Fucosylated haptoglobin is a novel marker for pancreatic cancer: detailed analyses of oligosaccharide structures

Changes in oligosaccharide structures have been reported in certain types of malignant transformation and thus can be used as tumor markers in certain types of cancer. In the case of pancreatic cancer (PC) cell lines, a variety of fucosylated proteins are secreted into the conditioned media. To identify fucosylated proteins in the sera of patients with PC, we performed Western blot analysis using Aleuria Aurantia Lectin (AAL), which is specific for fucosylated structures. An approximately 40 kD protein was found to be highly fucosylated in PC and N-terminal analysis revealed that it was the beta chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer, and colorectal cancer, the incidence was significantly higher in the case of PC. Fucosylated haptoglobin was observed more frequently at the advanced stage of PC and disappeared after operation. Haptoglobin has four sites of N-glycans and site-directed oligosaccharide analysis involving MS was performed. Site-specific increases in fucosylation of bi-antennary glycans of sites 2 and 4, and of tri-antennary glycans of all sites were observed in PC, compared to in normal volunteers and chronic pancreatitis. Therefore, increases in fucosylation seem to be not due to inflammation, but cancer itself. Coculturing of a human hepatoma cell line, Hep3B, with PC cells-induced production of fucosylated haptoglobin, suggesting that PC produces a factor that induces the production of fucosylated haptoglobin. On clinical investigation of 100 cases of colorectal cancer, cases in which it was located near the liver showed a higher positive rate of fucosylated haptoglobin, suggesting that the location of the cancer might also be an important factor for fucosylated haptoglobin if cancer tissues produce such inducible factors. Thus, fucosylated haptoglobin could become a novel tumor marker for PC and complicated mechanisms would be involved in its production.     

Site-specific and linkage analyses of fucosylated <Emphasis Type="Italic">N</Emphasis>-glycans on haptoglobin in sera of patients with various types of cancer: possible implication for the differential diagnosis of cancer

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 only in cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers.     

Mass spectrometric assay for analysis of haptoglobin fucosylation in pancreatic cancer

A mass spectrometric method was developed to elucidate the N-glycan structures of serum glycoproteins and utilize fucosylated glycans as potential markers for pancreatic cancer. This assay was applied to haptoglobin in human serum where N-glycans derived from the serum of 16 pancreatic cancer patients were compared with those from 15 individuals with benign conditions (5 normals, 5 chronic pancreatitis, and 5 type II diabetes). This assay used only 10 μL of serum where haptoglobin was extracted using a monoclonal antibody and quantitative permethylation was performed on desialylated N-glycans followed by MALDI-QIT-TOF MS analysis. Eight desialylated N-glycan structures of haptoglobin were identified where a bifucosylated triantennary structure was reported for the first time in pancreatic cancer samples. Both core and antennary fucosylation were elevated in pancreatic cancer samples compared to samples from benign conditions. Fucosylation degree indices were calculated and show a significant difference between pancreatic cancer patients of all stages and the benign conditions analyzed. This study demonstrates that a serum assay based on haptoglobin fucosylation patterns using mass spectrometric analysis may serve as a novel method for the diagnosis of pancreatic cancer.     

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.     

Analysis of polarized secretion of fucosylated alpha-fetoprotein in HepG2 cells

Fucosylated alpha-fetoprotein (AFP) is a more specific biomarker for hepatocellular carcinoma (HCC) than AFP. However, the mechanisms underlying the increase in fucosylated AFP in sera of HCC patients remain largely unknown. Recently, we reported that fucosylation is a possible signal for the secretion of hepatic glycoproteins into bile and that the fucosylation-based sorting machinery might be disrupted in the liver bearing HCC. In this study, we investigated the selective secretion of fucosylated AFP into bile canaliculus (BC) structures of the human hepatoma cell line HepG2. The proportion of fucosylated AFP in BC structures was higher than that in the medium, as judged by lectin affinity electrophoresis. Suppression of fucosylation by the double knock-down of GDP-mannose-4,6-dehydratase and the human homologue of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, which contribute to the synthesis of GDP-fucose, a donor substrate for fucosyltransferases, did not decrease the proportion of fucosylated AFP in BC structures but decreased this proportion in conditioned medium. Furthermore, increased AFP fucosylation was observed in medium, but not in BC structures, upon adding free fucose. These results suggest that saturation of fucosylated AFP in BC structures is accompanied by its increase in conditioned medium, probably leading to increased fucosylated AFP in sera of HCC patients.     

