Cell surface–expressed Thomsen-Friedenreich antigen in colon cancer is predominantly carried on high molecular weight splice variants of CD44

R. Singh2, B.J. Campbell2, L.-G. Yu2, D.G. Fernig3, J.D. Milton2, R.A. Goodlad4, A.J. FitzGerald5 and J.M. Rhodes1,2

2Department of Medicine, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK, 3School of Biological Sciences, University of Liverpool, Daulby Street, Liverpool L69 3BX, UK, 4Imperial Cancer Research Fund, Histopathology Unit, 35-43 Lincoln’s Inn Fields, London WC2A 3PN, UK, and 5Imperial College School of Science Medicine and Technology, Hammersmith Campus, Ducane Road, London W12 ONN, UK

Received on December 12, 2000; revised on February 12, 2001; accepted on February 12, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose ß1-3 N-acetylgalactosamine {alpha}-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[125I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, ~110–180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-{alpha}-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosamine{alpha}-). Incubation of PNA 15 µg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.

Key words: CD44/oncofetal carbohydrate/peanut agglutinin/sialyl-Tn/Thomsen-Friedenreich antigen


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Changes in O-glycosylation are very common in epithelial cancers, including colon cancer, and often result in expression of oncofetal blood group antigens, such as Thomsen-Friedenrich (TF), Tn, and sialyl-Tn (Brockhausen et al., 1998Go). These changes have often been identified using lectins as histochemical probes (Rhodes et al., 1986Go, 1998; Boland, 1988Go). It is not known whether they are determined by alterations in the relative activities of the relevant Golgi glycosyltransferases, altered substrate availability, or changes in the amino acid sequence of the glycoprotein, but it is becoming increasingly clear that they play a major role in determining the invasive and metastatic properties of tumor cells (Nicolson, 1982Go; Roos, 1984Go; Schirrmacher, 1985Go; Raz and Lotan, 1987Go).

Enhanced expression of the TF antigen (galactose ß1-3 N-acetylgalactosamine {alpha}-) by hyperplastic and neoplastic colon (Campbell et al., 1995Go) has been shown to allow interaction with luminal TF-binding lectins of dietary origin, such as peanut agglutinin (PNA), resulting in increased proliferation (Ryder et al., 1992Go). Previous studies have shown that PNA is mitogenic to HT29 but not Caco-2 colon cancer cells in vitro (Ryder et al., 1994Go) and that peanut ingestion causes increased proliferation of rectal epithelia in colonoscopically normal individuals with mucosal expression of PNA receptor (Ryder et al., 1998Go). Antibodies to TF antigen, which are universally present in human sera, also increase proliferation in human colon cancer cell lines in a dose-dependent manner (Yu et al., 1997Go). Despite the diverse biological effects of PNA, its cell-surface receptors have not yet been identified in colon cancer. The fact that PNA is mitogenic to HT29 but not Caco2 colonocytes in vitro (Ryder et al., 1994Go) also requires explanation because both cell lines express TF antigen on their cell surface.

In this study we have analyzed the profile of PNA-binding cell-surface glycoproteins in HT29 and Caco2 colon cancer cell lines. We report that PNA binds to TF antigen expressed by high molecular weight variant isoforms of CD44 (CD44v) in colon cancer. The standard CD44 from both normal and colon cancer tissues does not express TF. Both PNA and CD44v6 antibodies stimulate proliferation in HT29 cells, and their effect is not additive, suggesting that the proliferative effect of PNA is probably mediated via interaction with cell membrane CD44v. We also demonstrate coexpression of oncofetal carbohydrate antigens, TF with sialyl-Tn on CD44v. These findings provide a link between the altered glycosylation and alternative CD44 splicing that are commonly found in colorectal cancer and precancer.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Identification of CD44v as a PNA-binding and sialyl-Tn–expressing glycoprotein in HT29 cells
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography of [125I] radiolabeled PNA-affinity-purified cell-surface glycoproteins from HT29 and Caco-2 cells yielded very different profiles (Figure 1A). In addition to a broad high molecular weight glycoprotein band (110–160 kDa) accounting for 59.7% of the radiolabel incorporated into PNA-purified protein, a series of lower molecular weight glycoproteins (< 90 kDa) were present in HT29. In contrast, Caco-2 cells possessed just two glycoprotein bands of molecular weight greater than 200 kDa and one band corresponding to 75 kDa. Western blot analysis of PNA-agarose affinity-purified glycoproteins from HT29 and Caco-2 using pan CD44 showed that the broad band ranging from 110 to 180 kDa present in HT29 contained high molecular weight CD44 isoforms (Figure 1B). Autoradiographs of CD44 immunoprecipitated from HT29 and Caco2 cells metabolically labeled with [35S]-sulfate further confirmed the expression of high molecular weight CD44v in HT29 and its absence in Caco2 cells (Figure 1C). PNA blotting of CD44 immunoprecipitated from the HT29 cell extract showed that the high molecular weight CD44v expressed TF antigen (Figure 1D). CD44 immunoprecipitated both from crude or PNA-agarose purified HT29 membrane extracts was subjected to Western blot analysis using anti-sialyl-Tn antibodies (clone HB STn1). This showed a strong expression of sialyl-Tn on CD44v from both the extracts (Figure 1E). The PNA-binding, TF-expressing high molecular weight splice variants of CD44 therefore also express sialyl-Tn.




