5 Immunobiology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India; 6 Drug Design Development and Molecular Modelling, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India; 7 Biochemisches Institut, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany; 8 Biochemistry Center, Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany; 9 Schwerpunkt Tumorimmunologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany; and 10 Vivekananda Institute of Medical Sciences, Kolkata 700045, India
Received on January 21, 2004; revised on June 3, 2004; accepted on June 6, 2004
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Abstract |
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Key words: 9-O-acetylated sialoglycoconjugates / acute lymphoblastic leukemia / Achatinin-H / anti-Neu5,9Ac2 antibody / Neu5,9Ac2-binding lectin
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Introduction |
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Sialic acids are important constituents of the lymphocyte cell membrane and influence many biological reactions either by reacting with specific surface receptors or via masking of carbohydrate recognition sites (Angata et al., 2002; Kelm and Schauer, 1997
; Mandal et al., 2000
; Schauer, 2004
; Sinha et al., 2000
). Among the diverse derivatives of sialic acid, the most frequently occurring substitutions are O-acetylation at positions C-7, C-8, and C-9 to form N-acetyl-7-, -8-, and -9-O-acetyl sialic acids, respectively, leading to a family of O-acetylated sialoglycoconjugates (Klein and Roussel, 1998
; Schauer and Kamerling, 1997
). However, because O-acetyl esters from C-7 and C-8 positions are known to spontaneously migrate to C-9 even under physiological conditions, O-acetylation at C-9 is considered the most common biologically occurring modification (Vandamme-Feldhaus and Schauer, 1998
).
Lectins or lectin-like molecules have been used to predict changes in sialylation patterns (Angata et al., 2002; Mandal and Mandal, 1990
; Mandal et al., 2000
; Sinha et al., 1999a
). A lectin, Cancer antennarius agglutinin, which recognizes sialic acids that are O-acetylated at both C-4 and C-9 positions, has been used to identify an O-acetylated disialoganglioside, Neu5,9Ac2-GD3, as a biomarker in human melanoma cells (Ravindranath et al., 1988
). The enhanced presence of 9-O-acetylated GD3 has been reported in several tumors, including melanomas, basaliomas, breast cancer, and tumors of neuroectodermal origin (Fahr and Schauer, 2001
; Kohla et al., 2002
). In the gastrointestinal tract, the concentration of O-acetylated sialic acids of colonic mucin decreases in colorectal cancers and Hirschsprung's disease (Aslam et al., 1999
; Mann et al., 1997
).
9-O-acetylated sialoglycans are detectable at low levels on human B lymphocytes, (Kamerling et al., 1982) and altered expression in disease conditions has been found (Bandyopadhyay et al., 2004
; Chava et al., 2002
, 2004a
; Mandal et al., 2000
; Sharma et al., 1998
; Sinha et al., 1999a
). These sialoglycotopes have also been reported on parasites (Chatterjee et al., 2003
; Chava et al., 2004b
,c
). However, owing to the lack of detailed biochemical characterization, their biological significance, especially as a potential biomarker, remains obscure in ALL. The preferential specificity of a lectin, Achatinin-H, toward Neu5,9Ac2-
2,6-GalNAc (Mandal and Basu, 1987
; Mandal et al., 1989
; Sen and Mandal, 1995
) allowed us to identify these glycotopes on lymphoblasts of ALL patients. We have reported an increased amount of Neu5,9Ac2-GPs on erythrocytes (Mandal et al., 1997
) and peripheral blood mononuclear cells (PBMCs) of ALL patients (Mandal et al., 1997
; Pal et al., 2004a
; Sinha et al., 1999a
,b
,c
,d
). Subsequently, we detected an enhanced level of antibodies against Neu5,9Ac2-GPs in ALL patients as compared to normal individuals (Pal et al., 2000
, 2001
, 2004b
).
