Clinica Chimica Acta 446 (2015) 30–36
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Establishment of a novel lectin–antibody ELISA system to determine core-fucosylated haptoglobin Mayuka Shimomura a,1, Kotarosumitomo Nakayama a,1, Kanako Azuma a, Naoko Terao a, Kimihiro Nishino a, Shinji Takamatsu a, Miyako Nakano b, Shiro Takahashi b, Yuka Kobayashi c, Kohei Murata d, Yoshihiro Kamada a, Eiji Miyoshi a,⁎ a
Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, Japan Department of Molecular Biotechnology, Hiroshima University, Japan c J-Oil Mills Incorporation, Japan d Department of Surgery, Suita Municipal Hospital, Japan b
a r t i c l e
i n f o
Article history: Received 29 October 2014 Received in revised form 19 March 2015 Accepted 19 March 2015 Available online 7 April 2015 Keywords: Fucosylation Haptoglobin Lectin–antibody ELISA Core fucose PhoSL AAL
a b s t r a c t Background: Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker that increases in various pathological conditions. We previously established a Fuc-Hpt lectin–antibody assay using Aleuria aurantia lectin (AAL), and applied this to diagnose several diseases, including various cancers. AAL recognizes both α1-3/1-4 and α1-6 fucosylation on N/O-linked glycans. These fucosylation types differ in biological function, and in regulation by different fucosyltransferases. Recently, we identified a novel lectin, Pholiota squarrosa lectin (PhoSL), which specifically recognizes α1-6 fucosylation (core-fucosylation). Methods: We developed a lectin–antibody ELISA kit using PhoSL to determine core-Fuc-Hpt levels in sera from colorectal or pancreatic cancer patients. Results: Serum levels of AAL-reactive Hpt are higher in pancreatic cancer patients, whereas those of PhoSLreactive Hpt are higher in colorectal cancer patients. Mass spectrometry analyses of Hpt fucosylation levels were consistent with lectin–antibody ELISA results. Hpt-transfected colorectal cancer cell lines produced significant amounts of core-Fuc-Hpt, suggesting that colorectal cancer tissues produce core-Fuc-Hpt. Conclusions: These differences in Fuc-Hpt patterns might depend on cancer cells and the surrounding cells, which produce Hpt. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fucosylation is one of the most important glycosylation types involved in cancer and inflammation [1]. Several types of fucosylated glycoproteins have been used as cancer biomarkers. Previously, we found that serum levels of fucosylated haptoglobin (Fuc-Hpt) were increased in the sera of patients with pancreatic cancer [2]. Mass spectrometry analysis reveals that the fucosylation on haptoglobin (Hpt) contains a variety of linkages such as α1-3/α1-4 and α1-6 fucose. To develop a more convenient assay system for measuring Fuc-Hpt, we have established a lectin–antibody enzyme-linked immunosorbent assay (ELISA) for Fuc-Hpt [3] and evaluated this ELISA system in various conditions [4]. In this lectin-ELISA system, we used AAL (Aleuria aurantia Abbreviations: Fuc-Hpt, fucosylated haptoglobin; PhoSL, Pholiota squarrosa lectin; AAL, Aleuria aurantia lectin; MEF, mouse embryonic fibroblast; Fut8, α1-6 fucosyltransferase. ⁎ Corresponding author at: Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-oka Suita, 565-0871, Japan. Tel./fax: +81 6 6879 2590. E-mail address: [email protected] (E. Miyoshi). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cca.2015.03.037 0009-8981/© 2015 Elsevier B.V. All rights reserved.
lectin), which recognizes all types of fucosylation. Our previous study demonstrated that the type of Fuc-Hpt that showed the greatest increase in the sera of patients with pancreatic cancer was the Lewis-type of fucosylation (α1-3/α1-4 fucosylation), while core fucose (α1-6 fucosylation) was only slightly increased in those sera [5]. In contrast, it is known that core-fucosylation has characteristic oligosaccharide functions in antibody-dependent cellular cytotoxicity [6] and growth factor signaling [7,8]. Previously, we reported that Aspergillus oryzae l-fucose-specific lectin (AOL) could specifically recognize core fucose [9]. However, AOL does not bind to α1-3/1-4 fucose, but binds slightly to α1-2 fucose. Recently, we have identified PhoSL (Pholiota squarrosa lectin), which shows greater specificity towards core fucose [10]. Frontal chromatography revealed that PhoSL could only bind to glycans carrying core fucose. In this study, we investigated the ability of PhoSL to determine the presence of core-fucosylated haptoglobin using the lectin–antibody ELISA. We found that the positive rate of fucosylated haptoglobin was different between AAL ELISA and PhoSL ELISA. Here, we discuss the clinical significance of increases in core-fucosylated haptoglobin in cancer patients.
M. Shimomura et al. / Clinica Chimica Acta 446 (2015) 30–36
2. Materials and methods 2.1. Human serum samples Seventy-one patients with colorectal cancer (stage I/II/III/IV; 9/18/24/20) and 55 pancreatic cancer patients (stage I/II/III/IV; 5/27/14/9) who underwent primary resection at Osaka University-related hospitals from 1995 to 2005 were enrolled in this study. Sera from these patients before surgery, and of 60 age-matched healthy controls who received health check-ups, were collected and kept frozen at −80 °C until use. Written informed consent was obtained from all subjects in this study. This study was approved by the ethics committee of Osaka University Hospital. 2.2. Flow cytometry Mouse embryonic fibroblasts (MEFs) derived from α1-6 fucosyltransferase (Fut8)-deficient mice or wild-type mice were cultured in RPMI 1640 medium (Sigma, St. Louis) containing 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. After harvesting with 1 mmol/l EDTA/phosphate-buffered saline (PBS), 3.0 × 105 cells were incubated in 0.1 μg/ml fluorescein isothiocyanate (FITC)-labeled-PhoSL/PBS on ice for 10 min. Cells were washed twice with 0.1% BSA/PBS, followed by flow cytometric analysis with a BD AccuriTM C6 Flow Cytometer and the C6 Flow Cytometer Starter Kit (BD Biosciences). 2.3. Immunostaining MEFs derived from Fut8-deficient mice or wild-type mice were cultured on 24-well glass plates. Under subconfluent conditions, cells were
fixed with 4% paraformaldehyde/PBS (Wako). After blocking with 1% BSA/PBS for 30 min at room temperature, cells were stained with 0.1 μg/ml FITC-PhoSL/PBS for 1 h at room temperature. Cells were washed twice with PBS and stained with 4 ng/ml 4′,6-diamidino-2phenylindole (DAPI) (Invitrogen). Stained cells were evaluated with confocal microscopy (Fluoview FV10i, Olympus).
