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Glycobiomarker, fucosylated short-form secretogranin III levels are increased in the serum of patients with small cell lung carcinoma Akira Togayachi, Jun Iwaki, Hiroyuki Kaji, Hideki Matsuzaki, Atsushi Kuno, Yoshitoshi Hirao, Masaharu Nomura, Masayuki Noguchi, Yuzuru Ikehara, and Hisashi Narimatsu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00484 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Journal of Proteome Research

1

Glycobiomarker, fucosylated short-form secretogranin III levels are

2

increased in the serum of patients with small cell lung carcinoma

3 4

Akira Togayachi1, 2, †, Jun Iwaki1, †, Hiroyuki Kaji1, 2, Hideki Matsuzaki1, Atsushi Kuno1, 2, Yoshitoshi

5

Hirao1, Masaharu Nomura3, Masayuki Noguchi4, Yuzuru Ikehara1, 2, and Hisashi Narimatsu1, 2∗

6 7

1Research

Center for Medical Glycoscience, 2Biotechnology Research Institute for Drug Discovery,

8

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1

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Umezono, Tsukuba, Ibaraki 305-8568, Japan

10 11 12 13

3Department

of Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo

160-0023, Japan 4Department

of Pathology, Graduate School of Comprehensive Human Sciences, University of

Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

14 15

AUTHOR INFORMATION

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† These

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Author E-mail addresses:

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Akira

19

[[email protected]],

Hideki

20


Yoshitoshi

21

[[email protected]], Masayuki Noguchi [[email protected]], Yuzuru Ikehara

22

[[email protected]], and Hisashi Narimatsu [[email protected]]

authors contributed equally to this work and should be considered first authors.

Togayachi

[[email protected]], Matsuzaki Hirao

Jun

Iwaki


[[email protected]], [[email protected]],

Hiroyuki Atsushi

Masaharu

Kaji Kuno

Nomura

23 24

Author for all correspondence:

25

Hisashi Narimatsu, MD, PhD

26

Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science

27

and Technology (AIST), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan 1

ACS Paragon Plus Environment


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Tel: +81-29-861-3200; Fax: +81-29-861-3201; E-mail: [email protected]

2 3 4

NOTES

5

Potential conflicts of interest: The authors declare that they have no conflicts of interest.

6 7

FUNDING SOURCE

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This work was supported by the “Medical Glycomics: MG” project (grant number: 08061904) in New

9

Energy and Industrial Technology Development Organization (NEDO) in Japan.

10 11

ABBREVIATIONS

12

ChrA, chromogranin A; IGOT, isotope-coded glycosylation site-specific tagging; NCAM, neural cell

13

adhesion molecule; NSCLC, non-small cell lung carcinoma; PC 2 (PCSK 2), prohormone convertase 2;

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SCLC, small cell lung carcinoma; SgIII, secretogranin III

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2


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ABSTRACT

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Secretogranin III (SgIII) is a member of the chromogranin/secretogranin family of neuroendocrine

3

secretory proteins. Granins are expressed in endocrine and neuroendocrine cells and subsequently

4

processed into bioactive hormones. Although granin-derived peptide expression is correlated with

5

neuroendocrine carcinomas, little is known about SgIII. We previously identified SgIII by a comparative

6

glycoproteomics approach for elucidation of glycobiomarker candidates in lung carcinoma. Here, we

7

examined the expression, secretion, and glycosylation of SgIII to identify novel biomarkers of small cell

8

lung carcinoma (SCLC). In comparative immunohistochemical analysis and secretion profiling, SgIII

9

was observed in all types of lung cancer. However, low-molecular-weight SgIII (short-form SgIII) was

10

specifically found in SCLC culture medium. Glycoproteomics analysis showed that a fucosylated

11

glycan was attached to the first of three potential N-glycosylation sites and an unfucosylated glycan was

12

detected on the second site; however, the third site was not glycosylated. Next, we performed lectin

13

capture with a fucose-binding lectin and detected short-form SgIII specifically in the sera of patients

14

with SCLC. The results suggested an association between the fucosylated glycoform of short-form SgIII

15

and SCLC. Thus, fucosylated short-form SgIII may be a valuable biomarker for SCLC and could be

16

used to monitor development of the disease. All MS data are available via ProteomeXchange and jPOST

17

with identifiers PXD007626 and JPST000313.

18 19

Keywords:

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modification, secretogranin III, small cell lung carcinoma

biomarker,

fucosylation,

glycoprotein,

lectin-chromatography,

post-translational

21

3



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INTRODUCTION

2

With the development of recent biotechnologies, early detection of cancer with high reliability is

3

required for effective treatment and improvement of patients’ quality of life, and the need for effective,

4

noninvasive serum biomarkers has increased. Therefore, we have attempted to develop novel strategies

5

for the identification of glycobiomarkers using established technologies for glycan/glycoprotein

6

analyses.1 For decision making regarding treatment guidelines and monitoring of medical treatment, we

7

focused on detection of quantitative alterations in glycoprotein expression and qualitative changes in

8

glycan structures on glycoproteins derived from cancer tissues at an early stage and discovered a useful

9

glycobiomarker of liver fibrosis.2 Through the original strategy of glycobiomarker development by

10

glycoproteomics, various types of tissue-specific biomarkers for liver cirrhosis,3 cholangiocarcinoma,4, 5

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and clear cell ovarian carcinoma6 have also been identified and validated using clinical data. Moreover,

12

in our previous study, we performed glycoproteomics analysis to identify glycobiomarker candidates of

13

lung cancer, particularly lung adenocarcinoma.7 Lectin-captured isotope-coded glycosylation

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site-specific tagging (IGOT) analysis focusing on fucosylation revealed 1092 molecules from lung

15

cancer cell lines, and secretogranin III (SgIII) was found in the list of the small cell lung carcinoma

16

(SCLC)-specific glycobiomarker candidates.7

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Granins are a family of anionic glycoproteins found as constitutively secreted granules.

18

Members of the granin family are expressed in endocrine and neuroendocrine cells. To date,

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chromogranin A (ChrA), chromogranin B (ChrB), secretogranin II (SgII), SgIII, and secretogranins

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IV−VII (SgIV−VII) have been identified among diverse species. Interestingly, abnormal expression of

21

granins and granin-derived peptides is related to neuroendocrine carcinoma.8, 9 For example, ChrA

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shows high specificity as a histochemical marker of neuroendocrine cells,10 such as small cell

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neuroendocrine carcinoma (SCNEC)11, SCLC, and large cell neuroendocrine carcinoma of lung

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carcinoma (LCNEC).12,

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SCNEC, which is a type of malignant tumor accompanied by features of neuroendocrine cells.11

26

Moreover, Moss et al. reported that SgIII transcripts derived from circulating cells in blood could

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potentially be used as a biomarker of SCLC.14

13

Additionally, ChrA and/or cytokeratin are also used as biomarkers for

4


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The incidence of lung cancer has increased globally, and this disease is associated with high

2

mortality rates. Therefore, the development of useful markers for the diagnosis and prognosis of lung

3

cancer is needed in order to improve therapeutic outcomes. Approximately 20−25% of primary lung

4

cancer cases exhibit a spectrum of neuroendocrine differentiation perceived to be invasive

5

malignancy.15 The two most common types of neuroendocrine lung carcinoma are SCLC followed by

6

LCNEC, which are both histologically distinct from other non-small cell lung carcinomas (NSCLCs).

7

From a therapeutic viewpoint, these two cancers tend to respond well to chemotherapy and radiotherapy

8

and have high rates of immunohistochemical staining for useful markers, such as ChrA, neural cell

9

adhesion molecule (NCAM, CD56), and synaptophysin.16, 17 Neuron-specific enolase (NSE) is also

10

well-known as a marker of neuroendocrine carcinomas.18 Thus, several neuronal molecules are used as

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highly specific markers of SCLC.

12

Aberrant glycosylation of certain proteins is associated with a number of diseases and cancers.

13

For example, tetra-antennary N-glycans, possessing a β1,6-N-acetylglucosamine branch extended by

14

N-acetylglucosaminyltransferase V (GnT-V), are upregulated in various cancer tissues.19 This type of

15

branch facilitates elongation of poly-N-acetyllactosamine structures, which are capable of providing a

16

backbone for modification of Lewis epitopes. In particular, CA19-9 is a well-known glycobiomarker

17

targeting sialyl Lewis a epitopes derived from cancer cells.20 Fucosylation of this marker is closely

18

correlated with cancer progression and metastasis,19-21 and fucosylated glycans have also been found on

19

glycoproteins and glycolipids in lung cancer.22-25 In this context, specific protein glycoforms may

20

represent promising biomarkers for cancer.

21

In this study, we show that SgIII is secreted extracellularly after neuroendocrine-specific

22

post-translational modification in SCLC, which is characteristic of this type of lung carcinoma. We

23

carried out comparative analyses by immunohistochemistry of SgIII in several lung carcinoma tissues

24

and immunoblotting with a panel of culture media from various lung carcinoma cell lines established

25

from SCLC, large-cell carcinoma, adenocarcinoma, and squamous-cell carcinoma. Glycosylation of

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SgIII was verified based on the susceptibility to two types of fucose-binding lectins, and its

27

N-glycosylation sites were identified by the IGOT method26 with each of two selected culture media

28

(derived from either SCLC or adenocarcinoma cell lines). Two fucosylated peptides were obtained from 5



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two SCLC cell lines. Furthermore, we found an increase in fucosylated SgIII specifically in sera from

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patients with SCLC. Our findings showed that specific fucosylation of the N-glycans of SgIII could be

3

an advanced glycobiomarker of SCLC.

