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Molecular & Cellular Proteomics 8:99-108, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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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

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

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected 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) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-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 high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.

Recent advances in technologies such as mass spectrometry (MS), microarray, and laser microdissection have strongly advanced the proteomics-based biomarker discovery phase targeting both cultured cells and tissue specimens (9–14). This has resulted in the emergence of an extensive range of biomarker "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 more versatile and high-throughput technique, and has enabled multiplexed 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 critical glycosylation changes occurring on diverse glycoproteins, differential analysis of respective glycans is necessary in parallel with quantitative protein analysis. In this context, Chen et al. (20) have recently developed a lectin-overlay antibody microarray with the intention of discovering glycoprotein biomarkers. However, the proposed strategy requires repeated 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), was taken, 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 with the 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). Once a target glycoprotein (<100 ng) is enriched from a trace amount of crude samples by a microliter-scale immunoprecipitation procedure with the biotinylated antibody used for the 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. The combined procedure, consisting of antibody-enrichment, quantification and overlay detection of lectin microarray, is designated antibody-assisted lectin profiling (ALP). The system provides a feasible and powerful advance toward "focused differential glycoproteomics".

View larger version (33K): [in this window] [in a new window]   FIG. 1. A, alternative approach to fluorescent detection of lectin-glycan interaction. To observe lectin-glycan interactions in terms of fluorescent 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 and tissue extract. Therefore, this approach aiming at total glycan profiling is suited for glycomics-based analysis. In the latter case (indirect labeling), 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 is subjected to antibody-overlay lectin microarray. This sequential procedure is defined as a novel and feasible glyco-biomarker discovery pipeline.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Large-scale Purification of hPods—
hPod/CHO and hPod/LN319 were purified as described by Kato et al. (31). Briefly, cultured cells (1 x 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, the captured hPod/CHO was eluted with 100 µg/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 with appropriate 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 µg/ml) at room temperature for 1 h, then with biotin-conjugated secondary 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 cell line 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 solubilized with ProteoSOL Tissue Extraction System (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The hPods in these lysates were immunoprecipitated using 10 µg of NZ-1 antibody immobilized-Sepharose. After washing the resin with 1 ml of PBS, captured hPods were released with 20 µl 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 µl of streptavidin-immobilized magnetic beads, Dynabeads MyOne^TM Streptavidin T1 (DYNAL Biotech ASA, Oslo, Norway) conjugate. The beads thus capturing PSA were washed three times with 200 µl of PBSTx, and then the bound PSA was eluted with 10 µl 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 µl 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 was incubated with NZ-1 antibody, then with peroxidase-conjugated anti-rat antibody (1/1000 diluted; GE Healthcare) and developed for 1 min with 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 µl with PBSTx or TBSTx and then applied to the lectin array containing triplicate spots of 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 µg 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 µl 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 µl of Cy3-labeled streptavidin (GE Healthcare) solution in PBSTx was added to the array and then incubated at 20 °C for 30 min. The glass slide was rinsed with PBSTx and scanned by an evanescent-field fluorescence scanner, GlycoStation^TM (Moritex, Co., Tokyo, Japan). All data were analyzed 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 A^TM 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 A^TM and O-Glycanase^TM 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 was stirred at 1,000 rpm at 37 °C, and pre-incubated for 2 min, followed by the addition of 12 µl each of cells (2 x 10⁷ cells/ml). One to five minutes later, whole blood samples were sucked to detect aggregation pressure at a rate of 200 µl/6.4 s using a syringe containing screen microsieves made of nickel, 3.7 mm in diameter, with 300 openings of 20 x 20 µm² in a 1-mm diameter area. The final platelet aggregation pressure of each reaction tube was determined at the pressure rate (%) of a pressure sensor connected to the syringe.

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Fabrication of Antibody-overlay Lectin Microarray—
To achieve extremely high throughput and feasibility, the antibody-overlay lectin microarray is considered to be an ideal approach to achieve differential glycan analysis targeting a particular glycoprotein, by using a lectin microarray infrastructure (21). From a practical viewpoint, there is a critical issue to be 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 the antibody-overlay lectin microarray using hTf, which mainly contained 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 by Cy3-streptavidin detection as described under "Experimental Procedures". Signals were observed both in the presence and absence of hTf on extensive spots of 2–6 Sia-binders, SSA, SNA, and TJA-I, as well as a β-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) (in the absence of hTf), indicating the derived signals were not reliable. To suppress such undesirable background noise, attributed to the detection antibody, while preserving specific signals of hTf, effective blocking of the analyte (hTf)-bound microarray was attempted using an excess amount of non-labeled "human" polyclonal IgG (hpIgG) prior to detection by anti-hTf antibody (Fig. 2A, bottom). The blocker, hpIgG, having substantially the same glycoforms as those of the detection antibody ("rabbit" polyclonal antibody in this case), but lacking the epitope (biotin) for fluorescence detection, is expected to work for masking the residual binding sites on the array. Technical consideration was also taken to minimize the blocking time (30 min) to suppress the analyte exchange between the pre-captured analyte (hTf) and an excess amount of the blocker hpIgGs. However, no detectable signal reduction was observed even after 2 hours of incubation with the blocker hpIgG, as examined by direct probing of the array with Cy3-labeled hTf (Fig. 2C). As a result of blocking with 20 µg of hpIgG, the background signals totally disappeared in the absence of hTf, whereas intense (i.e. specific) signals were observed, e.g. on RCA120, in the presence of the analyte (Fig. 2B). The obtained signal pattern was closely correlated with the reported glycan structures of hTf (21).