2.4. Lectin–antibody ELISA for Fuc-Hpt The Fab fragment of anti-human Hpt IgG (Dako) was coated onto the bottom of a 96-well ELISA plate, because IgG has the fucosylated oligosaccharide in its Fc portion. Coated plates were blocked with PBS containing 3% bovine serum albumin for 1 h, followed by washing with PBS containing 0.1% Tween 20 (PBS-T). A 50-μl aliquot of sera was placed into each well and incubated for 1 h at room temperature. The plate was washed three times with PBS-T, using Immuno Wash (Bio-RAD Model 1517, Bio-RAD, Tokyo, Japan). To detect FucHpt, 1/1000 diluted biotinylated AAL or PhoSL was placed into each well, followed by incubation at room temperature for 1 h. After washing plates three times with PBS-T, peroxidase-conjugated avidin was added to each well, followed by incubation at room temperature for 1 h. After washing 4 times with PBS-T, tetramethylbenzidine was added to each well and allowed to develop for 15 min. To stop the development, 1 mol/l sulfuric acid was added to each well. A standard curve for FucHpt was obtained as described elsewhere [11], using conditioned medium from the pancreatic cancer cell line PK8 transfected with an expression vector for Hpt, which was prepared in Immuno Biological Laboratories (Suppl. 1). The concentration of Fuc-Hpt was expressed in relative units to account for lot-specific differences.
(A)
(B) 800
Cell Number
Cell Number
800 600 400
200
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0
0 10 1 10 2 10 3 10 4 10 5 10 6 10 7
10 1 10 2 10 3 10 4 10 5 10 6
Fluorescence Intensity
(C)
31
10 7
Fluorescence Intensity
(D)
Fig. 1. Flow cytometry and immunofluorescent analysis of PhoSL staining in wild-type and Fut8 knockout mouse fibroblasts. (A) and (B) Approximately 1 × 104 cells were analyzed by flow cytometry using FITC-labeled PhoSL (A, wild-type MEFs; B, Fut8 knockout MEFs). The background signal in the absence of FITC-PhoSL is represented by the black line. PhoSL staining is represented by the red line. (C) and (D) Wild-type (C) and Fut8-knockout (D) MEFs were stained with FITC-labeled PhoSL. The detailed procedure is described in Materials and methods. No staining of PhoSL was observed in Fut8-knockout fibroblasts. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.5. Evaluation of core-Fuc-Hpt lectin–antibody ELISA kit To calculate the coefficient of variation (CV), 6 patient serum samples (3 colorectal cancer and 3 pancreatic cancer patients) were assayed three times. To investigate the inhibitory effect of bilirubin, hemoglobin, and lipids as intermediate molecules on the core-Fuc-Hpt assay, we used Interference Check A Plus Kit (cat No. 79370, Sysmex Co., Ltd., Kobe, Japan), which includes free bilirubin (bilirubin F), hemoglobin, and formagine, according to directions. 2.6. Site-specific N-glycan analyses of Hpt by LC-ESI-MS The haptoglobin β chain contains four N-glycosylation sites: Asn184 (site 1), Asn207 (site 2), Asn211 (site 3), and Asn241 (site 4). For sitespecific analyses of N-glycans, desialo-glycopeptides of Hpt were prepared as described previously [5]. Briefly, Hpt was purified from each serum using an antibody affinity column. After digestion with trypsin, lysylendopeptidase, and endoprotease Glu-C, glycopeptides from the digests were enriched using affinity separation by partitioning with Sepharose CL4B. The enriched glycopeptides were treated with 2 mol/l acetic acid to remove sialic acids. The desialo-glycopeptides were analyzed using a new liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) method as follows. Desialo-glycopeptides containing sites 1–4 were separated using an octadecyl (C18 or ODS) column (Develosil 300 ODS-HG-5; 150 × 1.0 mm ID; Nomura Chemicals) under specific gradient conditions. The mobile phases were (A) 0.08% formic acid and (B) 0.15% formic acid in 80% acetonitrile. The column was eluted with solvent A for 5 min, at which point the concentration of solvent B was increased to 40% over 55 min at a flow rate of 50 μl/ min. The eluate was introduced continuously into an ESI source (LTQ Orbitrap XL; Thermo Fisher). For MS analyses, the voltage of the capillary source was set at 4.5 kV, and the temperature of the transfer capillary maintained at 300 °C. The capillary voltage and tube lens voltage were set at 15 and 50 V, respectively. MS data were obtained in positive ion mode over the mass range m/z 300 to 3000. Ratios of N-glycan at sites 1–4 were calculated based on the signal intensities of the corresponding glycopeptides obtained by LC-ESI-MS analyses.
show very high correlation values, and the absorbance was 10-fold higher with AAL lectin than with PhoSL lectin in the ELISA reaction. Next, we determined core-Fuc-Hpt levels using PhoSL ELISA in the sera of 60 normal volunteers, 71 patients with colorectal cancer, and 55 patients with pancreatic cancer (Fig. 2A). The cutoff value of coreFuc-Hpt was determined to be 160 units/ml (mean value + 1.5 SD) in healthy volunteers. Values above the cutoff value (160 U/ml) were taken as positive. Extremely high levels of core-Fuc-Hpt were seen in only one of the normal volunteers. Among the patients with colorectal cancer, 10 cases (12.7%) were positive for core-Fuc-Hpt. Five cases (9%) of patients with pancreatic cancer were positive for core-FucHpt. Fuc-Hpt levels determined using the AAL-reactive Fuc-Hpt assay were significantly higher in patients with pancreatic cancer. The levels of AAL-reactive Fuc-Hpt are shown in Fig. 2B. The efficacy of Fuc-Hpt and core-Fuc-Hpt for the diagnosis of colorectal or pancreatic cancer was determined using the ROC curve (Suppl. 3). There were no correlations among total Hpt levels, AAL-positive Fuc-Hpt levels, and PhoSLpositive Fuc-Hpt levels (Suppl. 4).
(A) *
500
400 300 200
100 0
3.1. Visualization of PhoSL by flow cytometry and cell staining Because Fut8 is the only enzyme involved in the synthesis of core fucose, MEFs from Fut8 knockout mice completely lack core-fucosylation on N-glycans [8]. MEFs of Fut8 knockout mice exhibit little binding to PhoSL, although strong binding to PhoSL was observed in MEFs from wild-type mice (Fig. 1A and B, Suppl. 2). A confocal microscopy analysis confirmed that PhoSL staining was absent in Fut8-deficient MEFs (Fig. 1C and D). These results indicate that PhoSL binds to core fucose on cell-surface glycoproteins. 3.2. Lectin–antibody ELISA to measure core-Fuc-Hpt The standard curves of lectin–antibody ELISA obtained using AAL and PhoSL are shown in Supplementary Data 1 (Suppl. 1). Both datasets
HV
colorectal cancer
pancreatic cancer
n=60
n=71
n=55
(B)
**
* p < 0.005 * * p < 0.0001
** Fuc-Hpt (Units/ml)
3. Results
*
600
2.7. Statistical analysis Statistical analysis was performed with JMP Pro 10.0 software (SAS Institute Inc.). Kruskal–Wallis tests and Wilcoxon tests were performed to assess the existence of any significant differences between groups. Spearman R correlations were used to assess the relationships between fucose/core fucose contents and the results of the lectin–antibody ELISA. The diagnostic performances of total Fuc-Hpt and core Fuc-Hpt were assessed by analyzing receiver operating characteristic (ROC) curves. A p b 0.05 was considered statistically significant.