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Fig. 1. (A) Autoradiograph of SDS–PAGE of radioiodinated PNA-affinity-purified cell-surface glycoproteins from human colon cancer cells HT29 and Caco2. An intense band ranging from 110 to 180 kDa, present in HT29 cells, is absent in Caco2. Molecular weight markers are indicated. (B) Western blot analysis of PNA-agarose affinity-purified cell-surface glycoproteins from human colon cancer cells HT29 and Caco2 using pan CD44 antibody (BBA10). The 110–180-kDa band is shown to contain CD44v. (C) Autoradiograph of [35S]-sulfate-labeled extracts from HT29 and Caco2 colonocytes following immunoprecipitation of CD44, using a pan CD44 antibody. (D) PNA blot of CD44 immunoprecipitated from HT29 extract confirms binding of PNA by CD44. (E) Western blot analysis of CD44 immunoprecipitated from crude and PNA-agarose affinity-purified HT29 membrane extract using the anti sialyl-Tn antibody (HBSTn1). In this and subsequent figures the blots shown are representative of at least two similar experiments. In panel A, a gradient (4–15%) polyacrylamide gel has been used. All other figures show blots from 7.5% polyacrylamide gels.

 
CD44v from human colon cancer tissue also binds PNA
SDS–PAGE and Western blot analysis showed that normal colon tissue expressed standard CD44 as a 85–90-kDa polypeptide, whereas colon cancer tissue contained standard as well as high and lower molecular weight variant isoforms of CD44 (Figure 2A). PNA-agarose affinity purification of these tissue extracts from normal and colon cancer tissue, followed by Western blot analysis using pan CD44 antibodies, showed that high molecular weight variant isoforms of CD44 were extractable by PNA-agarose affinity purification, but the standard CD44 was not (Figure 2B).



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Fig. 2. (A) Western blot of CD44 expression using pan CD44 mAb (BBA10) in human colon cancer tissue and distant macroscopically normal tissue. Unlike the normal colon, colon cancer tissue expresses high molecular weight variants of CD44. (B) Western blot analysis of PNA-affinity-purified glycoproteins from colon cancer (lane 1) and normal colon tissue (lane 2) using pan CD44 antibody (BBA10). Only the high molecular weight variants of CD44 are extractable by PNA-agarose affinity purification.

 
CD44 variants bind PNA via TF antigen
Removal of N-glycans from PNA-agarose-purified extracts of HT29 cells by treatment with Flavobacterium meningosepticum N-GlycanaseTM caused an increase in apparent overall PNA binding accompanied by a decrease in molecular weight (Figure 3A). A parallel Western blot analysis using pan CD44 antibodies was performed, and a similar downward shift in molecular weight of CD44 was observed (Figure 3B). Selective removal of TF antigen by O-GlycanaseTM treatment of the same extract, on the other hand substantially reduced PNA reactivity (70–75%, N = 3) as determined by densitometric analysis (Figure 4).



