Proteomic Analysis of Serum Associated Fucosylated Glycoproteins in the Development of Primary Hepatocellular Carcinoma
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Mary Ann Comunale†, Melissa Lowman,† Ronald E. Long,† Jonathan Krakover,‡,§ Ramila Philip,‡ Steven Seeholzer,| Alison A. Evans,| Hie-Won L. Hann,# Timothy M. Block,† and Anand S. Mehta*,† Drexel Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology, Drexel University College of Medicine, Doylestown, Pennsylvania 18901, The Institute for Virus and Hepatitis Research, Pennsylvania Commonwealth Institute, Doylestown, Pennsylvania 18901, Immunotope Inc., Doylestown, Pennsylvania 18901, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and Division of Gastroenterology and Hepatology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received September 29, 2005
Changes in N-linked glycosylation are known to occur during the development of cancer. For example, increased branching of oligosaccharides has been associated with metastasis and has been correlated to tumor progression in human cancers of the breast, colon and melanomas. Increases in core fucosylation have also been associated with the development of hepatocellular carcinoma (HCC). Chronic infection with the hepatitis B virus is associated with more than 55% of all cases of hepatocellular carcinoma. We show here that increased levels of core fucosylation can be observed via glycan analysis of total serum and are associated with the development of HCC. In a blinded study, the serum glycoproteins derived from people diagnosed with HBV induced liver cancer were found to possess a dramatically higher level of fucosylation. This change occurs on both immunoglobulin molecules and on other serum glycoproteins. Targeted glycoproteomic analysis was used to identify those glycoproteins that are hyperfucosylated in cancer. In total, 19 proteins were found to be hyperfucosylated in cancer. The potential of these proteins as biomarkers of cancer is discussed. Keywords: hepatocellular carcinoma • fucosylation • glycomics • biomarkers
Introduction Infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) is the major etiology of hepatocellular cancer (HCC).1-3 Both HBV and HCV cause acute and chronic liver infections and most chronically infected individuals remain asymptomatic for many years. Clinical disease (HCC and cirrhosis) is not apparent until decades later. 10-40% of all chronic HBV carriers eventually develop liver cancer, and it is estimated that over one million people worldwide die because of HBV/HCV associated liver cancer.4 Indeed, HBV and HCV infections are associated with over 80% of all HCC cases worldwide and can be as high as 96% in regions where HBV is endemic.5 The chronic infection of HBV or HCV is asymptomatic and monitored by regular (usually annual or biannual) physical assessments, serum liver function tests (LFTs) and ultrasound imaging for detection of small masses in the liver (Hepatitis B foundation, 1994). The effectiveness of ultrasound is limited, * To whom correspondence should be addressed. E-mail: anand.mehta@ drexel.edu. † Drexel University College of Medicine. ‡ Pennsylvania Commonwealth Institute. § Immunotope Inc. | Fox Chase Cancer Center. # Thomas Jefferson University.
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since the appearance of masses at least 3 cm in size are required for detection, and this often occurs at a stage when the prognosis is poor.6,7 The correlation between elevated serum concentrations of alpha feto protein (AFP) and the occurrence of HCC has provided a useful surrogate marker for disease.8 Levels of AFP exceeding 50 ng/mL occur in 30-60% of HCC cases at the time of diagnosis.9,10 However, AFP levels may fluctuate in chronically infected individuals and AFP is a poor biomarker for small tumors.9 As early surgical and chemotherapeutic intervention is an afflicted individual’s best hope, there is a clear and urgent need for noninvasive, reliable methods of detecting HCC as early as possible.11-13 Our previous work has utilized a targeted glycoproteomic approach in an effort to identify serum glycoproteins that correlate with the development of cancer. That is, in an animal model of virally induced liver cancer (woodchuck), total serum glycan analysis revealed a correlation between the development of HCC and an increase in the level of core fucosylation.14 Proteomic analysis of the fucosylated proteome revealed a protein, GP73, which was increased in animals with HCC. This protein was further analyzed in human samples and has shown to be a better marker of HCC than the currently used marker AFP.10,14 As this methodology proved successful in an animal model of HCC, it was of great interest to see if even more proteins could be identified in human samples. Thus, in this 10.1021/pr050328x CCC: $33.50
2006 American Chemical Society
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Serum Associated Fucosylated Glycoproteins Table 1. Profiles of the Clinical Populations (A) and HBV Infected Patients at Multiple Time Points (B) (a) Profiles of the Clinical Populations in This Study no. of individuals (N)
group
healthy
5
HBV infected HCCd
7 9
liver function testsb
HBV statusa
no evidence of HBV, HCC, or liver disease HBsAg+, HBV DNA positive HBsAg+, diagnosis of HCC
AFPc
age (( s.d.)
