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• W2044092574 abstract "Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40–50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Acα2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both di- and polySia were found to show distinctive changes during embryonic development. Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40–50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Acα2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both di- and polySia were found to show distinctive changes during embryonic development. α-2,8-linked polysialic acid N-acetyl-d-glucosamine N-acetyl-d-galactosamine N-acetylneuraminic acid N-acylneuraminic acid monosialic acid residue attached to the penultimate Gal residue disialic acid (Neu5Acα2→8Neu5Acα2→) residue attached to underlying Gal residue trisialic acid residues attached α2→3 to the Gal residue α-2,8-linked disialic acid, e.g.Neu5Acα2→8Neu5Ac α-2,8-linked polyNeu5Ac or (→8Neu5Acα2→) n neural cell adhesion molecule polysialyltransferase degree of polymerization bacteriophage-induced poly(→8Neu5Acylα2→) endo-N-acylneuraminidase asparagine-linked carbohydrate chain high performance liquid chromatography high performance anion-exchange chromatography high performance liquid chromatography with fluorometric detection high performance anion-exchange chromatography with pulsed electrochemical detection 1,2-diamino-4,5-methylenedioxybenzene embryonic day n after fertilization 2-(N-morpholino)ethanesulfonic acid Polysialic acid (polySia)1 is a structurally and functionally unique glycotope expressed on the surface of living cells (1Troy F.A. Glycobiology. 1992; 2: 5-23Crossref PubMed Scopus (316) Google Scholar). In higher vertebrates, the α2,8-linked homopolymer of Neu5Ac is the only reported structure of polySia, although diverse structures of polySia differing in C-5 substitution of Sia residues and in the inter-residue linkages have been discovered in bacteria, invertebrates, and lower vertebrates (2Inoue Y. Inoue S. Pure Appl. Chem. 1999; 71: 789-800Crossref Scopus (28) Google Scholar). In embryonic vertebrate brain, the neural cell adhesion molecule (N-CAM) is a major carrier protein of polySia. A hypothesis that the presence of polySia on N-CAM attenuates the adhesive function of this molecule (3Bonfanti L. Olive S. Poulain D.A. Theodosis D.T. Neuroscience. 1992; 49: 419-436Crossref PubMed Scopus (298) Google Scholar, 4Seki T. Arai Y. Neurosci. Res. 1993; 17: 265-290Crossref PubMed Scopus (374) Google Scholar) is supported by temporally regulated expression of polySia on embryonic N-CAM, its spatially limited expression in the olfactory bulb, hippocampus, and cerebellum of adult mammalian brain (where continuous plasticity is required). PolySia is also an oncodevelopmental antigen that is re-expressed on a number of human tumors, including neuroblastomas (5Livingston B.D. Jacobs J.L. Glick M.C. Troy F.A. J. Biol. Chem. 1988; 263: 9443-9448Abstract Full Text PDF PubMed Google Scholar) and Wilms tumor (6Bitter-Suermann D. Roth L. Immunol. Res. 1987; 6: 225-237Crossref PubMed Scopus (29) Google Scholar). The presence of polySia on N-CAM not only functions as a negative regulator of N-CAM-mediated homotypic cell-cell adhesion but also decreases interactions with other cells. Although the molecular details of how polySia affects cell interactions has not been fully elucidated, it has been hypothesized to depend on the physical properties of this negatively charged and heavily hydrated polymer (7Rutishauser, U. (1992) Dev. Suppl. 99–104Google Scholar,8Rutishauser U. Curr. Opin. Cell Biol. 1996; 8: 679-684Crossref PubMed Scopus (146) Google Scholar). Recent studies have shown that two polysialyltransferases (polySTs), designated PST-1 (PST/ST8SiaIV) and STX (ST8SiaII), catalyze the polysialylation of N-CAM. The genes encoding both enzymes have been cloned from several species and sequenced (9Eckhardt M. Muhlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 10Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 11Yoshida Y. Kojima N. Kurosawa N. Hamamoto T. Tsuji S. J. Biol. Chem. 1995; 270: 14628-14633Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Wood G.K. Liang J.