Research

Focused Differential Glycan Analysis with the
Platform Antibody-assisted Lectin Profiling for
Glycan-related Biomarker Verification*□
S

Atsushi Kuno‡, Yukinari Kato‡, Atsushi Matsuda, Mika Kato Kaneko, Hiromi Ito,
Koh Amano, Yasunori Chiba, Hisashi Narimatsu, and Jun Hirabayashi§
Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it isexpected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an
enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is
no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which
relates their specific expression to particular diseases.
Here, we present a new practical system for focused
differential glycan analysis in terms of antibody-assisted
lectin profiling (ALP). In the developed procedure, (i) atarget protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a coreprotein moiety; (ii) the target glycoprotein is quantified
by immunoblotting using the same antibody used in (i);and (iii) glycosylation difference is analyzed by means ofantibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As
model glycoproteins having either N-linked or O-linked
glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis.
As a result, specific signals corresponding to the target
glycoprotein glycans were obtained at a sub-picomole
level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes
could be observed in a rapid, reproducible, and highthroughput manner. Thus, the established systemshould provide a powerful pipeline in support of ongoing efforts in glyco-biomarker discovery. Molecular& Cellular Proteomics 8:99 –108, 2009.

Glycan synthesis in individual cells is regulated by harmonized expression of more than a hundred glycosyltransferases. Importantly, detectable dynamics occurring on eachcell surface during diverse biological events, e.g. differentiation, proliferation, and signal induction, indicate drastic
From the Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central 2,
1-1-1, Umezono, Ibaraki 305-8568, Japan
Received, July 7, 2008, and in revised form, August 11, 2008
Published, MCP Papers in Press, August 11, 2008, DOI 10.1074/
mcp.M800308-MCP200© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
This paper is available on line at http://www.mcponline.org

changes in the glyco-machinery (1). During the last few decades, there have been enormous advances in the findings ofglycosylation alterations related to oncogenesis. Cell surface
sialylation and !1– 6 branching of N-linked glycans arestrongly correlated with metastatic potential of cancer cells (2,3). Metastatic ability is also reflected by dramatic alteration ofcore structures of O-glycans (4). These observations suggestthat novel tumor-specific glycoproteins accompanying substantial structural changes in glycan moieties will become
reliable biomarkers with higher specificity than those established previously, e.g. CA19-9 (5), carcino-embryonic antigen
family (6), "-fetoprotein (AFP)1 (7), and prostate-specific antigen (PSA) (8).
Recent advances in technologies such as mass spectrometry (MS), microarray, and laser microdissection have strongly
advanced the proteomics-based biomarker discovery phasetargeting both cultured cells and tissue specimens (9 –14).
This has resulted in the emergence of an extensive range ofbiomarker “candidates”, many of which are glycoproteins.
However, these candidate molecules need to be subsequently subjected to a verification step prior to a much large
scale validation phase (e.g. treating with !1,000 incidences).
Antibody microarray has taken the place of ELISA as a moreversatile and high-throughput technique, and has enabledmultiplexed quantitative analysis for over a hundred proteins
with sufficient sensitivity (15). As an application of MS technology, multiple-reaction monitoring-MS has also been developed (16 –19). However, taking into consideration the criticalglycosylation changes occurring on diverse glycoproteins,differential analysis of respective glycans is necessary in parallel with quantitative protein analysis. In this context, Chen etal. (20) have recently developed a lectin-overlay antibody
microarray with the intention of discovering glycoprotein biomarkers. However, the proposed strategy requires repeated
1
The abbreviations used are: AFP, "-fetoprotein; ALP, antibodyassisted lectin profiling; PSA, prostate-specific antigen; "PSA, mouseanti-PSA monoclonal antibody; spPSA, seminal plasma PSA; CHO,
Chinese hamster ovary; hpIgG, human polyclonal IgG; LEC, lymphatic
endothelial cell; MS, mass spectrometry; TBS, Tris-buffered saline;
Pod, podoplanin; Sia, sialic acid; hTf, human serum transferrin; Tf,
transferrin; PBS, phosphate-buffered saline; ST, sialylated T; diST,disialylated T antigen; PBSTx, 1% Triton X-100 in PBS; AR, androgen
receptor; GalNAc, N-acetylgalactosamine; Tn, GalNAc"-Ser/Thr.

Molecular & Cellular Proteomics 8.1

99Differential Glycan Profiling for Biomarker Verification

FIG. 1. A, alternative approach to fluorescent detection of lectin-glycan interaction. To observe lectin-glycan interactions in terms offluorescent signals in the lectin microarray, there are two approaches to fluorescence labeling of glycoconjugates, i.e. direct and indirect
methods. In the former direct labeling method, as it means, appropriate fluorescent reagents (e.g. Cy3-SE) are used to label target
glyco-conjugates directly prior to a binding reaction. This method enables profiling of a mixture of glycoproteins from whole cell lysate andtissue extract. Therefore, this approach aiming at total glycan profiling is suited for glycomics-based analysis. In the latter case (indirectlabeling), a target glycoprotein is detected with the aid of both biotinylated specific antibody, raised against the core protein of the target
molecule, and fluorescently labeled streptavidin. B, schematic overview of focused differential glycan profiling using antibody-assisted lectin
microarray. Over 50% of proteins expressed in cell are glycosylated. A variety of glycoproteins exist in abundance, with different proportions
in crude protein samples such as serum and lysates from cells and tissue sections. These would interfere with the lectin-analyte interactions.
To profile the glycan structure of an extremely small amount of a target glycoprotein with the aid of antibody-overlay lectin microarray, (i) the
protein is enriched from such crude samples; (ii) glycoprotein thus enriched is quantified by Western blotting; and (iii) the residual protein issubjected to antibody-overlay lectin microarray. This sequential procedure is defined as a novel and feasible glyco-biomarker discovery
pipeline.

