Characterization of the Acid Stability of Glycosidically Linked Neuraminic Acid
2002; Elsevier BV; Volume: 277; Issue: 20 Linguagem: Inglês
10.1074/jbc.m110867200
ISSN1083-351X
AutoresJustin L. Sonnenburg, Herman van Halbeek, Ajit Varki,
Tópico(s)Protein Structure and Dynamics
ResumoThe glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that α2–3-linked Neu on chemically de-N-acetylated GM3 ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked α- and β-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neuα2Me is hydrolyzed after 3 h in 10 mm HCl at 80 °C, whereas Neu5Acα2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neuβ2Me is hydrolyzed even slower than Neuα2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides. The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that α2–3-linked Neu on chemically de-N-acetylated GM3 ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked α- and β-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neuα2Me is hydrolyzed after 3 h in 10 mm HCl at 80 °C, whereas Neu5Acα2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neuβ2Me is hydrolyzed even slower than Neuα2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides. Gangliosides are amphipathic glycosphingolipids that are mostly found in the outer leaflet of the plasma membrane (1Hakomori S. Annu. Rev. Biochem. 1981; 50: 733-764Crossref PubMed Scopus (1480) Google Scholar, 2Zeller C.B. Marchase R.B. Am. J. Physiol. Cell Physiol. 1992; 262: 1341-1355Crossref PubMed Google Scholar, 3Stults C.L.M. Sweeley C.C. Macher B.A. Methods Enzymol. 1989; 179: 167-214Crossref PubMed Scopus (234) Google Scholar, 4Van Echten G. Sandhoff K. J. Biol. Chem. 1993; 268: 5341-5344Abstract Full Text PDF PubMed Google Scholar). They are typically characterized by the presence of at least one sialic acid residue and a lactosyl ceramide core. Other features of a ganglioside oligosaccharide moiety, such as the number and branching pattern of monosaccharides and modifications such as O- acetylation, are regulated in a tissue-specific and temporal manner. The role of gangliosides in cell signaling (5Hanai N. Dohi T. Nores G.A. Hakomori S. J. Biol. Chem. 1988; 263: 6296-6301Abstract Full Text PDF PubMed Google Scholar, 6Weis F.M.B. Davis R.J. J. Biol. Chem. 1990; 265: 12059-12066Abstract Full Text PDF PubMed Google Scholar, 7Zhou Q. Hakomori S. Kitamura K. Igarashi Y. J. Biol. Chem. 1994; 269: 1959-1965Abstract Full Text PDF PubMed Google Scholar, 8Nagai Y. Behav. Brain Res. 1995; 66: 99-104Crossref PubMed Scopus (121) Google Scholar, 9Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and cell-cell and cell-matrix interactions (10Cheresh D.A. Pierschbacher M.D. Herzig M.A. Mujoo K. J. Cell Biol. 1986; 102: 688-696Crossref PubMed Scopus (265) Google Scholar, 11Probstmeier R. Pesheva P. Glycobiology. 1999; 9: 101-114Crossref PubMed Scopus (52) Google Scholar, 12Kojima N. Shiota M. Sadahira Y. Handa K. Hakomori S. J. Biol. Chem. 1992; 267: 17264-17270Abstract Full Text PDF PubMed Google Scholar, 13Vyas A.A. Schnaar R.L. Biochimie (Paris). 2001; 83: 677-682Crossref PubMed Scopus (81) Google Scholar) and host-pathogen interactions (14Markwell M.A. Svennerholm L. Paulson J.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5406-5410Crossref PubMed Scopus (214) Google Scholar, 15Walton K.M. Sandberg K. Rogers T.B. Schnaar R.L. J. Biol. Chem. 1988; 263: 2055-2063Abstract Full Text PDF PubMed Google Scholar, 16Karlsson K.-A. Annu. Rev. Biochem. 