 1 was approximately 80%, giving

some indication of the level of the losses of protein asso-

ciated with the depletion method. After removal of albumin

and IgG, depleted serum samples contained mainly serum

liver proteins including APP and other minor proteins.

3.2 SLex immunodetection in depleted serum

samples

Forty microgram of depleted serum samples from healthy

controls (HC 1–3), PaC (PaC 1–9) and CP (CP 1–3) patients

were electrophoresed, transferred onto a PVDF membrane

and subjected to SLex immunodetection. An immuno-

reactive band of approximately 50 kDa was observed for

stage IV PaC patients (PaC 1–5 and 8). Stages I, II and III

PaC patients (PaC 6, 7 and 9) showed no SLex immunode-

tection. Although this band was not detected in any of the

healthy controls analyzed, it did appear in some of the CP

patients CP 1 and 2 (two of three) (Fig. 1A). CP patients also

showed upper bands of more than 100 kDa that were posi-

tively stained for SLex, which were not further analyzed. We

focused the study on the 50 kDa band, as it was increased in

all advanced PaC patients.

In order to identify the glycoproteins carrying the

increased SLex epitope in the 50 kDa band, 60mg of depleted

PaC 8 sample was subjected to 2-DE followed by SLex

immunodetection. At approximately 50 kDa, a single SLex

immunoreactive spot was detected and showed a pI of 3.8

(Fig. 1B). A preparative 2-DE gel was run under the same

conditions but with 100 mg of depleted PaC 8 sample to

isolate and identify the proteins of the SLex immunoreactive

spot. For that the spot was excised from the

gel and subjected to the analysis of the peptide mass

fingerprints. AGP1 (score: 102, sequence coverage: 26%,

number of peptides: 9) and AGP2 (score: 119, sequence

coverage: 25%, number of peptides: 10) were the only

significant hits. AGP1 and AGP2 are two variants of the

same protein, AGP, that comigrate in the 2-DE due to their

high homology [28, 29]. Thus, AGP, one of the major

members of the positive APP [30], is the main protein

contributing to the increased SLex detection in the

approximately 50 kDa band.



3.3 N-glycan analysis of APP

The increased SLex found in AGP of stage IV PaC serum

compared with healthy controls suggested that other APP may

show an altered N-glycan moiety in tumour situations;

therefore, we analyzed them in detail by N-glycan sequencing.

2-DE was used to isolate different APP including AGP,

HPT, FET and AT (Fig. 2) from three healthy controls (HC

2, HC 4 and HC 5), four PaC patients from different stages

(PaC 9 (stage II), PaC 7 (stage III), PaC 5 (stage IVa), PaC 8

(stage IVb)) and two CP patients (CP 1 and CP 2) that had

been found SLex positive for AGP. These protein spots were

excised from the gel and screened for possible altered

glycosylation by N-glycan analysis. After N-glycan release,

the glycoproteins contained in each spot were identified

using mass spectrometric analysis, as AGP (spot 1), FET

(spots 2–4), AT (spots 5–7), HPT (spots 8–13) and TRF (spot

14) (Table 1). LC-ESI-Q-TOF analysis of spot 14 indicated

that apart from TRF (score 642, 18 peptides matched), Ig mu

chain (score 73, only two peptides matched) could also be

present. Taking into account that Ig mu chain was not

detected by MALDI-TOF-TOF, altogether indicates that this

glycoprotein could be present in this spot, but in much

minor proportion than TRF.

N-glycans from each of the sample spots were identified

using quantitative NP-HPLC combined with exoglycosidase

digestions and the structural assignments were checked

using Glycobase database matching (http://glycobase.nibrt.

ie/cgi-bin/public/glycobase.cgi). MALDI-TOF and negative

ion ESI-MS N-glycan analyses were also performed to

corroborate the N-glycan structures contained in each spot.

In addition, N-glycans from AGP from a pool of the patients

were subjected to WAX fractionation, prior to NP-HPLC, in

order to assign the number of sialic acids in each peak. NP-

HPLC of the N-glycan profiles of a representative spot of

each APP is shown in Fig. 3. N-glycans identified in each

protein spot are shown in Table 2. The relative percentages

of the N-glycan structures for each individual protein spot

were calculated in order to measure glycosylation changes

between the different groups of patients.