The present study reports (1) confirmation of the occurrence of Neu5,9Ac2 on PBMCs of ALL patients by fluorimetric high-performance liquid chromatography (HPLC) and flow cytometry using Achatinin-H as a probe; (2) affinity purification of Neu5,9Ac2-GPsALL from PBMC of ALL patients using Achatinin-H; (3) demonstration of three leukemia-specific Neu5,9Ac2-GPsALL by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE), western blotting, and isoelectric focusing (IEF); (4) examination of the structural aspects of the binding of Neu5,9Ac2-GPsALL with Achatinin-H using synthetic sialic acid analogs by inhibition enzyme-linked immunosorbent assay (ELISA); and (5) the potential application of Neu5,9Ac2-GPsALL to monitor leukemia-specific antibodies at different phases of treatment by an antigen ELISA. Given the importance of these disease-specific molecules, we propose that Neu5,9Ac2-GPsALL on lymphoblasts may serve as potential biomarkers for diagnosis and monitoring the disease status.
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Results |
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Purification of Neu5,9Ac2-GPs from PBMCs of ALL patients
Clinically confirmed, immunophenotyped children with B-ALL (n = 10) and T-ALL (n = 5) with >80% lymphoblasts (as measured using lineage-specific established anti-CD antibodies and Achatinin-H) were selected for purification of Neu5,9Ac2-GPsALL. Equal amounts of membrane proteins (0.8 mg) of PBMCs from ALL and normal donors (n = 5) were affinity-purified using Achatinin-H-Sepharose 4B as an affinity matrix. The yield of purified Neu5,9Ac2-GPs was 3.7-fold higher in ALL patients as compared to normal donors0.37 ± 0.15 mg and 0.10 ± 0.05 mg, respectively. The proportion of membrane proteins from ALL patients that bound to Achatinin-H was 46.54 ± 2.4% as compared with 12.50 ± 1.30% from normal individuals. An ELISA using equal amounts (0.4 µg) of purified Neu5,9Ac2-GPs from ALL patients (Neu5,9Ac2-GPsALL) and normal donors (Neu5,9Ac2-GPsN) as the coating antigen showed a threefold increase in lectin bindingOD405nm was 1.34 versus 0.45, which indicates the lower amount of Neu5,9Ac2-GPs in normal individuals.
Molecular analysis of purified Neu5,9Ac2-GPs
Three distinct leukemia-specific bands corresponding to 135, 120, and 90 kDa were demonstrated in purified Neu5, 9Ac2-GPsALL on SDSPAGE (Figure 3A) and western blot using Achatinin-H (data not shown). The presence of O-acetylation was strengthened by complete abolition of binding of Achatinin-H following de-O-acetylation with alkali treatment. In contrast, two common bands corresponding to 140 and 36 kDa (Figure 3) were visible both in Neu5,9Ac2-GPsN and Neu5,9Ac2-GPsALL.
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Characterization of the carbohydrate epitope of Neu5,9Ac2-GPsALL by inhibition-ELISA
To study the binding specificity of Neu5,9Ac2-GPsALL, an inhibition-ELISA was developed with synthetic analogs of sialic acid, using Achatinin-H as coating antigen (Table I). Three regions of sialic acid at C-4, C-2, and C-9 were modified keeping the common core intact. The percent inhibition (PI) for a particular inhibitor was calculated as follows:
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Replacement of the O-acetyl group of compound 1 with a hydroxyl group (2) and replacement of oxygen by sulfur (3) or NH (4) at C-9 decreased inhibition to 28%, 20%, and 13%, respectively. Interestingly, introduction of fluorine (F) into the 9-N-acetyl group of compound 4 at C-9, that is, compound 5, resulted in a fivefold reduction of inhibition. A striking difference was observed between compounds 6 and 7 being (71 ± 3.8% versus 2.0 ± 0.8%), where the O-acetyl group at C-9 of the former had been exchanged with the propionyl group-O.CO.CH2.CH3. No inhibition was observed with compounds 8 and 9, possibly due to the lack of O-acetylation at the C-9 position. Also, replacement of the CH3 group of compound 2 with hydrogen at C-2 (compound 8) made the carbohydrate noninhibitory.