assays with more than 30 lectins to search for critical differences in glycan structures. In the present study, an alternative
approach, i.e. “antibody-overlay lectin microarray” (21), wastaken, which is based on the lectin microarray platform originally developed in our laboratory (22–24). In this strategy, a
specific glycan profile of a target glycoprotein is acquired withthe aid of a specific antibody raised against the core protein
moiety, removing the need to covalently label each glycoprotein with a fluorescent reagent (Refs. 22–29 and Fig. 1A). Oncea target glycoprotein ("100 ng) is enriched from a trace
amount of crude samples by a microliter-scale immunoprecipitation procedure with the biotinylated antibody used forthe above detection, a precise glycan profile of the target
protein is obtained in a highly reproducible and high-throughput manner (see Fig. 1B). The system is unique in its ability to
distinguish critical differences in both core and terminal structures occurring on N- and O-glycans attached to the proteins.

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The combined procedure, consisting of antibody-enrichment,
quantification and overlay detection of lectin microarray, isdesignated antibody-assisted lectin profiling (ALP). The system provides a feasible and powerful advance toward “focused differential glycoproteomics”.
EXPERIMENTAL PROCEDURES

Reagents, Cell Lines, and Tissue Specimen—Reagents were purchased from Sigma, Wako Chemicals (Osaka, Japan), or Nacalai
Tesque (Kyoto, Japan) unless otherwise noted. Seminal plasma PSA
and human serum transferrin (hTf) were obtained from R&D Systems,
Inc. (Minneapolis, MN) and Athens Research & Technology (Athens,GA), respectively. Biotinylated mouse anti-PSA monoclonal antibody(bio-"PSA) and rabbit anti-hTf polyclonal antibody (bio-"hTf) wereobtained from R&D Systems, Inc. and Abcam, Inc. (Cambridge, MA),
respectively. Chinese hamster ovary (CHO) and prostate cancer celllines (LNCaP and PCa2b) were obtained from the American Type
Culture Collection (ATCC). Fifteen glioblastoma cell lines (LN18,
LN215, LN229, LNZ308, LN319, LN340, LN428, LN464, U87, U178,