Reevaluation of <Emphasis Type="Italic">Pholiota squarrosa</Emphasis> lectin-reactive haptoglobin as a pancreatic cancer biomarker using an improved ELISA system

An increase in Lewis- and core-type fucosylation of haptoglobin has been reported in patients with pancreatic cancer (PC), suggesting that fucosylated haptoglobin is a candidate PC biomarker. Previously, we developed a Pholiota squarrosa lectin antibody enzyme-linked immunosorbent assay (PhoSL-ELISA) system for the detection of core-fucosylated haptoglobin. However, with this methodology, positive results were only obtained for some patients with PC, demonstrating the need for a more sensitive detection system. In the current study, we developed an improved PhoSL-ELISA system with higher sensitivity to detect core-fucosylated haptoglobin using high-concentration urea as a denaturing agent with lectin to facilitate detection. We then reevaluated the performance of PhoSL reactive-core-fucosylated haptoglobin (PhoSL-HP) as a PC biomarker using the improved PhoSL-ELISA system. PhoSL-HP levels in the sera of patients with PC were significantly higher than those in healthy volunteers, with an area under the curve (AUC) value of 0.753. Furthermore, the AUC value of CA19–9 improved from 0.793 to 0.907 when combined with PhoSL-HP. Additionally, several CA19–9-negative cases among the patients with PC were diagnosed as positive for PhoSL-HP. In conclusion, PhoSL-HP detection using our improved ELISA system might allow PhoSL-HP to serve as a potential biomarker for PC and thus might be useful to complement the detection of CA19–9 in PC diagnosis.     

Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer

Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6-linked tri-sialylated triantennary glycan containing α1,3-linked fucose attached to the four-linked position of the three-arm mannose of N-linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression.     

Fucosylated glycoproteomic approach to identify a complement component 9 associated with squamous cell lung cancer (SQLC)

Human lung cancer is a major cause of cancer mortality worldwide. Understanding the pathophysiological features and the development of novel biomarkers for diagnosis as well as treatment are major tasks. In the present study, sera from ten SQLC patients and healthy control (HEC) were collected and pooled, respectively. The pooled sera were depleted via an immunoaffinity method and further subjected to fucosylation enrichment. Enriched fucosylated glycoproteins were resolved by SDS-PAGE and subsequently analyzed by LC-ESI-MS/MS. From comparative proteomic analysis, we selected the C9 protein. C9 protein levels were validated by Western blot, protein arrays and the fucosylation levels of C9 by hybrid lectin ELISA (HLE) in the sera of 120 HEC and 118 SQLC patients. The C9 protein level was 6.4-fold higher in SQLC patients compared to HEC, as determined by Western blot analysis. The results were concurrently confirmed by a protein array that showed a C9 level significantly higher in SQLC patients, as compared to HEC, with a sensitivity of 53% and a specificity of 89%. C9 fucosylation levels were significantly higher in SQLC patients compared to HEC (p<0.05) when tested by HLE. These findings suggest that C9 and fucosylated form could serve as a useful marker for SQLC.     

Targeting anticancer drug delivery to pancreatic cancer cells using a fucose-bound nanoparticle approach

Owing to its aggressiveness and the lack of effective therapies, pancreatic ductal adenocarcinoma has a dismal prognosis. New strategies to improve treatment and survival are therefore urgently required. Numerous fucosylated antigens in sera serve as tumor markers for cancer detection and evaluation of treatment efficacy. Increased expression of fucosyltransferases has also been reported for pancreatic cancer. These enzymes accelerate malignant transformation through fucosylation of sialylated precursors, suggesting a crucial requirement for fucose by pancreatic cancer cells. With this in mind, we developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specifically to cancer cells. L-fucose-bound liposomes containing Cy5.5 or Cisplatin were effectively delivered into CA19-9 expressing pancreatic cancer cells. Excess L-fucose decreased the efficiency of Cy5.5 introduction by L-fucose-bound liposomes, suggesting L-fucose-receptor-mediated delivery. Intravenously injected L-fucose-bound liposomes carrying Cisplatin were successfully delivered to pancreatic cancer cells, mediating efficient tumor growth inhibition as well as prolonging survival in mouse xenograft models. This modality represents a new strategy for pancreatic cancer cell-targeting therapy.     

An enzymatic method of analysis for GDP-L-fucose in biological samples,involving high-performance liquid chromatography

To investigate the biological significance of GDP-L-fucose, we established a unique method for the determination of GDP-L-fucose levels in microsomal fractions, using an HPLC assay of alpha 1-6-fucosyltransferase (alpha1-6-FucT), an enzyme that catalyzes the synthesis of core fucosylation in N-glycans. A microsomal protein and a large excess of fluorescence-labeled synthetic oligosaccharide (a substrate) were incubated with a large excess of alpha1-6-FucT. The fluorescent intensity of the fucosylated reaction product, which was analyzed by isocratic reverse phase HPLC, was proportional to the level of GDP-L-fucose in the microsomal fractions over the range 0.20-10 pmol. This assay is applicable to the determination of the GDP-L-fucose content in various cancer cell lines as well as rat liver and would be useful in developing a better understanding of the fucosylation potential of such cells and tissues.     