Bovine submandibular gland mucin (BSM) with a high Neu5,9Ac2 content was found to be a good inhibitor of Neu5,9Ac2-GPsALL binding to Achatinin-H. No inhibition was observed with de-O-acetylated BSM and asialo-BSM, indicating that the O-acetylated glycotope is the vital component for binding of Neu5,9Ac2-GPsALL with Achatinin-H. Other sialoglycoproteins, such as sheep submaxillary gland mucin (SSM), human chorionic gonadotropin (HCG), fetuin, and 1-acid glycoprotein, that have no O-acetylated sialic acids did not inhibit.
Potential use of purified Neu5,9Ac2-GPs for monitoring the disease status by an antigen-ELISA
Previous studies from our group have demonstrated increased antibody titers against Neu5,9Ac2 in sera of ALL patients (Pal et al., 2000, 2001
, 2004b
). Therefore, we wished to examine the reactivity of affinity-purified Neu5,9Ac2-GPsALL as a novel capture antigen to monitor the disease status. A strong binding with sera from untreated patients (n = 70) was observed in contrast to negligible binding with normal human serum (n = 21), mean ± SD of OD405nm being 1.14 ± 0.12 versus 0.16 ± 0.01, respectively (Figure 4A). With treatment, the binding gradually decreased in phases B, C, and D to 0.69 ± 0.01, 0.19 ± 0.01, and 0.12 ± 0.01, respectively (Figure 4B). In patients that relapsed (phase E, n = 10), absorbance increased again to 1.06 ± 0.06. In parallel, sera from patients with other hematological disorders (such as AML, CML, CLL, and NHL) were examined and showed no detectable levels of binding (Figure 4A). To minimize the false positivity, the cut-off value was selected as 0.2 based on the mean OD+3 SD obtained from normal controls. A good correlation (r = 0.92) was observed between antibody titers from the same set of patients as determined by both antigen-ELISA and BSM-ELISA using Neu5,9Ac2-GPALL and BSM as coating antigens, respectively (Figure 5). The data were subjected to a one-way analysis of variance with a post test for linear trend and confirmed a significant decrease of antibody titers with progress in treatment. Sensitivity, specificity, efficiency, positive predictive value, and negative predictive value of both assays are compared (Table II).
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Discussion |
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A major achievement of this investigation is the verification of the nature of the glycotope of Neu5,9Ac2-GPsALL (Table I) by inhibition-ELISA using synthetic sialic acid analogs that serve as inhibitors preventing binding between Neu5,9Ac2-GPsALL and Achatinin-H. A comparative analysis of the inhibitory potency of various sialic acid analogs showed that Neu5,9Ac2 is the most critical neuraminic acid derivative for competition, as compound 1 is the strongest inhibitor (Table I). Interestingly, a minor modification of this group caused drastic reduction in inhibition, thus confirming that O-acetylation at C-9 position is essential for binding. The introduction of a highly electronegative atom (F) at C-9 in compound 5 drastically reduced inhibition, suggesting hindrance of binding. The absence of inhibition with compounds lacking O-acetylation at C-9, or the 35.5-fold reduction in inhibition due to the introduction of a bulky group at C-9, suggests the existence of a smaller cleft size in the binding pocket. It can be envisaged that the cleft size around the binding site of C-9 is just right for an acetyl group, and even minor substitutions hinder binding. Introduction of a large group like C6H5 at position C-2 in place of a CH3 group reduces the inhibition only from 100% to 71%, indicating that the cleft size around C-2 is much bigger than that needed to accommodate CH3. At C-4 substitution of H with an acetyl group does not affect the inhibition properties, suggesting that adequate space for accommodation is present in this region. In summary, our data suggest that the region where C-9 is bound is highly specific for an O-acetyl group and does not allow any modification of this group, whereas binding criteria in other regions of the binding cleft are less stringent.