4

6


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EXPERIMENTAL SECTION

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Immunohistochemistry. Immunostaining for SgIII was carried out with anti-SgIII polyclonal antibody

3

27, 28

4

patients with lung carcinoma at the Tokyo Medical University. The institutional ethics committees of

5

AIST and Tokyo Medical University approved this study. Informed consent for the use of clinical

6

specimens was obtained from all patients. Formalin-fixed, paraffin-embedded lung tissue sections (5 µm

7

thick) were deparaffinized in accordance with an established procedure.29 Formalin-fixed,

8

paraffin-embedded tissue samples, which had been fixed with formalin within 48 h after collection and

9

stored for up to 3 years after embedding, were used. Before immunostaining, the sections were washed

10

with phosphate-buffered saline (PBS) and treated in 10 mM citrate buffer for 15 min at 121°C in an

11

autoclave for antigen retrieval. After pretreatment, the sections were returned to room temperature and

12

then washed three times with PBS (5 min per wash). The sections were incubated with methanol

13

containing 0.3% hydrogen peroxide for 10 min at room temperature to quench endogenous peroxidase

14

activity. The sections were again washed three times in PBS (5 min per wash) and then incubated with

15

goat primary antibody in a moist chamber for 2 h at 20°C. The sections were washed three times in PBS

16

(5 min per wash) and subsequently incubated with a horseradish-peroxidase (HRP)-conjugated anti-goat

17

secondary antibody. Once the blot had developed, the reaction was quenched by soaking in purified

18

water for 15 min (three 5-min washes). Finally, the nuclei were counterstained with hematoxylin for 1

19

min at room temperature followed by washing with tap water.

(C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Lung tissue sections were collected from

20 21

Cell culture of human lung carcinoma cell lines. Human SCLC cell lines (NCI-H524, NCI-H526,

22

NCI-H2171, Lu-130, Lu-134-A, Lu-134-B, Lu-135, Lu-139, Lu-165, SBC-1, and SBC-2), human lung

23

adenocarcinoma cell lines (A549, ABC-1, EHHA-9, NCI-H358, NCI-H441, NCI-H838, NCI-H1355,

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NCI-H1819, NCI-H1975, NCI-H3255, HAL-8, HAL-24, HCC827, PC-9, RERF-LC-MS, TMU2, and

25

TMU3), human lung large cell carcinoma cell lines (NCI-H661, NCI-H1155, NCI-H1299, and

26

NCI-H2126), and human lung squamous cell carcinoma cell lines (AOI, EBC1, OKaC1, and PC-10)

27

were used in this study. Human lung carcinoma cells (A549, Lu-134-A, Lu-134-B, Lu-135, Lu-139;

7



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HCC827, NCI-H524, NCI-H526, NCI-H2171, NCI-H838, NCI-H1355, NCI-H1819, NCI-H1975,

2

NCI-H358, NCI-H661, NCI-H1155, NCI-H1299, NCI-H2126, NCI-H3255, ABC-1, PC-9, EBC1,

3

OKaC1, and PC-10) were obtained from cell banks (RIKEN Bioresource Center Cell Bank, Ibaraki,

4

Japan; American Type Culture Collection, Rockville, MD; Japanese Collection of Research

5

Bioresources, Osaka, Japan; and Immuno-Biological Laboratories, Gunma, Japan). Other cell lines

6

(EHHA-9, HAL-8, HAL-24, RERF-LC-MS, SBC-1, SBC-2, TMU2, TMU3 and AOI) were obtained

7

from Kitano Hospital (Osaka, Japan) and Tokyo Medical University (Tokyo, Japan). Cells were

8

maintained as recommended by the respective institutions. Briefly, the cells were cultured until reaching

9

80–90% confluence in the appropriate medium (e.g., RPMI-1640, Dulbecco’s modified Eagle’s

10

medium containing high glucose or Ham’s F12) supplemented with 10% (v/v) fetal calf serum, 100

11

µg/mL streptomycin, and 100 IU/mL penicillin (Gibco, Grand Island, NY) in an atmosphere containing

12

5% CO2.

13 14

Immunoblotting. The human lung cell lines were incubated in serum-free medium for 48 h, and the

15

culture media were collected and concentrated 10-fold using Amicon Ultra-4 centrifugal filter units (30

16

kDa molecular-mass cut-off; Millipore, Bedford, MA, USA). The protein concentration of the culture

17

medium was determined using Bradford protein assays (BioRad Laboratories, Hercules, CA, USA). The

18

concentrated samples (500 µg each) and, if neccesary, recombinant SgIII (2.5 ng as a quantitative

19

reference; Proteintech, Manchester, UK) were subjected to sodium dodecyl sulfate polyacrylamide gel

20

electrophoresis (SDS-PAGE) using 17% gels and then transferred to polyvinylidene fluoride

21

membranes (Bio-Rad Laboratories). The membranes were blocked using 5% (w/v) nonfat milk in

22

Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature. After a wash step

23

with TBS-T for 5 min, the membrane was incubated with an anti-SgIII antibody27, 28 (C-19, 1:200

24

dilution) preliminarily biotinylated using a Biotin Labeling Kit-NH2 (Dojindo Molecular Technologies,

25

Kumamoto, Japan) or with an anti-prohormone convertase 2 polyclonal antibody (PC2; ALEXIS

26

Biochemicals, San Diego, CA, USA; 1:1000 dilution) for 1 h. After two 5-min wash steps with TBS-T,

27

the membranes were incubated with HRP-conjugated streptavidin (GE Healthcare, Piscataway, NJ,

28

USA; 1:3000 dilution) or with an HRP-conjugated anti-rabbit secondary antibody for 1 h followed by 8


Page 9 of 40

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1

three washes of 10 min each. Signals were developed with Western Lightning Chemiluminescence

2

Reagent Plus (Perkin-Elmer Life Sciences, Boston, MA, USA) and visualized using Amersham

3

Hyperfilm ECL (GE Healthcare, Little Chalfont, UK).

4 5

Deglycosylation of culture medium using PNGase F. A 500-µg aliquot of SBC-2 culture medium was

6

denatured for 3 min at 100°C as recommended by the manufacturer (PNGase F; TAKARA BIO Inc.,

7

Otsu, Japan). The denatured sample was solubilized and then treated with 1 mU PNGase F at 37°C for

8

18 h. Prior to the study, we confirmed the validity of N-glycan digestion in preparatory band-shift

9

examinations.

10 11

Identification of N-glycosylation sites by IGOT analysis. To identify the N-glycosylated proteins and

12

their glycosylation sites by the IGOT method, 500 mL culture medium was prepared from two selected

13

SCLC cell lines (H524 and H2171), and the proteins obtained from the culture medium were denatured,

14

reduced, alkylated, and digested with trypsin, as described previously.7 The tryptic digest was applied to

15

a column of Aleuria aurantia lectin (AAL, J-Oil Mills, Inc., Tokyo, Japan; 4.6 mm × 150 mm)

16

equilibrated with 10 mM HEPES-NaOH (pH 7.5). Glycopeptides were eluted with the same buffer

17

containing 5 mM fucose. The unbound fraction was applied on a Hippeastrum hybrid lectin (HHL)

18

column (Vector Laboratories, Burlingame, CA; 10 mm × 20 mm), and the bound glycopeptides were

19

eluted with 0.2 M methyl α-D-mannopyranoside (αMM). The unbound peptides were loaded similarly

20

on a Concanavalia ensiformis agglutinin (ConA) column, and the bound peptides were eluted with 0.2

21

M αMM. The glycopeptides were fractionated into three samples of two categories, i.e., fucosylated and

22

oligomannosylated glycopeptides, by serial lectin affinity chromatography. The obtained glycopeptides

23

were further purified by hydrophilic interaction chromatography on a agarose column as previously

24

described.26, 30 The glycopeptides were treated with PNGase F in H218O.

25 26

Identification of labeled peptides by nanoflow liquid chromatography-mass spectrometry

27

(LC-MS) analysis. Stable isotope-labeled peptides were analyzed by LC-MS, as described previously.7,

28

26, 30

Briefly, the peptide mixture was injected into a C18 trap column (0.5 mm × 1 mm). After washing, 9



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Page 10 of 40

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the column was connected to a nanoflow LC system (flow rate: 100 nL/min), and the peptides were

2

separated on a reverse-phase (C18) tip column (150 µm × 70 mm) by a linear gradient of MeCN (0–35%

3

in 0.1% formic acid) for 70 min. The eluted peptides were sprayed directly into a quadrupole

4

time-of-flight hybrid mass spectrometer (Q-TOF Ultima; Waters, Milford, MA, USA). The spectra were

5

obtained in the data-dependent MS/MS mode and processed using MassLynx software (version 4.0;

6

Waters) to create peak list files after smoothing by the Savitzky-Golay method (window channels, ± 3).