normal
normal
58.0 (6.5)
normal to elevated variable
variable elevated
51.2 (8.5) 55.1 (8.8)
(B) Profiles of HBV Infected Patients at Multiple Time Points. patient no.
age at time of HCC/sex
sample collection date 1 (precancer)
Dx at sample draw #1e
AFP level (ng/ML) at draw #1f
sample collection date 2 (cancer)g
Dx at sample draw #2
AFP level (ng/ML) at draw #2f
965 370 121 771 842 999 103 978
52/M 56/F 45/F 49/F 54/F 51/M 53/M 55/M
5/18/1994 3/25/2000 7/16/1998 10/4/1989 5/30/1990 12/19/1994 8/7/1995 8/15/1994
Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis
7.82 ND ND 2.2 2.7 ND 2.8 7.6
6/5/1996 3/18/2002 8/2/2000 4/11/2002 3/17/2000 2/15/1996 6/4/1997 10/30/1996
HCC HCC HCC HCC HCC HCC HCC HCC
5.0 6.8 ND 137.2 6.7 5.2 1488.6 2.4
a HBsAg and HBeAg reactivites (positive or negative) were determined by standard immunoassays (Abbott Labs). HBV DNA was detected by a “dot blot” method and has a detection limit of sensitivity of approximately 3 × 105 genome equivalents per ml. b ALT (alanine aminotransferase) levels were determined by an enzyme activity assay and the upper limit of normal was considered to be 50 IU/mL. c AFP (alpha fetoprotein) was determined using standard kits (Abbott Labs) and 20 ng/mL was considered the upper limit of normal. d Ultrasound imaging and biopsy confirmed HCC diagnosis. e The diagnosis of cirrhosis was made by ultrasound imaging and biopsy. f AFP (alpha fetoprotein) was determined using standard kits (Abbott Labs) and 20 ng/mL was considered the upper limit of normal. ND is not determined. g The diagnosis of HCC was made by ultrasound imaging and biopsy.
report we have determined that a significant increase in the level of core fucosylation on serum glycoproteins is associated with the development of human HCC. Using several proteomic methodologies, we have identified 19 glycoproteins that are altered in the fucosylated serum proteome from patients with HCC. The potential use of these markers in the detection of liver cancer is discussed.
Materials and Methods Human Subjects. Human serum samples used were obtained from two major sources. The first set was archived human serum from the Fox Chase Cancer Center (FCCC) that was obtained coded, and used for glycan analysis. These included samples from 7 patients who were chronically infected with HBV and 4 patients classified as having HCC as determined by ultrasound. Another set of samples, also from FCCC, which we have used previously, was used for the proteomic analysis. This sample set included normal patient sera from patients with no evidence of HBV, HCV, or other liver disease (5 patients); and serum from patients classified with HCC that were HBsAg+ with a positive identification of HCC by ultrasound (5 patients). Table 1a provides information regarding the samples obtained from FCCC. A separate sample set was obtained from Thomas Jefferson University and consisted of 16 samples from 8 patients either before or after the diagnosis of cancer. All patient samples before cancer were diagnosed with cirrhosis. The diagnosis of cirrhosis and/or HCC was determined by ultrasound and biopsy. See Table 1b for more detail. Glycan Analysis. Protein aliquots (1 mg/mL) were denatured in 1% SDS, 50 mM β-mercaptoethanol for 10 min at 100 °C. The solution was cooled and NP-40 was added to a concentration of 5.75% to mask the denaturing properties of the SDS. PNGase F (ProZyme, San Leandro, CA) was added to a final concentration of 1mU(IUB)/µL and the solution incubated at 37 °C for 24 h containing a cocktail of protease inhibitors. The released oligosaccharides were recovered and purified by solid