To show the involvement of the Neu5,9Ac2 moiety of Neu5,9Ac2-GPsALL in Achatinin-H binding, BSM was used as an inhibitor, which is known to contain this glycosidic group (Sen and Mandal, 1995). However, BSM is not a proper control, as Neu5,9Ac2
2-6GalNAc is only one of the oligosaccharide structures present along with mono-, di-, or tri-O-acetylated sialic acid. Ideally, to elucidate the sugar specificity one should use the disaccharides isolated from BSM. It is not possible to prepare the disaccharides with Neu5,9Ac2, because the alkali treatment of BSM required for the elimination of the O-glycosidically linked disaccharides would destroy the O-acetyl groups. Presently, no enzyme or process is available for releasing this glycoside from the sialoglycoprotein. Synthetic analogs are therefore the only alternative, but presently they are not available.
The detection of residual leukemic cells by FACS analysis mainly suffers from the lack of leukemia-specific CD markers. So far, progress in the identification of new leukemia-specific markers relied on testing the expression of known CD markers (Björklund et al., 2003). This approach, largely based on trial and error, is slow. We believe that purified and well-characterized Neu5,9Ac2-GPsALL will now be the new tool to reach the desired goal.
Previous observations suggested that a humoral response was directed specifically toward Neu5,9Ac2-GPsALL (Pal et al., 2000, 2001
). Accordingly, we developed a BSM-ELISA for the measurement of this antibody using BSM as a coating antigen that contains a mixture of mono-, di-, and tri-O-acetylated forms of Neu5,9Ac2. Therefore, it may be envisaged that not all forms of Neu5,9Ac2 present in BSM are equally available for binding with anti-Neu5, 9Ac2-GPs antibodies. Although a comparable correlation (r = 0.92) between antigen and BSM-ELISA was observed, antigen-ELISA revealed a higher specificity using a similar set of patients, and it may be considered as better capture antigen (Figure 5, Table II). The absence of cross-reactivity with other hematological diseases makes Neu5,9Ac2-GPsALL a suitable coating antigen for diagnosis and monitoring of ALL (Figure 4A). To the best of our knowledge, this is the first demonstration of the applicability of these unique Neu5,9Ac2-GPsALL molecules to monitor the clinical outcome of ALL (Figure 4B).
In a similar assay, following chemotherapy, anti-Neu5,9Ac2-GP antibody titers progressively decreased, reflecting clinical remission. However, a few children showed an increase in OD405nm, which correlated with clinical relapse. Therefore, monitoring of antibody titers using Neu5,9Ac2-GPsALL as capture antigen may be an alternate tool to assess the clinical status of patients.
Interestingly, we observed with a small population of children (n = 18) a good correlation of the antibody titers at early phase of treatment (phase B) with disease status (Figure 6). High antibody titers at phase B that showed close association with relapse (n = 10) were observed in 55.5% of patients. The relapse rate was 33% during maintenance therapy and 22% after completion of therapy. The higher incidence of relapse observed in these follow-up studies may be attributed to inclusion of patients irrespective of the risk factors (Riyat, 1995; Viana et al., 1994
). These patients may benefit from extensive treatment to avoid relapse. Therefore it may be recommended that patients with high antibody titers should be closely monitored for recurrence of disease. Additionally, a low-dose therapy may be considered for patients showing a persisting low antibody titer. In the population group of our study, we successfully correlated the antibody titers with the state of the disease, thus proving the potency of this assay for monitoring the disease. Of course, the study should be evaluated with a larger population group. Studies are ongoing to build a foundation for the predictive value of these antibodies.
The role of two different Neu5,9Ac2-GPsN, corresponding to 144 kDa and 36 kDa were detected on normal PBMCs, remains unclear. Earlier results suggest that they are constitutively present on normal PBMCs. This is corroborated by the presence of low anti-Neu5,9Ac2-GPs antibody in normal human serum, indicating that they are either less immunogenic or inadequately exposed on the cell surface of normal PBMC (Pal et al., 2000, 2001
).