-J. Flores G. Ahmad S. Quirion R. Srivastava L.K. Mol. Brain Res. 1997; 51: 69-81Crossref PubMed Scopus (30) Google Scholar). The availability of cDNAs encoding these enzymes has facilitated new approaches to study the function, mechanism, and regulation of polysialylation of N-CAM (13Wood G.K. Liang J.-J. Flores G. Ahmad S. Quirion R. Srivastava L.K. Mol. Brain Res. 1997; 51: 69-81Crossref PubMed Scopus (30) Google Scholar, 14Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Crossref PubMed Scopus (122) Google Scholar, 15Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Kudo M. Takayama E. Tashiro K. Funamachi H. Nakata T. Yadakuma T. Kitajima K. Inoue Y. Shiokawa K. Glycobiology. 1998; 8: 771-777Crossref PubMed Scopus (14) Google Scholar). The properties and developmentally regulated expression of polyST activity in the membrane fraction of embryonic chicken (17Oka S. Bruses J.L. Nelson R.W. Rutishauser U. J. Biol. Chem. 1995; 270: 19357-19363Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar,18Sevigny M.B. Ye J. Kitazume-Kawaguchi S. Troy F.A. Glycobiology. 1998; 8: 857-867Crossref PubMed Scopus (26) Google Scholar) and rat (19McCoy R.D. Vimr E.R. Troy F.A. J. Biol. Chem. 1985; 260: 12695-12699Abstract Full Text PDF PubMed Google Scholar) brain have been studied. Despite extensive studies on the expression and function of polySia on N-CAM, there is a dearth of structural information on the degree of polymerization (DP) and, importantly, how the chain length may change during embryonic development. The overall structure of polysialylated glycan chains is also poorly understood, although the structure of core glycans in the embryonic chicken brain N-CAM was extensively examined and shown to have several unusual features (20Kudo M. Kitajima K. Inoue S. Shiokawa K. Morris H.R. Dell A. Inoue Y. J. Biol. Chem. 1996; 271: 32667-32677Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The presence of α2,8-linked oligo/polySia in glycopeptides isolated from developing rat brain was initially established by the susceptibility to Vibrio cholerae sialidase and methylation analysis, coupled with gas chromatography-mass spectrometry (21Finne J. J. Biol. Chem. 1982; 257: 11966-11970Abstract Full Text PDF PubMed Google Scholar). In this pioneering work, it was shown that 8–12 Sia residues were linked to bi-, tri-, and tetra-antennary N-glycan chains. In more recent studies, evidence for the presence of polySia in neuronal tissues was based primarily on the susceptibility to a bacteriophage-induced poly(→8Neu5Acylα2→) endo-N-acylneuraminidase (Endo-N) (22Vimr E.R. McCoy R.D. Vollger H.F. Wilkinson N., C. Troy F.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1971-1975Crossref PubMed Scopus (146) Google Scholar) and reactivity to equine polyclonal antibody H.46 (23Finne J. Leinonen M. Makela P.H. Lancet. 1983; ii: 355-357Abstract Scopus (643) Google Scholar) or mouse monoclonal antibody 735 (24Finne J. Bitter-Suermann D. Goridis C. Finne U. J. Immunol. 1987; 138: 4402-4407PubMed Google Scholar). The functional and biosynthetic studies of polysialylation on N-CAM were stimulated and promoted by these sensitive and selective biological probes during past 15 years. However, although these specific reagents can be used for the diagnostic identification of polysialylated N-CAM, they are more effective for smaller oligoSia groups, i.e. 5 for Endo-N and 8–10 for H.46 and mouse monoclonal antibody. Consequently certain ambiguity is inevitable in the results obtained with these reagents when polySia chains are analyzed. The chemical and physicochemical determination of the DP of polySia chains contains many inherent problems which must be overcome. There are few