1989; 58: 309-350Crossref PubMed Scopus (622) Google Scholar) has been extensively studied and is largely dictated by the structure of the glycan component. Most sialic acids on gangliosides share a core neuraminic acid (Neu) 1The abbreviations used are: Neuneuraminic acidNeu5AcN-acetylneuraminic acidNeu5Acα2MeNeu5Ac α-methyl glycosideNeu5GcN-glycolylneuraminic acidNeu5PrN-propionylneuraminic acidNeuα2Meneuraminic acid α-methyl glycosideNeuβ2Meneuraminic acid β-methyl glycosideCHOChinese hamster ovaryDMB1,2-diamino-4,5-methylene dioxybenzeneELISAenzyme-linked immunosorbent assayHPTLChigh performance thin layer chromatographyHPLChigh performance liquid chromatographymAbmonoclonal antibodyPBSphosphate-buffered salinePLCphospholipase CSiasialic acid, type unspecifiedSiaQsialic acid quinoxalinone (DMB adduct)ESIelectrospray ionization structure and are N-acylated at the C-5 position with either an N-acetyl or an N-glycolyl group (giving Neu5Ac or Neu5Gc, respectively). It was originally thought that unsubstituted glycosidically linked Neu did not occur in nature (17Gottschalk A. The Chemistry and Biology of Sialic Acids and Related Substances. Cambridge University Press, Cambridge, UK1960Google Scholar). However, there have been several reports suggesting its presence in gangliosides (5Hanai N. Dohi T. Nores G.A. Hakomori S. J. Biol. Chem. 1988; 263: 6296-6301Abstract Full Text PDF PubMed Google Scholar,7Zhou Q. Hakomori S. Kitamura K. Igarashi Y. J. Biol. Chem. 1994; 269: 1959-1965Abstract Full Text PDF PubMed Google Scholar, 18Manzi A.E. Sjoberg E.R. Diaz S. Varki A. J. Biol. Chem. 1990; 265: 13091-13103Abstract Full Text PDF PubMed Google Scholar, 19Sjoberg E.R. Chammas R. Ozawa H. Kawashima I. Khoo K.-H. Morris H.R. Dell A. Tai T. Varki A. J. Biol. Chem. 1995; 270: 2921-2930Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Chammas R. Sonnenburg J.L. Watson N.E. Tai T. Farquhar M.G. Varki N.M. Varki A. Cancer Res. 1999; 59: 1337-1346PubMed Google Scholar, 21Hidari K.I.-P. J. Irie F. Suzuki M. Kon K. Ando S. Hirabayashi Y. Biochem. J. 1993; 296: 259-263Crossref PubMed Scopus (25) Google Scholar) and more recently in mucin-type glycoproteins (22Mitsuoka C. Ohmori K. Kimura N. Kanamori A. Komba S. Ishida H. Kiso M. Kannagi R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1597-1602Crossref PubMed Scopus (61) Google Scholar, 23Zanetta J.P. Pons A. Iwersen M. Mariller C. Leroy Y. Timmerman P. Schauer R. Glycobiology. 2001; 11: 663-676Crossref PubMed Scopus (78) Google Scholar). Hakomori and colleagues (5Hanai N. Dohi T. Nores G.A. Hakomori S. J. Biol. Chem. 1988; 263: 6296-6301Abstract Full Text PDF PubMed Google Scholar) first defined de-N-acetyl-gangliosides by suggestive evidence for a Neu residue that was presumed to have arisen from de-N-acetylation of Neu5Ac. A possible role of such gangliosides in signaling was also suggested based on the finding that synthetic de-N-acetyl-GM3 2Ganglioside nomenclature is based on the system of Svennerholm (41Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1313) Google Scholar). GM3, Neu5Acα2,3Galβ1,4Glcβ1,1-ceramide; GD3, Neu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ1,1-ceramide; GM1, Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1-ceramide. specifically enhanced epidermal growth factor receptor signaling when added to cells in culture, whereas the conventional N-acetylated GM3 had the opposite effect (5Hanai N. Dohi T. Nores G.A. Hakomori S. J. Biol. Chem. 1988; 263: 6296-6301Abstract Full Text PDF PubMed Google Scholar). However, proof for the natural occurrence of this monosialoganglioside was based upon the reactivity of a monoclonal antibody (mAb) DH5 that could recognize the synthetic molecule. neuraminic acid N-acetylneuraminic acid Neu5Ac α-methyl glycoside N-glycolylneuraminic acid N-propionylneuraminic acid neuraminic acid α-methyl glycoside neuraminic acid β-methyl glycoside Chinese hamster ovary 1,2-diamino-4,5-methylene dioxybenzene enzyme-linked immunosorbent assay high performance thin layer chromatography high performance liquid chromatography monoclonal antibody phosphate-buffered saline phospholipase C sialic acid, type unspecified sialic acid quinoxalinone (DMB adduct) electrospray ionization In related studies, our group used radiolabeling and pulse-chase techniques to indicate that ganglioside sialic acids were undergoing a de-/re-N-acetylation cycle in cultured human melanoma cells (18Manzi A.E. Sjoberg E.R. Diaz S. Varki A. J. Biol. Chem. 1990; 265: 13091-13103Abstract Full Text PDF PubMed Google Scholar). In collaboration with Tai's group (19Sjoberg E.R. Chammas R. Ozawa H. Kawashima I. Khoo K.