Changes in the fucose content of haptoglobin in breast and ovarian cancer: Association with disease progression

There is increasing evidence for changes in fucosylation in cancer. Previously, we showed that the fucose-specific lectin,Lotus tetragonolobus, extracts an abnormal form of haptoglobin (Hp) from cancer sera. This study investigates the monosaccharide content of Hp obtained from women with ovarian and breast cancer at different stages of their disease. In both cancers, Hp fucose was low when the disease was benign or in remission and much higher when the disease was progressive. This occurred whether the data was expressed per mole of protein or per three mannose residues. Changes in other monosaccharides were minor compared with fucose. There were small increases in theN-acetylglucosamine and galactose content (per three mannoses) in ovarian cancer, suggesting that some glycan chains have increased branching. The latter was independent of disease activity which may be due to some indirect cause such as cytotoxic therapy or an inflammatory response. When ovarian cancer patients were in remission, the number of glycosylation sites on Hp was reduced. Hp isolated from patients with early, but not advanced breast cancer also appeared to have increased glycan branching. The increased fucosylated Hp may interfere with fucose-mediated adhesion reactions of cancer cells.Part of this work was published in abstract form,Glycoconjugate J 1993;10; 318.     

The expression of core fucosylated E-cadherin in cancer cells and lung cancer patients： prognostic implications

It is well documented that the glycosylation of E-cadherin is correlated with cancer metastasis, but whether E-cadherin could be core fucosylated remains largely unknown. We found that E-cadherin was core fucosylated in highly metastatic lung cancer cells while absent in lowly metastatic lung cancer cells. Sinceα-1,6 Fucosyltransferase (α-1,6 FucT) is known to catalyze the reaction of core fucosylation, we investigated the biological function of core fucosylation on E-cadherin by α-1,6 FucT targeted RNAi and transfecting α-1,6 FucT expression vector. As a result, calcium dependent cell-cell adhesion mediated by E-cadherin was strengthened with the reduction of core fucosylation on E-cadherin after RNAi and was weakened with the elevated core fucosylation on E-cadherin after α-1,6 FucT over expression. Our data indicated that α-1,6 FucT could regulate E-cadherin mediated cell adhesion and thus play an important role in cancer development and progression. Computermodeling showed that core fucosylation on E-cadherin could significantly impair three-dimensional conformation of N-glycan on E-cadherin and produce conformational asymmetry so as to suppress the function of E-cadherin. Furthermore, the relationship between the expression of core fucosylated E-cadherin and clinicopathological background of lung cancer patients was explored in lung cancer tissue of patients. It turns out to demonstrate that core fucosylated E-cadherin could serve as a promising prognostic indicator for lung cancer patients.     

Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyer’s patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.     

Biological function of fucosylation in cancer biology

Fucosylation is one of the most common modifications involving oligosaccharides on glycoproteins or glycolipids. Fucosylation comprises the attachment of a fucose residue to N-glycans, O-glycans and glycolipids. O-Fucosylation, which is a special type of fucosylation, is very important for Notch signalling. The regulatory mechanisms for fucosylation are complicated. Many kinds of fucosyltransferases, the GDP-fucose synthesis pathway and GDP-fucose transporter are involved in the regulation of fucosylation. Increased levels of fucosylation have been reported in a number of pathological conditions, including inflammation and cancer. Therefore, certain types of fucosylated glycoproteins such as AFP-L3 or several kinds of antibodies, which recognize fucosylated oligosaccharides such as sialyl Lewis a/x, have been used as tumour markers. Furthermore, fucosylation of glycoproteins regulates the biological functions of adhesion molecules and growth factor receptors. Changes in fucosylation could provide a novel strategy for cancer therapy. In this review, the biological significance of and regulatory pathway for fucosylation have been described.     

Isolation and characterization of an N-linked oligosaccharide that is significantly increased in sera from patients with non-small cell lung cancer

The structures of N-linked oligosaccharides present in human sera from 12 healthy volunteers and from 14 patients with non-small cell lung cancer (NSCLC) were analyzed by our recently developed partially automated systematic method. Thirty different structures of oligosaccharides were deduced, and these accounted for 84.1% of the total N-linked oligosaccharides present in human sera. All of the quantified oligosaccharide levels in healthy human sera were within twice the standard deviation. The amount of a triantennary trigalactosylated structure with one outer arm fucosylation (A3G3Fo) was found to be markedly increased in NSCLC patients in comparison to that in healthy volunteers (p < 0.01). No significant positive correlation with other clinical data was found. Serum A3G3Fo levels can thus be a novel marker for the diagnosis of NSCLC.     