7

The files were processed by the MASCOT algorithm (version 2.4.1; Matrix Science, Boston, MA, USA)

8

to assign peptides using the RefSeq protein sequence database (71,826 entries, downloaded in July 2014,

9

http://www.ncbi.nlm.nih.gov/refseq/). The database search was performed with previously described

10

parameters.26, 30 Briefly, the parameters used in this study were as follows: enzyme, trypsin; maximum

11

missed cleavage, 2; fixed modification, carbamidomethylation (Cys); variable modifications,

12

deamination (pyroGlu, peptide N-terminal Gln), oxidation (Met), and IGOT (deamidation incorporating

13

18

14

peptide search were exported in CSV files and processed using Microsoft Excel. We first selected

15

peptides with a rank of 1 and an expectation value of less than 0.05. Next, we selected peptides that

16

contained one or more Asp moieties labeled with 18O atoms at the position of Asn in the consensus

17

sequence for N-glycosylation, Asn-Xaa-(Ser/Thr), where Xaa is any amino acid except Pro. False

18

discovery rates were confirmed using a decoy database by Mascot. All MS data are registered to

19

ProteomeXchange and jPOST with identifiers PXD007626 and JPST000313, respectively.

O, +3 Da, Asn); peptide mass tolerance, 200 ppm; fragment mass tolerance, 0.5 Da. All results of the

20 21

Lectin capture of fucosylated SgIII from the culture medium using AAL-agarose. In order to

22

fractionate fucosylated SgIII, we applied 30 µg of culture medium from lung carcinoma cell lines to a

23

500 µL aliquot of AAL-agarose slurry. After extensive washing, the bound material was eluted using 0.2

24

M L-fucose in PBS. The bound and unbound fractions were collected, and half of each sample was used

25

for immunoblotting, as described earlier.

26 27

Fractionation of serum by serial-lectin chromatography. Serum specimens were obtained from five

28

patients with SCLC and five patients with lung adenocarcinoma after obtaining approval from the 10


Page 11 of 40

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1

Ethical Committee of Tokyo Medical University Hospital. Written informed consent was obtained from

2

all patients before being included in the study. Normal healthy serum samples were obtained from five

3

healthy volunteers. Serial (or combinatorial) lectin affinity chromatography was performed with three

4

types of lectin agarose. Serum samples (500 µL each) from healthy volunteers and patients with lung

5

carcinoma were applied to a Lens culinaris lectin (LCA)-agarose column (0.70 × 1.3 cm) to remove

6

blood immunoglobulin G. The unbound fractions were collected and then isolated on an AAL-agarose

7

column (0.70 × 5.5 cm). The bound material was eluted using 0.2 M L-fucose in PBS, and the collected

8

eluates were concentrated to 50 µL with Ultrafree-0.5 centrifugal filter devices (30 kDa molecular-mass

9

cut-off; Millipore). The elution of LCA-agarose was carried out with 0.2 M L-fucose in PBS. The

10

fractions that passed through both the LCA- and AAL-agarose were applied to concanavalin A

11

(ConA)-agarose, and the bound fractions were eluted with 0.5 M α-methyl-D-mannoside in PBS-T.

12

Ten-microliter aliquots of the resulting sample were analyzed by immunoblotting.

13

11



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Page 12 of 40

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RESULTS

2

Screening of glycobiomarker candidates identified in the lectin-captured fractions of SCLC and

3

NSCLC cell lines. We performed comprehensive analysis of tissue-specific glycobiomarkers using

4

systematic

5

glycobiomarkers targeting lung cancer through lectin-assisted IGOT-LC-MS analysis to discriminate

6

between SCLC and NSCLC.7 Glycoproteomics analysis was performed using three types of lectin

7

columns

8

high-mannose/bi-antennary N-glycans, respectively. From these results, 61 glycoproteins were

9

identified by the AAL-captured IGOT method, and 110 glycoproteins were identified by the

10

leading-edge

(AAL-,

HHL-,

glycoproteomics

and

technology.

ConA-agarose)

Previously,

specific

for

we

reported

fucosylated,

potential

pouch-,

and

HHL/ConA-captured IGOT method.

11

Based on these results, glycoproteins as candidates for SCLC glycobiomarkers were selected

12

by biochemical analysis, according to the following conditions: (i) the candidate molecule was

13

specifically captured and identified by either fucose-binding lectin (AAL) or oligomannose-binding

14

lectin (HHL/ConA); (ii) sufficient expression of candidate molecules could be confirmed in lung cancer

15

cell lines or lung cancer tissues by western blotting analysis; (iii) antibodies used for western blotting

16

and immunoprecipitation were commercially available. In our previous study, we selected 31

17

glycoproteins for glycobiomarker candidates derived from the identified SCLC- and NSCLC-specific

18

glycoproteins described above (see Table 1 in our previous study7) and compared their profiles.

19

Subsequently, we focused on glycopeptide identification in NSCLC and selected candidate molecules.

20

In contrast, in this study, candidate molecules were selected by focusing on their expression in SCLC.

21

By IGOT analysis, 10 glycoproteins (neural cell adhesion molecule 1 [NCAM1], Thy-1 cell surface

22

antigen [THY1], sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 2 [SPOCK2],

23

cell adhesion molecule 4 [CADM4], antigen p97 [melanoma associated] identified by monoclonal

24

antibodies 133.2 and 96.5 [MFI2], subunit of the oligosaccharyltransferase complex (catalytic)

25

[STT3A], neuronal pentraxin receptor [NPTXR], sel-1 suppressor of lin-12-like [Caenorhabditis

26

elegans] [SEL1L], v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog [KIT], and SgIII

27

[SCG3]) were specifically identified in SCLC. These glycoproteins were not detected in NSCLC. In this

12


Page 13 of 40

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1

study, we focused on SCLC-specific fucosylation and narrowed down the list to four highly reliable

2

candidate molecules (NCAM1, THY1, CADM4, and SgIII). Antibodies for western blotting analysis

3

were commercially available for SgIII as well as NCAM1, which is a known neuron-specific marker.

4

Therefore, we selected SgIII as the most feasible candidate glycobiomarker and proceeded to analyze

5

SgIII in lung cancer.

6 7

Expression of SgIII in lung cancer tissues. Immunohistochemical analysis of SgIII in lung cancer

8

tissues has not yet been reported. To investigate the expression of SgIII in different lung tissues, its

9

immunoreactivity was examined with formalin-fixed, paraffin-embedded specimens of primary cancer

10

tissues derived from patients with lung cancer (Figure 1). Immunoreactivity to anti-SgIII antibody was

11

observed in epithelial cells of bronchial glands (Figure 1A) and a panel of lung cancer tissues, namely

12

SCLC (Figure 1B), squamous-cell carcinoma (Figure 1C), well-differentiated adenocarcinoma (Figure

13

1D), poorly differentiated adenocarcinoma (Figure 1E), and large-cell carcinoma (Figure 1F). As

14

previously reported by others, SgIII and several granins have also been identified in normal human

15

mammary glands, various lung neuroendocrine carcinomas, and medullary thyroid carcinomas.31, 32

16

Consistent with these reports, we found that proteins of the granin family (with the exception of ChrA 33)

17

were expressed in various tissues.

18

We also examined the secretion of SgIII into serum-free culture medium in 32 lung carcinoma

19

cell lines composed of eight SCLC, four large-cell carcinomas, 16 adenocarcinomas, and four squamous

20

carcinomas by immunoblotting. SgIII was detected in each of the total proteins secreted into the culture

21

medium (Figure 2A). Some SCLC cell lines expressed relatively high amounts of SgIII. Although the

22

culture medium of most of the cell lines contained a 70-kDa cross-reacting species, corresponding to the

23

uncleaved “pro-form”, a 60-kDa band (“short-form” of SgIII 34) was found in the culture medium of

24

SCLC cell lines. In addition, the short-form was found in the culture medium of one NSCLC cell line

25

(large-cell carcinoma, H1155). H1155 cells were established from metastatic LCNEC. Based on

26

densitometry analysis, the ratio of secreted short-form SgIII to the total amount of SgIII for the SCLC

27

cell lines was higher than that of pro-form SgIII but lower in lung adenocarcinoma cell lines (Figure

28

S1A). As a statistical validation, Mann-Whitney U tests of unpaired t-test data were used to compare the 13



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Page 14 of 40

1

expression of pro-form and short-form SgIII between SCLC and NSCLC cell lines, as shown in Figure

2

2B and Figure S1B. According to the analysis, the secretion of short-form SgIII expression and the ratio

3

of short-form SgIII to pro-form SgIII were specific to SCLC cell lines, whereas the pro-form did not

4

differ among cell line. Thus, the secreted short-form SgIII seemed to be characteristic of

5

neuroendocrine-like lung cancers, such as SCLC and LCNEC.