phase extraction using a porous graphite matrix (LudgerClean H, Ludger Limited, Oxford, UK). Released oligosaccharides were labeled with 2-aminobenzamide and cleaned using commercially available kits (Ludger Limited UK) Fluorescently labeled glycans were subsequently analyzed by HPLC using a normal phase column (TSK amide 80 column). The mobile phase consisted of Solvent A (50 mM ammonium formate, pH 4.4) and Solvent B (acetonitrile) and the gradient used was as follows: linear gradient from 20-58% Solvent A at 0.4 mL/ minute for 152 min followed by a linear gradient from 58100% Solvent A for the next 3 min. The flow rate was increased to 1.0 mL/minute and the column washed in 100% Solvent A for 5 min. Following the wash step, the column was equilibrated in 20% Solvent A for 22 min in preparation for the next sample run. HPLC analysis was performed using the Waters Alliance HPLC System, complemented with a Waters fluorescence detector, and quantified using the Millennium Chromatography Manager (Waters Corporation, Milford, MA). Glycan structures were identified by comparison to known standards and by sequential exoglycosidase digestion as has been done previously.14-17 Lectin Extraction and Analysis. Immunoglobulins were removed from the samples (media and serum) using a Protein A/G column (Pierce, Rockford, IL) prior to the lectin extraction as we have done previously.14 Samples were then supplemented with a lectin binding solution bringing the final concentration of the sample to 20 mM Tris buffered saline (TBS), 1 mM calcium chloride, 1 mM magnesium chloride, and 1 mM manganese chloride (pH 7.0). The samples were incubated for 16 h at 4 °C with an array of agarose bound fucose recognizing lectins.14 These lectins consisted of Lens culinaris (LCH), Pisum sativum (PSA) and Vicia faba (VFA) and recognize branched mannoses with the alpha fucose determinant (all purchased from EY laboratories, San Mateo, CA). Incubation was performed in an eppendorf tube prior to transfer to a Costar 0.45 µM Spin-X column (Corning, Acton, MA). The lectin column was washed thoroughly with lectin binding solution before the Journal of Proteome Research • Vol. 5, No. 2, 2006 309
research articles bound fraction was eluted using the appropriate inhibitory monosaccharides (200 mM methyl-R-D-glucopyranoside, 200 mM R-methyl-D-manno-pyranoside). The bound and unbound fractions were buffer exchanged into TBS using Milllipore YM-3 Centricon devices and subjected to glycan analysis or 2DE as described. Protein levels were monitored throughout all extractions. 2-Dimensional Gel Electrophoresis. The sample was diluted in a buffer containing 7M urea, 2M thiourea, 4% CHAPS, 65 mM DTT, 5 mM TBP, and 0.4% ampholytes, vortexed periodically for 1 h and applied to an 18 cm pH3-10NL IPG strip (Amersham, Piscataway, NJ). Gel rehydration was carried out for 14 h at 50 V, and focused using the Protean (Bio-Rad Laboratories Headquarters, Hercules, CA) IEF apparatus. After focusing, gel strips were reduced in 6 M urea, 2% SDS, 1.5% DTT, 30% glycerol, and 50 mM Tris pH 6.8 for 10 min and alkalated in 6 M urea, 2% SDS, 3% iodoacetamide, 30% glycerol and 50 mM Tris pH 6.8. The second dimension was resolved with an 8-18% acrylamide-0.8% PDA gradient gel on a Protean II xi cell (Bio-Rad) with the running conditions set to 20 mA/ gel for 20 min and 40 mA/gel for 4 h with gels cooled to 14 °C. Gels were fixed (30% EtOH/5% phosphoric acid) and stained with a colloidal coomassie brilliant blue stain. For all samples, gels were run in quadruplicate and only differences that were consistent in all gels were considered meaningful. Gel Imaging and Analysis. Gels were digitally imaged using a 16-bit cooled CCD camera (FluorChem 8000, AlphaInnotech, San Leandro, CA). TIFF files of the gel images were analyzed using NonLinear Dynamics Progenesis Workstation gel imaging software package (Nonlinear USA Inc., Durham, NC). The polypeptide “features” in each gel image were delineated and then the software determined the total intensity of pixels within each feature (the integrated intensity). Polypeptide features were normalized by using the integrated intensity of each feature and expressing it as a percent of the sum of integrated intensities of the entire gel. Mass Spectrometry. MALDI Analysis. Protein spots were excised from colloidal Coomassie blue stained gels, de-stained, and digested with trypsin as we have done previously.18 Recovered peptides were concentrated and desalted using Zip Tip C18 (Millipore, Bedford, MA) according to manufacturer’s directions and prepared for MALDI-TOF mass spectrometry