The N-glycans assigned in the AGP, HPT, AT and FET

spots (spots 1 to 13) were all complex type, and presented bi-,

tri- and tetra-antennary structures. TRF (spot 14) contained

only bi- and tri-antennary glycans together with bisected

Figure 1. Sialyl Lewis X immunodetection on HSA-IgG-depleted sera of healthy controls, PaC and CP patients. (A) 40 mg of total protein

was electrophoresed on a 10% polyacrylamide gel and transferred onto a PVDF to perform SLex immunodetection. (B) 2-DE of 100 mg of

total protein of sample PaC8. At approximately 50 kDa, a SLex immunoreactive spot (pI of 3.8) was detected and identified to be AGP.

Figure 2. Example of a 2-DE gel of an unde-

pleted serum (HC 2). Different protein spots

corresponding to AGP, HPT, AT, FET and TRF

were isolated by using 24 cm, pH 3–7 non-

linear immobiline dry strips and 10% poly-

acrylamide gels, followed by Coomassie

staining.



structures, which were not detected in the other APP. In

addition, TRF had high mannose type N-glycans in a

percentage that ranged from 3 to 11%, depending on the

sample. High-mannose-type structures have not been descri-

bed before for TRF. As spot 14 could include Ig mu chain,

which has been reported to contain only high mannose

structures [31], we cannot discard the possibility that these

N-glycan structures originated from the Ig mu chain. The rest

of the N-glycan structures of spot 14 can be attributed solely to

TRF. The proportion of bi-, tri-, tetra-antennary, bisected and

high-mannose structures were calculated for each APP spot.

In order to estimate these changes for HPT, AT and FET,

which are separated in several spots, the mean of the different

spots of the same protein was calculated (Fig. 4A). Several

N-glycans were outer-arm fucosylated and only one minor

structure, a disialylated biantennary glycan F(6)A2G(4)2S2 in

peak 7, present in all APP was core fucosylated. TRF

contained in addition other three core fucosylated structures

F(6)A2G(4)2S(6)1 in peak 4, F(6)A2BG(4)2S1 in peak 5 and

F(6)A2BG(4)2S2 in peak 7. No N-acetylgalactosamine was

detected. All galactoses were b-1-4-linked to N-acetyl-

glucosamine (the same HPLC profile was obtained after

digestion with bovine testes b-galactosidase, which hydrolyses

non-reducing b-1-3- and b-1-4-linked terminal galactose,

and with S. pneumoniae b-galactosidase, which hydrolyses

non-reducing b-1-4-linked terminal galactose only). All

outer-arm fucoses were determined to be a-1-3 linked,

because they were digested by almond meal a-fucosidase

(which releases a-1-3 and a-1-4 non-reducing terminal

fucose) but not by Xanthomonus sp. a-fucosidase (which

releases a-1-2 non-reducing terminal fucose). Substitution

at position 4 was discarded because it was occupied by

galactose. The summation of the relative peak areas contain-

ing sialylated structures with outer-arm fucose linked to

N-acetylglucosamine, which is simultaneously attached to

galactose (peaks 10, 13, 14 and 17–20), was taken as a measure

of the SLex antigen and was calculated for each spot. Although

peak 13 contained a mixture of two structures with and

without a-1-3 outer-arm fucosylation in AGP and HPT, the

major one was always the outer-arm fucosylated tri-antennary

glycan, A3F(3)1G(4)3S3 which was in a proportion of about

70% in AGP and 80% in HPT. In the rest of APP analyzed,

FET, AT and TRF, peak 13 contained only the

A3F(3)1G(4)3S3 structure.

3.4 Altered glycosylation of AGP

The HPLC profiles of the N-glycans obtained from spot 1

(AGP) from controls, PaC and CP patient sera are shown in

Fig. 3A. Most of the altered peaks were gradually increased or

decreased compared with healthy controls according the

following order: stages II–III PaC, stage IV PaC and CP.

Thus, stages II–III PaC profiles were more similar to controls,

while stage IV PaC and CP showed more marked differences

compared with controls. Peaks 10, 13, 14 and 17–20 were

increased in PCa stage IV and pancreatitis and contained tri-

or tetra-antennary structures with a-1-3 outer-arm fucosy-

lation. The rest of the peaks (usually the non-fucosylated form

of the increased ones) were either decreased or not altered.