Differential Glycan Profiling for Biomarker Verification
U251, U373, A1207, SF763, and T98G) were donated by Dr. Webster
K. Cavenee (Ludwig Institute for Cancer Research, San Diego, CA).
Two prostate cancer cell lines (PC3, DU145) were obtained fromTohoku University. Lymphatic endothelial cells (LECs) were purchased from AnioBio (Del Mar, CA). FLAG-tagged hPod-transfected
CHO cell line was prepared as described previously (30). These celllines were cultured at 37 °C in a humidified atmosphere of 5% CO2
and 95% air in RPMI 1640 medium (for CHO, PC3, and DU145) or
Dulbecco’s modified Eagle’s medium (for glioblastoma cell lines,
LNCaP and PCa2b) supplemented with 1 or 10% heat-inactivatedfetal bovine serum (Sigma), 2 mM L-glutamine (Invitrogen), and antibiotics (100 #g/ml of kanamycin for CHO, 100 IU/ml of penicillin, and100 #g/ml of streptomycin for PC3, DU145, LNCaP, and PCa2b). Fora pilot experiment of lectin microarray analysis using tissue sections,
glioblastoma cell line LN319 was transplanted into BALB/c nude
mice. The grown glioblastoma-like tumor was dissected at 5 #m for
immunohistochemical and lectin microarray analyses. Seminoma
specimen was purchased from Cybrdi, Inc. (Frederick, MD).
Large-scale Purification of hPods—hPod/CHO and hPod/LN319
were purified as described by Kato et al. (31). Briefly, cultured cells(1 # 1010 cells/preparation) were lysed with PBS containing 0.25%
Triton X-100. The lysate from CHO was applied on anti-FLAG antibody-conjugated-agarose gel (M2; Sigma). After washing the gel, thecaptured hPod/CHO was eluted with 100 #g/ml FLAG peptide(Sigma). To purify hPod/LN319, the cell lysate was applied on NZ-1
antibody-immobilized resin. After washing the gel, the captured
hPod/CHO was eluted with hpp38 –51 peptide (EGGVAMPGAEDDVV)(31).
Preparation of Recombinant hPods by a Yeast Genetic Engineering
System—A set of recombinant hPods with defined O-glycan structures were prepared as described by Amano et al. (32). Briefly, recombinant glutathione S-transferase and His6-tagged hPods with Tn
and Core1 structures were expressed in TN-1 and T-1 cells, respectively. The hPods purified by affinity chromatography were digested
with Precision protease. After removing glutathione S-transferase tag
fragments, both recombinant hPods were subsequently reacted withappropriate glycosyltransferases to build up STn and Core3 structures on the hPod having Tn and Core2, sialylated T (ST) and disialylated T antigen (diST) structures on the hPod having T (Core1),
respectively.
Immunohistochemical Analysis—Tissue sections on glass slides
were deparaffinized and rehydrated. Testicular tumor specimens
were incubated first with NZ-1 for Pod (a rat monoclonal antibody; 1#g/ml) at room temperature for 1 h, then with biotin-conjugatedsecondary anti-rat IgG antibody (Dako, Glostrup, Denmark) for 1 h,
and finally with peroxidase-conjugated biotin-streptavidin complex(Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA) for
1 h. Color was developed using 3,3$-diaminobenzene tetrahydrochloride tablet sets (Dako) for 3 min. In the case of PSA, prostate cancer
or benign prostate hyperplasia specimens were incubated with bio"PSA at room temperature for 1 h.
Immunoprecipitation—For analysis of endogenous hPod, each cellline was solubilized with lysis buffer (1% Triton X-100 in PBS(PBSTx)). Immunohistochemically hPod-positive cell populations
from tissue sections were excised (about 3.5 mm3) and solubilizedwith ProteoSOL Tissue Extraction System (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The hPods in these lysates were
immunoprecipitated using 10 #g of NZ-1 antibody immobilizedSepharose. After washing the resin with 1 ml of PBS, captured hPods
were released with 20 #l of SDS-PAGE sample buffer excluding
bromphenol blue, reducing agent and glycerol by heat denaturation.
For PSA analysis, PSA was immunoprecipitated from culture supernatant concentrate (20 times) of each cell line using 500 ng of biotinylated anti-PSA monoclonal antibody pre-conjugated to 10 #l ofstreptavidin-immobilized magnetic beads, Dynabeads MyOneTM
Streptavidin T1 (DYNAL Biotech ASA, Oslo, Norway) conjugate. The
beads thus capturing PSA were washed three times with 200 #l of
PBSTx, and then the bound PSA was eluted with 10 #l of the elution
buffer (TBS containing 0.2% SDS) by heat denaturation. After dilution
by an equal volume of 1% Triton X-100 in TBS, the contaminant
antibody was completely depleted by addition of 20 #l of the streptavidin-immobilized magnetic beads.
Western blot Analysis—The purified hPods were electrophoresed
under reducing conditions on 10 –20% polyacrylamide gels. The separated proteins were transferred to a polyvinylidene difluoride membrane. After blocking with 4% skim milk in PBS, the membrane wasincubated with NZ-1 antibody, then with peroxidase-conjugated antirat antibody (1/1000 diluted; GE Healthcare) and developed for 1 minwith ECL reagents (GE Healthcare) using Kodak X-Omat AR film. In
the case of PSA, the membrane was incubated with biotinylated
anti-PSA monoclonal antibody, then with alkaline phosphatase-conjugated streptavidin (1/5000 diluted with TBST; ProZyme, Inc., San
Leandoro, CA). The treated membrane was reacted with Western
Blue" stabilized substrate for alkaline phosphatase (Promega).
Antibody-overlay Lectin Microarray—Antibody-overlay lectin microarray was basically performed as described previously (22–24).
Briefly, the purified proteins were diluted to 60 #l with PBSTx or
TBSTx and then applied to the lectin array containing triplicate spotsof 43 lectin (see supplemental Fig. S1) into each of 8-divided incubation baths on the epoxy-coated glass slide from Schott AG (Mainz,
Germany). After incubation at 20 °C for 12 h, 20 #g of human serum
polyclonal IgG was added to the glass slide followed by 30-min
incubation. The reaction solution was discarded, and the glass slide
was washed three times with PBSTx. 60 #l of biotinylated antibody
solution in PBSTx was applied to the array and then incubated at
20 °C for 1 h. After washing three times with PBSTx, 60 #l of Cy3labeled streptavidin (GE Healthcare) solution in PBSTx was added tothe array and then incubated at 20 °C for 30 min. The glass slide wasrinsed with PBSTx and scanned by an evanescent-field fluorescencescanner, GlycoStationTM (Moritex, Co., Tokyo, Japan). All data wereanalyzed with the Array-Pro analyzer version 4.5 (Media Cybernetics,Inc.). The net intensity value for each spot was calculated by subtracting background value from signal intensity values of three spots.
Glycosidase Treatment—To remove Sia residues, hPods were incubated with Sialidase ATM from Arthrobacter ureafaciens (ProZyme,
Inc.) or "-2,3-Neuraminidase from Macrobdella decora expressed in
Escherichia coli (ProZyme, Inc.) at 37 °C for 2 h. hPods were also
digested with both Sialidase ATM and O-GlycanaseTM from Streptococcus pneumoniae expressed in E. coli (ProZyme, Inc.) at 37 °C for
12 h to remove Core1-related O-glycans completely.
Platelet Aggregation Assay by WBA Carna—Platelet aggregating
activity was measured by WBA Carna (IMI, Saitama, Japan) as described previously (31). Heparinized mouse whole blood was drawn
from BALB/c mice. Two hundred microliters each of mouse whole
blood samples and NZ-1 or control rat IgG in four reaction tubes wasstirred at 1,000 rpm at 37 °C, and pre-incubated for 2 min, followedby the addition of 12 #l each of cells (2 # 107 cells/ml). One to fiveminutes later, whole blood samples were sucked to detect aggregation pressure at a rate of 200 #l/6.4 s using a syringe containing
screen microsieves made of nickel, 3.7 mm in diameter, with 300
openings of 20 # 20 #m2 in a 1-mm diameter area. The final platelet
aggregation pressure of each reaction tube was determined at thepressure rate (%) of a pressure sensor connected to the syringe.
RESULTS

