Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue.

We have recently discovered unusual sugar chains (xylose (Xyl)-glucose (Glc) and (Xyl)2-Glc) linked to a serine residue in the epidermal growth factor (EGF)-like domains of human and bovine clotting factors VII (Ser-52), IX (Ser-53), and protein Z (Ser-53), in addition to bovine platelet glycoprotein thrombospondin. We now have evidence of another modification in the first EGF-like domain of human factor IX, which proved to be a tetrasaccharide O-fucosidically linked to Ser-61. Two large peptides containing Ser-61 (positions 44-63), named hIX-GP1 and hIX-GP2, were first isolated from the lysyl endopeptidase-digest of human factor IX, by reversed-phase high performance liquid chromatography (HPLC). Data on the component sugar analysis after pyridylamination (PA) and sialic acid analysis of the isolated peptides indicated that they contained 1 mol each of galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (NeuAc), in addition to Glc and Xyl. hIX-GP1 was further digested with asparaginyl endopeptidase, and two glycopeptides containing Ser-61, named N-3 (positions 59-63) and N-9 (positions 55-63), were isolated, respectively. These glycopeptides were both composed of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc but did not contain Xyl and Glc. Moreover, the data on beta-elimination for N-9 and of the fast atom bombardment mass spectrometric analysis on peptide N-3 suggested the presence of a tetrasaccharide linked to Ser-61. An analysis of the PA-oligosaccharide released from hIX-GP1 by hydrazinolysis followed by pyridylamination revealed that the reducing end was PA-Fuc. All the results support the proposal that human factor IX has a novel tetrasaccharide consisting of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc, which is O-glycosidically linked to Ser-61 through the Fuc residue.     

Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z

We have recently described a unique trisaccharide linked to a serine residue in the first epidermal growth factor-like domains of bovine blood coagulation factors VII (Ser-52) and IX (Ser-53) (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The sugar chain identified in these clotting factors consists of 1 mol of hexose (glucose (Glc] and 2 mol of pentose (xylose (Xyl]. We report here that human factors VII and IX and protein Z and bovine protein Z also contain such carbohydrate moieties linked to a serine residue at the same position found in bovine factors VII and IX. A glycopeptide derived from each of these proteins was subjected to amino acid sequence and component sugar analyses and fast atom bombardment mass spectrometric analysis. The results indicate that the glycopeptide derived from human factor IX contains 1 mol each of Glc and Xyl. The reducing end of this disaccharide was identified as Glc by analyzing the disaccharide generated by hydrazinolysis. In contrast, human factor VII and protein Z yielded two different glycopeptides which contained Glc and Xyl at molar ratios of 1:1 and 1:2, respectively, suggesting microheterogeneity of these O-linked sugar chains. Bovine protein Z glycopeptide contained 1 mol of Glc and 2 mol of Xyl. These sugar compositions were confirmed by analyses of the intact proteins. In relation to the trisaccharide sugar chain previously discovered in bovine factors VII and IX, these findings indicate the existence of a Xyl2-Glc-Ser and a Xyl-Glc-Ser structure in the first epidermal growth factor-like domains of human factors VII and IX and protein Z in addition to that of bovine protein Z. Whether these carbohydrate moieties contribute to the biological activities of these proteins is unknown.     

Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells

The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1 Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3 Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that alpha(1-->3) fucosylation of N-glycans does occur but beta(1-->4) galactosylation of the sugar chains does not in the tobacco cultured cells.     

A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX

A new trisaccharide sugar chain was identified in bovine blood coagulation factors VII and IX. A pentapeptide isolated from factor VII contained Ser-52, which could not be identified with a gas-phase sequencer, suggesting an unknown substituent on the serine residue (Takeya, H. et al. (1988) J. Biol. Chem., in press). The same results were obtained for a pentapeptide containing Ser-53 of factor IX. Component sugar analysis revealed that the peptide contained 1 mol of glucose and 2 mol of xylose. This sugar component was also confirmed by high-resolution fast atom bombardment mass spectrometric analysis of the pentapeptide. The trisaccharide was released from the peptides by means of beta-elimination reaction and its reducing end was coupled with 2-aminopyridine. The fluorescent pyridylamino (PA-) derivative of the trisaccharide was purified by gel-filtration and reversed-phase HPLC. The sugar composition of the PA-trisaccharide was found to be 2 mol of xylose and 1 mol of PA-glucose. These results indicate the existence of a (Xyl2)Glc-Ser structure in factors VII and IX.     