Taking into account that peak 13 of AGP contains a mixture of

tri-antennary A3F(3)1G(4)3S3 (13a) and tetra-antennary

A4G(4)4S3 (13b) in a proportion of about 70:30, the percen-

tage of a-1-3 fucosylation (SLex) was calculated first by

Table 1. Identification of the proteins in 2-DE spots by MS analysis

Spot Equipment Identificationa) Accessionno.

Proteinscoreb)

Sequencecoverage (%)

Peptidesmatched

1 MALDI-TOF-TOF AGP1 P02763 201 42 71 MALDI-TOF-TOF AGP2 P19652 78 34 72 LC-ESI-Q-TOF a-2-HS-glycoprotein/fetuin P02765 110 8 33 LC-ESI-Q-TOF a-2-HS-glycoprotein/fetuin P02765 99 13 34 LC-ESI-Q-TOF a-2-HS-glycoprotein/fetuin P02765 67 2 15 MALDI-TOF-TOF a-1-antitrypsin P01009 525 66 266 MALDI-TOF-TOF a-1-antitrypsin P01009 722 70 317 MALDI-TOF-TOF a-1-antitrypsin P01009 771 72 318 MALDI-TOF-TOF Haptoglobin P00738 185 22 79 MALDI-TOF-TOF Haptoglobin P00738 237 46 13

10 MALDI-TOF-TOF Haptoglobin P00738 250 33 911 MALDI-TOF-TOF Haptoglobin P00738 230 43 1312 MALDI-TOF-TOF Haptoglobin P00738 230 43 1313 MALDI-TOF-TOF Haptoglobin P00738 287 33 914 MALDI-TOF-TOF Transferrin P02787 547 40 2314 LC-ESI-Q-TOF Transferrin P02787 642 34 1814 LC-ESI-Q-TOF Ig mu chain C region P01871 73 5 2

a) Only glycoproteins identified are listed (unglycosylated proteins are not listed).b) Only proteins with a significant MASCOT score (456) are listed for the MALDI analysis. For the ESI analysis, all the proteins identified

with significant peptides scores (428) are listed.



Figure 3. NP-HPLC chromatograms of N-glycans released from (A) AGP spot 1, (B) HPT spot 8, (C) AT spot 5, (D) FET spot 2 and (E) TRF

spot 14 from sera of healthy controls, PaC and CP patients.



Table 2. N-glycan structures identified on the APP

Peaka) GUb) Structurec) Spot 1(AGP)

Spots 2–4(FET)

Spots 5–7(AT)

Spots 8–13(HPT)

Spot 14(TRF)

1 6.12 M5 �d)

2 6.94 M6 �d)

3 7.86 3a A2G(4)2S1 � ��

3b M7 �d)

4 8.43 4a A2G(4)2S(3)2 � ��

4b F(6)A2G(4)2S(6)1 �

5 8.67 5a F(6)A2BG(4)2S1 �

5b M8 (�)d)

6 8.93 6a A2G(4)2S(6)2 � ��

6b A2G(4)2S(3,6)2 � � � �

7 9.40 7a F(6)A2G(4)2S2 � ��

7b F(6)A2BG(4)2S2 �

8 9.69 8a A3G(4)3S(3)2 � � �

8b A3G(4)3S(3,6)2 � �

9 10.16 A3G(4)3S(6)2 � �

10 10.30 A3F(3)1G(4)3S2 � � �

11 10.56 A3G(4)3S3 � ��

12 10.93 A4G(4)4S2 � �

13 11.11 13aA3F(3)1G(4)3S3

� �70% �� 80% �



adding the relative peak areas of A3F(3)1G(4)1, A4 F(3)1G(4)1,

A4F(3)2G(4)2 and A4F(3)3G(4)3 generated after A. ureafacienssialidase 1 bovine testes b-galactosidase digestion of each of

the patients’ AGP N-glycans. Then, it was compared with that

from the summation of the undigested peaks 10, 13, 14 and

17–20 relative areas. The change in the proportion of SLex

Table 2. Continued.