reports on the determination of the DP of polySia on N-CAM by HPLC-based methods, and the published values vary widely, depending on the technique used. The initial evidence for the presence of extended polySia chains was based on the HPLC on a MonoQ column for the glycopeptides isolated from [3H]GlcNAc-labeled human neuroblastoma cells after brief treatment with Endo-N (5Livingston B.D. Jacobs J.L. Glick M.C. Troy F.A. J. Biol. Chem. 1988; 263: 9443-9448Abstract Full Text PDF PubMed Google Scholar). Although the chromatograms seem to indicate the presence of extended polySia chains up to DP of 55, the resolution for DP > 45 was poor, and furthermore, the peaks at high DP region were not explicitly identified as polyNeu5Ac chains. In contrast, average DP obtained for a sample of N-CAM from embryonic chick brain, based on the separation and quantitation of non-reducing terminal and internal sialic acid residues, was 18 (25Ashwell G. Berlin W.K. Gabriel O. Anal. Biochem. 1994; 222: 495-502Crossref PubMed Scopus (14) Google Scholar). However, this value may be an underestimate, as the molecule contains monoSia residues in addition to polySia chains. Since the chain length-dependent physicochemical properties of polySia may determine its physiological role, a more accurate estimation of the DP of polySia chain expressed on embryonic N-CAM and the change in DP, if any, during development are essential for understanding the regulatory effects of polySia residues on N-CAM-associated physiological events. Information on the range of DP of polySia chains is also useful in understanding the biosynthetic reactions of polysialylation, and in clarifying how many sialyltransferases are involved in the formation of polySia N-CAM. To gain understanding to these problems, we addressed the following issues. First, a new analytical method was used to determining the DP of polySia chains of DP > 50 more accurately. Second, new methods were developed for isolation of polySia-N-CAM that eliminate or minimize unwanted cleavage of the inter-residue linkages of extended polySia chains, which are known to be more labile than shorter oligo/polySia (26Manzi A.E. Higa H.H. Diaz S. Varki A. J. Biol. Chem. 1994; 269: 23617-23624Abstract Full Text PDF PubMed Google Scholar). Third, two highly sensitive analytical methods were used for selective detection of monoSia, diSia, oligoSia, and polySia residues. The advantages of these recently developed HPLC-based analytical methods are twofold. First, high performance anion-exchange chromatography with pulsed electrochemical detector (HPAEC-PED) (27Zhang Y. Inoue Y. Inoue S. Lee Y.C. Anal. Biochem. 1997; 250: 245-251Crossref PubMed Scopus (39) Google Scholar,28Lin S.-L. Inoue Y. Inoue S. Glycobiology. 1999; 9: 807-814Crossref PubMed Scopus (29) Google Scholar) method accomplishes a highly efficient separation of underivatized oligo/polySia chains with DP ranging from 2 to as high as 80. Second, high performance liquid chromatography on a MonoQ column with fluorometric detection (HPLC-FD) method (28Lin S.-L. Inoue Y. Inoue S. Glycobiology. 1999; 9: 807-814Crossref PubMed Scopus (29) Google Scholar, 29Sato C. Inoue S. Matsuda T. Kitajima K. Anal. Biochem. 1999; 266: 102-109Crossref PubMed Scopus (63) Google Scholar) is a highly sensitive and selective method to measure fluorescence-tagged oligo/polySia residues (DP up to about 30). In the present study, these methods were used in tandem with an improved method for isolating and purifying polySia glycopeptides from chicken brain, so that stage-dependent changes in the DP and level of polySia expressed in embryonic and adult chicken brains can be determined. We thus can conclude that the DP narrowly ranges between 40 and 50 Sia residues (average DP = ∼45) in embryonic chicken brain. Surprisingly, both the DP range and the average DP values showed little variation during developmental stages, E5 to E21. On the other hand, the total amount of polySia expressed per brain exhibited large differences, with maximum expression around E14, as reported previously (18Sevigny M.B. Ye J. Kitazume-Kawaguchi S. Troy F.A. Glycobiology. 