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Fig. 3. (A) PNA blot of PNA-agarose-purified glycoproteins from HT29 following removal of N-glycans with N-GlycanaseTM (+) compared to control (–). Note the lower molecular weight bands seen after N-GlycanaseTM treatment. (B) Western blot analysis of PNA-agarose-purified proteins from HT29 using anti-CD44s mAb BBA10 with (+) or without (–) N-GlycanaseTM treatment. The presence of lower molecular weight forms of CD44 is confirmed.

 


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Fig. 4. PNA blot of PNA-agarose affinity-purified glycoproteins from HT29 colonocytes following specific removal of TF antigen with O-GlycanaseTM (lane 3) compared to control (lane 4). Lanes 1 and 2 contain asialofetuin (a TF antigen expressing glycoprotein) following treatment with and without O-GlycanaseTM, respectively. Removal of TF antigen markedly decreases PNA binding.

 
PNA has no additional mitogenic effect in the presence of the anti CD44v6 mAb
In HT29 cells PNA (15 µg/ml) produced a 44 ± 8% (mean ± SD) increase in thymidine incorporation (P = 0.0005, analysis of variance [ANOVA]; N = 3). A similar increase of 33 ± 2% was observed (P = 0.0014, ANOVA; N = 3) with mAb anti CD44v6 (BBA13). When both PNA and antibody to CD44v6 were added together DNA synthesis was reduced (26 ± 5%) compared to PNA alone (P = 0.0398, ANOVA; N = 3), but similar to that observed in the presence of the antibody alone (P = 0.300, ANOVA; N = 3) (Figure 5).



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Fig. 5. Effect of PNA and anti CD44v6 mAb (BBA13) on HT29 [3H]-thymidine incorporation into HT29 cells. The results represent means ± SD of triplicate determinations. Control (1% v/v FCS) compared to PNA (15 µg/ml) [P = 0.0005, ANOVA; N = 3]; control compared to BBA13 [P = 0.0014, ANOVA; N = 3]; BBA13 plus PNA compared to PNA [P = 0.0398, ANOVA; N = 3].

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this study, high molecular weight variant isoforms of CD44 (CD44v) are shown to be major PNA-binding cell-surface glycoproteins in colon carcinoma and in HT29 colon cancer cells, accounting for over half of the PNA binding by cell-surface glycoproteins. Lesser degrees of PNA binding were shown by other cell-surface membrane glycoproteins. It is worth noting though that in whole cell HT29 preparations the major PNA-binding ligand is an unidentified 90-kDa protein (Yu et al., 2001Go). Subsequent Western blots of PNA-agarose affinity-purified glycoproteins from HT29 and colon tissue samples (data not shown) confirm MUC1 as a PNA-binding glycoprotein (Karlsson et al., 1983Go; Swallow et al., 1987Go) but accounting for a much lower proportion of PNA binding given that MUC1 from these sources is included within SDS–PAGE gels run under the conditions used for Figure 1A and that its two alleles are clearly separable from CD44 as higher molecular weight proteins. TF antigen expression by CD44v was confirmed by reduction in PNA binding to CD44 following treatment with a Streptococcal endo-{alpha}-N-acetylgalactosaminidase (O-GlycanaseTM) that is specific for unsubstituted TF (Umemoto et al., 1978Go). CD44v expressed in colon cancer tissue also bound PNA. Because these CD44 variants are present in colon cancer and inflammatory bowel disease tissue and are absent from normal mucosa (Rosenberg et al., 1995Go; Yamao et al., 1998Go), these results could also explain lack of PNA reactivity in normal colon and enhanced PNA binding in colon cancer and inflammatory bowel disease (Rhodes et al., 1986Go, 1988; Boland, 1988Go).

A 250-kDa CD44 containing the product of all known splice variant exons has been shown to be the major PNA-binding glycoprotein in human epidermal keratinocytes (Hudson et al., 1995Go). Selective antisense suppression of CD44 in mouse keratinocytes disrupts hyaluronate metabolism in skin and impairs keratinocyte proliferation (Kaya et al., 1997Go). Because the proliferation of HT29 cells but not Caco2 colon cancer cells is stimulated by PNA (Ryder et al., 1998Go) this suggests the possibility that the proliferative response to PNA may be dependent on expression of CD44v which is lacking in Caco2. This is further supported by the fact that although both PNA and monoclonal antibodies to CD44v6 enhance proliferation in HT29 colon cancer cells, PNA has no additive effect in the presence of mAb to CD44v. The proliferation increase seen in response to the CD44 antibody alone is assumed to be due to receptor clustering triggering protein kinase activity in a way analogous to that seen with antibodies to epidermal growth factor receptor (Spaargaren et al., 1991Go).