-H. Morris H.R. Dell A. Tai T. Varki A. J. Biol. Chem. 1995; 270: 2921-2930Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), we also described monoclonal antibody SGR37 raised against synthetic de-N-acetyl-GD3, which was used to suggest that this disialoganglioside is specifically expressed in some human melanomas and lymphomas (20Chammas R. Sonnenburg J.L. Watson N.E. Tai T. Farquhar M.G. Varki N.M. Varki A. Cancer Res. 1999; 59: 1337-1346PubMed Google Scholar). Despite all of these suggestive reports, no one has provided definitive structural proof for the natural existence of de-N-acetyl-gangliosides. Although Hidari et al.(21Hidari K.I.-P. J. Irie F. Suzuki M. Kon K. Ando S. Hirabayashi Y. Biochem. J. 1993; 296: 259-263Crossref PubMed Scopus (25) Google Scholar) provided 1H NMR data defining traces of de-N-acetyl-GM1 in bovine brain gangliosides, these preparations had been subjected to alkaline saponification for phospholipid degradation under conditions that would have caused some chemical de-N-acetylation. Previous attempts to purify de-N-acetyl-gangliosides from natural sources have also been unsuccessful. Contaminating molecules in total lipid extracts, such as phospholipids and more abundant N-acylated gangliosides, can interfere with purification and detection by mAbs. Additionally, cell lines and cell line-based tumors have proven to be unreliable sources of de-N-acetyl-gangliosides because the expression level based on monoclonal antibody detection is low and variable. Furthermore, as reported here, at least one of these mAbs shows nonspecific cross-reactivity to a peptide. In unrelated studies we have reported an acid-stable, mono-carboxylated modification of as yet unknown structure on the N-linked glycans of bovine lung (24Norgard-Sumnicht K.E. Roux L. Toomre D.K. Manzi A.E. Freeze H.H. Varki A. J. Biol. Chem. 1995; 270: 27634-27645Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 25Srikrishna G. Toomre D.K. Manzi A. Panneerselvam K. Freeze H.H. Varki A. Varki N.M. J. Immunol. 2001; 166: 624-632Crossref PubMed Scopus (33) Google Scholar). In seeking to understand the nature of this carboxylated moiety, we considered the possibility that it might be a modified type of sialic acid. It is known that the glycosidic linkage of amino sugars like glucosamine is more stable to acid hydrolysis when the amino group is unsubstituted (26Moggridge, R. C. G., and Neuberger, A. (1938) J. Chem. Soc. 745–750Google Scholar). Although sialic acids are generally among the most acid labile of glycosidically linked sugars, we considered the possibility that the glycosidic linkage of Neu with its unsubstituted amino group might be acid-resistant. As it turned out, the carboxylate in question was not part of a sialic acid, and further studies are currently under way to define its true nature. However, in the course of exploring this issue, we discovered that the glycosidic linkage of Neu is indeed quite resistant to acid. Employing this knowledge, we report here a new approach for detection of naturally occurring Neu in de-N-acetyl-gangliosides. Hybridoma cells secreting mAbs SGR37 or SMR36 were prepared in collaboration with Dr. Tadashi Tai as previously described (19Sjoberg E.R. Chammas R. Ozawa H. Kawashima I. Khoo K.