by mixing 0.5 µL of peptide mixture with 0.5 µL of 10 mg/mL of alpha-cyano 4-hydroxy cinnamic acid, 1% formic acid in 50% acetonitrile and allowing the droplet to dry on the MALDI plate. Peptide mass maps were obtained using a Voyager-DE Pro Mass Spectrometer (PE Biosystems, Foster City, CA) operated in positive ion reflectron mode. Proteins were identified from the peptide mass maps using the MASCOT online database www.matrixscience.com to search the nonredundant protein database. LC MS/MS Analysis. Peptide identification was performed on a ThermoFinnigan LCQ ion trap mass spectrometer (Thermo Electron Corporation, CA) equipped with on-line microcapillary HPLC (Eldex, Napa, CA) and microspray ionization source (home-built). The microspray consists of a microcapillary column, picotip 360 × 75 µm, with an integrated 15 µm tip (New objective, Woburn MA) self-packed with Reliasil C18 resin (Column Engineering, Ontario, CA) to a length of 10 cm. An aliquot of the sample was loaded via a pressure bomb into a capillary sample trap (Upchurch, WA) self-packed with C18 then placed just upstream of the microcapillary column. The HPLC is programmed to produce a 3 h gradient (5%-65%B) at 310
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30 µL/min. Prior to the trap, passive flow splitting is used to reduce the flow down to ∼500 nl/ minute. Buffer A consists of 5% acetonitrile + 1% acetic acid, buffer B consists of 90% acetronitrile + 1% acetic acid. The LCQ was programmed to perform a full scan from 450 to 2000 m/z, followed by 3 data dependent MS/MS scans set to pick the most abundant ion species from the full scan. Data Analysis and Interpretation. Mass spectrometry data (spectra) was searched against the nonredundant human database using SeQuest (Thermo Electron Corporation). Peptides that score above the threshold value of 1.5 XCorr (singly)/ 2.0 XCorr (Doubly)/2.5 XCorr (triply) were then manually verified. In all cases, the bound peptides should contain the N-linked glycosylation sequon, N-X-S/T that has been converted to a D-X-S/T by the action of the PNGase F enzyme. This results in a Mass difference of 0.9840 Da as compared to the glycosylated sequence. Immunoblotting. Equal volumes of patient sera from either total serum (0.5 µL/lane), non lectin bound (unbound) or lectin bound (bound) were resolved by SDS-PAGE on 4-20% polyacrylamide gradient gels. The proteins were transferred to a PVDF membrane by immunoblotting. The membranes were blocked by incubating with a blocking buffer of 1× TBS (50 mM Tris-HCl, pH 7.6, 150 mM sodium chloride) made 5% nonfat dried milk, and 0.1% Tween 20 for 1 h at room temperature. The blots were then incubated overnight with desired antibody as we have done previously and developed using chemiluminescent detection system (“ECL Plus”, Amersham Pharmacia Biotech, Arlington Heights, IL). Blots were visualized using an AlphaInnotech FluorChem CCD camera with AlphaEase spot densitometry software (AlphaInnotech Corporation San Leandro, CA).14
Results We have developed a targeted glycoproteomic methodology that allows for the comparative analysis of both glycans and glycoproteins in serum. The first step in this methodology is to identify the glycan modification that occurs with the development of cancer. Subsequently, those glycoproteins containing the altered glycan are extracted using lectins and the proteins resolved using multiple proteomic methodologies. In our previous work with the woodchuck model of HBV induced HCC, we observed a significant increase in the level of core fucosylation on serum glycoproteins associated with HCC and identified a potential biomarker of HCC that is currently being tested for use in human liver cancer.10,14 However, proteomic analysis in the woodchuck animal model was difficult as homology to other rodent species is low and protein databases for the woodchuck do not exist.14 Thus, we wondered if a similar increase in core fucosylation (or any other specific glycan) could be seen in human patients diagnosed with HCC. To determine this, we performed N-linked glycan analysis on the total serum