Fabrication of Antibody-overlay Lectin Microarray—To
achieve extremely high throughput and feasibility, the antibody-overlay lectin microarray is considered to be an ideal

Molecular & Cellular Proteomics 8.1

101

Differential Glycan Profiling for Biomarker Verification

FIG. 2. Optimization of experimental conditions for antibody-overlay lectin microarray. A, a concept for noise reduction without loss of
specific signals for an analyte. Glycans connected to a detection antibody potentially interact with lectins immobilized on the glass slide (top).
To avoid this, the analyte-bound microarray is blocked with hpIgG prior to incubation with a detection antibody (bottom). B, effect of theblocking reagent on the background noise reduction because of detection antibody. A scan image of antibody-overlay lectin microarray for hTf(top). Positive signal intensities (P; in the presence of hTf) are subtracted by negative one (N; in the absence of hTf). The resultant intensities
with or without blocking treatment are indicated as closed or open bars, respectively (bottom). C, lectin microarray analysis of Cy3-labeled hTf
in the presence (black) or absence (white) of hpIgG. Cy3-labeled hTf-bound lectin microarray was incubated with an excess amount of hpIgG
for 1 h followed by scanning (top). The obtained signals on SNA (F), SSA (f), TJA-I (!), and RCA120 (Œ) are elevated in a dose-dependent
manner (bottom). No substantial signal because of the blocker hpIgG was observed upon binding of Cy3-labeled hTf.

approach to achieve differential glycan analysis targeting aparticular glycoprotein, by using a lectin microarray infrastructure (21). From a practical viewpoint, there is a critical issue tobe overcome when using antibodies for glycoprotein detection, since the antibody glycans potentially interact with lectins immobilized on the glass slide (Fig. 2A, top). To examine
this possibility, we first carried out a pilot experiment of theantibody-overlay lectin microarray using hTf, which mainlycontained sialylated biantennary, complex-type N-glycans at
two N-glycosylation sites (22), as a model glycoprotein. Lectin
microarray comprising 43 lectins (supplemental Fig. S1) was
incubated first with or without 1 ng of hTf and then with 20 ng
of biotinylated rabbit anti-hTf polyclonal antibody followed byCy3-streptavidin detection as described under “Experimental
Procedures”. Signals were observed both in the presence andabsence of hTf on extensive spots of "2– 6 Sia-binders, SSA,
SNA, and TJA-I, as well as a !-Gal-binder, RCA120 (Fig. 2B).
This indicates a typical glycan profile to serum antibody, i.e.
partially "2– 6 sialylated biantennary complex type N-glycan.
In fact, positive signal intensities (P) (in the presence of hTf)
were almost comparable with those of negative ones (N) (inthe absence of hTf), indicating the derived signals were notreliable. To suppress such undesirable background noise,
attributed to the detection antibody, while preserving specificsignals of hTf, effective blocking of the analyte (hTf)-boundmicroarray was attempted using an excess amount of nonlabeled “human” polyclonal IgG (hpIgG) prior to detection byanti-hTf antibody (Fig. 2A, bottom). The blocker, hpIgG, having substantially the same glycoforms as those of the detec-

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Molecular & Cellular Proteomics 8.1

tion antibody (“rabbit” polyclonal antibody in this case), butlacking the epitope (biotin) for fluorescence detection, is expected to work for masking the residual binding sites on thearray. Technical consideration was also taken to minimize theblocking time (30 min) to suppress the analyte exchange
between the pre-captured analyte (hTf) and an excess amountof the blocker hpIgGs. However, no detectable signal reduction was observed even after 2 hours of incubation with theblocker hpIgG, as examined by direct probing of the arraywith Cy3-labeled hTf (Fig. 2C). As a result of blocking with 20#g of hpIgG, the background signals totally disappeared in
the absence of hTf, whereas intense (i.e. specific) signals wereobserved, e.g. on RCA120, in the presence of the analyte (Fig.
2B). The obtained signal pattern was closely correlated withthe reported glycan structures of hTf (21).
Focused Differential Glycan Profiling of an N-glycosylated
Protein, PSA—To profile a relatively small amount ("100 ng)
of a target glycoprotein with the aid of antibody-overlay
lectin microarray, the target should be enriched in advancefrom crude samples. Here, a crucial point is how ALP is
designed to realize an effective procedure for the verification of glyco-biomarker candidates. In our development
concept, a single antibody plays the following three roles; (i)prior enrichment by immunoprecipitation; (ii) quantification
by Western blotting; and (iii) glycan profiling by antibodyoverlay lectin microarray (Fig. 1B). Based on these criteria,
we attempted differential glycan profiling of prostate-specific antigen (PSA), which is known to be specifically expressed in AR-positive prostate cancer cells, and has aDifferential Glycan Profiling for Biomarker Verification