CD44 is a transmembrane glycoprotein encoded by a complex gene composed of 20 exons, 10 of which are always expressed to produce a heavily glycosylated 85–90-kDa isoform known as standard or hematopoetic CD44 (CD44s or CD44H). The remaining exons are alternatively spliced in various combinations to generate high molecular weight variant isoforms. CD44 is a receptor for hyaluronic acid and is involved in cell–cell adhesion (Koopman et al., 1990Go), cell–matrix adhesion (Jalkanen and Jalkanen, 1992Go), lymphocyte homing (Jalkanen et al., 1987Go), and lymphocyte activation (Huet et al., 1989Go).

Several studies have suggested a link between CD44 splicing, altered cell surface glycosylation, and tumor cell behavior. Thus, transfection of a poorly tumorigenic rat colon cancer cell line with human H blood group antigen-forming {alpha}(1-2) fucosyltransferase cDNA resulted in cell-surface expression of H antigens selectively born on CD44v6 and conferred increased mobility and tumorigenicity to the transfected cells (Goupille et al., 1997Go). Studies in melanoma cell lines have shown that treatment with benzyl-alpha-N-acetylgalactosamine resulted in increased PNA binding on CD44 (Nakano et al., 1996Go) and treatment with ginsenoside Rh2 (Rh2) increased PNA binding to cell-surface glycoproteins (Ota et al., 1993Go); both reagents also caused an increase in experimental metastatic ability. Human melanoma cell populations enriched for PNA-binding cells also generated a higher frequency of metastases when xenografted into immune-suppressed neonatal rats (Dore et al., 1994Go). Neuraminidase treatment of tumor cells (mouse sarcoma-1) alters in vivo (Balb/c-mice) the organotropic distribution of metastases; instead of being found exclusively in the lung, they are found both in lung and liver. However, preinjection and regular application of D-galactose or arabinogalactan prevents the setting of metastases in the liver (Uhlenbruck et al., 1987Go). Terminal expression of galactose by CD44 variants, as shown by their PNA reactivity, might therefore contribute to organotropic distribution of metastases in colon cancer. High molecular weight CD44 variants have also been found in inflammatory bowel disease (Rosenberg et al., 1995Go), a condition that is also associated with increased expression of TF (Campbell et al., 1995Go) and sialyl-Tn (Karlen et al., 1998Go) and with increased cancer risk (Podolsky, 1991Go).

The demonstration that cell-surface TF antigen expression by colon cancer epithelial cells largely depends on the presence of high molecular weight splice variants of CD44 provides a link between the extensive evidence for disease-related alterations in CD44 splice variants and the equally extensive evidence for disease-related alteration in cell surface expression of TF antigen. Furthermore, it suggests that the nature of O-glycosylation may be determined, at least in part, by the amino-acid sequence of the protein (CD44) undergoing glycosylation.

TF antigen–expressing CD44v is also shown to bear the related oncofetal antigen sialyl-Tn. Mucin-associated sialyl-Tn (STn) antigen is a marker of poor prognosis for patients with colon and gastric cancer and of cancer risk for those with ulcerative colitis (Ma et al., 1993Go; Yamada et al., 1995Go; Itzkowitz et al., 1995Go, 1996). Furthermore, mucins bearing sialyl-Tn have been shown to mediate inhibition of natural killer cell cytotoxicity, thereby providing protection for cancer cells (Ogata et al., 1996Go).