glycoproteins using methods that we had previously developed.14 In contrast to the woodchuck, human sera contains large quantities of immunoglobulins that normally contain fucose residues. Thus, glycan analysis was performed on both total serum and on IgG depleted sera. These results are shown in Figure 1A,B. Figure 1A shows the glycan analysis of serum from three representative patients who were clinically diagnosed as being normal, HBV infected or HBV infected with HCC. As this figure shows, there is an increase in the level of fucosylation that occurs in patients with cancer. The main structure that becomes core fucosylated with cancer
Serum Associated Fucosylated Glycoproteins
research articles protein A/G column. While the FcA2G0 structure is no longer present, an increase in R-1,6 linked core fucosylation is still seen on the biantennary glycan (FcA2G2), which is increased in the patient with HCC. Figure 1C shows the level of the FcA2G2 structure in a small set of patients. These samples were provided to us as “coded” samples by collaborators at the FCCC. Glycan analysis was performed and the level of the FcA2G2 structure determined as a function of the total glycan pool. Figure 1C shows the results of the complete study in which samples fell into two categories: HBV infected individuals with active hepatitis and cirrhosis who had lower levels of the FcA2G2 glycan (similar to purchased normal serum) and patients diagnosed with HCC with higher levels of the FcA2G2 glycan. These results suggested that, like the situation in the animal model, total serum glycan analysis could reveal changes in glycosylation and more specifically, increased fucosylation correlates with the development of liver cancer. Figure 1D shows the level of the FcA2G2 structure in patients either before or after the development of cancer. This small sample set consisted of 16 samples from 8 patients at two time points: before the development of cancer or after the diagnosis of cancer. In this case, all patients classified as before were clinically diagnosed with cirrhosis. Samples classified as after were clinically diagnosed with HCC by ultrasound and biopsy. As this figure shows, patients before the diagnosis of HCC have much lower levels of total FcA2G2 glycan as compared to after the diagnosis of HCC. For example, patient 121 had 7.23% of the FcA2G2 glycan before the diagnosis of HCC and 13.2% after the diagnosis of HCC. As seen in Figure 1D, this upward trend was true of all patients examined. Consistent with the results obtained in Figure 1C the only patients with >10% of the FcA2G2 glycan were those diagnosed with HCC. Again, these results highlight the fact that increased levels of R-1,6 linked core fucosylation are associated with the development of HCC. This is true in samples that were AFP positive (>20 ng/ML) or AFP negative. That is, as shown in Figure 1D and Table 1b, patients 965, 307, 842, 999, and 978 were all AFP negative, yet had increased levels of the FcA2G2 glycan. The results also suggest that the periodic surveillance of HBV+ positive individuals for elevations in their serum associated FcA2G2 glycan could potentially lead to the detection of HCC.
Figure 1. Level of core fucosylated alpha 1,6 linked bi-antennary (FcA2G2) glycan in normal individuals and in individuals that have developed HCC. (A) Total serum glycan profiles from representative normal and HCC+ individuals with the FcA2G0 peak indicated. (B) Glycan analysis of IgG depleted serum from a representative normal and HCC+ individuals with the FcA2G2 peak indicated. (C) The levels of the FcA2G2 glycan in a small patient cohort. Sample 377244, 374539, 377525, and 375609 are HCC positive (right side of chart). All other patients are from either HBV infected patients (387170, 586792, 387323, 376159, 376991, 377226, 605745) or from purchased sources (Sigma). Values are the average of 5 runs. (D) Levels of the FcA2G2 structure in 8 individuals either before (B) or after the diagnosis of cancer (A). For graph, Y axis is the % of FcA2G2 structure in each individual as a function of total released glycan.