spPSA
bTf
αPSA

2 ng 20 ng
20 ng
+
+
+

4

Net intensity

LCA
PSA
UEA-I
AOL
AAL
MAL
SNA
SSA
TJA-I
PHA(L)ECL
RCA120PHA(E)
DSA

3
2
1
0

[spPSA] (ng/well)
LNCaP

spPSA

WGA
WFA
UDA
STL
LEL
BPL
HHL
ConA
DSA
PHA(E)
RCA120
ECA
TJA-I
SSA
SNA
MAL
AAL
AOL
LCA
PSA
0

1

0

PCa2b

1

0

1

FIG. 3. Focused differential glycan profiling of PSA. A, dose-dependent fluorescent signals of the spPSA for 14 lectins (left). No
signal was obtained for bovine serum transferrin (bTf) as a negative
control. The binding isotherms of interaction of eight positive lectinswith spPSA (right) are shown except for SNA, SSA, and TJA-I; AAL(#), AOL (f), LCA ("), Pisum sativum agglutinin (!), PHA(E) (‚),
RCA120 (Œ), PHA(L) (E), and MAL (F), respectively. B, differential
glycan profiling among PSAs from different origin. The microarray was
incubated with PSAs enriched from seminal plasma and culture supernatants of LNCaP and PCa2b cells. Relative intensities of 20
lectins (i.e. maximal fluorescent intensity defined as 1 on each lectin
array) are indicated as horizontal bars. Typical glycan epitopes (structural units) relevant to the present analysis are also indicated by theConsortium for Functional Glycomics format.

single N-glycosylation site (33, 34). Firstly, various concentrations (2%20 ng/60 #l) of normal seminal plasma PSA(spPSA) were applied to the lectin microarray followed by
hpIgG blocking as described, and detection with 50 ng of
biotinylated mouse anti-PSA monoclonal antibody (bio"PSA). As shown in Fig. 3A, specific signals were obtainedin a dose-dependent manner, whereas essentially no signal
was obtained for bovine serum transferrin as a negative
control. The resultant glycan profile of spPSA provides thefollowing information: (i) modification of biantennary N-glycan with Core "1– 6 fucosylation as evident in signals on
LCA, Pisum sativum agglutinin, AOL, and AAL; (ii) heavy"2– 6 sialylation associated with strong signals on SNA,
SSA, and TJA-I; and (iii) partial "2–3 sialylation attributed toa weak signal on MAL. All data are consistent with a recent
analysis by high performance liquid chromatography andMS (34).
Glycan analysis was then performed with the optimized
procedure toward PSA enriched from the culture supernatantof a prostate cancer cell line, LNCaP. Following 20-fold concentration of the supernatant by ultrafiltration, PSA secreted
from LNCaP (designated hereafter PSA/LNCaP) was immunoprecipitated from 20 #l of the concentrate with 500 ng ofbio-"PSA, which had previously been conjugated with 10 #lof streptavidin-coated magnetic beads. After extensive washing, the captured PSA was released by heat treatment in thepresence of 0.2% SDS, and the denatured bio-"PSA coeluted with PSA was selectively recaptured with 20 #l of thestreptavidin-coated beads. Thus enriched PSA was quantified
by Western blotting to yield about 20 ng (1 ng/#l). Subsequently, the residual fraction of enriched PSA (5 ng) was
subjected to antibody-overlay lectin microarray using thesame bio-"PSA. Intense signals were observed on some lectin spots. No substantial signal was obtained by direct probing (i.e. without enrichment) the microarray even with 20-foldconcentrated crude culture supernatant (supplemental Fig.
S2). Although a residual amount of bio-"PSA (i.e. 10 ng or 2%of the input antibody estimated by Western analysis) actuallycontaminated the analyte, it did not cause substantial background noise nor interference with the specific signals. PSA/LNCaP showed notable differences compared with spPSA
described above (Fig. 3B and supplemental Fig. S2). (i) Drastic
reduction in sialylation signals on both "2– 6 Sia-binders,
SNA, SSA, and TJA-I and an "2–3 Sia-binder, MAL. (ii) Complementary increment of terminal Lac/LacdiNAc structuresassociated with signals on RCA120, ECA, WGA and WFA. (iii)
A higher terminal fucose content represented by Lewis antigens (Fuc"1–3Gal or Fuc"1– 4Gal) and H-antigen (Fuc"1–
2Gal) evident on AAL and AOL signals. These structural features correlate well with results in previous reports (33, 34).
Thus, the developed system allows differential glycan profilingof a target glycoprotein even in a small quantity contained ina cultured supernatant.
To extend this approach, three more human prostate cancer cell lines; AR-positive MDA PCa2b, AR-negative PC3 andDU145, were analyzed. Twenty-microliter of each concentrated culture supernatant was used for enrichment as described. Western blot quantification revealed that PCa2b gave
%3 times lower amount (i.e. %0.33 ng PSA/#l of elution fraction) of the analyte (PSA) compared with LNCaP. Thus, 15 #lof the elution fraction of PSA/PCa2b (5 ng) was applied to thelectin microarray (supplemental Fig. S3). It was found that thesignal pattern of PSA/PCa2b was clearly different from thoseof spPSA and PSA/LNCaP (Fig. 3B). Although there has been
no report on glycan structures of PSA/PCa2b, notable features of PSA/PCa2b can be documented as regards comparison with psPSA: (i) drastic decrease in "2– 6 sialylation as
observed in PSA/LNCaP; (ii) corresponding increment of ter-Molecular & Cellular Proteomics 8.1