p = 0.0002
700
Core-Fuc-Hpt (Units/ml)
32
*
10000 8000 6000 4000 2000
0 HV
colorectal cancer
pancreatic cancer
n=60
n=71
n=55
Fig. 2. Serum levels of core-Fuc-Hpt and total Fuc-Hpt in healthy volunteers and in colorectal cancer and pancreatic cancer patients. Serum levels of core-Fuc-Hpt (A) and total Fuc-Hpt (B) were determined with lectin–antibody ELISA using PhoSL and AAL, respectively. Sixty healthy volunteers, 79 patients with colorectal cancer, and 55 patients with pancreatic cancer were investigated. The grey bars indicate the average Fuc-Hpt level, and the green bars indicate the median Fuc-Hpt level in each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
M. Shimomura et al. / Clinica Chimica Acta 446 (2015) 30–36
(A)
(C) 30
BPC
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36.52
Patient #3
NLFLNHSE
80
Site 2 VVLHPNYSQVD
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37.25 38.30
Site 4
40
2.87
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VSHHNLTTGATLINE
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20 25 Time (min)
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877.38 z= 4
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0
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(%) Relative Abundance
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9 10 Core Fuc-Hpt (PhoSL-ELISA)
Fuc-Hpt (Units/ml)
Fig. 3. Determination of the contents of fucosylated N-glycans in haptoglobin purified from the sera of 10 patients with colorectal cancer by LC-ESI MS. (A) Base peak chromatogram of desialo-glycopeptides containing sites 1–4 of Hpt (patient #3). (B) Averaged mass spectra of desialo-glycopeptides containing site 1 (patient #3 and #7). (C) Ratio of peak intensity of fucosylated N-glycans to total peak intensity of all N-glycans on Hpt (patient #1–#10). 2-0, biantennary N-glycan; 3-0, triantennary N-glycan; 4-0, tetraantennary N-glycan; 2-F, mono-fucosylated biantennary N-glycan; 3-F, mono-fucosylated triantennary N-glycan; 4-F, mono-fucosylated tetraantennary N-glycan; 3-FF, di-fucosylated triantennary N-glycan.
1
2 3
0
5
4 5 6 7
8
9 10
300 250 200 150 100 50
0 10 15 20 25 30 p < 0.0001 r = 0.945
Fig. 4. Correlation between Fuc-Hpt levels determined with lectin–antibody ELISA, and fucose content determined by LC-ESI MS. The sera from Fig. 4 were used in this analysis. Core fucose content was evaluated as the level of fucose content in 2-F. Spearman R correlations were used to assess the relationships between fucose/core fucose contents and the results of lectin– antibody ELISA.
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3.3. Site-specific N-glycan analyses of Hpt by LC-ESI-MS Ten cases of colorectal cancer exhibiting high levels of core-Fuc-Hpt, as measured using PhoSL-antibody ELISA, were analyzed by LC-ESI MS. The base peak chromatogram, shown in Fig. 3A, demonstrated the separation of glycopeptides containing sites 1–4. The averaged mass spectra of site 1 N-glycans on Hpt for the patients #3 and #7 are shown in Fig. 3B. Based on peak intensity of each N-glycan, the ratio of fucosylated N-glycans to total N-glycans on Hpt in 10 cases is shown in Fig. 3C. All cases showed a high proportion of fucosylated glycans on site 1 of Hpt glycans (2.2–28.2%). This ratio was significantly higher than that of healthy volunteers (2.8–4.3%). Next, we compared the ratio of fucosylated glycans to total glycans on Hpt, with Fuc-Hpt levels measured using AAL/PhoSL-antibody ELISA. As shown in Fig. 4, Fuc-Hpt levels measured with AAL-antibody ELISA were strongly correlated with fucosylation ratios, as determined by mass spectrometry, with the exception of one case; this case showed a high level of core-Fuc-Hpt, as determined with PhoSL-antibody ELISA. Furthermore, the proportion of fucosylated glycans on bi-antennary sugar chains correlated with core-Fuc-Hpt levels, as determined using PhoSL-antibody ELISA. 3.4. Clinical significance of core-Fuc-Hpt in colorectal cancer We investigated the clinical significance of serum levels of core-FucHpt, as reflected by the clinical stage of colorectal cancer. As shown in Fig. 5A and B, we evaluated the number of core-Fuc-Hpt-positive patients among the patients with stage I (0 cases), stage II (2 cases), stage III (5 cases), and stage IV (3 cases) colorectal cancer. Although we observed no statistical significance between core-Fuc-Hpt levels and clinical stage, most of the core-Fuc-Hpt-positive cases (8/10 cases) were in the advanced stages of colorectal cancer (stage III and IV). However, there
Disease
PhoSL-Hpt (U/ml)
CV value (%)
Colorectal cancer Colorectal cancer Colorectal cancer Pancreatic cancer Pancreatic cancer Pancreatic cancer
94.9 ± 16.5 141.4 ± 46.4 310.9 ± 17.1 145.1 ± 27.8 268.1 ± 24.1 536.2 ± 42.0
17.4 32.8 5.5 19.2 9 7.8
PhoSL-Hpt values are presented as the mean ± SD.
were no significant differences in mean disease-free time between core-Fuc-Hpt-positive and negative patients. 3.5. Evaluation of core-Fuc-Hpt lectin–antibody ELISA kit in various conditions We evaluated the efficacy of our developed core-Fuc-Hpt lectin– antibody ELISA kit in various conditions. At first, we assessed the reproducibility of the core-Fuc-Hpt assay. As shown in Table 1, the CV values were 5.5 to 32.8%. Secondly, we investigated the effects of serum dilution on the measurement values of our ELISA kit (Fig. 6). When we compared data from the 25-fold dilution with those from other dilutions, relatively high correlations were observed among assay results obtained using 10-, 20-, 50-, and 100-fold dilutions. Thirdly, we evaluated the effects of icterus, hemolysis, and lipids on the lectin–antibody ELISA kit. We added free bilirubin (bilirubin F), hemoglobin, and formagine to the samples (pooled sera of colorectal cancer patients) for ELISA. As shown in Fig. 7, bilirubin and formagine
***
5000
*
*
**
Core-Fuc-Hpt (Units/ml)
(A) Fuc-Hpt (Units/ml)
Table 1 Reproducibility of PhoSL-Hpt assay at different times, using patient serum samples (colorectal and pancreatic cancer patients).
4000 3000 2000 1000 0 I
II
III
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100 0
IV
I
II
Stage
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*
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Fuc-Hpt (Units/ml)
8000 4000 0 II
IV
Stage
12000
I
III
III
Stage
IV
700 600 500 400 300 200 100 0
I
II
III
IV
Stage
Fig. 5. Correlation between each type of Fuc-Hpt and clinical stage of colorectal cancer and pancreatic cancer. The serum levels of total (AAL-reactive) and core (PhoSL-reactive) Fuc-Hpt determined using lectin–antibody ELISA were investigated in terms of clinical stage of colorectal cancer (A) and pancreatic cancer (B). Wilcoxon test was used for statistical analysis. * p b 0.005, ** p b 0.02, *** p b 0.05.