Peaka) GUb) Structurec) Spot 1(AGP)

Spots 2–4(FET)

Spots 5–7(AT)

Spots 8–13(HPT)

Spot 14(TRF)

13bA4G(4)4S(3,?,?)3

� �30% � �20%

14 11.39 A4F(3)1G(4)4S2 � ��

15 11.74 15a A4G(4)4S(6)3 �

15b A4G(4)4S(3,?,?,?)4

� �

15cA3F(3)2G(4)3S3

�

16 11.83 A4G(4)4S(6)4 �

17 12.00 17aA4F(3)1G(4)4S3

� � �

17b A4F(3)1G(4)4S(3,?,?,?)4

� � �

17c A4F(3)2G(4)4S2

�

18 12.37 18aA4F(3)1G4S(6)4

� � �

18b A4F(3)2G4S3 �

19 12.75 A4F(3)2G4S4 � � �

20 ? A4F(3)3G4S4 � �

a) Only peaks marked with a dot were detected on each protein.b) GU are mean values of the N-glycan peaks from all the glycoproteins analyzed.c) The N-glycan structures were assigned to each peak using quantitative HPLC, exoglycosidase digestion and Glycobase (http://

glycobase.nibrt.ie/cgi-bin/public/glycobase.cgi) combined with MALDI-TOF and ESI-MS. Structures not confirmed by MS are markedwith an asterisk. Fetuin samples failed to give any spectra neither by ESI nor MALDI-TOF.

d) High mannose structures could not be unambiguously assigned to transferrin. Minor structures are in brackets.



between the groups of patients was practically the same using

the two methods, indicating that the structure 13b hardly

influences the differences between the groups. Thus, in order

to simplify the comparison with the results of the other APP,

only the data of the undigested peaks are shown. The

percentage of outer-arm fucosylation (SLex) was clearly

increased in stage IV PaC (69.370.6) and CP (72.870.8)

groups compared with controls (45.773.7), whereas the

stages II–III PaC group showed a lower increase in outer-arm

fucosylation (54.978.9) (Fig. 4B). Branching was also

increased in stage IV PaC and CP compared with controls.

Thus, the percentage of tetra-antennary structures was

increased in both stage IV PaC (40.874.0) and CP (42.770.8)

compared with controls (29.871.7). Interestingly, stage IV

PaC showed a decrease of tri-antennary structures compared

with controls (43.670.1 versus 52.970.7), whereas the CP

group showed a decrease in biantennary glycans compared

with controls (6.370.1 versus 17.372.2) (Fig. 4A).

Peak 7 was the only peak containing core fucosylated

structures (F(6)A2G(4)2S2). In spite of its low relative

abundance, it was specifically increased in stage IV PCa

(about 50%) compared with the other groups (Fig. 4C).

3.5 Altered glycosylation of HPT

In all HPT spots (8–13), there was a gradual increase in the

percentage of tri- and tetra-antennary structures with a-1-3

outer-arm fucosylation (peaks 10, 13 and 14), compared with

controls according the following order: stages II–III PaC,

stage IV PaC and CP. However, these differences were more

easily observed in spots 8–11, as spots 12 and 13 contain

mainly biantennary structures, both mono- or disialylated

and hardly any tri- or tetra-antennary structures. Generally

speaking, the higher the isoelectric point of the protein spot,

the lower the sialic acid content of the glycoprotein. In HPT

spots, the most acidic spot (8) contained the lowest

proportion of monosialylated structures and the highest

proportion of tri- and tetra-sialylated structures, whereas the

least acid spot (13) contained the highest proportion of

monosialylated structures. N-glycan profiles of spot 8 are

shown as an example of the changes observed (Fig. 3B). The

percentage of outer-arm fucosylation (SLex) was increased in

the CP group (31.370.4) compared with controls

(22.072.1); however, it was just slightly increased in the

stage IV PaC group (25.371.3) and not increased for the

stages I–II PaC group (Fig. 4B). The changes in branching

showed the same behaviour as for spot 1 (AGP): the

percentage of tetra-antennary structures was increased in

both stage IV PaC and CP compared with controls (9.370.4

and 11.970.1 versus 8.572.0), and tri-antennary structures

were decreased in stage IV PaC (26.370.6 versus 28.872.4),

while biantennary ones where decreased in CP (55.270.4

versus 62.772.3) (Fig. 4A). Regarding core fucosylation, the

low abundant peak 7 (F(6)A2G(4)2S2) was again increased

in the stage IV PCa group compared with the rest of groups;

however, the differences were not as pronounced as in AGP

(Fig. 4C).