-H. Morris H.R. Dell A. Tai T. Varki A. J. Biol. Chem. 1995; 270: 2921-2930Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Expired clinical grade mAb R24 (Celltech Ltd.) (27Pukel C.S. Lloyd K.O. Travassos L.R. Dippold W.G. Oettgen H.F. Old L.J. J. Exp. Med. 1982; 155: 1133-1147Crossref PubMed Scopus (493) Google Scholar) was obtained from the National Cancer Institute; mAb DH2 (28Dohi T. Nores G. Hakomori S. Cancer Res. 1988; 48: 5680-5685PubMed Google Scholar) was kindly provided by Dr. Sen-itiroh Hakomori. All hybridomas were cultured in RPMI 1640, 10% heat-inactivated fetal calf serum, 1 ng/ml IL-6. B16 mouse melanoma cells were maintained in Dulbecco's modified Eagle's medium (regular glucose), 10% heat-inactivated fetal calf serum, and human U937 cells in RPMI 1640, 10% heat-inactivated fetal calf serum. Chinese hamster ovary (CHO) cells (K1 strain) were cultured in α-minimum essential medium, with 10% heat-inactivated fetal calf serum. Human tissue samples were kindly provided by Dr. Nissi Varki through the University of California, San Diego Cancer Center Histology Core Service. Neu5Acα2Me was purchased from Sigma, and Neuβ2Me was purchased from ICN Biomedicals. Unless otherwise stated, all other chemicals were of reagent grade or higher and purchased from commercial sources. Synthetic de-N-acetyl-gangliosides were prepared as described (19Sjoberg E.R. Chammas R. Ozawa H. Kawashima I. Khoo K.-H. Morris H.R. Dell A. Tai T. Varki A. J. Biol. Chem. 1995; 270: 2921-2930Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29Nores G.A. Hanai N. Levery S.B. Eaton H.L. Salyan E.K. Hakomori S. Carbohydr. Res. 1988; 179: 393-410Crossref PubMed Scopus (34) Google Scholar,30Nores G.A. Hanai N. Levery S.B. Eaton H.L. Salyan M.E.K. Hakomori S. Methods Enzymol. 1989; 179: 242-252Crossref PubMed Scopus (14) Google Scholar). Briefly, 1 mg of GM3 or GD3 (Matreya) was dissolved in 14.3 ml of 1 m tetramethylammonium hydroxide in butanol and heated to 100 °C in an oil bath for 3 h with constant stirring. After adding 175 ml of water, the solution was neutralized with 14.3 ml of 1 m acetic acid and dried on a rotary evaporator. Small volumes of water were added throughout to ensure complete evaporation of butanol. The de-N-acetyl-gangliosides were recovered after dialysis and lyophilization, dissolved in methanol, and stored at –20 °C. The extent of de-N-acetylation was determined by high performance thin layer chromatography (HPTLC) to be ∼50%. CHO cells were plated on tissue culture slide chambers (LabTek) and grown to 70–90% confluence. The media was removed, and cells were washed once in PBS and fixed in 2% paraformaldehyde in PBS for 30 min. Blocking was performed by incubating the slides in PBS containing 1% bovine serum albumin, and 3% goat serum for 30 min. Slides were incubated with primary antibody solutions for 30 min. Primary antibodies included R24 (5 μg/ml final) and SGR37 (50% solution of hybridoma supernatant) and were prepared in the same solution that had been used for blocking. Slides were washed three times with PBS, and the horseradish-conjugated secondary antibody was added (goat-anti-mouse IgG-horseradish peroxidase; 1:50) in PBS. Slides were washed again in PBS 3 times and developed using the AEC (3-amino-9-ethylcarbazole) substrate system (Vector Labs). The substrate solution was removed, slides were washed twice, and nuclei were counterstained with hematoxylin. Images were captured using the 40× objective on a Zeiss Axiophot microscope fitted with a Sony DKC-5000 using NIH Image software. CHO and Melur cells were grown to 80% confluence in large culture dishes (15-cm diameter) and harvested using a cell scraper. 