103Differential Glycan Profiling for Biomarker Verification
minal "2–3 sialylation and LacNAc structures evident by signals on an "2–3 Sia-binder, MAL, and LacNAc-binders,
RCA120 and ECA, respectively; and (iii) higher frequency of
core-fucosylation on biantennary complex type N-glycans revealed by signals on LCA and Pisum sativum agglutinin.
These signals also imply an increase in high-mannose structures together with other mannose-binders, UDA, HHL and
ConA. On the other hand, when compared with the previous
cell line, LNCaP; (i) drastic reduction in signals on WGA as wellas WFA (both having affinity to N-acetyl group); and in contrast (ii) dramatic increase in PHA(E) signal suggesting thepresence of bisecting GlcNAc are depicted. Throughout theanalysis, "2– 6 de-sialylation of N-glycan is a consistently
evident feature shared by the AR-positive prostate cancer celllines (LNCaP and PCa2b). However, their profiles did notnecessarily show particular similarity to the one reported recently for serum PSA derived from prostate cancer (PC) patients, except for "2–3 Sia increment. It should be mentioned,
however, that serum PSA level is abnormally high (!10 #g/ml
serum) in these patients (35). From a clinical viewpoint, it isimportant to differentiate PSA glycoforms of malignant prostate cancer from benign prostate hyperplasia. With the ultrasensitivity and preciseness of the developed system, it will bepossible to achieve differential profiling using patient sera
containing even a small amount ("100 ng/ml serum) of PSA.
Focused Differential Glycan Profiling of a Mucin-like Glycoprotein, Podoplanin—Glycosylation change in mucin has also
been a potential target for a glyco-biomarker in the context oftumor progression and metastatic potential, particularly because it represents a major component of epithelia, the major
origin of tumorigenesis. Human platelet-aggregating factor
podoplanin (hPod) was chosen as a model mucin-like glycoprotein from both practical and biological points of view. hPod
is expressed in many tumor cells, such as testicular germ celltumors, brain tumors, several squamous cell carcinomas, andmalignant mesothelioma (31, 36 –39), while it is also known as
a lymphatic endothelium marker (40). Recent structural analysis of glioblastoma-derived hPod demonstrated that it mainly
bears fully sialylated Core1, i.e. diST (30, 31, 41). More recently, we produced recombinant hPods by a yeast genetic
engineering system, which have homogenous O-glycan structures (32). In this study, we prepared a set of recombinant
hPods with defined O-glycan structures, i.e. Tn, T (Core1),
Core2, Core3, sialylated Tn (STn), ST, or diST. As a result ofantibody-overlay lectin microarray analysis with the aid of anestablished monoclonal antibody, NZ-1 (31), the seven recombinant glycoproteins (each 10 ng) gave totally different
signal patterns from one another, whereas Core1 and its
structural derivatives were in part contaminated with Tn. The
results of ten lectins showing critical difference are summarized in Fig. 4, where a unique representation format allows us
to follow structural changes easily (for details, see legend to
Fig. 4). For instance, 6-OH modification of the reducing terminal GalNAc is rejected by a Core1/GalNAc-binder, Jacalin

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FIG. 4. Focused differential glycan profiling of podoplanins(Pods). Relation of signal patterns of ten lectins with alterations ofO-glycan structure in hPod. hPod with a specific O-glycan, i.e. either
Tn or T (Core1), was over-expressed in yeast as described previously(32). Both recombinant hPods were subsequently reacted with appropriate glycosyltransferases (each step was indicated by an arrow)to build up Core3 and STn structures on the hPod having Tn, orCore2, ST, and diST structures on the hPod having T (Core1), respectively. Glycan changes by Sialidase ATM treatment were noted bydashed arrows. O-glycan structures are shown by the Consortium for
Functional Glycomics format. Each of the engineered hPods wasanalyzed by antibody-overlay lectin microarray with biotinylated NZ-1
antibody. The binding signals of the seven analytes observed on
selected ten lectin spots are shown as a patterning diagram with
ten-divided columns: positive and negative interactions are indicatedas black and white boxes, respectively. For instance, Jacalin (column3) cannot bind to STn and diST, of which positions are colored inwhite. Thus, Jacalin rejects 6-OH modification of reducing terminal
GalNAc residue. In other words, Jacalin works as a “zone-discriminator” regarding 6-OH modification, showing no binding to modified
structures. On the other hands, PNA (column 4) works as an “epitope
detector”, indicating the presence of Core 1 (T). ABA can react with allof the O-glycan structures relevant to this study. Core3 and ST
structures, which show identical pattern diagrams to each other, canbe clearly differentiated after Sialidase ATM treatment, which specifically works on ST.