In summary, our results indicate that leukemia-specific Neu5,9Ac2-GPsALL recognized by Achatinin-H are novel lymphoblastoid antigens. It may be envisaged that these antigens will allow designing primers for RT-PCRbased detection of minimal residual disease. In the future, development of monoclonal antibodies against these Neu5,9Ac2-containing glycoproteins will be useful for immunophenotyping and drug targeting. Studies on the production of humanized monoclonal antibodies are under way and will be helpful for future immunotherapy in childhood ALL.
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Materials and methods |
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Children had been entered into UK ALL X (Eden et al., 2000) with addition of further drugs, that is, etoposide and cytosar, for intensification. They were broadly grouped as follows: induction of remission (phase A, n = 70), consolidation/early intensification (phase B, 48 weeks, n = 58), period of maintenance therapy (phase C, 8 weeks2.5 years, n = 40), follow-up case (phase D, 2.5 years onward, n = 33), and patients who relapsed (phase E, n = 10). A few patients (n = 18) were longitudinally monitored for 224 weeks. Normal healthy individuals (n = 21) of both sexes and different blood groups and patients with other hematological disorders (n = 50) that included CML (n = 15), AML (n = 20), CLL (n = 10), and NHL (n = 5) served as controls.
Venous blood (34 ml) or bone marrow was collected at Vivekananda Institute of Medical Science (Kolkata, India) and then sent to the Indian Institute of Chemical Biology, where PBMCs were separated by Ficoll-Hypaque density centrifugation; sera or plasma was stored at 20°C. Informed consent was obtained from donors, patients, and parents or guardians. The study was approved by the institutional human ethical committee as per protocol of the Indian Council of Medical Research.
Probes and reagents
Achatinin-H, a lectin, purified from the hemolymph of the African giant land snail Achatina fulica, has been shown to preferentially bind to glycoconjugates with terminal Neu5,9Ac2-2,6-GalNAc residues mainly based on inhibition study with several monosaccharides and sialoglycoproteins, for example, BSM, SSM, HCG, fetuin and
1-acid glycoprotein. BSM having terminal Neu5,9Ac2 and a subterminal GalNAc in an
2,6-linkage showed highest inhibition (Sen and Mandal, 1995
). SSM, HCG, fetuin, and
1-acid glycoprotein with terminal sialic acid either in
2,6- or
2,3-linkage did not show any inhibition, reconfirming the specificity of Achatinin-H toward Neu5,9Ac2
2,6GalNAc glycotope (Mandal and Basu, 1987
; Mandal et al., 1989
; Sen and Mandal, 1995
).
The cell lines used included B-ALL (Nalm-6 and REH), T-ALL (CEM-C7, MOLT 3, MOLT-4, and JURKAT), JOK-1 (derived from peripheral blood of a patient with hairy cell leukemia), U266 (derived from peripheral blood of a patient with an IgE myeloma), U937 (histiocytic lymphoma), TF-1 (erythroleukemia), and KG-1a (AML). All cell lines were maintained in RPMI-1640 supplemented with glutamine (2 mM), gentamycin, and 10% heat-inactivated human AB serum (medium A). Other chemicals and biological reagents were from Sigma (St. Louis, MO) unless otherwise stated.
Fluorimetric HPLC analysis of sialic acids
PBMCs (5 x 108) of ALL patients were extensively washed in phosphate buffered saline, and the cell pellet was resuspended in 1 ml double-distilled water. Cell lysis was completed by sonication (three pulses of 16 s each, keeping samples on ice in between). Glycosidically bound sialic acids were then subjected to acid hydrolysis with an equal volume of 4 M propionic acid. Samples were heated to 80°C for 4 h, cooled on ice for 10 min, separated into three portions, and then lyophilized. Controls included (1) saponification of sialic acids by placing one of the lyophilized samples in an ammonium atmosphere overnight and (2) treatment by sialate-pyruvate lyase by resolving a sample in 250 mM phosphate buffer, pH 7.2, containing 25 mU lyase and incubation for 2 h at 37°C. All samples were then derivatized with 1,2-diamino-4,5-methylenedioxybenzene for fluorimetric reverse-phase HPLC analysis (Chatterjee et al., 2003).