30 million cells were washed in PBS twice, and 300 μl of reducing SDS-PAGE sample buffer was added. DNA was sheared by repeated pipetting using a 20-guage needle. The samples were boiled at 100 °C for 5 min followed by a 5-min spin at 15,000 × g. 10 μl of the samples were used per lane on a 7.5% SDS-PAGE that was subsequently run at 100 V for approximately 1 h. The proteins in the gel were transferred to a polyvinylidene difluoride membrane at 50 V for 3 h. Scissors were used to cut the membrane for separation of each lane. Strips of polyvinylidene difluoride were rocked gently in either 2 mm sodium periodate in PBS for 30 min at 4 °C (mild periodate) or 25 mm sodium periodate in 0.1m sodium acetate buffer, pH 5.0, for 30 min at room temperature (strong periodate) or left untreated. After washing the periodate-treated lanes in PBS, all strips were soaked in blocking buffer (PBS, 1% powdered milk) to block nonspecific binding sites. Primary antibodies R24 (10 μg/ml) and SGR37 (50% hybridoma supernatant) were diluted in blocking buffer and incubated with the polyvinylidene difluoride strips for 1 h. The polyvinylidene difluoride strips were washed 3 times for 5 min and incubated with the alkaline phosphatase-conjugated secondary antibody (Goat-anti-mouse IgG-AP; 1:4000) in blocking buffer. After three washes, the blots were developed using the alkaline phosphatase-conjugate substrate system (Bio-Rad). Aliquots of gangliosides (600 ng/well) in methanol were added to wells of 96-well plates (NUNC) and allowed to dry overnight at room temperature. All washes and incubations were performed at room temperature in ELISA buffer (PBS, 1% bovine serum albumin). Nonspecific binding sites were blocked with ELISA buffer for 2 h followed by a 2-h primary antibody incubation. Primary antibodies used include DH2 (hybridoma supernatant) and SMR36 (ammonium sulfate precipitate in 50% saturated salt solution). mAbs were titrated to determine the optimal dilution. Wells were washed 3 times for 5 min each in ELISA buffer before a 1-h incubation with secondary antibody conjugated to horseradish peroxidase (1:4000) (goat-anti-mouse IgG DH2; goat-anti-mouse IgM for SMR36) (Bio-Rad). Wells were washed again 3 times for 5 min each and developed in citrate phosphate buffer, pH 5.0, containing 400 μg/ml o- phenylenediamine and 0.12% hydrogen peroxide. Reactions were allowed to proceed for several minutes until a yellow color was visible and then quenched with the addition of 1/3 volume 9m sulfuric acid. Absorbance was measured at 490 nm on a SpectraMax-250 96-well plate reader (Molecular Devices). Samples in methanol were applied 1 cm above the bottom of an activated silica gel 60 glass-backed HPTLC plate (Merck) using a 1-μl Hamilton syringe (4 μg of ganglioside/lane; phospholipid extract originating from 2 × 106 B16 cells/lane). Plates were developed in glass TLC tanks pre-equilibrated with chloroform:methanol:water (65:25:4) for phospholipid separation or chloroform, methanol, 0.02% CaCl2 (60:40:9) for separation of gangliosides. Phospholipids were visualized using phosphomolybdate spray reagent (Sigma) according to the manufacturer's directions. Individual gangliosides were visualized using the appropriate monoclonal antibody in an immuno-overlay assay. This procedure was performed as described elsewhere (19Sjoberg E.R. Chammas R. Ozawa H. Kawashima I. Khoo K.-H. Morris H.R. Dell A. Tai T. Varki A. J. Biol. Chem. 1995; 270: 2921-2930Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 31Magnani J.L. Brockhaus M. Smith D.F. Ginsburg V. Methods Enzymol. 