M. Shimomura et al. / Clinica Chimica Acta 446 (2015) 30–36
(A)
(B) 500
400 R = 0.703 P = 0.0158
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Fig. 6. Comparison of serum dilution and core-Fuc-Hpt levels in the lectin–antibody ELISA. Serum core-Fuc-Hpt levels in 11 subjects including 2 healthy volunteers, 5 colorectal cancer patients, and 4 pancreatic cancer patients. (A–D) Comparison of the values of each dilution (×10, ×20, ×50, ×100) in the ELISA assay. The x-axis represents data from the 25-fold dilution in all cases and the individual y-axis represents data from the 10- to 100-fold dilutions.
decreased the measurement values of core-Fuc-Hpt. Surprisingly, we found that hemoglobin increased the measured core-Fuc-Hpt levels in a dose-dependent manner. 4. Discussion We investigated the possibility of using the lectin–antibody ELISA as a tool to measure core-Fuc-Hpt in the sera of patients with cancer. As expected, most of the Fuc-Hpt increase in the sera of cancer patients consisted of Lewis-type fucosylation, and the PhoSL-antibody ELISA was positive for significantly fewer cases than the AAL-antibody ELISA.
(A)
However, the positive rate of core-Fuc-Hpt in colorectal cancer was higher than in pancreatic cancer, although the positive rate of Fuc-Hpt determined with AAL was higher in the sera of patients with pancreatic cancer [2]. Fuc-Hpt in pancreatic cancer patients might be produced in metastatic lesions in the liver, because pancreatic cancer cell lines scarcely expressed Hpt mRNA [2], and interleukin-6 (IL6) produced by pancreatic cancer cell lines induced the production of AAL-reactive Fuc-Hpt from co-cultured hepatoma cells [11]. Fucosylated glycoproteins produced in hepatocytes are secreted into bile, but not into blood in a normal liver [12], and the loss of polarity of hepatocytes in hepatocarcinogenesis or liver metastasis of other cancers could result
(B)
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0 0 2.5 10 Bilirubin F conc. (mg/dl)
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200
0
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150 100
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Formagine conc. (mg/dl)
Fig. 7. Effects of bilirubin, hemoglobin, and formagine contamination on the core-Fuc-Hpt assay. Sera of colorectal cancer patients were measured using core-Fuc-Hpt lectin–antibody ELISA kit with various concentrations of free bilirubin (bilirubin F) (A), hemoglobin (B), and formagine (C).
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in the secretion of fucosylated glycoproteins into blood, which might be detected as a cancer biomarker [13]. In contrast, core-Fuc-Hpt is scarcely produced in normal hepatocytes, which do not express α1-6 fucosyltransferase, an enzyme involved in the process of corefucosylation. Therefore, core-Fuc-Hpt could be produced in cancer cells themselves, or in lymphocytes around cancer tissue. An immunohistochemical study has shown the production of Hpt in colon cancer tissue and demonstrated that serum Fuc-Hpt levels are increased in colorectal cancer [14]. Furthermore, the expression of α1-6 fucosyltransferase is dramatically increased in colorectal cancer tissue [15]. The expression of Hpt mRNA was detected in several kinds of colon cancer cell lines (data not shown). These results suggest that discrimination of core and Lewis-type Fuc-Hpt could indicate the origin of Fuc-Hpt producing cells. In contrast, the biological/clinical significance of Hpt remains unknown, and the genotype of Hpt is involved in several diseases [16]. The biological functions of characteristic Fuc-Hpt are not proven compared to non-fucosylated/total Hpt. In clinical analyses, cases with high levels of core-Fuc-Hpt are frequently observed at the advanced clinical stages of colorectal cancer (Fig. 5A), and a combination of carcinoembryonic antigen (CEA) and AAL-reactive Fuc-Hpt is a marker for poor prognosis following surgery [17]. Nevertheless, cases with high levels of core-Fuc-Hpt showed relatively long survival/diseasefree time compared to core-Fuc-Hpt-negative and AAL-reactive FucHpt-positive cases. However, compared to AAL-reactive Fuc-Hpt, coreFuc-Hpt has little clinical significance in pancreatic cancer (Fig. 5B). These results might suggest that core-Fuc-Hpt has biological functions such as promoting cancer immune surveillance in the microenvironment of colorectal cancer, but not in that of pancreatic cancer; further studies are warranted to investigate this hypothesis. PhoSL lectin, as well as PhoSL-antibody ELISA, may prove to be effective tools for this purpose. Furthermore, we evaluated the efficacy of our developed lectin–antibody ELISA kit in various conditions (Figs. 6, 7) like previous our study. Our findings indicated that serum dilution from 10 to 100-fold can be measured in PhoSL-antibody ELISA kit accurately. The contamination of bilirubin and lipids had inhibitory effects on the ELISA measurements. In contrast, the contamination of hemoglobin increased values of measured core-Fuc-Hpt levels, although the contamination of hemoglobin decreased values of Fuc-Hpt levels measured with AALantibody ELISA kit [4]. This discrepancy might be conformational changes of haptoglobin and hemoglobin, resulting in changes of each lectin binding. These data indicated that the measurements of coreFuc-Hpt using PhoSL-antibody ELISA kit require some attention in the patients with dyslipidemia, icterus, and especially hemolysis. Taken together, we have demonstrated the development and utility of a core-Fuc-Hpt lectin-ELISA kit using PhoSL. Disclosure statement Yuka Kobayashi is an employee of J-Oil mills Incorporation. None of the other authors have any conflict of interest to declare.
Acknowledgments This study was performed as a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2015.03.037.