6 7

SCLC-specific size reduction of SgIII through post-translational processing. SgIII (gene: SCG3,

8

Gene ID: 29106, genomic location: chromosome 15q21.2, UniProt ID: Q8WXD2, Evidence at protein

9

level: PE1, protein length: 468 aa) is an acidic glycoprotein composed of 468 amino acids, including 93

10

acidic residues (Figure 3A, underlined font). The acidic nature of the protein seems to be important for

11

the biogenesis of secretory granules associated with other granins and processing enzymes, e.g.,

12

prohormone convertases (PCs) and carboxypeptidase E, for sorting and condensing of peptide

13

hormones.35 In previous studies, Sasaki and colleagues reported that processing enzymes digest SgIII at

14

specific processing sites to generate several SgIII-derived peptide fragments (enzyme recognition site:

15

R/KXnR/K motif, n = 2, 4, or 6; Figure 3A, asterisks) 36 These processing enzymes can be categorized

16

into the serine protease family and are found in neural and endocrine cells, in which they cleave

17

prohormones in secretory granules.37 In lung cancers, PC2 is preferentially expressed in SCLC

18

compared with other lung tissues and cell lines.38, 39 To clarify the correlation between the generation of

19

short-form SgIII and PC2 in neuroendocrine tissues, we examined expression of PC2 at the protein level

20

in lung carcinoma cell lines (Figure 3B). We focused on two cell lines: SBC-2 from SCLC, in which

21

both pro-form and short-form SgIIIs were secreted, and PC-9 from lung adenocarcinoma expressing the

22

pro-form only. Substantial expression of PC2 (66 kDa) was found in representative SBC-2 cells and

23

other SCLC cell lines, not in PC-9 cells or any other NSCLC cell line (Figure S2). This observation

24

suggests that the size reduction of SgIII may be dependent on PC2 processing. Nonetheless, the

25

molecular weight of short-form SgIII was further reduced after digestion with PNGase F; therefore the

26

lack of N-glycosylation in SBC-2 culture medium was not the reason for the presence of short-form

27

SgIII (Figure 3C). The molecular mass of the deglycosylated short-form SgIII identified as a 60-kDa

28

protein was significantly higher than that anticipated from theoretical predictions based on the amino 14


Page 15 of 40

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1

acid sequence (53 kDa). However, the surfeit of acidic residues (Asp and Glu) in SgIII may have caused

2

anomalous migration of the protein during SDS-PAGE.40

3 4

Identification of the N-glycosylation site at which fucosylated glycan was attached to SgIII. As

5

shown in Figure 3A, there are three potential N-glycosylation sites (NX[S/T]) in the SgIII polypeptide.

6

To identify the actual N-glycosylated sites of SgIII secreted from lung carcinoma cell lines,

7

glycopeptides were captured by serial lectin affinity chromatography using two types of lectin columns

8

with

9

high-mannose/bi-antennary N-glycans); the peptides were then identified by IGOT-LC-MS. From this

10

analysis, two types of SgIII-derived peptides were identified from the culture medium of H524 and

11

H2171 cell lines (Figure S3). One peptide, 56KTYPPENKPGQSNYSFVDNLNLLK79, was identified as

12

the AAL-bound glycopeptide of both cells, in which Asn68 (in NYS) was stable-isotope labeled,

13

indicating that the residue was glycosylated. The second peptide,

14

identified as a ConA-bound glycopeptide in both cell lines; Asn346 (in the NAT sequence italicized and

15

underlined above) was labeled. Labeling of Asn350 in the NIS sequence was not detected. No SgIII

16

peptide was identified from the culture medium of NSCLC cells.7

different

binding

specificities

(AAL

for

fucosylated

N-glycans

341

and

ConA

for

NKLEKNATDNISK353, was

17 18

Fucosylation of short-form SgIII derived from the culture medium of SCLC cell lines. To further

19

characterize the SCLC-specific glycopeptide from SgIII as a SCLC biomarker, its fucosylated

20

glycoform was elucidated using a fucose-binding lectin. We verified the affinity of short-form SgIII to

21

AAL immobilized on agarose. Culture medium from large-cell adenocarcinoma, adenocarcinoma,

22

squamous adenocarcinoma, and SCLC cell lines was applied to the AAL-agarose column. The

23

glycopeptides included in the flow-through and elution fractions were analyzed by immunoblotting

24

(Figure 4). The short-form SgIII from SCLC was apparently bound to AAL-agarose. In contrast, SgIII

25

from the three other NSCLC cell lines was detected in the flow-through fractions. The fucosylated

26

glycoform was specifically found as the short-form SgIII from SCLC.

27 28

Increased terminal fucosylation of short-form SgIII in the serum of patients with SCLC. In order 15



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Page 16 of 40

1

to detect the fucosylated short-form SgIII specific to SCLC, serum samples from five patients with

2

SCLC and five patients with adenocarcinoma were fractionated with lectin agarose. First, a depletion

3

step was performed with LCA-agarose to remove a considerable amount of core-fucosylated

4

immunoglobulin from the serum samples. The resulting flow-through fractions were applied to an

5

AAL-agarose column, and bound glycoproteins were subsequently eluted with 0.2 M fucose. The

6

fucosylated short-form SgIII was identified by immunoblotting of the elution fractions (Figure 5). As a

7

result, AAL-susceptible short-form SgIII was significantly enriched from SCLC sera. Short-form SgIII

8

was also detected in the serum of normal volunteers and patients with lung adenocarcinoma, but at much

9

lower levels. The fucosylated glycoform of pro-form SgIII was not detected in these samples.

10

16


Page 17 of 40

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1

DISCUSSION

2

SgIII is expressed in endocrine and neuroendocrine cells and interacts with cholesterol in the membrane

3

of secretory granules under acidic conditions in association with ChrA, SgII, and carboxypeptidase

4

E.41-43 This glycoprotein has been shown to serve as an anchored adapter molecule, forming a bridge

5

between aggregated hormones and the membrane of secretory granules.35 Until now, glycosylation of

6

granins has never been examined in relation to lung cancer. Furthermore, few granins have been found

7

through comprehensive glycoproteomics with several lectin columns.44, 45 Granins are difficult to detect

8

because an enrichment step is necessary to identify minor constituents from abundant serum

9

components. While their physiological function remains unclear, it is assumed that the pro-form of SgIII

10

plays a pivotal role in assembling components of the secretory granules. However, unlike other granins,

11

the pro-form of SgIII may not function as a precursor of the functional peptide hormone. Thus, SgIII

12

does not appear to be secreted into the circulatory system.32, 46, 47 Short-form SgIII can be produced via

13

processing by proteases, such as PCs, localized in secretory granules. In PC-12 cells, derived from the

14

rat adrenal medulla, the cells lack PC1/3 and PC2,48 and SgIII was detected as the pro-form.49 Hence,

15

SgIII is specifically processed by endocrine specific PCs in endocrine cells, such as SCLC and LCNEC

16

cells. Furthermore, SgIII and PC2 were expressed concomitantly in SCLC but not NSCLC (Table S6).

17

Short-form SgIII was only detected from specific cells in which PCs were expressed.36 According to

18

previous reports by Sasaki et al., C-terminal peptide regions of SgIII (IEWLKKHDKK and/or

19

TEAYLEAIRK) can be generated by cleavage at the recognition sites of PCs (i.e., [(R/K)Xn(R/K)], n =

20

0, 2, 4, or 6) under stimulated conditions.36

21

Our histochemical analysis showed that SgIII was generally expressed in normal bronchial

22

glands and various lung cancers. Indeed, these findings are consistent with previous reports on normal

23

islet cells50 and mammary glands.31 However, secreted short-form SgIII was observed in the culture

24

medium from SCLC and LCNEC cell lines, and the fucosylation sites of the derived glycopeptides were

25

identified by the IGOT method.26 Our analysis revealed that only two N-glycosylation sites were labeled

26

with stable isotope, despite the presence of three potential sites (Asn60, Asn346, and Asn350).51 In the

27

IGOT method, the N-terminal region of the SgIII peptide, including Asn60, was captured by

17



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Page 18 of 40

1

AAL-agarose. Intriguingly, the C-terminal peptide, which includes two closely located potential

2

N-glycosylation sites (Asn346 and Asn350), bound to ConA-agarose. Our analysis showed that Asn346 was

3

glycosylated, whereas Asn350 was not. This binding interface of the association between SgIII and ChrA

4

is located near each glycosylated site.49 Interestingly, the glycosylation of ChrA has been reported to

5

contribute to the polarity of organelles in MDCK cells.52 Thus, the C-terminal glycosylation of SgIII

6

may have a similar function. In contrast, the N-terminal glycosylation of SgIII is thought to be related to

7

cholesterol binding.43 It is interesting to speculate whether the glycosylation status determines if the

8

molecule is anchored onto the cholesterol-rich membrane or is secreted. Because SCLC secretes various

9

peptide hormones, including adrenocorticotropic hormone, antidiuretic hormone, and physiologically

10

active substances, such as estrogen, these hormones may promote the proliferation and metastasis of

11

cancers through an autocrine/paracrine secretion mechanism.

12

The biological functions of SgIII in lung small cell carcinoma are still unclear; however, this

13

protein may be have some functions in cancer cells derived from neuroendocrine cells. According to the

14

data from public database (Expression Atlas website, https://www.ebi.ac.uk/gxa/home/), both PCSK2

15

(PC2) and SCG3 genes are expressed in many cell lines derived from small cell lung carcinoma. In

16

contrast, the expression of these two genes is not observed in NSCLC cell lines (Table S6). Notably,

17

DNA microarray analysis has been performed to clarify underlying mediators for neuroendocrine

18

differentiation and tumorigenesis in human prostate cancer.53 The results showed that the expression of

19

32 genes, including chromogranin A (Chga), SgIII (SCG3), prohormone convertase 1 (PCSK1), and

20

secretory granule neuroendocrine protein 1 (7B2: Sgne1) was upregulated by more than 2-fold at the

21

mRNA level. Moreover, Sgne1 promotes the maturation and activation of prohormone convertase 2

22

(PC2, PCSK2). Since SgIII expression and PC2 maturation (expression) were linked, we believe that

23

SgIII may have any biological functions in neuroendocrine cells. Further studies are required to

24

elucidate the biological functions of changes in the expression of short-form SgIII and its glycosylation

25

in lung small cell carcinoma.