1982; 83: 235-241Crossref PubMed Scopus (59) Google Scholar). Briefly, HPTLC plates were allowed to air dry after development and then plasticized by immersing the plate for 1 min in hexane, 2% polyisobutylmethacrylate in chloroform (84:16). The plate was allowed to air dry, placed horizontally in a humidified chamber, covered with primary antibody in overlay buffer (PBS, 1% bovine serum albumin), and incubated at 4 °C overnight. Antibodies were used as described for ELISA. After primary antibody incubation, the surface of the plate was washed with overlay buffer 3 times for 5 min each and then covered with a 1:1000 dilution of secondary antibody. Secondary antibodies were identical to those used for ELISAs. The secondary incubation was allowed to proceed at room temperature for 1 h. After three 5-min washes, the gangliosides were visualized using 400 μg/ml o- phenylenediamine in citrate phosphate buffer, pH 5.0, with 0.12% hydrogen peroxide. Once bands were visible, plates were rinsed with water and dried with a blow drier. Tissue samples were sliced into small pieces with a scalpel and homogenized in 10 mm HEPES, pH 7.4, using a Polytron homogenizer (Brinkmann Instruments). Ten volumes of chloroform:methanol (1:1) were added, samples were sealed under nitrogen gas, and the extraction was allowed to proceed at room temperature for 12 h with gentle agitation. Protein precipitates were pelleted by centrifugation (10,000 × g for 15 min), supernatants were collected and stored at 4 °C under nitrogen, and the extraction was repeated using the same volume of chloroform:methanol (1:1). The organic extracts were pooled and dried under a stream of nitrogen. Dried lipids were resuspended in 50 mm potassium phosphate buffer, pH 7.4, using probe sonication and/or bath sonication. Phospholipase C (PLC) (Sigma; P-9439) was added at 5 milliunits/mg of tissue extracted and incubated with vigorous agitation at 37 °C in 3-h increments until degradation was complete (as determined by HPTLC, showing that excess PLC failed to further degrade a test aliquot of a given sample). Dry samples containing known or putative de-N-acetyl sialic acids were dissolved in saturated sodium bicarbonate and treated with 3.3% acetic anhydride or propionic anhydride for 15 min at room temperature. An identical mixture of sodium bicarbonate and the acyl anhydride was added 2 more times and allowed to react for 15 min each time. Samples were neutralized by adding an appropriate volume of 1 m HCl. Control samples contained amounts of acetic or propionic acids equivalent to the amounts of the anhydride used. Samples containing native or chemically acylated sialic acids were incubated at 80 °C for 3 h in 2 m acetic acid. In some cases, samples were then passed over a cation exchange column (Dowex AG50W-X2, H+ form), and the resulting volatile acids were removed by lyophilization. Free sialic acids from some samples were finally bound to an anion exchange column (Dowex AG1-X8, formate form) (Bio-Rad), eluted with 1 m formic acid, and lyophilized to remove the acid. Aliquots of free sialic acids were derivatized in 8 mm DMB, 1.5 m acetic acid, 0.8 mβ-mercaptoethanol, 14 mm sodium hydrosulfite for 2.5 h at 50 °C in the dark (32Hara S. Yamaguchi M. Takemori Y. Furuhata K. Ogura H. Nakamura M. Anal. Biochem. 1989; 179: 162-166Crossref PubMed Scopus (249) Google Scholar). DMB-derivatized sialic acids were resolved using a reverse phase C18 Microsorb-MV column (Varian, 4.6-mm internal diameter × 25 cm, 5 μm) on a Rainin Dynamax SD-200 HPLC. Samples were eluted at a flow rate of 0.9 ml/min using either a 50-min isocratic elution in 8% acetonitrile, 7% methanol in water or a 70-min isocratic elution in 7% acetonitrile, 7% methanol in water. A Spectrovision FD-300 on-line fluorescence detector was used to visualize the sialic acid derivatives as they eluted (excitation at 373 nm, emission at 448 nm). DMB derivatives of sialic acids were validated by mass spectrometry. Fractions eluting from the C18 column were collected based on the elution position of known standards, dried down using a speed-vac and/or shaker-evaporator and stored in the dark. The