3.6 Altered glycosylation of AT

In all AT spots, there was also a gradual increase in the size

of peaks 13 and 14 (tri- and tetra-antennary structures with

outer-arm fucosylation) compared with controls as follows:

stages II–III PaC, stage IV PaC and CP. However, these

differences were more easily observed in spots 5 and 6. The

N-glycan profiles of spot 5 are shown as an example of the

changes observed (Fig. 3C). The percentage of a-13 outer-

arm fucosylation (SLex) was slightly increased in the CP

(21.870.0) compared with controls (17.171.9) and it was

not increased for any of the PaC groups (Fig. 4A). There

were no evident changes of branching (Fig. 4A). Regarding

core fucosylation, the low abundant peak 7 (F(6)A2G(4)2S2)

was again not only increased in the stage IV PCa group

(10.872.6), but also in the CP group (10.070.1) compared

with controls (5.670.6) (Fig. 4C).

3.7 Altered glycosylation of FET

In all FET spots, a gradual increase in the size of

peaks 13 and 14 (tri- and tetra-antennary structures with

outer-arm fucosylation) was detected compared with

controls as follows: stages II–III PaC, stage IV PaC and CP.

However, these differences were more easily observed in

spots 2 and 3. The N-glycan profiles of spot 2 are shown as

an example of the changes observed (Fig. 3D). The

percentage of a-1-3 outer-arm fucosylation (SLex) was

increased in the CP group around 2.5-fold compared with

controls (32.278.5 versus 12.473.6) and it was not increased

for any of the PaC groups. The percentage of tri- and tetra-

antennary structures was increased in CP compared with

controls (21.172.6 versus 13.573.9 and 15.377.0 versus4.87.0.9), while biantennary structures were decreased in

CP (63.679.6 versus 81.774.7) (Fig. 4B). There

were no significant changes regarding core fucosylation

although an increased tendency in stage IV PaC was

observed (Fig. 4C).

3.8 Altered glycosylation of TRF

In TRF, only one peak (13) was found to contain a-1-3-

fucosylated structures; thus, its relative percentage was

taken as a measure of outer-arm fucosylation (SLex) and

gradually increased compared with controls as follows:

stages II–III PaC, stage IV PaC and CP (Fig. 4B). Following

the same gradation, the percentage of high mannose

structures, bisected structures and tri-antennary structures

gradually increased concomitant with a decrease in bi-

antennary structures (Fig. 4A). Interestingly, and in a



similar way to the other APP studied, peak 7 showed an

increase in stage IV PaC compared with the rest of the

groups, although, in this case, when comparing with CP,

this increase was very small. When considering all peaks

containing core-fucosylated structures (peaks 4, 5 and 7), CP

was the group that showed the highest increase compared

with controls (Fig. 4C).

4 Discussion

4.1 Altered glycosylation of APP in PaC and

pancreatitis

APP are plasma proteins secreted mostly by the liver and

their plasma concentration increases (positive APP) or

decreases (negative APP) by at least 25% in response to

inflammation [5]. Most of these proteins are glycosylated

and changes in their glycosylation pattern can be associated

with disease [28, 32, 33]. Positive APP fulfil a variety

of functions but principally are thought to help in the

body response to infection and may also have a role in

healing and repair [5, 34]. In our study, we have focused

on the possible changes in the N-glycans of some of the

major APP AGP, HPT, FET, AT and TRF in the serum

of PaC patients. As controls, we have chosen serum

from healthy people and also from CP patients that have a

chronic inflammatory status, as changes of APP N-glycans

have been described in inflammation [32]. Our results

have shown several N-glycan modifications in stage IV

PaC and in CP on the different APP analyzed. The main

changes included increased a-1-3 fucosylation (SLex) and

Figure 4. Representation of

branching (A), a-1-3 fucosyla-

tion (B) and core fucosylation

(C) changes on AGP, HPT, AT,

FET and TRF in healthy

controls, stages II–III PaC, stage

IV PaC and CP patients. �High

mannose structures could not

be unambiguously assigned to

TRF.



branching in both stage IV PaC and CP and an increase of

core fucosylation in stage IV PaC on most of the APP

analyzed.

4.2 Changes in N-glycan branching

An increase in the amount of tetra-antennary structures on

AGP and HPT was observed in CP and in stage IV PaC. A

concomitant decrease in tri-antennary structures was

observed in stage IV PaC patients, while in CP patients, the

percentage of biantennary glycans was decreased. FET

showed the same changes in CP patients as AGP and HPT

but more significantly, and in addition, an increase of tri-

antennary structures was also observed.