1998; 8: 857-867Crossref PubMed Scopus (26) Google Scholar). One of the most unexpected findings was that no glycopeptides bearing short (5 to ∼30) polySia chains were isolated from embryonic chicken brains, although diSia residues were present in a fraction separated from polySia glycopeptides. Thus, polysialylation profile in the embryonic brain was in sharp contrast to that of the adult chicken brain, which showed polySia glycopeptides with polydispersity ranging from DP ∼15 to 35. In addition, we also isolated glycopeptide fractions expressing the α2,8-linked diSia glycotope and proportionally lower levels of triSia and tetraSia residues in adult brain. Fertilized eggs were purchased from Taiwan Animal Health Research Institute and incubated at 38 °C under humidified conditions. Lyophilized homogenates of the brain prepared from chicken embryo at early developmental stages were prepared at University of California, Davis (18Sevigny M.B. Ye J. Kitazume-Kawaguchi S. Troy F.A. Glycobiology. 1998; 8: 857-867Crossref PubMed Scopus (26) Google Scholar). Adult brains were purchased at the local market at Taipei soon after chicken (3 months old) were sacrificed. Brain tissues were stored at −30 °C or −80 °C for less than 1 month before further processing. Brains were homogenized at 4 °C by either of the following methods: (i) with a 2-ml Kontes glass homogenizer in 50 mm MES buffer (pH 6.1) containing 500 kallikrein-inactivating unit/ml aprotinin, 40 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride: (ii) with a Polytron homogenizer (Kinematica, Littau, Switzerland) in 10 mm Tris-HCl buffer (pH 8.0). No detectable difference was found between these two methods. Homogenates (fresh or after lyophilization) were delipidated with chloroform-methanol as described previously (20Kudo M. Kitajima K. Inoue S. Shiokawa K. Morris H.R. Dell A. Inoue Y. J. Biol. Chem. 1996; 271: 32667-32677Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The delipidated material was air-dried and exhaustively digested with bacterial protease, Streptomyces griseus proteinase (type XIV, Sigma) as described previously (20Kudo M. Kitajima K. Inoue S. Shiokawa K. Morris H.R. Dell A. Inoue Y. J. Biol. Chem. 1996; 271: 32667-32677Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). After digestion, an equal volume of cold acetone was added and the mixture was kept at −20 °C overnight, and precipitate (50% acetone precipitate) that contained high molecular weight compounds) was separated by centrifugation. Small glycopeptides remaining in the supernatant were precipitated by adding one more volume of acetone (75% acetone precipitate). Both the 50% and 75% acetone precipitates were subjected to size fractionation on Sephacryl S-200 columns (1.6 × 134 cm) equilibrated and eluted with 10 mm Tris-HCl (pH 8.0) containing 0.1 m NaCl. The elution was monitored by A230 nm and by determination of Neu5Ac using the fluorometric HPLC method, after hydrolysis in 0.1 n HCl for 2 h at 80 °C. Sialoglycopeptides in the 50% acetone precipitate were separated into fractions H (tube numbers 38–48, Mr100,000–20,000), and l (tube numbers 52–70,Mr 12,000–3,000). Sialoglycopetides in the 75% acetone precipitate were eluted at a position similar to thel fraction. All fractions were dialyzed against MilliQ water in the cold and lyophilized. Purification and further fractionation of the sialoglycopeptides were carried out on a MonoQ HR 10/10 column (Amersham Pharmacia Biotech, Uppsala, Sweden), equilibrated with 10 mm Tris-HCl (pH 8.0) and eluted with a 0–0.7 m NaCl gradient in 10 mm Tris-HCl (pH 8.0) at 2 ml/min. Neu5Ac-containing fractions (monitored by the 1,2-diamino-4,5-methylenedioxybenzene (DMB) method after hydrolysis) were pooled and desalted on a Sephadex G-10 column. Free Neu5Ac was quantitated by the fluorometric HPLC method after derivatization for 2.5 h at 55 °C with DMB (Dojindo Laboratories, Kumamoto, Japan) as described previously (30Hara S. Takemori Y. Yamaguchi M. Nakamura M. Ohkura Y. Anal. Biochem. 