References [1] Miyoshi E, Moriwaki K, Nakagawa T. Biological function of fucosylation in cancer biology. J Biochem 2008;143:725–9. [2] Okuyama N, Ide Y, Nakano M, et al. Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation. Int J Cancer 2006;118:2803–8. [3] Matsumoto H, Shinzaki S, Narisada M, et al. Clinical application of a lectin–antibody ELISA to measure fucosylated haptoglobin in sera of patients with pancreatic cancer. Clin Chem Lab Med 2010;48:505–12. [4] Kamada Y, Kinoshita N, Tsuchiya Y, et al. Reevaluation of a lectin antibody ELISA kit for measuring fucosylated haptoglobin in various conditions. Clin Chim Acta 2013; 417:48–53. [5] Nakano M, Nakagawa T, Ito T, et al. Site-specific analysis of N-glycans on haptoglobin in sera of patients with pancreatic cancer: a novel approach for the development of tumor markers. Int J Cancer 2008;122:2301–9. [6] Shinkawa T, Nakamura K, Yamane N, et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complextype oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 2003;278:3466–73. [7] Wang X, Inoue S, Gu J, et al. Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucosedeficient mice. Proc Natl Acad Sci U S A 2005;102:15791–6. [8] Wang X, Gu J, Ihara H, Miyoshi E, Honke K, Taniguchi N. Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling. J Biol Chem 2006;281:2572–7. [9] Matsumura K, Higashida K, Ishida H, et al. Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose. J Biol Chem 2007;282:15700–8. [10] Kobayashi Y, Tateno H, Dohra H, et al. A novel core fucose-specific lectin from the mushroom Pholiota squarrosa. J Biol Chem 2012;287:33973–82. [11] Narisada M, Kawamoto S, Kuwamoto K, et al. Identification of an inducible factor secreted by pancreatic cancer cell lines that stimulates the production of fucosylated haptoglobin in hepatoma cells. Biochem Biophys Res Commun 2008;377:792–6. [12] Nakagawa T, Uozumi N, Nakano M, et al. Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts. J Biol Chem 2006;281:29797–806. [13] Nakagawa T, Moriwaki K, Terao N, et al. Analysis of polarized secretion of fucosylated alpha-fetoprotein in HepG2 cells. J Proteome Res 2012;11:2798–806. [14] Park SY, Lee SH, Kawasaki N, et al. alpha1-3/4 fucosylation at Asn 241 of betahaptoglobin is a novel marker for colon cancer: a combinatorial approach for development of glycan biomarkers. Int J Cancer 2012;130:2366–76. [15] Muinelo-Romay L, Vazquez-Martin C, Villar-Portela S, Cuevas E, Gil-Martin E, Fernandez-Briera A. Expression and enzyme activity of alpha(1,6)fucosyltransferase in human colorectal cancer. Int J Cancer 2008;123:641–6. [16] Nakhoul FM, Miller-Lotan R, Awaad H, Asleh R, Levy AP. Hypothesis—haptoglobin genotype and diabetic nephropathy. Nat Clin Pract Nephrol 2007;3:339–44. [17] Takeda Y, Shinzaki S, Okudo K, Moriwaki K, Murata K, Miyoshi E. Fucosylated haptoglobin is a novel type of cancer biomarker linked to the prognosis after an operation in colorectal cancer. Cancer 2012;118:3036–43.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Establishment of a novel lectin–antibody ELISA system to determine core-fucosylated haptoglobin Mayuka Shimomura a,1, Kotarosumitomo Nakayama a,1, Kanako Azuma a, Naoko Terao a, Kimihiro Nishino a, Shinji Takamatsu a, Miyako Nakano b, Shiro Takahashi b, Yuka Kobayashi c, Kohei Murata d, Yoshihiro Kamada a, Eiji Miyoshi a,⁎ a
Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, Japan Department of Molecular Biotechnology, Hiroshima University, Japan c J-Oil Mills Incorporation, Japan d Department of Surgery, Suita Municipal Hospital, Japan b
a r t i c l e
i n f o
Article history: Received 29 October 2014 Received in revised form 19 March 2015 Accepted 19 March 2015 Available online 7 April 2015 Keywords: Fucosylation Haptoglobin Lectin–antibody ELISA Core fucose PhoSL AAL
a b s t r a c t Background: Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker that increases in various pathological conditions. We previously established a Fuc-Hpt lectin–antibody assay using Aleuria aurantia lectin (AAL), and applied this to diagnose several diseases, including various cancers. AAL recognizes both α1-3/1-4 and α1-6 fucosylation on N/O-linked glycans. These fucosylation types differ in biological function, and in regulation by different fucosyltransferases. Recently, we identified a novel lectin, Pholiota squarrosa lectin (PhoSL), which specifically recognizes α1-6 fucosylation (core-fucosylation). Methods: We developed a lectin–antibody ELISA kit using PhoSL to determine core-Fuc-Hpt levels in sera from colorectal or pancreatic cancer patients. Results: Serum levels of AAL-reactive Hpt are higher in pancreatic cancer patients, whereas those of PhoSLreactive Hpt are higher in colorectal cancer patients. Mass spectrometry analyses of Hpt fucosylation levels were consistent with lectin–antibody ELISA results. Hpt-transfected colorectal cancer cell lines produced significant amounts of core-Fuc-Hpt, suggesting that colorectal cancer tissues produce core-Fuc-Hpt. Conclusions: These differences in Fuc-Hpt patterns might depend on cancer cells and the surrounding cells, which produce Hpt. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fucosylation is one of the most important glycosylation types involved in cancer and inflammation [1]. Several types of fucosylated glycoproteins have been used as cancer biomarkers. Previously, we found that serum levels of fucosylated haptoglobin (Fuc-Hpt) were increased in the sera of patients with pancreatic cancer [2]. Mass spectrometry analysis reveals that the fucosylation on haptoglobin (Hpt) contains a variety of linkages such as α1-3/α1-4 and α1-6 fucose. To develop a more convenient assay system for measuring Fuc-Hpt, we have established a lectin–antibody enzyme-linked immunosorbent assay (ELISA) for Fuc-Hpt [3] and evaluated this ELISA system in various conditions [4]. In this lectin-ELISA system, we used AAL (Aleuria aurantia Abbreviations: Fuc-Hpt, fucosylated haptoglobin; PhoSL, Pholiota squarrosa lectin; AAL, Aleuria aurantia lectin; MEF, mouse embryonic fibroblast; Fut8, α1-6 fucosyltransferase. ⁎ Corresponding author at: Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-oka Suita, 565-0871, Japan. Tel./fax: +81 6 6879 2590. E-mail address: [email protected] (E. Miyoshi). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cca.2015.03.037 0009-8981/© 2015 Elsevier B.V. All rights reserved.
lectin), which recognizes all types of fucosylation. Our previous study demonstrated that the type of Fuc-Hpt that showed the greatest increase in the sera of patients with pancreatic cancer was the Lewis-type of fucosylation (α1-3/α1-4 fucosylation), while core fucose (α1-6 fucosylation) was only slightly increased in those sera [5]. In contrast, it is known that core-fucosylation has characteristic oligosaccharide functions in antibody-dependent cellular cytotoxicity [6] and growth factor signaling [7,8]. Previously, we reported that Aspergillus oryzae l-fucose-specific lectin (AOL) could specifically recognize core fucose [9]. However, AOL does not bind to α1-3/1-4 fucose, but binds slightly to α1-2 fucose. Recently, we have identified PhoSL (Pholiota squarrosa lectin), which shows greater specificity towards core fucose [10]. Frontal chromatography revealed that PhoSL could only bind to glycans carrying core fucose. In this study, we investigated the ability of PhoSL to determine the presence of core-fucosylated haptoglobin using the lectin–antibody ELISA. We found that the positive rate of fucosylated haptoglobin was different between AAL ELISA and PhoSL ELISA. Here, we discuss the clinical significance of increases in core-fucosylated haptoglobin in cancer patients.
M. Shimomura et al. / Clinica Chimica Acta 446 (2015) 30–36
2. Materials and methods 2.1. Human serum samples Seventy-one patients with colorectal cancer (stage I/II/III/IV; 9/18/24/20) and 55 pancreatic cancer patients (stage I/II/III/IV; 5/27/14/9) who underwent primary resection at Osaka University-related hospitals from 1995 to 2005 were enrolled in this study. Sera from these patients before surgery, and of 60 age-matched healthy controls who received health check-ups, were collected and kept frozen at −80 °C until use. Written informed consent was obtained from all subjects in this study. This study was approved by the ethics committee of Osaka University Hospital. 2.2. Flow cytometry Mouse embryonic fibroblasts (MEFs) derived from α1-6 fucosyltransferase (Fut8)-deficient mice or wild-type mice were cultured in RPMI 1640 medium (Sigma, St. Louis) containing 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. After harvesting with 1 mmol/l EDTA/phosphate-buffered saline (PBS), 3.0 × 105 cells were incubated in 0.1 μg/ml fluorescein isothiocyanate (FITC)-labeled-PhoSL/PBS on ice for 10 min. Cells were washed twice with 0.1% BSA/PBS, followed by flow cytometric analysis with a BD AccuriTM C6 Flow Cytometer and the C6 Flow Cytometer Starter Kit (BD Biosciences). 2.3. Immunostaining MEFs derived from Fut8-deficient mice or wild-type mice were cultured on 24-well glass plates. Under subconfluent conditions, cells were
fixed with 4% paraformaldehyde/PBS (Wako). After blocking with 1% BSA/PBS for 30 min at room temperature, cells were stained with 0.1 μg/ml FITC-PhoSL/PBS for 1 h at room temperature. Cells were washed twice with PBS and stained with 4 ng/ml 4′,6-diamidino-2phenylindole (DAPI) (Invitrogen). Stained cells were evaluated with confocal microscopy (Fluoview FV10i, Olympus).