The coexpression of oncofetal carbohydrate blood group antigens on CD44 has functional consequences that seem highly likely to contribute to the malignant potential of epithelial cells thus confirming oncofetal carbohydrate antigens as appropriate targets for immunotherapy (Springer et al., 1995Go; MacLean et al., 1996Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Monoclonal antibodies against CD44s (BBA10, clone 2C5) and CD44v6 (BBA13, clone 2F10) were obtained from R&D systems (Abingdon, UK). Monoclonal antibodies against sialyl Tn (clone HB STn1) were obtained from Dako (Ely, UK). [35S]-sulfate and carrier-free Na[125I] were purchased from Amersham-Pharmacia Biotech (Little Chalfont, UK). PNA-agarose (2–4 mg lectin/ml) was obtained from Sigma (Poole, UK). Peptide-N-glycosidase F (N-GlycanaseTM EC 3.2.2.18) and S. pneumoniae endo-{alpha}-N-acetylgalactosaminidase (O-GlycanaseTM EC 3.2.1.97) were obtained from Oxford Glycosciences (Abingdon, UK). All other reagents were of analytical grade.

Cell culture
The HT29 and Caco-2 cell lines were obtained from the European Collection of Animal Cell Cultures at the Public Health Laboratory Service (Porton Down, Wiltshire, UK). The cell lines were grown in monolayer and maintained at 37°C in a humidified atmosphere of 5% CO2 95% air. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (v/v) fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin, 4 mM glutamine, and 50 ng/ml insulin. DNA synthesis assays were performed as described (Ryder et al., 1998Go). Briefly, cells were seeded at a density of 2 x 104/well into 24 well plates and 48 h later the culture medium was replaced with DMEM containing 1% (v/v) FCS alone (control) or containing either PNA (15 µg/ml), anti-CD44v6 antibody BBA13 (10 µg/ml) or PNA plus BBA13. After 24 h, 0.5 µCi [3H]-thymidine was added to each well for 3 h and the cells were prepared for scintillation counting. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons of means (Newman and Keuls). Differences were considered significant when P < 0.05.

Preparation of human tissue extracts
Tissues obtained from the Cancer Tissue Bank (Department of Pathology, University of Liverpool) were taken from human colon cancers and macroscopically normal colon at least 10 cm distant from tumor, and homogenized in 50 mM Tris (pH 8) containing 150 mM NaCl, 0.02% (w/v) sodium azide, 100 µg/ml phenyl methylsulfonyl fluoride (PMSF), 100 µg/ml aprotinin, and 1% (v/v) Triton X-100. Total protein concentration was measured with the bicinchoninic acid assay (Sigma).

PNA-agarose affinity chromatography of radioiodinated cell surface proteins
Iodination of cell surface proteins in HT29 and Caco2 cells was performed by the method of Bresalier et al. (1990)Go using lactoperoxidase and glucose oxidase. A confluent 75-cm2 culture plate was washed three times in phosphate buffered saline (PBS), pH 7.4, and once with PBS containing 3 mM glucose. Tyrosine residues of cell surface proteins were labelled in situ by the addition of 5 U lactoperoxidase, 0.5 mCi carrier-free Na[125I], and 10 U glucose oxidase in 2 ml PBS containing 3 mM glucose. The flask was then incubated at 25°C for 30 min. Cells were then washed four times with PBS containing 1 mM unlabeled sodium iodide and 5 mM PMSF. Cells were then scraped into 4 ml PBS-PMSF and centrifuged at 2000 x g for 10 min. The cell pellet was resuspended in 1 ml PBS-PMSF, sonicated on ice using three 20-s pulses to lyse the cells, and centrifuged at 100,000 x g for 1 h. The membrane-rich cell pellet was solubilized by sonication in PBS containing 1% (v/v) Nonidet P40 (NP40) and 5 mM PMSF using five 20-s pulses, left on ice for 2 h, and then centrifuged at 100,000 x g for 1 h. The supernatant was collected and loaded onto a 2-ml PNA-agarose column, which was then washed with PBS until the absorbance of the wash-through at 280 nm returned to the baseline and the bound proteins were then eluted with PBS containing 0.2 M galactose. The eluate was then desalted on a PD-10 Sephadex GM25 column (Amersham-Pharmacia Biotech), and the void volume fraction was freeze-dried. This fraction, containing PNA-affinity-purified, radiolabeled, cell-surface glycoproteins was then subjected to SDS–polyacrylamide gradient (4–15%) gel electrophoresis followed by autoradiography using Hyperfilm. Of the HT29 cell preparations, 2.3% of cell-surface radiolabeled material adhered to the PNA agarose column and 0.3% was eluted with galactose; of the Caco2 cell preparations, 14.7% of cell-surface radiolabeled material adhered to the PNA agarose column and 0.9% was eluted with galactose. Protein bands were quantified after scanning using Quantity One software (Biorad, Hemel Hemstead, UK).