View larger version (29K): [in this window] [in a new window]   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 the blocking 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 (•), SSA (), TJA-I (), and RCA120 () are elevated in a dose-dependent manner (bottom). No substantial signal because of the blocker hpIgG was observed upon binding of Cy3-labeled hTf.

View larger version (24K): [in this window] [in a new window]   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 lectins with spPSA (right) are shown except for SNA, SSA, and TJA-I; AAL (), AOL (), LCA (), Pisum sativum agglutinin (), PHA(E) (), RCA120 (), PHA(L) (), and MAL (•), 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 the Consortium for Functional Glycomics format.

To extend this approach, three more human prostate cancer cell lines; AR-positive MDA PCa2b, AR-negative PC3 and DU145, 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/µl of elution fraction) of the analyte (PSA) compared with LNCaP. Thus, 15 µl of the elution fraction of PSA/PCa2b (5 ng) was applied to the lectin microarray (supplemental Fig. S3). It was found that the signal pattern of PSA/PCa2b was clearly different from those of 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 terminal 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 well as WFA (both having affinity to N-acetyl group); and in contrast (ii) dramatic increase in PHA(E) signal suggesting the presence of bisecting GlcNAc are depicted. Throughout the analysis, 2–6 de-sialylation of N-glycan is a consistently evident feature shared by the AR-positive prostate cancer cell lines (LNCaP and PCa2b). However, their profiles did not necessarily 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 µg/ml serum) in these patients (35). From a clinical viewpoint, it is important to differentiate PSA glycoforms of malignant prostate cancer from benign prostate hyperplasia. With the ultra-sensitivity and preciseness of the developed system, it will be possible 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 of tumor 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 cell tumors, brain tumors, several squamous cell carcinomas, and malignant 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 of antibody-overlay lectin microarray analysis with the aid of an established 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 (42), while it is accepted by another Core1-binder, ACA. It is known that difference in sialylation frequency affects the platelet-aggregating activity of hPod (31) (also see Fig. 5A). This fact is easily traced with the combination of five lectins, i.e. Jacalin, BPL, PNA, MAH and WGA, where their fluorescence intensities are normalized relative to Jacalin (also see Fig. 5, B and C).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Focused differential glycan profiling of podoplanins (Pods). Relation of signal patterns of ten lectins with alterations of O-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, or Core2, ST, and diST structures on the hPod having T (Core1), respectively. Glycan changes by Sialidase ATM treatment were noted by dashed arrows. O-glycan structures are shown by the Consortium for Functional Glycomics format. Each of the engineered hPods was analyzed 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 indicated as black and white boxes, respectively. For instance, Jacalin (column 3) cannot bind to STn and diST, of which positions are colored in white. 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 all of the O-glycan structures relevant to this study. Core3 and ST structures, which show identical pattern diagrams to each other, can be clearly differentiated after Sialidase ATM treatment, which specifically works on ST.

View larger version (31K): [in this window] [in a new window]   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 was evaluated 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 to the fluorescent intensity on Jacalin are indicated with black (intact), gray (with 2,3-Neuraminidase), and white bars (with Sialidase ATM), respectively.

View larger version (69K): [in this window] [in a new window]   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 SDS-PAGE 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.

View larger version (35K): [in this window] [in a new window]   FIG. 7. Differential glycan profiling among hPod/CHO, hPod/LN319, and hPod/LEC. The three hPods were analyzed as their sialylated and desilalylated forms (for details, see Fig. S4). Net intensities of three representative lectins (Jacalin, MAH, and ABA) with sialylated 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 the relevant lectins, i.e. Jacalin/ABA and MAH/ABA, were found to be statistically significant based on the Student's t test (p < 0.01) in the microarray analyses (n = 4) (B).

View larger version (69K): [in this window] [in a new window]   FIG. 8. Challenge to glycan profiling of hPod enriched from a tissue lysate. Podoplanin-positive area on the counterpart sections was selectively dissected (excised volume estimated to be 3.6 mm3). hPod was enriched with NZ-1-immobilized-Sepharose and was 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.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 abundant target proteins (21). Our recently improved lectin microarray system 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- and O-glycoproteins, was demonstrated using a small amount of sample comparable with Western blotting analysis. This significant improvement will help to make the developed ALP system widespread 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 profiling technology. 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. lectin-overlay antibody microarray (20). For realization of this, selection of appropriate lectin probes is critical to differentiate disease-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.

	FOOTNOTES

 
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

¹ The abbreviations used are: AFP, -fetoprotein; ALP, antibody-assisted lectin profiling; PSA, prostate-specific antigen; PSA, mouse anti-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.

² A. Kuno, H. Narimatsu, and J. Hirabayashi, unpublished results.

^* This work was supported in part by a grant from New Energy and Industrial Technology Development Organization, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^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: 81-29-861-3125. E-mail: jun-hirabayashi{at}aist.go.jp

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