is a de-galactosylated biantennary structure (FcA2G0). This structure is associated with IgG (and IgA) as removal of these components removes this signal (Figure 1B). This finding is consistent with a recent report that found this structure to increase with cirrhosis and possibly HCC.19 Figure 1B shows glycan analysis of samples after the removal of IgG using a
As the level of R-1,6 linked core fucosylation was increased in patients with liver cancer, it was of interest to determine those proteins that had increased fucosylation. Therefore, the fucosylated glycoproteins associated with sera from either pooled composite normal or HCC positive individuals was extracted using lectins specific for core fucosylated glycoproteins and analyzed via 2-dimensional gel electrophoresis (2DE).14 The rationale for using pooled patient samples (composite samples) as a primary screen for biomarkers is provided in another publication,18,20 and relates to the goal of simplifying the simultaneous analysis of samples from multiple origins and identifying biomarkers robust enough to be detected by 2-DE of multiple samples. Figure 2 shows the fucosylated 2DE proteome from the composite sera of 5 normal (A) and 5 HCC positive individuals (B) after the removal of immunoglobulins using a protein A/G column. Figure 2A shows the 2DE profile from the total serum proteome (minus immunoglobulins), the unbound non fucosylated serum proteins and the fucosylated glycoproteins from the pooled normal sera. Figure 2B shows the same analysis from the sera from pooled cancer sera. For Figure 2A,B the glycan analysis with each fraction is shown on top of each gel image with the position of the FcA2G2 glycan Journal of Proteome Research • Vol. 5, No. 2, 2006 311
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Figure 2. Lectin extraction and glycan analysis of fractionated glycoproteins from pooled serum from either normal individuals or individuals with cancer. (A) The proteins associated with total pooled normal sera depleted of immunoglobulin (left); found in the flow through of the fucose specific lectin column (unbound, middle) or associated with the fucose blinding lectin (bound, right). (B) The proteins associated with total pooled HCC sera depleted of immunoglobulin (left); found in the flow through of the fucose specific lectin column (unbound, middle) or associated with the fucose blinding lectin (bound, right). The glycan profiles from each fraction are shown on the top of the 2DE image and highlight the extraction efficiency (>95%). (C) The fucosylated proteome from the composite cancer sample with specific areas of the gel highlighted. The relative change in expression levels of specific fucosylated glycoproteins from pooled serum from normal individuals (Normal, left column) or from individuals diagnosed with HCC (HCC, right column) is shown in the panels to the right of the main figure. Panel 1: Alpha-1-acid glycoprotein; panel 2: haptoglobin; panel 3: ceruloplasmin; panel 4: alpha-2 macroglobulin; panel 5: serotransferrin.
indicated. The efficiency of extraction is observed in the level of the FcA2G2 glycan present in the unbound fraction. As this figure shows less than 1% of the FcA2G2 glycan (as compared to total) was found in the unbound fraction. The presence of the non fucosylated A2G2 structure in the bound fraction is the result of glycans heterogeneity. That is, a glycoprotein may have multiple sites of glycosylation but only contain fucosylation on a limited number of them. Figure 2C shows the fucosylated proteome from the composite cancer sample with specific areas of the gel highlighted and these areas are shown in the inset from both the composite normal and cancer samples. As this figure shows, there are 312
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Figure 3. LC MS/MS analysis of the human fucosylated proteome. Briefly, after immunoglobulin extraction (step 1), human serum protein will be fragmented with trypsin (step 2) and the fucosylated glycopeptides extracted using a mixture of lectins (step 3). Extraction efficiency is measured by the glycan analysis of the bound and unbound fraction (step 4). The unbound fraction contains no fucosylated peptides and the bound fraction contains primarily fucosylated glycan. MS fragmentation analysis is used to assign protein identity. In all cases, the bound peptides should contain the N-linked glycosylation sequon, N-X-S/T that has been converted to a D-X-S/T by the action of the PNGase F enzyme.
several features that can be observed in the fucosylated proteome. Many of these are increased in the HCC sample. Proteins such as alpha-1-acid glycoprotein (AGP; panel 1), ceruloplasmin (panel 3), alpha-2-macroglobulin (panel 4) and sero-transferrin (panel 5) increase dramatically, whereas the levels of haptoglobin (panel 2) decrease dramatically in the fucosylated proteome of those patients with cancer. A large albumin spot is also present in all samples and is a contamination of the system. To further examine the difference in the fucosylated proteome as a function of cancer, we employed the use of a simple LC MS/MS following lectin extraction of fucosylated glycopeptides. Although this method would not allow for quantitative differences (in abundance) of the identified glycoproteins, it would rather be useful in the identification of all the fucosylated proteins in a given pool. In addition, it would allow for the identification of those fucosylated proteins that are present in one sample but not another. Briefly, as shown in Figure 3, after immunoglobulin depletion, proteins are digested with trypsin and the fucosylated peptides extracted with fucose specific lectins. The fucosylated peptides are then treated with PNGase F, to remove N-linked glycan and analyzed by LC MS/MS. As summarized in Tables 2 and 3, this method allowed for the identification of many of the proteins identified in Figure 2C and the identification of glycoproteins that were masked by the albumin contamination. For example, both hemopexin and AFP have molecular weights and isoelectric points similar to
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Serum Associated Fucosylated Glycoproteins
Figure 4. Level of fucosylated R-1 acid glycoprotein (AGP) in pooled serum from patients infected with HBV and positive for liver cancer (C) or just infected with HBV (N) as detected by immunoblotting. From left to right:(+) is the level of AGP in total serum purchased from Sigma Chemicals. The position of AGP is indicated. The total lanes contain the level of AGP in the unfractionated Cancer + and Normal serum. The unbound fraction contains the level of non fucosylated AGP in the cancer and normal serum while the bound fraction contains only the fucosylated AGP. As this figure shows, only the fucosylated level of AGP changes with disease.