Evaluation of the context of downstream N- and free N-glycomic alterations induced by swainsonine in HepG2 cells

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.     

Acceptor requirements for GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase activity solubilized from pea epicotyl membranes.

GDP-fucose:xyloglucan (XG) fucosyltransferase from growing Pisum epicotyl tissue was solubilized in detergent and used to examine the capacity of intact XG from Tamarindus seeds, and its partial hydrolysis products, to act as fucose acceptors with GDP-[14C]fucose as donor. Native seed XG (Mr greater than 10(6) Da) was partially depolymerized by incubation with Trichoderma cellulase for various periods of time. Cellulase was inactivated and reaction mixtures were incubated with GDP-[14C]fucose plus solubilized pea fucosyltransferase and then fractionated on columns of Sepharose CL-6B or Bio-Gel P4. Specific activities (Bq/microgram carbohydrate) of fragments with Mr ranging from 10(6) to 10(4) Da were constant throughout the size ranges, indicating that all stretches of the XG chains were available for fucosylation. More complete cellulase hydrolysis yielded subunit oligosaccharides that chromatographed in a cluster of hepta-, octa-, and nonasaccharides, none of which acted as fucosyl acceptors when incubated with pea fucosyltransferase. However, a substantial amount (up to half of hydrolysate) of larger transient oligosaccharides was also formed with a size equivalent to three of the oligosaccharide subunits. Octasaccharide subunits in this trimer were readily fucosylated. This fucosyltransfer was inhibited by uncombined (free) subunit oligosaccharides, which implies that the latter could bind to the transferase and displace at least part of the trimer, even though they could not themselves be fucosylated. Reduction of the trimer oligosaccharide with NaB3H4, followed by further hydrolysis with cellulase, resulted in tritiated nonasaccharide and unlabeled octasaccharide in a concentration ratio of 1:2. The tamarind XG trimer which accepts fucose is therefore composed mainly of the subunit sequence: octa-octa-nonasaccharide (reducing). One of the terminal oligosaccharide subunits in this trimer, probably the nonasaccharide, appears to be required as a recognition (binding) site in fucosyltransferase in order for adjacent octasaccharide(s) to be fucosylated by the active (catalytic) enzyme site.     

N-linked glycan changes of serum haptoglobin β chain in liver disease patients

Human haptoglobin is a serum glycoprotein secreted by the liver with four potential N-glycosylation sites on its β chain. Many studies have reported glycan changes of haptoglobin in diseases such as breast cancer and pancreatic cancer. The objective of our study is to analyze N-linked glycan alterations of serum haptoglobin β chain obtained from patients with the hepatitis B virus (HBV), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). MALDI-QIT-TOF mass spectrometry revealed the intensity of m/z 1809.6, identified as a fucosylated glycan, was much higher in samples from patients with LC and HCC relative to the patients with HBV and healthy controls. Compared with LC patients, triantennary glycan was elevated and the biantennary structure was decreased in the haptoglobin β chain of HCC patients. Thus, alterations in the glycan structure of the haptoglobin β chain may constitute significant spectral signatures of cirrhosis and HCC disease.     

Carbohydrate structures of haptoglobin in sera of healthy people and a patient with congenital disorder of glycosylation

Haptoglobin is one of acute phase glycoproteins often used as markers in glycopathology studies. In this work the oligosaccharide structures of haptoglobin from 'healthy' subjects have been studied in detail, taking into consideration the possible dependence of glycosylation on the phenotype. About 75% of charged haptoglobin glycans were of biantennary complex structure, and some of them lacked one terminal sialic acid molecule. Triantennary structures made up almost 25% of the charged glycans pool, and highly branched tetrasialylated oligosaccharides did not exceed 1%. The main difference between haptoglobin derived from the sample of pooled 44 sera and from the 2-2 phenotype individual concerned the relative content of trisialylated oligosaccharide with one 2-3 linked sialic acid residue. The oligosaccharide profile of haptoglobin derived from serum of a patient suffering from congenital disorder of glycosylation was compared to 'healthy' controls. It was shown, that four main glycans are identical in patient and 'normal' haptoglobins. Some alterations were found in the relative content of mono-, bi-, and trisialylated glycans as well as in the appearance of some tracely abundant oligosaccharides in haptoglobin of the patient with congenital disorder of glycosylation.