1987; 164: 138-145Crossref PubMed Scopus (294) Google Scholar, 31Inoue S. Kitajima K. Inoue Y. J. Biol. Chem. 1996; 271: 24341-24344Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The reaction mixture contained 2.7 mDMB, 9 mm sodium hydrosulfite, 0.5 mβ-mercaptoethanol, and 0.02 m trifluoroacetic acid. Samples were hydrolyzed in 0.1 m trifluoroacetic acid for the time required to obtain the maximum yield of free Neu5Ac, usually 4 h for polySia, and 1 h for diSia, and evaporated under vacuum to remove the acid. To monitor the elution profile of Neu5Ac-containing material after column chromatography, samples were hydrolyzed in 0.1 n HCl for 2 h at 80 °C. For oligo/polySia analysis by the HPLC-FD method, samples (100–500 ng of total Neu5Ac) were derivatized with the DMB reagent for 2 h at 50 °C without pre-hydrolysis, and the reaction mixture was neutralized with 1 m NaOH before injection. A Hewlett-Packard HPLC system series 1100 with a fluorescence detector 1046 (set at 373 nm for excitation and 448 nm for emission) was used with a MonoQ HR 5/5 column. Samples were eluted with 10 mmTris-HCl (pH 8.0) containing a 0–0.7 m NaCl gradient, at 0.5 ml/min. Analysis of polySia by HPAEC with pulsed electrochemical detector (PED) was as described previously (28Lin S.-L. Inoue Y. Inoue S. Glycobiology. 1999; 9: 807-814Crossref PubMed Scopus (29) Google Scholar). To improve the yield of higher polymers, lyophilized samples (12 μg of Neu5Ac) were first treated with 10 μl of 1 m HCl at ambient temperature for 2 h to facilitate lactonization of polySia (32Zang Y. Lee Y.C. J. Biol. Chem. 1999; 274: 6183-6189Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), dried on a centrifugal vacuum evaporator, and then subjected to controlled hydrolysis in 100 μl of 0.1 m acetic acid for 15 min at 60 °C. To the hydrolysate, 50 μl of 0.5 m NaOH was added and a 100-μl portion (8 μg of Neu5Ac) was injected into a CarboPac PA-100 column. A DX ion chromatography system (Dionex, Sunnyvale CA) with an ED-40 electrochemical detector was operated under conditions as described previously (28Lin S.-L. Inoue Y. Inoue S. Glycobiology. 1999; 9: 807-814Crossref PubMed Scopus (29) Google Scholar). Carbohydrate composition was determined by gas-liquid chromatography analysis after methanolysis and trimethylsilylation (33Chaplin M.F. Chaplin M.F. Kennedy J.F. Carbohydrate Analysis: A Practical Approach. IRL Press, Oxford1994: 27-34Google Scholar) using a Shimadzu gas chromatograph GC-17A. Amino acid analysis was carried out after hydrolysis in 6 n HCl at 105 °C for 20 h under N2 and precolumn derivatization with phenylisothiocyanate (34Heinrikson R.L. Meredith S.C. Anal. Biochem. 1984; 136: 65-74Crossref PubMed Scopus (1347) Google Scholar) A Pico-Tag amino acid analysis system (Waters) was used. α2,8-Linked dimer and tetramer of Neu5Ac were obtained from Nihon Gaishi (Handa, Japan). Higher oligomers and polymers of Neu5Ac were prepared from colominic acid purchased from Nacalai Tesque (Kyoto, Japan) by controlled hydrolysis and ion-exchange chromatographic separation on a DEAE-Sephadex A-25 (35Nomoto H. Iwasaki M. Endo T. Inoue S. Inoue Y. Matsumura G. Arch. Biochem. Biophys. 