(42), while it is accepted by another Core1-binder, ACA. It is
known that difference in sialylation frequency affects theplatelet-aggregating activity of hPod (31) (also see Fig. 5A).
This fact is easily traced with the combination of five lectins,

Differential Glycan Profiling for Biomarker Verification

FIG. 5. Effect of sialylation of hPod on its platelet aggregating
activity. A sialylated mucin-like glycoprotein hPod/CHO was purified
as described previously (30), then reacted with Sialidase ATM or"2,3-Neuraminidase to obtain desilalylated hPods with different degree of sialylation. The platelet aggregating activity of each hPod wasevaluated as described under “Experimental Procedures” (A). The
obtained hPods, together with hPod/CHO deglycosylated by O-glycanase, were qualified by antibody-overlay lectin microarray as described under “Experimental Procedures” (B). Relative intensities of
eleven lectins with hPod/CHO derivatives determined from the ratio tothe fluorescent intensity on Jacalin are indicated with black (intact),gray (with "2,3-Neuraminidase), and white bars (with Sialidase ATM),
respectively.

i.e. Jacalin, BPL, PNA, MAH and WGA, where their fluorescence intensities are normalized relative to Jacalin (also see
Fig. 5, B and C).
To prove the ultra-sensitivity of ALP from a more practical
viewpoint, we extended the approach to LEC, because
glycosylation features of hPod/LEC have never been characterized because of its low natural abundance. hPod/LEC
was immunoprecipitated from 1-dish cultured cells (%5 #106) with 10 #l of NZ-1-Sepharose, and the obtained glycan
profile was compared with those of both recombinant hPod
over-expressed in CHO cells (hPod/CHO) and endogenous
hPod from human glioblastoma LN319 cell line (hPod/
LN319), the latter being the most over-expressing cell line of

FIG. 6. Differential analysis of fifteen kinds of glioblastoma
cells. Every cell line was cultured, harvested and lysed with PBS
containing 0.25% Triton X-100. Each lysate was incubated with NZ-1
antibody immobilized resin and captured hPods were eluted in SDSPAGE sample buffer with heating denature. The obtained hPods were
quantified by Western blotting (top), and subjected to antibody-overlay lectin microarray (bottom). Asterisks (* and **) denote the bands
corresponding to heavy chain of Nz-1 antibody and hPod, respectively. Evidently the signal intensity of each cell line was correlated
well with the expression level of hPod revealed by Western analysis.

glioblastoma (see Refs. 30, 31 and Fig. 6). Notably, glycan
profile of the hPod/CHO thus enriched differed from that of
hPod/CHO obtained by a large scale preparation describedabove. This is probably because of the difference in elution
conditions: i.e. in the former procedure, the bound antigen
was eluted by heat denaturation in the presence of SDS,
while in the latter, it was eluted more mildly by adding acompetitive peptide having affinity to immobilized Nz-1 antibody. This may affect the binding pattern on less stablelectins, e.g. WGA. Nevertheless, we confirmed that the resultant signal patterns of desialylated forms of the three hPod
preparations were closely similar to one another (supplemental
Fig. S4A). However, when untreated hPods were compared, aglycan profile of hPod/LEC considerably differed from hPod/
CHO, while it closely resembled that of hPod/LN319 (supplemental Fig. S4A). The observed differences are interpreted as
higher frequency of "2– 6 sialylation at the reducing terminal
GalNAc, which is evident from both increment of the MAH
signals and complementary decrement of the Jacalin signals(Figs. 4 and 7).

Molecular & Cellular Proteomics 8.1

105Differential Glycan Profiling for Biomarker Verification

FIG. 8. Challenge to glycan profiling of hPod enriched from atissue lysate. Podoplanin-positive area on the counterpart sections
was selectively dissected (excised volume estimated to be %3.6mm3). hPod was enriched with NZ-1-immobilized-Sepharose andwas analyzed by antibody-overlay lectin microarray. For comparison,
hPod enriched from tumor section of LN319 cell-transplanted mice
was also analyzed as a positive control. Desilalylated hPods were also
subjected to antibody-overlay lectin microarray.
FIG. 7. Differential glycan profiling among hPod/CHO, hPod/
LN319, and hPod/LEC. The three hPods were analyzed as theirsialylated and desilalylated forms (for details, see Fig. S4). Net intensities of three representative lectins (Jacalin, MAH, and ABA) withsialylated and desialylated forms are indicated with close (sialidase
minus) and open bars (sialidase plus), respectively (A). Regarding
hPod/CHO and hPod/LEC, differences in the signal ratios of therelevant lectins, i.e. Jacalin/ABA and MAH/ABA, were found to bestatistically significant based on the Student’s t test (p " 0.01) in the
microarray analyses (n & 4) (B).

As mentioned above, expression of endogenous hPod has
been found histologically in an extensive range of tumors. A
comprehensive glycan analysis of hPod from various types oftumor cells should give a direct answer to whether or not the
sialylated O-glycans are actually involved in platelet aggregating-activity of hPod, which might lead to enhanced metastatic potential. It was therefore decided to apply the developed ALP system to hPod extracted from a relevant tissue
section. For this purpose, hPod-positive seminoma cells(%3.6 mm3) were scratched from a deparaffinized and formalin-fixed testicular tissue section followed by extraction of
membrane proteins. After immunoprecipitation as described,
a 1/10 aliquot of the enriched protein was subjected to antibody-overlay lectin microarray to obtain a unique signal pattern to hPod derived from seminoma (supplemental Fig. S5).
Although significant signals were observed on ABA and Jacalin in the desialylated form of hPod/seminoma as well as
xenograft LN319, intact hPod showed strong signals on ACA
and WGA. Since signals on both ABA and Jacalin were greatly
reduced, it strongly implies a high degree of 6-OH modification of the reducing terminal GalNAc residue of Core1, e.g.
diST (Fig. 8). Notably, the WGA signal remained even after
desialylation (Figs. 4 and 8). By comparison with the patterning diagrams obtained for the engineered hPods (Fig. 4),
where WGA signal was specifically observed in Core2-hPodprobe, the Core2 structure is the most likely glycoform expressed on hPod/seminoma. Furthermore, the binding signals