26

Alterations in glycoforms have been shown to be valuable markers for cancer. We have

27

established a progressive strategy to effectively explore the distinction between normal and aberrant

28

glycoforms.1 Evanescent-field fluorescence-assisted lectin microarray is a useful technology for glycan 18


Page 19 of 40

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1

profiling,54-56 and antibody-assisted lectin profiling was developed for glycan-related biomarker

2

verification.57 Furthermore, serial lectin chromatography of serum samples for a glycobiomarker,

3

α1-acid glycoprotein, is available for staging of hepatic fibrosis without immunoprecipitation.58. Using

4

this strategy, specific glycoforms (e.g., core-fucose and terminal fucose of glyco-epitopes) are classified

5

by the serial use of two types of fucose-binding lectins, LCA followed by AAL. In fact, the total amount

6

of fucosylated SgIII in sera without a depletion step using LCA lectin made no difference among the

7

three tissue types of lung cancers (Figure S4). Linkage-specific fucosylation provides in-depth

8

information of slight variations in glycosylation in terms of glycobiomarkers. 59, 60 In this study, we

9

found that the level of fucosylated short-form SgIII increased in the sera of patients with SCLC, as

10

revealed by serial lectin chromatography. Multivalent lectin fractionates linkage-specific glycoforms

11

and concentrates small amounts of the target glycoprotein, whereas antibody-based strategies could not

12

detect SgIII.47, 61 Moreover, fucosylated short-form SgIII was detected in all five serum samples from

13

patients with SCLC and was upregulated to a greater extent than that in healthy volunteers and patients

14

with lung adenocarcinoma. Herein, false-positive detection of the fucosylated glycoform of short-form

15

SgIII was found in one adenocarcinoma serum sample. Further studies are needed to investigate the

16

relationship between the expression of fucosylated short-form SgIII and pathological significance.

17

Additionally, high-throughput detection of the specific SgIII form subjected to post-translational

18

modification should be established for comprehensive analysis for sera from patients with lung cancer.

19

Fucosylation increases in SCLC and many other cancers.23, 62, 63 Notably, Lewis x (LeX) is a

20

well-known cancer marker generated by myeloid α1,3-fucosyltransferase 4 (Fut4) and

21

α1,3-fucosyltransferase 9 (Fut9). To identify a specific synthetase to the neural LeX epitope, Kudo et al.

22

discovered neuron-specific fucosyltransferase (Fut9), whereas Fut4 was not detected; Fut9 was

23

specifically expressed in neurons but not glia. This fucosyltransferase is generally located in the

24

trans-Golgi apparatus as well as PCs in secretory granules.37, 40 Therefore, processing and fucosylation

25

of SgIII may concomitantly occur at the trans-Golgi apparatus of SCLC cells. In this context, hormone

26

production, including glycosylated granin peptides, could be related to the development of SCLC

27

because SgIII secretion has never been reported in NSCLC cells.

19



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Page 20 of 40

1

Table S7 shows some tumor markers for lung cancer and biomarkers related to sugar chains.

2

Currently, tumor markers primarily used for the diagnosis of lung cancer include SLX, CYFRA 21.1,

3

SCC (squamous cell antigen), NCAM, Pro-gastrin-releasing peptide (ProGRP), NSE, and

4

carcinoembryonic antigen (CEA), as described above. Many markers have been approved for clinical

5

examinations in Japan; however, few have been approved by the US Food and Drug Administration. All

6

tumor markers show increases specific to lung cancer and may also increase in cancers in other organs.

7

Among these tumor markers, SLX specific to adenocarcinoma is related to the reaction to carbohydrate

8

antigens. This epitope is sialyl Lewis x (sLeX) antigen and contains fucose residues.23 In cancers of other

9

organs, many tumor markers related to carbohydrate antigens are known, and in particular, tumor

10

markers related to upregulation of fucosylation have been extensively studied. For example, in the liver,

11

α-fetoprotein (AFP) is a major tumor marker dependent on detection of the amount of protein present; in

12

contrast, AFP-L3 (%), which measures the fucosylation value of AFP, has higher specificity for liver

13

cancer than AFP.21 Therefore, combining proteins with disease-specific glycan structures improves

14

specificity. In addition to fucosylation, many biomarkers that are reacted with WFA lectin has been

15

found in recent years (Table S7). Concurrent changes in glycan structure and protein cleavage were

16

specifically occured on SgIII in small cell lung carcinoma. Therefore, we speculate that fucosylated

17

short-form SgIII is a stable mature-form unlike an unstable pro-form such as ProGRP, and may have the

18

potential to show more specificity for lung small cell carcinoma. In the future, we plan to construct a

19

sandwich enzyme-linked immunosorbent assay system using AAL and anti-SgIII antibody and to

20

conduct validation assays using many clinical samples.

21

Similar to small cell lung carcinoma, SCNEC in other organs (e.g., digestive organs) is also

22

neuroendocrine carcinoma. Neuroendocrine carcinoma arising from peptidergic neurons and occurs in

23

various organs/tissues, such as the digestive tract, lungs, nasopharynx, pharynx, mediastinum, thymus,

24

breast, and uterus. The Ki-67 index, cytokeratin, chromogranin, NCAM, synaptophisin, and NSE are

25

known biomarkers for neuroendocrine carcinoma.11 Currently, we have no data for fucosylated SgIII

26

expression in SCNEC other than SCLC. However, PC2 is expressed in neuroendocrine tissue.64

27

Therefore, there is a possibility that short-form SgIII is secreted from SCNEC. If similar expression of

28

glycogenes in SCNEC is observed, short-form SgIII may be an effective SCNEC biomarker. Further 20


Page 21 of 40

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1

studies are needed for validation in SCLC and determination of the effectiveness of short-form

2

fucosylated SgIII as biomarker for SCNEC.

3

Although proteomics relates to quantitative protein expression, glycoproteomics takes into

4

account both quantitative and qualitative alterations in the glycoforms of glycoproteins. Recently, there

5

has been rapid progress in the development of new analytical technologies. Therefore, in the near future,

6

even trace amounts of an aberrant glycoform will become detectable. Identification of distinctive

7

glycoforms with glyco-epitopes (e.g., various lectins) is now feasible using highly sensitive analytical

8

procedures. Notwithstanding an extremely low amount of target molecule in blood, SgIII can be

9

captured by lectin enrichment. In summary, our findings demonstrated that fucosylated short-form SgIII

10

(AAL-reactive short-form SgIII) may have potential applications as a noninvasive discriminative

11

molecule of SCLC in biological fluids. Indeed, this potential glycobiomarker could be used to monitor

12

the prognosis, diagnosis, and recurrence of SCLC.

13

21



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Page 22 of 40

1

ACKNOWLEDGEMENTS

2

We thank Mr. Furuya of Translational Research and Resource Core in University of Tsukuba Hospital,

3

for assistance with the immunohistochemistry analysis of SgIII in various lung tissues. We would like to

4

give special thanks to members of the Research Center for Medical Glycoscience in AIST; Drs. Minako

5

Abe, Takashi Ohkura, and Jun Hirabayashi for thier helpful advice; Ms. Azusa Yanagida-Tomioka,

6

Mika Fujita, Yuki Tsunoda, Nami Suzuki, Maki Sogabe, and Minami Kai for the preparation of culture

7

medium derived from lung carcinoma cell lines and for excellent technical assistance; Ms. Fukuda for

8

the collection of serum samples from patients with lung cancer; and Ms. Tomomi Nakagawa and Kozue

9

Saito for fractionation of serum samples with lectin-agarose.

10 11

22


Page 23 of 40

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1

REFERENCES

2

1.

Narimatsu, H.; Sawaki, H.; Kuno, A.; Kaji, H.; Ito, H.; Ikehara, Y. A strategy for discovery of

3

cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics.

4

FEBS J. 2010, 277, 95-105.

5

2. Kuno, A.; Ikehara, Y.; Tanaka, Y.; Ito, K.; Matsuda, A.; Sekiya, S.; Hige, S.; Sakamoto, M.; Kage,

6

M.; Mizokami, M.; Narimatsu, H. A serum "sweet-doughnut" protein facilitates fibrosis

7

evaluation and therapy assessment in patients with viral hepatitis. Sci. Rep. 2013, 3, 1065.

8

3.

Ocho, M.; Togayachi, A.; Iio, E.; Kaji, H.; Kuno, A.; Sogabe, M.; Korenaga, M.; Gotoh, M.;

9

Tanaka, Y.; Ikehara, Y.; Mizokami, M.; Narimatsu, H. Application of a glycoproteomics-based

10

biomarker development method: alteration in glycan structure on colony stimulating factor 1

11

receptor as a possible glycobiomarker candidate for evaluation of liver cirrhosis. J. Proteome

12

Res. 2014, 13, 1428-1437.

13

4.

Matsuda, A.; Kuno, A.; Kawamoto, T.; Matsuzaki, H.; Irimura, T.; Ikehara, Y.; Zen, Y.;

14

Nakanuma, Y.; Yamamoto, M.; Ohkohchi, N.; Shoda, J.; Hirabayashi, J.; Narimatsu, H.

15

Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human

16

cholangiocarcinoma. Hepatology 2010, 52, 174-182.