2.4. Lectin–antibody ELISA for Fuc-Hpt The Fab fragment of anti-human Hpt IgG (Dako) was coated onto the bottom of a 96-well ELISA plate, because IgG has the fucosylated oligosaccharide in its Fc portion. Coated plates were blocked with PBS containing 3% bovine serum albumin for 1 h, followed by washing with PBS containing 0.1% Tween 20 (PBS-T). A 50-μl aliquot of sera was placed into each well and incubated for 1 h at room temperature. The plate was washed three times with PBS-T, using Immuno Wash (Bio-RAD Model 1517, Bio-RAD, Tokyo, Japan). To detect FucHpt, 1/1000 diluted biotinylated AAL or PhoSL was placed into each well, followed by incubation at room temperature for 1 h. After washing plates three times with PBS-T, peroxidase-conjugated avidin was added to each well, followed by incubation at room temperature for 1 h. After washing 4 times with PBS-T, tetramethylbenzidine was added to each well and allowed to develop for 15 min. To stop the development, 1 mol/l sulfuric acid was added to each well. A standard curve for FucHpt was obtained as described elsewhere [11], using conditioned medium from the pancreatic cancer cell line PK8 transfected with an expression vector for Hpt, which was prepared in Immuno Biological Laboratories (Suppl. 1). The concentration of Fuc-Hpt was expressed in relative units to account for lot-specific differences.
(A)
(B) 800
Cell Number
Cell Number
800 600 400
200
600 400 200
0
0 10 1 10 2 10 3 10 4 10 5 10 6 10 7
10 1 10 2 10 3 10 4 10 5 10 6
Fluorescence Intensity
(C)
31
10 7
Fluorescence Intensity
(D)
Fig. 1. Flow cytometry and immunofluorescent analysis of PhoSL staining in wild-type and Fut8 knockout mouse fibroblasts. (A) and (B) Approximately 1 × 104 cells were analyzed by flow cytometry using FITC-labeled PhoSL (A, wild-type MEFs; B, Fut8 knockout MEFs). The background signal in the absence of FITC-PhoSL is represented by the black line. PhoSL staining is represented by the red line. (C) and (D) Wild-type (C) and Fut8-knockout (D) MEFs were stained with FITC-labeled PhoSL. The detailed procedure is described in Materials and methods. No staining of PhoSL was observed in Fut8-knockout fibroblasts. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.5. Evaluation of core-Fuc-Hpt lectin–antibody ELISA kit To calculate the coefficient of variation (CV), 6 patient serum samples (3 colorectal cancer and 3 pancreatic cancer patients) were assayed three times. To investigate the inhibitory effect of bilirubin, hemoglobin, and lipids as intermediate molecules on the core-Fuc-Hpt assay, we used Interference Check A Plus Kit (cat No. 79370, Sysmex Co., Ltd., Kobe, Japan), which includes free bilirubin (bilirubin F), hemoglobin, and formagine, according to directions. 2.6. Site-specific N-glycan analyses of Hpt by LC-ESI-MS The haptoglobin β chain contains four N-glycosylation sites: Asn184 (site 1), Asn207 (site 2), Asn211 (site 3), and Asn241 (site 4). For sitespecific analyses of N-glycans, desialo-glycopeptides of Hpt were prepared as described previously [5]. Briefly, Hpt was purified from each serum using an antibody affinity column. After digestion with trypsin, lysylendopeptidase, and endoprotease Glu-C, glycopeptides from the digests were enriched using affinity separation by partitioning with Sepharose CL4B. The enriched glycopeptides were treated with 2 mol/l acetic acid to remove sialic acids. The desialo-glycopeptides were analyzed using a new liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) method as follows. Desialo-glycopeptides containing sites 1–4 were separated using an octadecyl (C18 or ODS) column (Develosil 300 ODS-HG-5; 150 × 1.0 mm ID; Nomura Chemicals) under specific gradient conditions. The mobile phases were (A) 0.08% formic acid and (B) 0.15% formic acid in 80% acetonitrile. The column was eluted with solvent A for 5 min, at which point the concentration of solvent B was increased to 40% over 55 min at a flow rate of 50 μl/ min. The eluate was introduced continuously into an ESI source (LTQ Orbitrap XL; Thermo Fisher). For MS analyses, the voltage of the capillary source was set at 4.5 kV, and the temperature of the transfer capillary maintained at 300 °C. The capillary voltage and tube lens voltage were set at 15 and 50 V, respectively. MS data were obtained in positive ion mode over the mass range m/z 300 to 3000. Ratios of N-glycan at sites 1–4 were calculated based on the signal intensities of the corresponding glycopeptides obtained by LC-ESI-MS analyses.
show very high correlation values, and the absorbance was 10-fold higher with AAL lectin than with PhoSL lectin in the ELISA reaction. Next, we determined core-Fuc-Hpt levels using PhoSL ELISA in the sera of 60 normal volunteers, 71 patients with colorectal cancer, and 55 patients with pancreatic cancer (Fig. 2A). The cutoff value of coreFuc-Hpt was determined to be 160 units/ml (mean value + 1.5 SD) in healthy volunteers. Values above the cutoff value (160 U/ml) were taken as positive. Extremely high levels of core-Fuc-Hpt were seen in only one of the normal volunteers. Among the patients with colorectal cancer, 10 cases (12.7%) were positive for core-Fuc-Hpt. Five cases (9%) of patients with pancreatic cancer were positive for core-FucHpt. Fuc-Hpt levels determined using the AAL-reactive Fuc-Hpt assay were significantly higher in patients with pancreatic cancer. The levels of AAL-reactive Fuc-Hpt are shown in Fig. 2B. The efficacy of Fuc-Hpt and core-Fuc-Hpt for the diagnosis of colorectal or pancreatic cancer was determined using the ROC curve (Suppl. 3). There were no correlations among total Hpt levels, AAL-positive Fuc-Hpt levels, and PhoSLpositive Fuc-Hpt levels (Suppl. 4).