106

Molecular & Cellular Proteomics 8.1

on terminal GalNAc binders, WFA and VVA (see pattern diagrams of STn and Tn in Fig. 4), rather than that on a Core1binder, PNA, were specifically observed in desialylated hPod/seminoma. These results strongly suggest that hPod/seminoma expresses STn structure, i.e. the 6-OH sialylated
form of an immature Core1 (Tn), as well as diST and Core2.
This structural diversity of hPod/seminoma is considered torepresent the heterogeneous nature of the tissue specimens.
It should be emphasized that such information has never beenobtained by conventional procedures requiring a much larger
amount of pure samples. The developed system ALP therefore constitutes a great practical advance toward the realization of ultra-sensitive, highly precise glycan profiling of marker
candidate glycoproteins.
DISCUSSION

Glycosylation alteration occurring on glycoproteins hasoften been discussed in relation to oncogenesis and tumor
progression and provides potential targets for serologicalprotein biomarker development. Recent advances in glycoproteomics produced a number of glycoproteins, whichhave been nominated as disease-related biomarker candidates (14, 43– 47). As a standard procedure employed inconcurrent proteomics-based biomarker development, thecandidates are first quantified with the samples used in
the candidate identification phase, then verified via a hundred clinical samples before the last validation phase (10). In
general, serum concentrations of diagnostic glycoproteins
are too low to detect their glycosylation changes in an
unambiguous manner. In the case of hepatocellular carcinoma (HCC), the presence of !35% AFP-L3 fraction, i.e.
LCA-reactive AFP, among the total AFP glycoforms, hasbeen established as a diagnostic and prognostic marker
with 100% specificity (7). Nevertheless, for this diagnosis,
total AFP values in patient serum are in the range 10 –200
ng/ml (48). Other glycosylation alterations include a serological prostate cancer marker, PSA, for which a gray zoneDifferential Glycan Profiling for Biomarker Verification
concentration to distinguish prostate cancer from benign
prostate hyperplasia resides between 4 and 10 ng/ml inserum. A reliable technology, from sample preparation to
analysis, enabling a nanogram-scale analysis is urgently
required for these low-abundance target molecules. To improve the present diagnostic approach, ALP is considered
to be the best candidate as a verification system. Its feasibility depends on dual focusing of analytes in crude glycoprotein solution with antibody, i.e. nanogram-scale immunoprecipitation and subsequent antibody-overlay detection,
which has proven to be an ultrasensitive system for “focused differential glycan analysis”.
Technical points to be refined in the above profiling system
are sensitivity and noise reduction because of the antibodies
used. In fact, previous investigators attempting a similar approach used up to a microgram order of glycoproteins, which
severely restricted an analytical range to relatively abundanttarget proteins (21). Our recently improved lectin microarraysystem assures ultra-high sensitive analysis, i.e. a nanogram
order of glycoproteins (24). The problem of background noise
was solved by an extremely simple masking procedure; i.e.
using non-labeled heterospecies IgG. Successful analysis of
endogenous glycoproteins, PSA and Pod, representing N- andO-glycoproteins, was demonstrated using a small amount ofsample comparable with Western blotting analysis. This significant improvement will help to make the developed ALP systemwidespread over the relevant fields including molecular, cellular,
and tissue biologies, as well as clinical sciences.
In conclusion, a highly feasible system, ALP, has been
proposed as the best-focused differential glycan profilingtechnology. With this system, a glyco-biomarker discovery
pipeline targeting proteomics-based biomarker discovery as
described by Rifai et al. (10) will be rapidly established. Protocols for nanogram-scale verification have been constructed
for over twenty kinds of glycoprotein marker candidates toward the forthcoming validation phase2, where in turn an
immune-based sandwich assay should work better, i.e. lectinoverlay antibody microarray (20). For realization of this, selection of appropriate lectin probes is critical to differentiatedisease-specific glycan alterations. Both microarray technologies are necessary and should be combined to achieve rapid
glyco-biomarker development in the near future.
Acknowledgments—We thank N. Uchiyama, Y. Kubo, J. Murakami,
and S. Koizumi for technical assistance. We are very grateful to Dr.
Kazuhiko Mishima, Dr. Isoji Sasagawa, and Dr. Webster K. Cavenee
for their generous donation of materials.
* This work was supported in part by a grant from New Energy andIndustrial Technology Development Organization, Japan. The costs ofpublication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2

A. Kuno, H. Narimatsu, and J. Hirabayashi, unpublished results.

□
S The on-line version of this article (available at http://www.
mcp.org) contains Supplemental Figs. S1–S5. All lectin abbreviations
are given in Supplemental Table 1 and Supplemental Fig. S1.
‡ Both authors contributed equally to this study.
§ To whom correspondence should be addressed: Research Center for Medical Glycoscience, National Institute of Advanced Industrial
Science and Technology (AIST), AIST Tsukuba Central 2, 1-1-1 Umezono, Ibaraki 305-8568, Japan. Ph.: 81-29-861-3124; Fax: 81-29861-3125. E-mail: jun-hirabayashi@aist.go.jp.
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