Metabolic labeling with [35S]-sulfate and preparation of extracts
Subconfluent cultures of HT29 and Caco-2 cells were incubated for 18 h in culture medium containing 50 µCi/ml [35S]-sulfate. The medium was removed and cells were rinsed three times with PBS. Cells were then left on ice for 15 min in 1 ml 10 mM Tris, 66 mM EDTA, 1 mM PMSF (pH 7.4) containing 1% (v/v) NP40 and 0.4% (w/v) sodium deoxycholate. The extracts were centrifuged at 16,000 x g for 15 min at 4°C and the pellets discarded.

Immunoprecipitation and western blotting
Labeled or unlabeled cell extracts were precleared with 20 µl Protein A-agarose beads to remove nonspecific binding material. The supernatants were then incubated for 3 h with pan CD44 antibody (BBA10) at 4°C on a shaker. Protein A-agarose beads were then added and the mixture incubated in an end-over-end shaker overnight at 4°C. Beads and associated immune complexes were collected by centrifugation and washed once in PBS containing 0.5% (v/v) Triton X-100, 0.1% (w/v) SDS; once in PBS containing 0.5% (v/v) Triton X-100 and 0.5 M NaCl; and twice more in the former buffer. Pellets were resuspended in SDS–PAGE sample buffer (62.5 mM Tris [pH 6.8] containing 2% [w/v] SDS, 10% [v/v] glycerol, 5% [v/v] mercaptoethanol, and 0.001% [w/v] bromophenol blue) and boiled for 5 min. Immunoprecipitated polypeptides were then separated on a 7.5% or 4–15% gradient polyacrylamide gels. The separated polypeptides were electrotransferred to nitrocellulose membrane for 1 h at 100 V in 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol. After transfer the membrane was blocked with PBS containing 1% (w/v) bovine serum albumin (BSA), 0.1% (v/v) Tween 20 for 1 h and then incubated with primary antibody, followed by peroxidase-conjugated rabbit anti-mouse IgG, each for 1 h at room temperature in PBS with 1% (w/v) BSA. Bound antibody was detected using an enhanced chemiluminescence kit (Amersham-Pharmacia Biotech).

Selective removal of TF antigen and N-glycans
Selective removal of TF antigen was achieved by treatment of PNA-agarose-purified HT29 membrane extract with S. pneumoniae endo-{alpha}-N-acetylgalactosaminidase (O-GlycanaseTM). Briefly, lyophilized PNA-agarose-purified HT29 membrane extract (25 µg protein) was incubated with 2 mU O-GlycanaseTM in 50 µl 100 mM citrate/phosphate buffer, pH 6, containing 100 µg/ml BSA and 0.02% (w/v) sodium azide for 18 h at 37°C. Enzymatic release of TF was confirmed using asialofetuin (Sigma) as a control TF-expressing substrate.

N-linked oligosaccharides were removed using Peptide-N-Glycosidase F (N-GlycanaseTM). PNA-agarose purified HT29 membrane extract (20 µg protein) was dissolved in 20 µl of 20 mM sodium phosphate, pH 7.5, containing 50 mM EDTA, 0.02% (w/v) sodium azide, 0.5% (w/v) SDS, 5% (v/v) ß-mercaptoethanol, and denatured by heating at 100°C for 2 min. To the denatured extract were added 10 µl 5% (v/v) NP-40, 20 µl incubation buffer and 6 µl (3 U) N-GlycanaseTM and the mixture was incubated for 18 h at 37°C.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by grants from the Wellcome Trust (R.S.), Biotechnology and Biological Sciences Research Council (D08258 R.S., R.A.G., B.J.C., and J.M.R.), and a European Union Concerted Action Award (BMH4-CT98–3222).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ANOVA, analysis of variance; BSA, bovine serum albumin; CD44v, CD44 splice variants; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; mAb, monoclonal antibody; NP40, Nonidet P-40; PBS, phosphate buffered saline; PMSF, phenyl methylsulfonyl fluoride; PNA, Peanut agglutinin; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TF, Thomsen-Friedenreich.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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