Flow cytometric analysis
The expression of Neu5,9Ac2-GPs on lymphoblasts of ALL patients was evaluated by flow cytometry (FACS Calibur flow cytometer) using FITC-Achatinin-H. Briefly, PBMCs (1 x 106 cells/100 ml) from both T- and B-ALL patients and several established cell lines in medium A was blocked with goat serum (10%) and individually labeled with FITC-Achatinin-H or phycoerythrin-conjugated anti-CD10, anti-CD19, anti-CD7 (Pharmingen, San Diego, CA) in ice for 1 h (Pal et al., 2000). The cells were washed, fixed in paraformaldehyde (1%), and analyzed. FITCbovine serum albumin (BSA) or FITC-Achatinin-H in the presence of the inhibitor (BSM) or unconjugated lectin control (preincubation of cells with Achatinin-H followed by incubation with FITC-Achatinin-H) was used as different sets of controls. The % positive cells were recorded based on the threshold or background fluorescence provided by all these sets of controls, which gave a similar level of background fluorescence.
Alternatively, cells were incubated with Achatinin-H followed by rabbit anti-Achatinin-H polyclonal antibody, and the bound complex was detected by FITC-conjugated second antibody. The percentage of lymphoblasts that bind to this template was calculated relative to appropriate isotype matched antibodies, which served as background fluorescence.
The binding was also measured using FITC-labeled anti-CD60b, a 9-O-acetyl GD3-specific monoclonal antibody. FITC-IgM served as an isotype control. Analysis and calculations were performed using Cell Quest software.
Esterase treatment of lymphoblasts of ALL patients
The presence of O-acetyl sialoglycan groups on PBMC of ALL membrane was demonstrated by taking advantage of the 9-O-acetyl hemagglutinin esterase of influenza C virus (Chatterjee et al., 2003). It had been originally cloned in an SV40 vector (Vlasak et al., 1987
) to construct a gene consisting of the influenza C virus HE1 domain fused to the eGFP gene. Briefly, the entire HE1 coding region was isolated as a Sac I/Cla I restriction fragment. The Cla I site was filled in to allow blunt end ligation with the filled-in BamH I site immediately upstream of the eGFP gene derived from plasmid pEGFP-N3 (Clontech Laboratories, Austria). The resulting chimeric gene contains the entire HE1 domain and the first four codons of the HE2 domain linked via a five-codon spacer to the coding region of eGFP. This construct was ligated into the recombination vector pBakPAK8. The resulting plasmid pBacPAK-CHE1-eGFP was cotransfected with baculovirus DNA (Pharmingen) into Sf9 cells. Recombinant baculovirus Bak-CHE1-eGFP was plaque-purified and used to express the recombinant HE1-eGFP fusion protein. The expression of the HE-1 domain was sufficient to obtain a specific 9-O-acetyl esterase activity. Accordingly, cells (1 x 106) were incubated with the culture supernatant (100 µl) containing recombinant protein for 1 h at 2025°C, washed, and processed for flow cytometric analysis as described.
Purification of Neu5,9Ac2-GPs
PBMC membranes from clinically confirmed, immunophenotyped children of B-ALL (n = 10) and T-ALL (n = 5), at presentation of disease, that is, before any drug treatment, having >80% lymphoblasts (as measured using lineage specific established anti-CD antibodies and Achatinin-H) and normal donors (n = 5) were prepared according to Weissman et al. (1988). In brief, cells (1 x 107) were washed in ice-cold phosphate buffered saline (1.9 mM disodium hydrogen phosphate, 154 mM sodium chloride, pH 7.2) and suspended in lysis buffer containing TrisHCl (50 mM, pH 7.6), NaCl (300 mM), Triton X-100 (0.5%), phenylmethyl sulfonyl fluoride (0.01 M), and iodoacetamide (1.8 mg/ml). Following incubation for 45 min on ice, the nuclear pellet was discarded by centrifugation at 15,000 x g for 15 min at 4°C. To the supernatant, SDS (10%) and sodium deoxycholate (10%) were added to a final concentration of 0.2% each, centrifuged, and the supernatant containing membrane-enriched fraction was stored at 70°C. The purity of membrane fractions was confirmed by measuring 5' nucleotidase activity, and the protein concentration was determined using BSA as the standard.