TRF showed a different repertoire of N-glycan structures

than the other studied APP; apart from the main disialylated

biantennary and minor tri-antennary N-glycans, it contained

no tetra-antennary structures, but possessed bisected

glycans, in agreement with other data previously reported

[35–38]. In our work, we also found minor high mannose

structures in each of the samples analyzed (from 3 to 11%);

these probably originate from the Ig mu chain protein that

appeared to contaminate the TRF sample. Interestingly, a

change in branching pattern was also detected, namely a

decrease in biantennary structures in both stage IV PaC and

CP patients concomitant with an increase in bisected and

tri-antennary glycans.

Substantial increases in AGP glycoforms expressing

biantennary glycans are apparent in the early phase of an

acute-phase reaction. However, these are decreased

compared with control levels after the second day following

surgical trauma [39]. There is no agreement regarding

branching changes observed in chronic inflammation.

Some publications described an increase of biantennary

glycans on AGP [40]. Others reported that AGP concanava-

lin A (ConA) reactivity showed a transition from initially

elevated to decreased levels as disease became chronic,

which indicates an increase in branching in chronic

inflammation [41], consistent with the increased branching

we observe in CP.

HPT has previously been described as having an

increased branching in ovarian cancer compared with

controls [42] and in prostate cancer compared with benign

prostatic hyperplasia [43]. Increase in TRF branching has

been reported for HCC compared with healthy controls [38]

and for chronic inflammation, in particular in different

types of rheumatoid arthritis [44].

4.3 Changes in SLex expression

AGP showed an increased SLex content in pancreatitis and

stage IV PaC patients compared with controls by two

different approaches, Western blot and N-glycan sequen-

cing. This APP has been described to carry an increased

a-1-3 fucosylation in patients with acute inflammation

[39, 40] and chronic inflammation such as rheumatoid

arthritis and diabetes mellitus [40, 45, 46]. An increase in

SLex expression on AGP has also been reported in both

acute and chronic inflammations [47].

a-1-3 fucosylation on AGP was proposed as a marker of

progression and prognosis in different types of malig-

nancies in a study with 214 patients [48]. In both advanced

ovarian and breast cancers an increase of SLex in serum

AGP have been described and indeed proposed as a

marker of disease progression in the case of breast cancer

[49, 50].

The rest of APP, such as HPT, FET, AT and TRF, showed

an increased a-1-3 fucosylation (SLex) for the CP patients

compared with controls, which was much more pronounced

on FET. Taking into account that the CP group showed also

other immunoreactive bands for SLex detected by Western

blot, altogether these data suggest that this sialylated anti-

gen is closely linked to an inflammatory condition of the

pancreas.

In the case of HPT, stage IV PaC patients also showed an

increased SLex compared with controls. Increases in fuco-

sylated HPT have been observed in both acute (severe

trauma) and chronic inflammations (rheumatic arthritis and

inflammatory bowel disease) [42, 47] and in different types

of cancers including advanced ovarian cancer [49, 51],

mammary carcinoma [52], prostate cancer [43] and lung

cancer [53]. An increase of total fucosylation on HPT has

been described in PaC [50] and, in agreement with our

results, the incidence of positive HPT fucosylation (Aleuriaaurantia lectin affinity) increased in advanced stages [54]. In

another work [55], the authors described that SLex-type

fucosylation on HPT also increased in pancreatitis patients

but to a lower extent than in PaC patients. On the contrary,

we have observed a higher HPT SLex-type fucosylation in

pancreatitis than in PaC patients. Interestingly, we observed

a significant increase in the a-1-3 fucosylation of FET (2.5-

fold) in the pancreatitis patients that should be further

investigated with more serum samples to define a possible

clinical implications.

Although an increased fucosylation on AT has been

described in other types of cancer (breast and ovarian) [56],

we did not observe an increase in PaC patients and only

pancreatitis patients showed a slight increase of a-1-3

fucosylation on AT. The increase in a-1-3 fucosylation of

TRF has been detected in the tri-antennary structure present

in peak 13. a-1-3 fucosylation increase has also been

reported in the tri-antennary N-glycans of TRF for HCC

patients using lectin and Bio-Gel P4 chromatography [38].

4.4 Changes in core fucosylation

While our results indicate that increase in branching and

a-1-3 fucosylation on APP is a common feature of both

advanced PaC and pancreatitis, increase of the minor-core-



fucosylated biantennary structure F(6)A2G(4)2S2 seems to

be a more cancer-specific modification. Although the

percentage of this glycan is increased in all the APP, it is

only significantly augmented at advanced stages of PaC

patients on AGP and HPT.