1982; 218: 335-341Crossref PubMed Scopus (82) Google Scholar) or a MonoQ column. A freshly dissolved sample of colominic acid in water was subjected to fractionation on a MonoQ HR 10/10 column using an NaCl gradient (0–0.7 m) in 0.01m Tris-HCl (pH 8.0). A series of resolved peaks that were identified as DP 2 to DP 32 were eluted under conditions used: 0.3 m NaCl (20 min) to 0.5 mNaCl (80 min), at a flow rate of 2 ml/min. A major broad peak that eluted after the DP 32 peak was used as a high molecular weight colominic acid sample throughout this study. A published method (20Kudo M. Kitajima K. Inoue S. Shiokawa K. Morris H.R. Dell A. Inoue Y. J. Biol. Chem. 1996; 271: 32667-32677Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) was modified to minimize hydrolytic cleavage of polySia chains that often occurs during prolonged treatments such as solubilization of polySia-containing glycopeptides from the precipitate with quaternary pyridinium ion (21Finne J. J. Biol. Chem. 1982; 257: 11966-11970Abstract Full Text PDF PubMed Google Scholar), and concentration of the compounds from dilute solutions. As it is imperative to keep polySia chains intact and to accurately quantify the polySia, we exhaustively digested membrane-associated polySia-containing glycopeptides from delipidated homogenates of intact whole chicken brains with nonspecific bacterial protease to bring about solubilization. During proteolysis (0.1 m Tris-HCl, pH 8.0), chemical and enzymatic cleavage of sialyl linkages was negligible, as no significant amounts of free Sia or free oligo/polySia chains were detected in any step of purification. Addition of equal volume of acetone to the material solubilized by proteolysis effectively precipitated all polySia-containing glycopeptides, which were readily re-solubilized in the small volume of buffer used in the next step. The polySia glycopeptides in the 50% acetone-precipitated fraction at each developmental stage was fractionated by Sephacryl S-200 chromatography. The chromatographic profile (monitored for total Neu5Ac content) revealed two peaks H (high Mr) and L (low Mr) (Fig.1). The column was calibrated using sialoglycoproteins of known Mr isolated from fish eggs (36Inoue S. Inoue Y. J. Biol. Chem. 1986; 261: 5256-5261Abstract Full Text PDF PubMed Google Scholar, and S. Inoue, unpublished results). The peak H (Mr range 100,000–20,000, peak 45,000) increased during the early stages of development and reached a maximum value around day E14 after fertilization (designated E14) and then gradually decreased. This finding confirms the previous findings based on a polySia antibody (18Sevigny M.B. Ye J. Kitazume-Kawaguchi S. Troy F.A. Glycobiology. 1998; 8: 857-867Crossref PubMed Scopus (26) Google Scholar). The H fraction from adult chicken brain eluted in a lower molecular weight range than that of embryonic H fractions (range 72,000–15,000, peak 27,000). Oligo/polySia analysis by the HPLC-FD method of H and L fractions showed that polySia was present only in the H fraction. In L fractions and the fraction soluble in 50% acetone, diSia residues were present at all developmental stages, although the major portion of Neu5Ac occurred as monoSia residue. It is noted that the colominic acid sample was eluted in a more polydisperse peak than the H fractions, with Mr values ranging from 72,000 down to 5,000, with a peak at 15,000. The HPLC-FD method is a sensitive and selective method (28Lin S.-L. Inoue Y. Inoue S. Glycobiology. 1999; 9: 807-814Crossref PubMed Scopus (29) Google Scholar, 29Sato C. Inoue S. Matsuda T. Kitajima K. Anal. Biochem. 1999; 266: 102-109Crossref PubMed Scopus (63) Google Scholar), and was used in this study. Under the conditions required for derivatization with the DMB reagent, some polySia underwent partial hydrolysis to result in the characteristic elution ladders of (Sia) n (n = 1 − n) useful in detection and identification of oligo/polySia. After such treatment, an authentic higher oligomers of Neu5Ac showed a parent peak, which is always of higher yield than the rest of the oligomer peaks produced during derivatization (Fig. 2, a–c). Thus, the peak of the highest DP can be regarded as the maximum size of the polySia chain under study. In contrast, for polydisperse materials like colominic acid, no distinct highest peak was yielded, but a range of peaks up to DP ∼ 25 was shown (Fig. 2 d). Several other conditions of acid and temperature for derivatization examined did not improve the yield of highest DP peak. When colominic acid was treated under similar