The purified Achatinin-H was covalently linked to Sepharose-4B (1.0 mg of Achatinin-H/ml of gel). Equal amount of membrane fractions (0.8 mg) from each patient or normal donors were separately passed through this affinity column (1 x 2 cm) previously equilibrated with Tris-buffered saline (TBS) containing TrisHCl (0.05 M), NaCl (0.15 M), and sodium azide (0.02%), pH 7.2, with CaCl2 (0.03 M) at 4°C. After nonspecific washing, bound Neu5,9Ac2-GPs were eluted with TBS containing sodium citrate (0.04 M, pH 7.2), dialyzed against TBS at 4°C and stored at 70°C. The biological activity of Neu5,9Ac2-GPsALL and Neu5,9Ac2-GPsN was compared by an ELISA where equal amounts of purified fractions (0.4 mg/100 µl/well) were allowed to bind with Achatinin-H on a 96-cell plates, probed with anti-Neu5,9Ac2-GP antibodies and processed as described in inhibition-ELISA (see later discussion).
Molecular analysis of purified Neu5,9Ac2-GPs by SDSPAGE, western blot, and IEF
Affinity-purified Neu5,9Ac2-GPs were separated by SDSPAGE (7.5%) according to the method of Laemmli (1970) and stained with Coomassie brilliant blue R-250. For western blot analysis, Neu5,9Ac2-GPs were transferred after SDSPAGE onto nitrocellulose at 100 V for 2 h. After blocking the nonspecific binding sites with BSA (10%) in TBS (0.1 M, pH 7.4) the membranes were probed with Achatinin-H (160 µg/ml) in TBS-BSA-Ca2+ (0.03 M). After washing, the blot was incubated with rabbit anti-Achatinin-H (diluted 1:500) at 4°C and washed, and the antigen-antibody complex was detected using horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (Cappel, St. Louis, MO; 1:10,000). For de-O-acetylation, the blots were incubated with NaOH (0.1 N) for 45 min at 4°C, neutralized, and processed similarly.
Following SDSPAGE, the 90- and 120-kDa Neu5,9Ac2-GPALL bands were gel-eluted using an Electro-Eluter (Model 422; BioRad, Hercules, CA) according to the manufacturer's instructions and analyzed by IEF on ampholine polyacrylamide gel (4%) using a Mini-Protean II tube cell apparatus (BioRad) at a constant voltage of 400 V for 6 h. The gels were washed with sulfosalicylic acid (5%), methanol (30%), and acetic acid (10%) solution, fixed in trichloroacetic acid (10%), and stained with silver nitrate. The isoelectric point (pI) of individual proteins was determined as a function of their migration from the cathode using standard pI markers ranging from 3.5 to 10 (Bio Rad).
Synthesis of sialic acid analogs
Me--Neu5,9Ac2 (1) and benzyl-
-Neu4,5,9Ac3 (6) were obtained by partial acetylation of the respective Neu5Ac glycoside using acetimidazole. Me-
-Neu5Ac (2) was prepared as reported earlier (Meindl and Tuppy, 1965
; Kuhn et al., 1966
). Me-
-Neu5Ac9-SAc (3) and Me-
-Neu5Ac9-NHAc (4) were synthesized as previously described (Brossmer and Gross, 1994
; Isecke and Brossmer, 1995
). Conversion of Me-
-9-amino-Neu5Ac with nitrophenyl fluoroacetate gave the corresponding N-fluoroacetyl derivative (5). Reaction of benzyl-
-Neu5Ac with propionic anhydride afforded after purification the corresponding 9-O-propionic ester (7). Catalytic hydrogenation of benzyl-
-Neu4,5Ac2 produced Neu4,5Ac2 (9). All analogs were characterized by nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectroscopy.