17

5.

Matsuda, A.; Kuno, A.; Matsuzaki, H.; Kawamoto, T.; Shikanai, T.; Nakanuma, Y.; Yamamoto,

18

M.;

19

Glycoproteomics-based cancer marker discovery adopting dual enrichment with Wisteria

20

floribunda agglutinin for high specific glyco-diagnosis of cholangiocarcinoma. J. Proteomics

21

2013, 85, 1-11.

22

6.

Ohkohchi,

N.;

Ikehara,

Y.;

Shoda,

J.;

Hirabayashi,

J.;

Narimatsu,

H.

Sogabe, M.; Nozaki, H.; Tanaka, N.; Kubota, T.; Kaji, H.; Kuno, A.; Togayachi, A.; Gotoh, M.;

23

Nakanishi, H.; Nakanishi, T.; Mikami, M.; Suzuki, N.; Kiguchi, K.; Ikehara, Y.; Narimatsu, H.

24

Novel glycobiomarker for ovarian cancer that detects clear cell carcinoma. J. Proteome Res.

25

2014, 13, 1624-1635.

26

7.

Hirao, Y.; Matsuzaki, H.; Iwaki, J.; Kuno, A.; Kaji, H.; Ohkura, T.; Togayachi, A.; Abe, M.;

27

Nomura, M.; Noguchi, M.; Ikehara, Y.; Narimatsu, H. Glycoproteomics approach for

28

identifying glycobiomarker candidate molecules for tissue type classification of non-small cell

29

lung carcinoma. J. Proteome Res. 2014, 13, 4705-4716.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

8.

2 3 4

Page 24 of 40

Conlon, J. M. Granin-derived peptides as diagnostic and prognostic markers for endocrine tumors. Regul. Pept. 2010, 165, 5-11.

9.

Portela-Gomes, G. M.; Grimelius, L.; Wilander, E.; Stridsberg, M. Granins and granin-related peptides in neuroendocrine tumours. Regul. Pept. 2010, 165, 12-20.

5

10. Campana, D.; Nori, F.; Piscitelli, L.; Morselli-Labate, A. M.; Pezzilli, R.; Corinaldesi, R.;

6

Tomassetti, P. Chromogranin A: is it a useful marker of neuroendocrine tumors? J. Clin. Oncol.

7

2007, 25, 1967-1973.

8

11. Korse, C. M.; Taal, B. G.; Vincent, A.; van Velthuysen, M. L.; Baas, P.; Buning-Kager, J. C.;

9

Linders, T. C.; Bonfrer, J. M. Choice of tumour markers in patients with neuroendocrine

10

tumours is dependent on the histological grade. A marker study of Chromogranin A, Neuron

11

specific enolase, Progastrin-releasing peptide and cytokeratin fragments. Eur. J. Cancer 2012,

12

48, 662-671.

13

12. Travis, W. D.; Linnoila, R. I.; Tsokos, M. G.; Hitchcock, C. L.; Cutler, G. B., Jr.; Nieman, L.;

14

Chrousos, G.; Pass, H.; Doppman, J. Neuroendocrine tumors of the lung with proposed criteria

15

for large-cell neuroendocrine carcinoma. An ultrastructural, immunohistochemical, and flow

16

cytometric study of 35 cases. Am. J. Surg. Pathol. 1991, 15, 529-553.

17

13. Takei, H.; Asamura, H.; Maeshima, A.; Suzuki, K.; Kondo, H.; Niki, T.; Yamada, T.; Tsuchiya,

18

R.; Matsuno, Y. Large cell neuroendocrine carcinoma of the lung: a clinicopathologic study of

19

eighty-seven cases. J. Thorac. Cardiovasc. Surg. 2002, 124, 285-292.

20

14. Moss, A. C.; Jacobson, G. M.; Walker, L. E.; Blake, N. W.; Marshall, E.; Coulson, J. M. SCG3

21

transcript in peripheral blood is a prognostic biomarker for REST-deficient small cell lung

22

cancer. Clin. Cancer. Res. 2009, 15, 274-283.

23 24 25 26

15. Travis, W. D. Lung tumours with neuroendocrine differentiation. Eur. J. Cancer 2009, 45 Suppl 1, 251-266. 16. Lloyd, R. V.; Wilson, B. S. Specific endocrine tissue marker defined by a monoclonal antibody.

Science 1983, 222, 628-630.

27

17. Kibbelaar, R. E.; Moolenaar, C. E.; Michalides, R. J.; Bitter-Suermann, D.; Addis, B. J.; Mooi, W.

28

J. Expression of the embryonal neural cell adhesion molecule N-CAM in lung carcinoma.

29

Diagnostic usefulness of monoclonal antibody 735 for the distinction between small cell lung

24


Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3


cancer and non-small cell lung cancer. J. Pathol. 1989, 159, 23-28. 18. Schmechel, D.; Marangos, P. J.; Brightman, M. Neurone-specific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 1978, 276, 834-836.

4

19. Fernandes, B.; Sagman, U.; Auger, M.; Demetrio, M.; Dennis, J. W. β1-6 branched

5

oligosaccharides as a marker of tumor progression in human breast and colon neoplasia.

6

Cancer Res. 1991, 51, 718-723.

7

20. Itzkowitz, S. H.; Yuan, M.; Fukushi, Y.; Palekar, A.; Phelps, P. C.; Shamsuddin, A. M.; Trump, B.

8

F.; Hakomori, S.; Kim, Y. S. Lewisx- and sialylated Lewisx-related antigen expression in

9

human malignant and nonmalignant colonic tissues. Cancer Res. 1986, 46, 2627-2632.

10 11 12 13

21. Miyoshi, E.; Moriwaki, K.; Nakagawa, T. Biological function of fucosylation in cancer biology. J.

Biochem. 2008, 143, 725-729. 22. Fukushima, K. Expression of Lewis(x), sialylated Lewis(x), Lewis(a), and sialylated Lewis(a) antigens in human lung carcinoma. Tohoku J. Exp. Med. 1991, 163, 17-30.

14

23. Togayachi, A.; Kudo, T.; Ikehara, Y.; Iwasaki, H.; Nishihara, S.; Andoh, T.; Higashiyama, M.;

15

Kodama, K.; Nakamori, S.; Narimatsu, H. Up-regulation of Lewis enzyme (Fuc-TIII) end

16

plasma-type

17

expression of sialyl Lewis x antigen in non-small cell lung cancer. Int. J. Cancer 1999, 83,

18

70-79.

α1,3fucosyltransferase

(Fuc-TVI)

expression

determines

the

augmented

19

24. Tokuda, N.; Zhang, Q.; Yoshida, S.; Kusunoki, S.; Urano, T.; Furukawa, K. Genetic mechanisms

20

for the synthesis of fucosyl GM1 in small cell lung cancer cell lines. Glycobiology 2006, 16,

21

916-925.

22

25. Zhang, S.; Zhang, H. S.; Cordon-Cardo, C.; Reuter, V. E.; Singhal, A. K.; Lloyd, K. O.;

23

Livingston, P. O. Selection of tumor antigens as targets for immune attack using

24

immunohistochemistry: II. Blood group-related antigens. Int. J. Cancer 1997, 73, 50-56.

25

26. Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.;

26

Takahashi, N.; Isobe, T. Lectin affinity capture, isotope-coded tagging and mass spectrometry

27

to identify N-linked glycoproteins. Nat. Biotechnol. 2003, 21, 667-672.

28

27. Coppinger, J. A.; Cagney, G.; Toomey, S.; Kislinger, T.; Belton, O.; McRedmond, J. P.; Cahill, D.

29

J.; Emili, A.; Fitzgerald, D. J.; Maguire, P. B. Characterization of the proteins released from

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 40

1

activated platelets leads to localization of novel platelet proteins in human atherosclerotic

2

lesions. Blood 2004, 103, 2096-2104.

3

28. Tanabe, A.; Yanagiya, T.; Iida, A.; Saito, S.; Sekine, A.; Takahashi, A.; Nakamura, T.; Tsunoda,

4

T.; Kamohara, S.; Nakata, Y.; Kotani, K.; Komatsu, R.; Itoh, N.; Mineo, I.; Wada, J.; Funahashi,

5

T.; Miyazaki, S.; Tokunaga, K.; Hamaguchi, K.; Shimada, T.; Tanaka, K.; Yamada, K.;

6

Hanafusa, T.; Oikawa, S.; Yoshimatsu, H.; Sakata, T.; Matsuzawa, Y.; Kamatani, N.;

7

Nakamura, Y.; Hotta, K. Functional single-nucleotide polymorphisms in the secretogranin III

8

(SCG3) gene that form secretory granules with appetite-related neuropeptides are associated

9

with obesity. J. Clin. Endocrinol. Metab. 2007, 92, 1145-1154.

10

29. Sakashita, S.; Li, D.; Nashima, N.; Minami, Y.; Furuya, S.; Morishita, Y.; Tachibana, K.; Sato,

11

Y.; Noguchi, M. Overexpression of immunoglobulin (CD79a) binding protein1 (IGBP-1) in

12

small lung adenocarcinomas and its clinicopathological significance. Pathol. Int. 2011, 61,

13

130-137.

14

30. Kaji, H.; Yamauchi, Y.; Takahashi, N.; Isobe, T. Mass spectrometric identification of N-linked

15

glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable

16

isotope tagging. Nat. Protoc. 2006, 1, 3019-3027.