conditions (0.02m trifluoroacetic acid for 2 h at 50 °C), oligo/polymers of Neu5Ac up to DP 40 were detected by HPAEC-PED. In the chromatographic separation on a MonoQ column (similar to HPLC-FD method) for underivatized polySia, resolution of peaks in the region of DP 30–40 could be improved by changing salt gradient. However, for DMB-polySia, no such improvement has so far been achieved. Thus, the HPLC-FD method cannot be used for the determination of DP values >30. The DP of the Sia chains in the H and L fractions from embryonic and adult chicken brains was determined by HPLC-FD method. First, sialoglycopeptides (containing 300–700 ng of total Neu5Ac) isolated from H fraction after Sephacryl S-200 chromatography (Fig. 1) were treated with the DMB reagent and analyzed on a MonoQ HR 5/5 column. Fig. 3 shows a representative profile for samples obtained at three stages of development: E5, E14, and adult. Profiles nearly identical to that shown for E14 (panel b) were observed for E8, E12, E16, E18, and E21. These profiles, when compared with that of colominic acid (Fig. 2 d), indicate that polySia chains in the samples from these developmental stages are large in DP. The results indicate that polySia chains were also present in as early a developmental stage as E5 (panel a), and in the adult (panel c). It is noted, however, that the proportion of monoSia with respect to the higher DPs was large in the E5 and adult samples. In contrast to H fraction, no polySia chains were detected in L fraction when examined by the same HPLC-FD technique. Rather, this fraction contained a small amount of diSia, which as described below, was present in larger amount in the soluble fraction of the 50% acetone fractionation. To obtain information on the DP range of the polySia chains, the H and L fractions from each developmental stage were subjected to HPLC on a MonoQ HR 10/10 column, pre-equilibrated with 0.01 mTris-HCl (pH 8.0), and eluted with a NaCl gradient. Essentially all sialoglycopeptides in the H fractions obtained from E5 to E21 were eluted in a peak at 0.45–0.55 m NaCl (Fig.4). This elution position was slightly delayed in comparison to that of colominic acid, which eluted at 0.4–0.5 m NaCl (Fig. 4). It is noted that in samples obtained from late (>E18) stages, a small proportion of sialyl compounds eluted earlier than the major peak (e.g. Q2 for E21). In contrast, sialoglycopeptides isolated from adult brain showed a different elution profile (Fig. 4). In addition to peak Q1, which eluted slightly before the embryonic Q1 material, a larger portion of the adult derived sialoglycopeptides eluted over a broad region under partially separated multiple peaks (Q2–Q4). The DP analysis of these peaks by HPLC-FD showed that Q1 and Q2 contained polySia as expected from the elution position (Fig. 3 c). The Q3 and Q4 fractions, eluted at lower NaCl concentration, also contained polySia but with DPs of 15–20 (Fig. 5), significantly shorter than the chains derived from embryonic polySia. When the L fractions were fractionated on a MonoQ HR 10/10 column, the major proportion of Neu5Ac-containing molecules eluted with NaCl at less than 0.2 m, and much smaller proportions (depending on the developmental stage) eluted at NaCl concentration between 0.3 and 0.55 m. Interestingly, those fractions eluted at the lower NaCl concentrations contained only monoSia residues, as expected, whereas the fractions that eluted at the higher NaCl concentrations contained significant levels of diSia (Fig.6). In some fra" @default.
• W2044092574 created "2016-06-24" @default.
• W2044092574 creator A5025720845 @default.
• W2044092574 creator A5030736937 @default.
• W2044092574 creator A5079718541 @default.
• W2044092574 date "2000-09-01" @default.
• W2044092574 modified "2023-09-30" @default.
• W2044092574 title "Chemical Analysis of the Developmental Pattern of Polysialylation in Chicken Brain" @default.
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