Competitive binding of purified Neu5,9Ac2-GPsALL with synthetic sialic acid analogs by inhibition-ELISA
To substantiate the binding specificity of purified Neu5,9Ac2-GPsALL, an inhibition-ELISA was developed. Several sialic acid analogs (Troncoso et al., 2000) were used as inhibitors to compare their potency to inhibit binding of Neu5,9Ac2-GPsALL to immobilized Achatinin-H. A microtiter plate was coated with Achatinin-H (1 µg/100 µl/well in 0.05 M TBS, pH 7.4) and incubated overnight at 4°C. Following three washes, the wells were blocked with TBS2% BSA for 2 h at 25°C. A constant amount of purified Neu5,9Ac2-GPsALL (1 µg/50 µl TBS-BSA) was preincubated separately with inhibitor (50 µl, 1068 mM) in the presence of 30 mM Ca2+ at 4°C for 30 min. The mixture (100 µl) was added to lectin-coated wells, incubated overnight at 4°C, and washed thrice with TBS containing 0.1% Tween-20 (TBS-T). The binding of Neu5,9Ac2-GPsALL to Achatinin-H was detected by incubating anti-Neu5,9Ac2-GPsALL antibodies (1 µg), affinity-purified from sera of ALL patients (Pal et al., 2000
), overnight at 4°C. The plate was washed thrice with TBS-T, and the specific antigenantibody complex was measured using HRP-conjugated protein A (diluted 1:10,000, Cappel) and azino-bis-thio-sulfonic acid. The absorbance was recorded at 405 nm in an ELISA reader.
Based on the long-standing evidence that BSM contains a high percentage of 9(8)-O-acetylated sialic acid derivatives as estimated fluorimetrically (Sharma et al., 1998) and by fluorimetric HPLC (Chatterjee et al., 2003
) it was also used as an inhibitor in the inhibition-ELISA. In parallel, de-O-acetylated BSM, desialylated BSM, and other sialoglycoproteins (such as SSM, HCG, fetuin, and
1-acid glycoprotein) having no O-acetylated sialic acids were also used.
Monitoring disease status by measuring the reactivity of Neu5,9Ac2-GPsALL with ALL serum in an antigen-ELISA
Purified Neu5,9Ac2-GPsALL (0.5 µg/100 µl TBS/well) were coated overnight on 96-well plate at 4°C. Following three washes with TBS-T, the wells were blocked with 2% BSA. Sera (diluted 1:10) from ALL patients at different stages of treatment, and from patients with other hematological disorders and normal donors were added and incubated overnight at 4°C. After washing thrice with TBS-T, the antigenantibody complex was detected using HRP-protein A as described. In addition, anti-Neu5,9Ac2-GPsALL present in ALL serum was quantified by BSM-ELISA using BSM (1 µg/100 µl/well) as coating antigen and binding of anti-Neu5,9Ac2-GPsALL was measured as previously described (Pal et al., 2000).
Statistical analysis
Statistical analysis was performed using the Graph-Pad Prism statistics software program (Graph-Pad Software, San Diego, CA). Student's unpaired or paired t-tests were used. Reported values are two-tailed and p-values lower than 0.05 were considered statistically significant. The Spearman correlation test was used for the comparison of independent variables. Life table analysis according to Kaplan and Meier were performed for relapse-free intervals and overall survival.
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Acknowledgements |
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Footnotes |
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1 These authors contributed equally to this work.
2 Present address: Gurudas College, Department of Botany, Kolkata 700054, India
3 Present address: Chess GmbH, D-68526 Ladenburg, Germany
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Abbreviations |
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References |
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