albumin and would be in the area of the 2D gel covered by the albumin. However, this LC MS/MS method identified both these glycoproteins as being present in the pooled cancer serum, but not in the serum of normal individuals. Other proteins that were not detected in the pooled normal sera but were present in the sera from pooled from HCC patients included Apo-D, fetuin A, IgM, Kininogen, and GP73. Surprisingly, in all cases, only a single peptide for each glycoprotein was identified as containing a core fucosylated glycan, suggesting glycan heterogeneity on the identified glycoproteins. This is consistent with the results obtained from the glycan sequencing of the lectin extracted fractions in Figure 2A,B. The relative increase or decrease of all identified glycoproteins was confirmed by 1-dimension gel electrophoresis of the fucosylated protein pool followed by an immunoblot for the specific protein. An example of this is presented in Figure 4, which shows the level of fucosylated alpha 1-acid glycoprotein (AGP) in the pooled cancer and normal sera as detected by immunoblotting. As this figure shows, there is a dramatic increase in the level of only fucosylated alpha 1-acid glycoprotein in patients diagnosed with cancer. That is, while total levels of AGP do not change (compare C and N “total” lanes in Figure 4) the levels in the bound fucosylated AGP change dramatically. Thus, while the total level of AGP may not make a good biomarker of cancer, the level of the fucosylated AGP might. Figure 4 also shows that only a small portion of AGP becomes fucosylated. In this case, it is 10% of the total amount. This is consistent with the hypothesis that the tumor tissue primarily secretes fucosylated proteins.
a rapid, highly sensitive and quantitative method of glycan sequencing to identify specific changes that occur with the development of liver cancer. Indeed, as Figure 1 shows, specific changes in the glycosylation of total serum can be seen in patients with the development of cancer. This is true in both total serum and serum that has been depleted of IgG. An increase in the R-1,6 linked core fucosylation of the immunoglobulins (IgG) is interesting and consistent with recent reports.19 Further proteomic analysis of either fucosylated peptides, or protein A/G associated fucosylated peptides, indicated that the Fc domain of IgG, IgA, and IgM had increased fucosylation in HCC sera. Why these immunoglobulins are hyper fucosylated in HCC is unclear. Investigations are underway to determine if specific immunoglobulins are altered or if it is a global effect. Changes in glycosylation were also observed in serum that was depleted of IgG. A consistent increase in the level of the FcA2G2 glycan was observed in patients with HCC. While all patients diagnosed with HCC had >10% of the FcA2G2 glycan, there was considerable variation between HCC patients. It is unclear if this difference is related to the actual size of the tumor or other factors. It should also be noted that many of the patients shown in Figure 1 were AFP negative but still contained elevated levels of core fucosylation. Thus, the discovery of fucosylated proteins that alter with the development of HCC may be useful in combination with AFP.
Discussion
The identity of the other proteins that became fucosylated was determined using a targeted glycoproteomic methodology. By this method, we have confirmed the up-regulation of several glycoforms that have been previously reported to be elevated and fucosylated in the serum of people with liver cancer. The complete list of fucosylated glycoproteins is presented in Table 3. For example, AFP, alpha-1-acid glycoprotein, alpha-1-antitrypsin and transferrin were all hyperfucosylated in the samples with HCC.21-23 In many ways, the detection of these glycoproteins by our system is a validation of our approach. In addition, several other major glycoproteins were found to have altered fucosylation patterns (in the sample with HCC) such as ceruloplasmin, alpha 1 antichymotrypsin, hemopexin, fibrinogen gamma chain precursor, Apo-D, fetuin A, IgM, Kininogen, the HBsAg and GP73. It is important to note that not all proteins in the fucosylated proteome increased with cancer, as the levels of fucosylated haptaglobin actually decreased. The majority of those proteins identified in this study consisted of the acute phase proteins, which are highly abundant. It is understood that the presence of these proteins may “mask” other less abundant proteins. Hence, methods are underway to remove those proteins prior to glycan analysis, lectin extraction and proteomic analysis. Indeed, removal of these abundant proteins prior to proteomic analysis may allow for the detection of more proteins that correlate with cancer.