AGP and HPT showed an increased core fucosylation in

lung cancer patients compared with healthy controls [57]. In

HCC, an increase of core fucosylation in AGP, AT, FET and

TRF has been described compared with controls and other

chronic liver diseases [38, 36, 58, 59]. An increase of core

fucosylation on HPT has also been reported in HCC

compared with chronic liver disease [60], although another

study showed an opposite tendency when comparing HPT

from HCC and control serum [58]. The increase in core

fucosylation for most APP described in HCC has been usually

detected using specific lectins, namely Lens culinaris aggluti-

nin. Increase of core-fucosylated HPT has been described in

PaC patients (Aspergillus oryzae lectin affinity) compared with

controls [54] in agreement with our results. In our work, we

have performed detailed N-glycan sequencing, which can

discriminate between core and outer-arm fucosylation and

gives more precise information than lectin-binding studies. In

addition, we have also included the pancreatitis patients and

have shown that increased core fucosylation is a cancer-

specific modification of AGP and HPT. Other glycoproteins

show an increased core fucosylation in cancer. Core fucosy-

lated a-fetoprotein, called AFP-L3, is very specifically increased

in HCC and was approved as a tumour marker by the food

and drug administration in 2005 [61]. Serum ribonuclease 1

was also much more core fucosylated in PaC sera than in

control patients [62] which suggests that quantification of core

fucosylation of some PaC serum glycoproteins might be

useful for diagnostic or prognosis purposes.

4.5 Regulation of glycosylation in APP

Cytokines involved in the induction of the inflammatory

reaction have been described to regulate both APP synthesis

and glycosylation [5]. Their effect has been studied in vitro

using hepatoma cell lines. Stimulation of the hepatic carci-

noma cells HUH-7 with proinflammatory cytokines such as

IL-1b and/or IL-6 for 2 days increased AGP production and

ConA reactivity (biantennary glycoforms). However, there

was a decrease on AGP ConA reactivity after 5 days of

stimulation, probably linked to a decrease of biantennary

structures (increase of branching) [63], consistently with the

present study where an increase of branching is observed in

chronic inflammation (pancreatitis). However, the

mechanism by which these cytokines cause the branching

modifications of APP has not been studied. Increases in

tetra-antennary structures have been related to an upregu-

lation of N-acetylglucosaminyltransferase V (GnT-V), the

enzyme responsible for the b-1-6 branching [64]. Thus,

this enzyme may be overexpressed in liver cells of both

pancreatitis and stage IV PaC patients, as both illnesses

show an increase of tetra-antennary structures in AGP and

HPT. In stage IV PaC patients, a concomitant decrease in

tri-antennary structures was observed while in pancreatitis

biantennary glycans were decreased. These results suggest

that N-acetylglucosaminyltransferase IV (GnT-IV), the

enzyme responsible for the addition of the b-1-4 branch-

ing (third antennae), may be upregulated in CP.

Stimulation of HUH-7 cells with IL-1b also increased

SLex on secreted AGP by enhancing the expression of the

b-galactoside a-2,3-sialyltransferase IV (ST3 Gal IV) and

fucosyltransferase VI (FUT VI) [16, 63]. Stimulation of

hepatoma cells with IL-6 increase both HPT synthesis and

fucosylation, which was linked to an enhanced expression of

fucosylation-related genes such as FUT6 and FUT8, GDP-

fucose synthase and GDP-mannose-4,6-dehydratase [65].

Cytokines might play a similar role in vivo and modify APP

glycosylation. The most important sources of cytokines are

macrophages and monocytes at the inflammatory sites [5]. In a

chronic inflammatory condition such as CP, these cytokines

would be produced in the damaged pancreas and produce

various systemic effects including liver stimulation to increase

APP synthesis and specific glycosyltransferases responsible for

the increase of APP branching and SLex-type fucosylation. A

similar mechanism may take place in cancer, as tumours and

Clinical Relevance

PaC lacks any specific and sensitive tumour marker.