17 18

31. Gronberg, M.; Amini, R. M.; Stridsberg, M.; Janson, E. T.; Saras, J. Neuroendocrine markers are expressed in human mammary glands. Regul. Pept. 2010, 160, 68-74.

19

32. Portela-Gomes, G. M.; Grimelius, L.; Stridsberg, M. Secretogranin III in human

20

neuroendocrine tumours: a comparative immunohistochemical study with chromogranins A

21

and B and secretogranin II. Regul. Pept. 2010, 165, 30-35.

22

33. Lai, M.; Lu, B.; Xing, X.; Xu, E.; Ren, G.; Huang, Q. Secretagogin, a novel neuroendocrine

23

marker, has a distinct expression pattern from chromogranin A. Virchows Arch. 2006, 449,

24

402-409.

25

34. Prasad, P.; Yanagihara, A. A.; Small-Howard, A. L.; Turner, H.; Stokes, A. J. Secretogranin III

26

directs secretory vesicle biogenesis in mast cells in a manner dependent upon interaction with

27

chromogranin A. J. Immunol. 2008, 181, 5024-5034.

28 29

35. Hosaka, M.; Watanabe, T. Secretogranin III: a bridge between core hormone aggregates and the secretory granule membrane. Endocr. J. 2010, 57, 275-286.

26


Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2


36. Sasaki, K.; Satomi, Y.; Takao, T.; Minamino, N. Snapshot peptidomics of the regulated secretory pathway. Mol. Cell. Proteomics 2009, 8, 1638-1647.

3

37. Seidah, N. G.; Day, R.; Marcinkiewicz, M.; Benjannet, S.; Chretien, M. Mammalian neural and

4

endocrine pro-protein and pro-hormone convertases belonging to the subtilisin family of serine

5

proteinases. Enzyme 1991, 45, 271-284.

6

38. Mbikay, M.; Sirois, F.; Yao, J.; Seidah, N. G.; Chretien, M. Comparative analysis of expression

7

of the proprotein convertases furin, PACE4, PC1 and PC2 in human lung tumours. Br. J.

8

Cancer 1997, 75, 1509-1514.

9 10 11 12

39. Rounseville, M. P.; Davis, T. P. Prohormone convertase and autocrine growth factor mRNAs are coexpressed in small cell lung carcinoma. J. Mol. Endocrinol. 2000, 25, 121-128. 40. Aarnoudse, C. A.; Garcia Vallejo, J. J.; Saeland, E.; van Kooyk, Y. Recognition of tumor glycans by antigen-presenting cells. Curr. Opin. Immunol. 2006, 18, 105-111.

13

41. Hotta, K.; Hosaka, M.; Tanabe, A.; Takeuchi, T. Secretogranin II binds to secretogranin III and

14

forms secretory granules with orexin, neuropeptide Y, and POMC. J. Endocrinol. 2009, 202,

15

111-121.

16

42. Hosaka, M.; Watanabe, T.; Sakai, Y.; Kato, T.; Takeuchi, T. Interaction between secretogranin

17

III and carboxypeptidase E facilitates prohormone sorting within secretory granules. J. Cell

18

Sci. 2005, 118, 4785-4795.

19

43. Hosaka, M.; Suda, M.; Sakai, Y.; Izumi, T.; Watanabe, T.; Takeuchi, T. Secretogranin III binds

20

to cholesterol in the secretory granule membrane as an adapter for chromogranin A. J. Biol.

21

Chem. 2004, 279, 3627-3634.

22

44. Heo, S. H.; Lee, S. J.; Ryoo, H. M.; Park, J. Y.; Cho, J. Y. Identification of putative serum

23

glycoprotein

24

chromatography and LC-MS/MS. Proteomics 2007, 7, 4292-4302.

biomarkers

for

human

lung

adenocarcinoma

by

multilectin

affinity

25

45. Ueda, K.; Takami, S.; Saichi, N.; Daigo, Y.; Ishikawa, N.; Kohno, N.; Katsumata, M.; Yamane,

26

A.; Ota, M.; Sato, T. A.; Nakamura, Y.; Nakagawa, H. Development of serum glycoproteomic

27

profiling technique; simultaneous identification of glycosylation sites and site-specific

28

quantification of glycan structure changes. Mol. Cell. Proteomics 2010, 9, 1819-1828.

29

46. Holthuis, J. C.; Jansen, E. J.; Martens, G. J. Secretogranin III is a sulfated protein undergoing

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 40

1

proteolytic processing in the regulated secretory pathway. J. Biol. Chem. 1996, 271,

2

17755-17760.

3

47. Stridsberg, M.; Eriksson, B.; Janson, E. T. Measurements of secretogranins II, III, V and

4

proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours. Regul. Pept.

5

2008, 148, 95-98.

6 7

48. Seidah, N. G.; Chretien, M.; Day, R. The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. Biochimie 1994, 76, 197-209.

8

49. Han, L.; Suda, M.; Tsuzuki, K.; Wang, R.; Ohe, Y.; Hirai, H.; Watanabe, T.; Takeuchi, T.;

9

Hosaka, M. A large form of secretogranin III functions as a sorting receptor for chromogranin A

10

aggregates in PC12 cells. Mol. Endocrinol. 2008, 22, 1935-1949.

11

50. Stridsberg, M.; Grimelius, L.; Portela-Gomes, G. M. Immunohistochemical staining of human

12

islet cells with region-specific antibodies against secretogranins II and III. J. Anat. 2008, 212,

13

229-234.

14

51. Rong, Y. P.; Liu, F.; Zeng, L. C.; Ma, W. J.; Wei, D. Z.; Han, Z. G. Cloning and characterization of

15

a novel human secretory protein: secretogranin III. Shanghai Acta Biochim.Biophys.Sin. 2002,

16

34, 411-417.

17 18

52. Kuhn, U.; Cohn, D. V.; Gorr, S. U. Polarized secretion of the regulated secretory protein chromogranin A. Biochem. Biophys. Res. Commun. 2000, 270, 631-636.

19

53. Hu, Y.; Ippolito, J. E.; Garabedian, E. M.; Humphrey, P. A.; Gordon, J. I. Molecular

20

characterization of a metastatic neuroendocrine cell cancer arising in the prostates of

21

transgenic mice. J. Biol. Chem. 2002, 277, 44462-44474.

22

54. Ito, H.; Kuno, A.; Sawaki, H.; Sogabe, M.; Ozaki, H.; Tanaka, Y.; Mizokami, M.; Shoda, J.;

23

Angata, T.; Sato, T.; Hirabayashi, J.; Ikehara, Y.; Narimatsu, H. Strategy for glycoproteomics:

24

Identification of glyco-alteration using multiple glycan profiling tools. J. Proteome Res. 2009, 8,

25

1358-1367.

26

55. Kuno, A.; Uchiyama, N.; Koseki-Kuno, S.; Ebe, Y.; Takashima, S.; Yamada, M.; Hirabayashi, J.

27

Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling.

28

Nat. Methods 2005, 2, 851-856.

29

56. Matsuda, A.; Kuno, A.; Ishida, H.; Kawamoto, T.; Shoda, J.; Hirabayashi, J. Development of an

28


Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

all-in-one technology for glycan profiling targeting formalin-embedded tissue sections.

2

Biochem. Biophys. Res. Commun. 2008, 370, 259-263.

3

57. Kuno, A.; Kato, Y.; Matsuda, A.; Kaneko, M. K.; Ito, H.; Amano, K.; Chiba, Y.; Narimatsu, H.;

4

Hirabayashi, J. Focused differential glycan analysis with the platform antibody-assisted lectin

5

profiling for glycan-related biomarker verification. Mol. Cell. Proteomics 2009, 8, 99-108.

6

58. Kuno, A.; Ikehara, Y.; Tanaka, Y.; Saito, K.; Ito, K.; Tsuruno, C.; Nagai, S.; Takahama, Y.;

7

Mizokami, M.; Hirabayashi, J.; Narimatsu, H. LecT-Hepa: A triplex lectin-antibody sandwich

8

immunoassay for estimating the progression dynamics of liver fibrosis assisted by a bedside

9

clinical chemistry analyzer and an automated pretreatment machine. Clin. Chim. Acta 2011,

10

412, 1767-1772.

11

59. Comunale, M. A.; Rodemich-Betesh, L.; Hafner, J.; Wang, M.; Norton, P.; Di Bisceglie, A. M.;

12

Block, T.; Mehta, A. Linkage specific fucosylation of alpha-1-antitrypsin in liver cirrhosis and

13

cancer patients: Implications for a biomarker of hepatocellular carcinoma. PLoS One 2010, 5,

14

e12419.

15

60. Liu, Y.; He, J.; Li, C.; Benitez, R.; Fu, S.; Marrero, J.; Lubman, D. M. Identification and

16

confirmation of biomarkers using an integrated platform for quantitative analysis of

17

glycoproteins and their glycosylations. J. Proteome Res. 2010, 9, 798-805.

18

61. Madera, M.; Mechref, Y.; Klouckova, I.; Novotny, M. V. High-sensitivity profiling of

19

glycoproteins from human blood serum through multiple-lectin affinity chromatography and

20

liquid chromatography/tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed.

21

Life Sci. 2007, 845, 121-137.