Changes in N-linked glycosylation have long been associated with the development of disease. In this study, we have utilized
The usefulness of these proteins as biomarkers of liver cancer will require the screening of hundreds if not thousands of
Table 2. Additional Proteins Identified by LC-MS/MS Analysis of Fucosylated Peptides from Pooled Normal and Cancer Sera peptide
match (accession no.)
HCC
normal
FTKVNFTEIQK VC*QDC*PLLAPLBNDTR ADGTVNQIEGEATPVNLTEPAKLEVK NLFLNHSENATAK SWPAVGNCSSALR GLTFQQNASSMC*VPDQDTAIR LNAENNATFYFK
AFP (P02771) FETUIN-A (P02765) APO-D (P05090) HAPTOGLOBIN (P00738) HEMOPEXIN (P02790) Ig M (P01871) KININOGEN (P03952)
YES YES YES NO YES YES YES
NO YES YES YES NO NO NO
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Table 3. Proteins Identified as Part of the Fucosylated Proteome from Pooled Sera from Either Normal Individuals or Individuals with Cancer Identified fucosylated proteina
Fc-GP-73d Fc-Hemopexin Fc-HBsAge Fc-AFP Fc-Alpha-acid glycoprotein Fc-Alpha-1-antichymotrypsin Fc-Alpha-1-antitrypsin Fc-Serotransferrin Fc-Ceruloplasmin Fc-Alpha-2-macroglobulin Fc-Alpha-2-HS-glycoprotein (Fetuin A) Fc-Haptoglobin Fc-Fibrinogen gamma chain precursor (P)4 Fc-Complement factor B Fc-IgG Fc-IgA Fc-APO-D Fc-Ig M Fc-Kininogen
present in normalb
present in cancerc
No No No No Yes Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes (Elevated) Yes (Elevated) Yes (Elevated) Yes (Elevated) Yes (Elevated) Yes (Elevated) Yes (Elevated)
Yes No
No Yes
Yes Yes Yes Yes No No
Yes (Elevated) Yes (Elevated) Yes (Elevated) Yes (Elevated) Yes Yes
a Proteins identified either through the 2DE or LC MS/MS. b The presence (yes) or absence (no) of the fucosylated glycoprotein in pooled normal sera. c The presence (yes) or absence (no) of the fucosylated glycoprotein in pooled cancer sera. d Identified in Block et al., 2005.14 e Identified in Steel et al., 2003.20
samples consisting of many disease states, including cirrhotic and those with other cancers. In addition, an assay that is more suitable for high throughput use, such as a Lectin-ELISA, will have to be developed. These methods and studies are underway. It is noted that while we have utilized this methodology for the identification of glycoproteins that change with the development of HCC, it is applicable to other diseases as well. For example, changes in glycosylation have been associated with rheumatoid arthritis,24 prostate cancer,25 breast cancer,26 and colo-rectal cancer27,28 to name but a few. More importantly, this methodology can be utilized to identify novel changes in glycosylation that are yet unknown and target those specific glycoproteins. This research describes the application of a new, but proven technology for the rapid quantitative analysis of the fucosylated glycoproteome. The disease of interest, HCC, has been shown by many laboratories to exhibit altered glycosylation in serum glycoproteins in addition to other molecular changes. However, no laboratory has been able to apply a suitable technology to truly quantify and evaluate these glycan observations within systematic study. Thus, we have attempted to identify all the serum glycoproteins that have increased levels of core R-1,6 fucosylation in HCC. Now that a panel of proteins that alter with cancer has been identified, their suitability for biomarker use will be tested using a Lectin-ELISA based approach. In addition, it will be important to determine how early the changes occur in the development of cancer and if they could be used in a way to monitor treatment.
Acknowledgment. This work was supported by The Hepatitis B Foundation, the National Cancer Institute (NCI) Early Research Detection Network (EDRN), the NCI Innovative Molecular Analysis Technologies Program (IMAT), and an appropriation from The Commonwealth of Pennsylvania. 314
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Anand Mehta is the Bruce Witte Research Scholar of the Hepatitis B Foundation. Dr. Ender Simsek is thanked for his careful reading of the manuscript.
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