(A) *
500
400 300 200
100 0
3.1. Visualization of PhoSL by flow cytometry and cell staining Because Fut8 is the only enzyme involved in the synthesis of core fucose, MEFs from Fut8 knockout mice completely lack core-fucosylation on N-glycans [8]. MEFs of Fut8 knockout mice exhibit little binding to PhoSL, although strong binding to PhoSL was observed in MEFs from wild-type mice (Fig. 1A and B, Suppl. 2). A confocal microscopy analysis confirmed that PhoSL staining was absent in Fut8-deficient MEFs (Fig. 1C and D). These results indicate that PhoSL binds to core fucose on cell-surface glycoproteins. 3.2. Lectin–antibody ELISA to measure core-Fuc-Hpt The standard curves of lectin–antibody ELISA obtained using AAL and PhoSL are shown in Supplementary Data 1 (Suppl. 1). Both datasets
HV
colorectal cancer
pancreatic cancer
n=60
n=71
n=55
(B)
**
* p < 0.005 * * p < 0.0001
** Fuc-Hpt (Units/ml)
3. Results
*
600
2.7. Statistical analysis Statistical analysis was performed with JMP Pro 10.0 software (SAS Institute Inc.). Kruskal–Wallis tests and Wilcoxon tests were performed to assess the existence of any significant differences between groups. Spearman R correlations were used to assess the relationships between fucose/core fucose contents and the results of the lectin–antibody ELISA. The diagnostic performances of total Fuc-Hpt and core Fuc-Hpt were assessed by analyzing receiver operating characteristic (ROC) curves. A p b 0.05 was considered statistically significant.
p = 0.0002
700
Core-Fuc-Hpt (Units/ml)
32
*
10000 8000 6000 4000 2000
0 HV
colorectal cancer
pancreatic cancer
n=60
n=71
n=55
Fig. 2. Serum levels of core-Fuc-Hpt and total Fuc-Hpt in healthy volunteers and in colorectal cancer and pancreatic cancer patients. Serum levels of core-Fuc-Hpt (A) and total Fuc-Hpt (B) were determined with lectin–antibody ELISA using PhoSL and AAL, respectively. Sixty healthy volunteers, 79 patients with colorectal cancer, and 55 patients with pancreatic cancer were investigated. The grey bars indicate the average Fuc-Hpt level, and the green bars indicate the median Fuc-Hpt level in each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
M. Shimomura et al. / Clinica Chimica Acta 446 (2015) 30–36
(A)
(C) 30
BPC
100
36.52
Patient #3
NLFLNHSE
80
Site 2 VVLHPNYSQVD
60
37.25 38.30
Site 4
40
2.87
20
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(B)
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10
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20 25 Time (min)
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35
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Relative Abundance
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45
969. 41 z= 4
96 8.66 z=4
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40
927.07 z=3
0.87 2-0 84z=4
969
80
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60
877.38 z= 4
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932. 15 96 8.6 6 z=4 z=4
40 20
970
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1005.18 z= 4 1023.43 z= 4
971 m/z
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0
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80
927.07 z=3
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850
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969 970 932. 15 z=4 96 8.6 6 z=4 969. 42 1023.43 z=3 z= 4
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content of fucosylated N-glycans at site1 (%)
(%) Relative Abundance
33
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CoreFuc-Hpt (Units/ml)
6000
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0
5
10 15 20 25 30 p = 0.0415 r = 0.621
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PhoSL
600 400 200 0
9 10 Core Fuc-Hpt (PhoSL-ELISA)
Fuc-Hpt (Units/ml)
Fig. 3. Determination of the contents of fucosylated N-glycans in haptoglobin purified from the sera of 10 patients with colorectal cancer by LC-ESI MS. (A) Base peak chromatogram of desialo-glycopeptides containing sites 1–4 of Hpt (patient #3). (B) Averaged mass spectra of desialo-glycopeptides containing site 1 (patient #3 and #7). (C) Ratio of peak intensity of fucosylated N-glycans to total peak intensity of all N-glycans on Hpt (patient #1–#10). 2-0, biantennary N-glycan; 3-0, triantennary N-glycan; 4-0, tetraantennary N-glycan; 2-F, mono-fucosylated biantennary N-glycan; 3-F, mono-fucosylated triantennary N-glycan; 4-F, mono-fucosylated tetraantennary N-glycan; 3-FF, di-fucosylated triantennary N-glycan.
1
2 3
0
5
4 5 6 7
8
9 10
300 250 200 150 100 50
0 10 15 20 25 30 p < 0.0001 r = 0.945
Fig. 4. Correlation between Fuc-Hpt levels determined with lectin–antibody ELISA, and fucose content determined by LC-ESI MS. The sera from Fig. 4 were used in this analysis. Core fucose content was evaluated as the level of fucose content in 2-F. Spearman R correlations were used to assess the relationships between fucose/core fucose contents and the results of lectin– antibody ELISA.
34
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3.3. Site-specific N-glycan analyses of Hpt by LC-ESI-MS Ten cases of colorectal cancer exhibiting high levels of core-Fuc-Hpt, as measured using PhoSL-antibody ELISA, were analyzed by LC-ESI MS. The base peak chromatogram, shown in Fig. 3A, demonstrated the separation of glycopeptides containing sites 1–4. The averaged mass spectra of site 1 N-glycans on Hpt for the patients #3 and #7 are shown in Fig. 3B. Based on peak intensity of each N-glycan, the ratio of fucosylated N-glycans to total N-glycans on Hpt in 10 cases is shown in Fig. 3C. All cases showed a high proportion of fucosylated glycans on site 1 of Hpt glycans (2.2–28.2%). This ratio was significantly higher than that of healthy volunteers (2.8–4.3%). Next, we compared the ratio of fucosylated glycans to total glycans on Hpt, with Fuc-Hpt levels measured using AAL/PhoSL-antibody ELISA. As shown in Fig. 4, Fuc-Hpt levels measured with AAL-antibody ELISA were strongly correlated with fucosylation ratios, as determined by mass spectrometry, with the exception of one case; this case showed a high level of core-Fuc-Hpt, as determined with PhoSL-antibody ELISA. Furthermore, the proportion of fucosylated glycans on bi-antennary sugar chains correlated with core-Fuc-Hpt levels, as determined using PhoSL-antibody ELISA. 3.4. Clinical significance of core-Fuc-Hpt in colorectal cancer We investigated the clinical significance of serum levels of core-FucHpt, as reflected by the clinical stage of colorectal cancer. As shown in Fig. 5A and B, we evaluated the number of core-Fuc-Hpt-positive patients among the patients with stage I (0 cases), stage II (2 cases), stage III (5 cases), and stage IV (3 cases) colorectal cancer. Although we observed no statistical significance between core-Fuc-Hpt levels and clinical stage, most of the core-Fuc-Hpt-positive cases (8/10 cases) were in the advanced stages of colorectal cancer (stage III and IV). However, there
Disease
PhoSL-Hpt (U/ml)
CV value (%)
Colorectal cancer Colorectal cancer Colorectal cancer Pancreatic cancer Pancreatic cancer Pancreatic cancer
94.9 ± 16.5 141.4 ± 46.4 310.9 ± 17.1 145.1 ± 27.8 268.1 ± 24.1 536.2 ± 42.0
17.4 32.8 5.5 19.2 9 7.8
PhoSL-Hpt values are presented as the mean ± SD.
were no significant differences in mean disease-free time between core-Fuc-Hpt-positive and negative patients. 3.5. Evaluation of core-Fuc-Hpt lectin–antibody ELISA kit in various conditions We evaluated the efficacy of our developed core-Fuc-Hpt lectin– antibody ELISA kit in various conditions. At first, we assessed the reproducibility of the core-Fuc-Hpt assay. As shown in Table 1, the CV values were 5.5 to 32.8%. Secondly, we investigated the effects of serum dilution on the measurement values of our ELISA kit (Fig. 6). When we compared data from the 25-fold dilution with those from other dilutions, relatively high correlations were observed among assay results obtained using 10-, 20-, 50-, and 100-fold dilutions. Thirdly, we evaluated the effects of icterus, hemolysis, and lipids on the lectin–antibody ELISA kit. We added free bilirubin (bilirubin F), hemoglobin, and formagine to the samples (pooled sera of colorectal cancer patients) for ELISA. As shown in Fig. 7, bilirubin and formagine
***
5000
*
*
**
Core-Fuc-Hpt (Units/ml)
(A) Fuc-Hpt (Units/ml)
Table 1 Reproducibility of PhoSL-Hpt assay at different times, using patient serum samples (colorectal and pancreatic cancer patients).