22

62. Kudo, T.; Ikehara, Y.; Togayachi, A.; Morozumi, K.; Watanabe, M.; Nakamura, M.; Nishihara,

23

S.; Narimatsu, H. Up-regulation of a set of glycosyltransferase genes in human colorectal

24

cancer. Lab. Invest. 1998, 78, 797-811.

25

63. Miyoshi, E.; Moriwaki, K.; Terao, N.; Tan, C. C.; Terao, M.; Nakagawa, T.; Matsumoto, H.;

26

Shinzaki, S.; Kamada, Y. Fucosylation is a promising target for cancer diagnosis and therapy.

27

Biomolecules 2012, 2, 34-45.

28

64. Ohagi, S.; LaMendola, J.; LeBeau, M. M.; Espinosa, R., 3rd; Takeda, J.; Smeekens, S. P.; Chan,

29

S. J.; Steiner, D. F. Identification and analysis of the gene encoding human PC2, a prohormone

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 40

1

convertase expressed in neuroendocrine tissues. Proc. Natl. Acad. Sci. U. S. A. 1992, 89,

2

4977-4981.

3 4 5

30


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1

FIGURE LEGENDS

2

Figure 1. Immunohistochemistry of SgIII in lung cancer tissue sections. Thin sections were prepared

3

from formalin-fixed and paraffin-embedded normal bronchial glands and lung cancer tissues. The

4

sections were stained with an anti-SgIII antibody. A, Normal bronchial glands; B, small cell lung

5

carcinoma (SCLC); C, squamous-cell carcinoma; D, well-differentiated adenocarcinoma; E, poorly

6

differentiated adenocarcinoma; F, large-cell carcinoma. Original magnification: 100×.

7 8

Figure 2. Secretion of SgIII from lung cancer cell lines. A, Secretion of SgIII in culture medium from 32

9

lung carcinoma cell lines (SCLC, large cell carcinoma, adenocarcinoma, and squamous carcinoma) was

10

examined by immunoblotting. B, Densitometry analysis of secreted SgIII using ImageJ software. The

11

amount of SgIII expression was calculated with a reference sample (recombinant SgIII).

12 13

Figure 3. Verification of size-reduction of secreted SgIII. A, Typical PC cleavage sites are indicated

14

with asterisks; underlined amino acids indicate acidic residues. Triplets of N-glycosylation potential

15

sites are boxed. B, Expression levels of secreted SgIII (left panel) and PC2 (right panel) were compared

16

between culture medium from an SCLC cell line (SBC-2) and an adenocarcinoma cell line (PC-9). C,

17

SgIII from the culture medium from SBC-2 cells was deglycosylated with PNGase F. The band shift of

18

SgIII was detected by immunoblotting with anti-SgIII antibody (C-19). The results shown are

19

representative of two independent experiments.

20 21

Figure 4. Confirmation of short-form SgIII modification with fucosylation. The fucosylated glycoform

22

of SgIII was elucidated by lectin-capture with AAL-agarose. Culture medium from lung carcinoma cell

23

lines from H661 cells: large-cell carcinoma (LC), H1975 cells: adenocarcinoma (AD), AOI cells:

24

squamous carcinoma (SQ), and SBC1 cells: small cell lung carcinoma (SC) was fractionated with

25

AAL-agarose. SgIII enriched from the flow-through fractions (T) and elution fractions (E) was detected

26

by immunoblotting. The results shown here are representative of three independent experiments of

27

comparisons between SCLS and NSCLC cell lines.

31



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Page 32 of 40

1 2

Figure 5. Specific-fucosylation of short-form SgIII in the sera from patients with SCLC. Serum samples

3

from healthy volunteers (Hv) and patients with SCLC (Sc) and adenocarcinoma (Ad) were fractionated

4

by serial lectin chromatography. Elution fractions of AAL-agarose were compared with immunoblotting

5

using anti-SgIII antibody. The results shown are representative of a preliminary study from three

6

independent experiments with small sample sets composed of sera from Hv, Sc, and Ad.

7 8

32


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1

ASSOCIATED CONTENT

2

Supporting Information

3 4

Figure S1. (A) Comparative analysis of secreted SgIII with data of the ratio (short-form SgIII/total

5

SgIII) from three independent experiments (for Lu165, H441, and TMU3: two independent

6

experiments) among four lung carcinoma types, performed as described for Figure 2. (B) As a statistical

7

validation of the data in Figure 2, Mann-Whitney U tests of unpaired t test data comparing the pro-form

8

and short-form SgIII between SCLC and NSCLC cell lines were carried out.

9 10

Figure S2. Expression of PC2 was examined in selected lung cancer cell lines by immunoblotting using

11

anti-PC2 antibody.

12 13

Figure S3. Identification of glycosylated SgIII peptides by IGOT analysis. Glycopeptides captured by

14

serial lectin capturing (AAL- and ConA-agarose) were analyzed by IGOT LC/MS analysis. Peptide

15

sequences and glycosylation sites of identified glycopeptide signals were determined by MS/MS.

16 17

Figure S4. Fucosylated SgIII was captured by AAL-agarose from sera without a depletion step.

18 19

Tables S1. List of ideal fragmentation-ion values obtained by AAL-captured peptide sequencing of

20

SgIII from H524.

21 22

Tables S2. List of ideal fragmentation-ion values obtained by AAL-captured peptide sequencing of

23

SgIII from H2171.

24 25

Tables S3. List of ideal fragmentation-ion values obtained by ConA-captured peptide sequencing of

26

SgIII from H524.

27 33



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Page 34 of 40

1

Tables S4. List of ideal fragmentation-ion values obtained by ConA-captured peptide sequencing of

2

SgIII from H2171.

3 4

Tables S5. Clinical and pathological characteristics of the samples used in this study.

5 6

Table S6. Gene expression level of SCG3 and PCSK2 (FPKM, cutoff: 0.5 FPKM); data from

7

Expression Atlas database.

8 9

Tables S7. Carbohydrate antigen-related and glycoprotein-related biomarkers (representative list)

10 11

34


Page 35 of 40

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1


TOC only

2

3

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 1 A

B

C

D

E

F


Page 36 of 40

PC-10 OkaC1 EBC1 AOI TMU3 H441 HAL8 RERF-LCMS TMU2 PC-9 HCC827 HAL24 H1975 H1819 H1355 H838 H358 EHHA-9 ABC-1 A549 H2126 H1299 H661 H1155 Lu165 Lu135 Lu134B Lu130 H2171 H524 SBC-2 SBC-1 20

B

Pro-form

Short-form

10 Expression (ng)

Fig. 2

PC-10 OkaC1 EBC1 AOI TMU3 H441 HAL8 RERF-LCMS TMU2 PC-9 HCC827 HAL24 H1975 H1819 H1355 H838 H358 EHHA-9 ABC-1 A549 H2126 H1299 H661 H1155 Lu165 Lu135 Lu134B Lu130 H2171 H524 SBC-2 SBC-1

Cell lines of lung carcinoma


SQ AD LC SC

A

Pro-form Short-form

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research Page 37 of 40


Fig. 3 A

MGF L GT GT WI L VL VL PI QAF PKPGGS QDKS L HNRE L S AE R PL NE QI AE AE

0 50

E DKI KKT YPP E NKPGQS NYS F VDNL NL L KA I T E KE KI E KE RQS I RS S PL D

100

NKL NVE DVDS T KNRKL I DDY DS T KS GL DHK F QDDPDGL HQ L DGT PL T AE D

150

I VHKI AARI Y E E NDRAVF DK I VS KL L NL GL I T E S QAHT L E DE VAE VL QKL

200

I S KE ANNYE E DPNKPT S WT E NQAGKI PE KV T PMAAI QDGL AKGE NDE T VS

250

NT L T L T NGL E RRT KT YS E DN F RDF QYF PNF YAL L KS I DS E KE AKE KE T L I

300

T I MKT L I DF V KMMVKYGT I S PE E GVS YL E N L DE MI AL QT K NKL E KNAT DN

350

I S KL F PAPS E KS HE E T DS T K E E AAKME KE Y GS L KDS T KDD NS NPGGKT DE

400

PKGKT E AYL E AI RKNI E WL K KHDKKGNKE D YDL S KMRDF I NKQADAYVE K * * * GI L DKE E AE A I KRI YS S L 468

N

Signal

Cholesterol-binding domain

ChrA-binding domain CPE-binding domain

: Glycosylation potential site

B

IB: SGIII

450

C

: Putative cleavage site

C

IB: PC2

IB: SGIII

100 kD

100 kD 70 kD

Pro-form Short-form

50 kD 35 kD

100 kD 70 kD

PC2

70 kD

50 kD

50 kD

35 kD

35 kD

Pro-form Short-form

- +

PNGase F

SBC-2

PC-9

SBC-2

PC-9

SBC-2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Fig. 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

LC T

E

AD T

E

SQ T

E

SC T

E

140 kD 100 kD 70 kD

Pro-form Short-form

50 kD 35 kD



Fig. 5 Healthy volunteer Small cell carcinoma Adenocarcinoma

Ad-5

Ad-4

Ad-3

Ad-2

Ad-1

Sc-5

Sc-4

Sc-3

Sc-2

Sc-1

Hv-5

Hv-4

50 kD

Hv-3

140 kD 100 kD 70 kD

Hv-2

Hv-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Short -form

35 kD 20 kD 15 kD


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Glycobiomarker, Fucosylated Short-Form ... - ACS Publications

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