fractions were reconstituted in water and run on a Finnigan MAT HPLC with online Mass Spectrometer model LCQ-Mass Spectrometer System. A Varian C18 column was used and eluted at 0.9 ml/min in the isocratic mode with 8% acetonitrile, 7% methanol, and 0.1% formic acid in water over 50 min. The eluant was simultaneously monitored by absorbance at 373 nm and by electrospray ionization (ESI) mass spectrometry. The following ESI settings were used. Capillary temperature was set at 210 °C, the capillary voltage was set at 31 V, and the lens offset voltage set at 0 V. The spectra were acquired by scanning from m/z150–2000 in the positive-ion mode. Tandem mass spectrometry spectra were acquired by selecting the parent mass and using a 20% normalized collision energy. Data analysis was performed using the Xcalibur data analysis program from the instrument manufacturer. De-N-acetylation of Neu5Acα2Me was accomplished using hydrazine. Two micromoles of lyophilized Neu5Acα2Me were dissolved in 0.2–0.3 ml anhydrous hydrazine, capped tightly in a nitrogen atmosphere, and incubated at 100 °C for 6 h. To remove the hydrazine, the sample was brought to room temperature, uncapped, and placed in an evacuated chamber containing a beaker of concentrated sulfuric acid and allowed to sit overnight. After hydrazine evaporation, 0.2 ml of toluene was added to the sample twice and blown off under a stream of nitrogen. The sample was resuspended in 0.5 ml of water, and Neuα2Me was separated from surviving Neu5Acα2Me by loading onto a 0.5-ml column of Dowex AG50W-X2 (H+ form) resin, washing with 2 ml of water, and eluting the de-N-acetylated material with 2 ml of 1 m HCl. The HCl was evaporated using a shaker-evaporator, and the sample was resuspended in water and stored at –20 °C. Solutions of methyl glycosides (∼100 nmol) in D2O (0.7 ml; 99.9% D; Aldrich) were transferred into 5-mm NMR tubes (Wilmad; 528PP), acidified with HCl in H2O (a few μl) to the desired molarity (i.e. pH), and sealed. The pH of the stock solutions (without the glycosides) was measured using a Corning 240 pH meter. Time courses of glycoside hydrolysis were monitored discontinuously as follows. The tubes were placed in a water bath at 80 °C and removed for NMR analysis at specified time points. The time that samples spent at room temperature during NMR data collection or between 80 °C incubations was determined to be insignificant with regard to sample degradation. NMR experiments were carried out using a 500-MHz Varian Unity Inova spectrometer controlled by a SUN MicroSystems Ultra-10 computer running Varian VNMR software (version 6.1B). 1H NMR spectra were acquired at 27 °C in 512 transients each; the samples were not spun. The residual HDO signal was suppressed by low power presaturation. Data processing included line-broadening (lb = 0.5) and zero-filling (from 16 K to 32 K complex points) before Fourier transformation followed by base-line correction and integration. Chemical shifts (δ) are reported relative to TSP-d4 (sodium 3-(trimethylsilyl)-propionate-2,2,3,3-d4) (Aldrich); chemical shifts were actually measured using acetic acid (δ 2.081 ppm at pH < 4) or acetone (δ 2.217 ppm; pH-independent) as internal standards. All prior reports of detection of de-N-acetylated gangliosides have used monoclonal antibodies. However, data using such antibodies cannot be considered definitive, since reactivity can be abrogated by interfering molecules (e.g. phospholipids). 3R. Chammas and J. L. S., unpublished observations. Additionally, antibodies directed against carbohydrates can sometimes show nonspecific cross-reactivity with other epitopes. Indeed, we have observed such cross-reactivity when staining CHO cells with mAb SGR37, which was thought to b
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