4000 3000 2000 1000 0 I
II
III
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100 0
IV
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(B)
*
*
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Fuc-Hpt (Units/ml)
8000 4000 0 II
IV
Stage
12000
I
III
III
Stage
IV
700 600 500 400 300 200 100 0
I
II
III
IV
Stage
Fig. 5. Correlation between each type of Fuc-Hpt and clinical stage of colorectal cancer and pancreatic cancer. The serum levels of total (AAL-reactive) and core (PhoSL-reactive) Fuc-Hpt determined using lectin–antibody ELISA were investigated in terms of clinical stage of colorectal cancer (A) and pancreatic cancer (B). Wilcoxon test was used for statistical analysis. * p b 0.005, ** p b 0.02, *** p b 0.05.
M. Shimomura et al. / Clinica Chimica Acta 446 (2015) 30–36
(A)
(B) 500
400 R = 0.703 P = 0.0158
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200 0
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0
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Fig. 6. Comparison of serum dilution and core-Fuc-Hpt levels in the lectin–antibody ELISA. Serum core-Fuc-Hpt levels in 11 subjects including 2 healthy volunteers, 5 colorectal cancer patients, and 4 pancreatic cancer patients. (A–D) Comparison of the values of each dilution (×10, ×20, ×50, ×100) in the ELISA assay. The x-axis represents data from the 25-fold dilution in all cases and the individual y-axis represents data from the 10- to 100-fold dilutions.
decreased the measurement values of core-Fuc-Hpt. Surprisingly, we found that hemoglobin increased the measured core-Fuc-Hpt levels in a dose-dependent manner. 4. Discussion We investigated the possibility of using the lectin–antibody ELISA as a tool to measure core-Fuc-Hpt in the sera of patients with cancer. As expected, most of the Fuc-Hpt increase in the sera of cancer patients consisted of Lewis-type fucosylation, and the PhoSL-antibody ELISA was positive for significantly fewer cases than the AAL-antibody ELISA.
(A)
However, the positive rate of core-Fuc-Hpt in colorectal cancer was higher than in pancreatic cancer, although the positive rate of Fuc-Hpt determined with AAL was higher in the sera of patients with pancreatic cancer [2]. Fuc-Hpt in pancreatic cancer patients might be produced in metastatic lesions in the liver, because pancreatic cancer cell lines scarcely expressed Hpt mRNA [2], and interleukin-6 (IL6) produced by pancreatic cancer cell lines induced the production of AAL-reactive Fuc-Hpt from co-cultured hepatoma cells [11]. Fucosylated glycoproteins produced in hepatocytes are secreted into bile, but not into blood in a normal liver [12], and the loss of polarity of hepatocytes in hepatocarcinogenesis or liver metastasis of other cancers could result
(B)
(C)
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Formagine conc. (mg/dl)
Fig. 7. Effects of bilirubin, hemoglobin, and formagine contamination on the core-Fuc-Hpt assay. Sera of colorectal cancer patients were measured using core-Fuc-Hpt lectin–antibody ELISA kit with various concentrations of free bilirubin (bilirubin F) (A), hemoglobin (B), and formagine (C).
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in the secretion of fucosylated glycoproteins into blood, which might be detected as a cancer biomarker [13]. In contrast, core-Fuc-Hpt is scarcely produced in normal hepatocytes, which do not express α1-6 fucosyltransferase, an enzyme involved in the process of corefucosylation. Therefore, core-Fuc-Hpt could be produced in cancer cells themselves, or in lymphocytes around cancer tissue. An immunohistochemical study has shown the production of Hpt in colon cancer tissue and demonstrated that serum Fuc-Hpt levels are increased in colorectal cancer [14]. Furthermore, the expression of α1-6 fucosyltransferase is dramatically increased in colorectal cancer tissue [15]. The expression of Hpt mRNA was detected in several kinds of colon cancer cell lines (data not shown). These results suggest that discrimination of core and Lewis-type Fuc-Hpt could indicate the origin of Fuc-Hpt producing cells. In contrast, the biological/clinical significance of Hpt remains unknown, and the genotype of Hpt is involved in several diseases [16]. The biological functions of characteristic Fuc-Hpt are not proven compared to non-fucosylated/total Hpt. In clinical analyses, cases with high levels of core-Fuc-Hpt are frequently observed at the advanced clinical stages of colorectal cancer (Fig. 5A), and a combination of carcinoembryonic antigen (CEA) and AAL-reactive Fuc-Hpt is a marker for poor prognosis following surgery [17]. Nevertheless, cases with high levels of core-Fuc-Hpt showed relatively long survival/diseasefree time compared to core-Fuc-Hpt-negative and AAL-reactive FucHpt-positive cases. However, compared to AAL-reactive Fuc-Hpt, coreFuc-Hpt has little clinical significance in pancreatic cancer (Fig. 5B). These results might suggest that core-Fuc-Hpt has biological functions such as promoting cancer immune surveillance in the microenvironment of colorectal cancer, but not in that of pancreatic cancer; further studies are warranted to investigate this hypothesis. PhoSL lectin, as well as PhoSL-antibody ELISA, may prove to be effective tools for this purpose. Furthermore, we evaluated the efficacy of our developed lectin–antibody ELISA kit in various conditions (Figs. 6, 7) like previous our study. Our findings indicated that serum dilution from 10 to 100-fold can be measured in PhoSL-antibody ELISA kit accurately. The contamination of bilirubin and lipids had inhibitory effects on the ELISA measurements. In contrast, the contamination of hemoglobin increased values of measured core-Fuc-Hpt levels, although the contamination of hemoglobin decreased values of Fuc-Hpt levels measured with AALantibody ELISA kit [4]. This discrepancy might be conformational changes of haptoglobin and hemoglobin, resulting in changes of each lectin binding. These data indicated that the measurements of coreFuc-Hpt using PhoSL-antibody ELISA kit require some attention in the patients with dyslipidemia, icterus, and especially hemolysis. Taken together, we have demonstrated the development and utility of a core-Fuc-Hpt lectin-ELISA kit using PhoSL. Disclosure statement Yuka Kobayashi is an employee of J-Oil mills Incorporation. None of the other authors have any conflict of interest to declare.
Acknowledgments This study was performed as a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2015.03.037.
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