As other carcinomas, it is associated with changes in

cellular glycosylation and release of inflammatory

cytokines. Serum APP, which are secreted by the

liver, may modify their glycosylation in tumours and

could be potential sensors of this disease. The

glycosylation analysis of major APP in PaC, CP and

control patients has shown that some N-glycan

changes were associated to an inflammatory

condition, as they were detected in advanced stages

of PaC and in CP, while others (core fucosylation)

could only be detected in advanced PaC patients and

therefore could be regarded as cancer specific. The

evaluation of a larger cohort of patients is required

to define the potential clinical utility of increased

core fucosylation of AGP and HPT as PaC markers.

However, these results highlight the importance of

determining the glycosylation changes of serum

proteins, such as APP, as this post-translational

modification is modulated in pathophysiological

conditions and therefore it may be useful as a

disease marker.



tumour microenvironments are a rich source of cytokines [15].

This fact has indeed been observed in PaC tissues and cell lines

[18, 19]. In addition, it was described that IL-6 from the

conditioned media of the PSN-1 PaC cells increase production

of fucosylated HPT from HepG2 hepatoma cells [65].

Different serum cytokines are increased in PaC patient

serum compared with healthy controls, although most of

them are increased even more in CP [66]. This would explain

why increase of SLex and branching on APP is more evident

in pancreatitis and in stage IV PaC. Stages II–III PaC show

very little APP glycan alterations, probably due to a lower

serum level of cytokines. However, the presence of other

cancer-related regulatory factors of APP glycosylation cannot

be ruled out. In this regard, the increase in core fucosylation

seems to be a more specific cancer glycan alteration than SLex-

type fucosylation or branching. FUT8 is the only known FUT

involved in the addition of core fucose [61]. Thus, upregulation

of this enzyme, which expression is very low in normal liver

[67], might take place in the liver of PaC patients from

advanced stages but not in pancreatitis patients. Other

mechanisms such as the enhancement in the levels of GDP-

fucose synthase or GDP-mannose-4,6-dehydratase proteins,

which both contribute to the synthesis of GDP-fucose, or even

in the levels of GDP-fucose transporter, could also occur and

would contribute to the increase of the core-fucosylated glycan

structures on AGP and HPT in the liver of PCa patients.

4.6 Biological function and diagnostic significance

of APP-altered glycosylation

It is not clear which biological functions are affected by the

altered glycosylation of APP in cancer and in inflammation.

It has been proposed that the increase in SLex-substituted

glycans on AGP might represent a mechanism for feedback

inhibition of leukocyte extravasation into inflamed tissues.

AGP expressing SLex may interact with E-selectin expressed

at the surface of endothelial cells and compete with leuko-

cytes also expressing SLex [30]. Regarding the diagnostic

significance of the APP glycan alterations, increases of

branching and SLex on APP might reflect the inflammatory

status of the patient, and may only be used as markers of

progression and prognosis [48, 50]. However, core fucosy-

lation of APP seems to be a cancer-specific modification not

related to inflammation, as it was detected in our study at

advanced stages of PaC and also reported in other cancers

such as HCC. The increase in core fucosylation in several

serum APP has been described using several methods in

different sets of samples, which makes it difficult to draw

solid conclusions relating to its potential clinical applica-

tions. An overall study that could analyze this N-glycan

modification in a larger number of samples and in different

types of malignancies would be required to establish the

clinical utility of APP core fucosylation changes.

In conclusion, glycosylation changes in serum APP could

act as sensors of disease. We have shown that increased

branching and SLex-type fucosylation on APP is a common

feature of advanced PaC and CP. Thus, these glycan changes

may be associated with the cancer-related inflammation and

not just the cancer itself. Increase of core fucosylation on

AGP and HPT seems to be a more cancer-specific modifi-

cation, although only detectable at advanced stages of PaC.

It should be investigated in a larger cohort of patients

including other cancers, to define its potential clinical utility

as a PaC diagnostic or monitoring tool.

A. S. acknowledges University of Girona for a pre-doctoralfellowship and for a short-term mobility fellowship. The authorsthank the Proteomics Platform of Barcelona Science Park; amember of ProteoRed network for the MS analysis of the proteinsand the Wellcome Trust for an equipment grant to purchase theQ-TOF Ultima Global mass spectrometer. This work was supportedby Spanish Ministerio de Ciencia e Innovacion (grant BIO 2007-61323, awarded to R. P.), La Marato de TV3 foundation (grant050932, awarded to R. P.), the Government of Catalonia (grant2005SGR00065, awarded to R. L.) and National Institute forBioprocessing Research and Training, Ireland.

The authors have declared no conflict of interest.

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Glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis.

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