The epidermal growth factor-like domains of factor IX. Effect on blood clotting and endothelial cell binding of a fragment containing the epidermal growth factor-like domains linked to the gamma-carboxyglutamic acid region

The binding of factor IX to cultured bovine endothelial cells was characterized using isolated domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like domain(s) also contributes to the interaction of the enzyme with its cofactor.     

N-linked oligosaccharides of glycoproteins from Ginkgo biloba pollen, an allergenic pollen.

The pollen of Ginkgo biloba is one of the allergens that cause pollen allergy symptoms. The plant complex type N-glycans bearing beta1-2 xylose and/or alpha1-3 fucose residue(s) linked to glycoallergens have been considered to be critical epitopes in various immune reactions. In this report, the structures of N-glycans of total glycoproteins prepared from Ginkgo biloba pollens were analyzed to confirm whether such plant complex type N-glycans occur in the pollen glycoproteins. The glycoproteins were extracted by SDS-Tris buffer. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine and the resulting pyridylaminated (PA-)N-glycans were purified by a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, IS-MS, and MS/MS. The plant complex type structures (GlcNAc2Man3Xyl1Fuc1GlcNAc2 (31%), GlcNAc2Man3Xyl1GlcNAc2 (5%), Man3Xyl1Fuc1GlcNAc2 (13%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (8%), and GlcNAc1Man3Xyl1GlcNAc2 (17%)) have been found among the N-glycans of the glycoproteins of Ginkgo biloba pollen, which might be candidates for the epitopes involved in Ginkgo pollen allergy. The remaining 26% of the total pollen N-glycans have the typical high-mannose type structures: Man8GlcNAc2 (11%) and Man6GlcNAc2 (15%).     

Conversion of brain-specific complex type sugar chains by N-acetyl-beta-D-hexosaminidase B

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.     

Structural analysis of the sugar chains of human urinary thrombomodulin

The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/-Siaalpha2-3Galbeta1-3(+/-Siaalpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.     

Structures of the sugar chains of interferon-gamma produced by human myelomonocyte cell line HBL-38

Interferon-gamma produced by the human myelomonocyte cell line HBL-38 contained galactose, mannose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid as sugar components. Sugar chains were liberated from interferon-gamma by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine under new reaction conditions in which no sialic acid residue was hydrolyzed. The pyridylamino (PA-) derivatives of the sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Seven major PA-sugar chains were isolated and the structure of each purified PA-sugar chain was identified by stepwise exoglycosidase digestion and 500-mHz 1H-NMR spectroscopy. The results indicated that the structures of the major PA-sugar chains were of the biantennary type, to which 0 to 2 mol of fucose and 1 to 2 mol of N-acetylneuraminic acid were linked as shown below. (formula; see text)     

Novel proteoglycan linkage tetrasaccharides of human urinary soluble thrombomodulin, SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl

O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were liberated by hydrazinolysis followed by N-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1+ ++-4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.     

Structures of sugar chains of a p-nitrophenyl acetate-hydrolyzing esterase from the microsomes of rat liver

The structures of sugar chains of a p-nitrophenyl acetate-hydrolyzing esterase from the microsomes of rat liver were established. The enzyme contained mannose and glucosamine as sugar components. Asparagine-linked sugar chains of the esterase were liberated by hydrazinolysis. After N-acetylation of the hydrazinolysate, the reducing ends of the sugar chains were coupled with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Eleven PA-sugar chains were obtained. The structures of the PA-sugar chains were first identified by HPLC using two series of separation systems by which 11 PA-oligomannose-type sugar chains with known structures could be separated. Further elucidation of the structures of each PA-sugar chain was performed by exoglycosidase digestions and partial acetolysis. The structures of two of the PA-sugar chains were further confirmed by 500 mHz 1H-NMR spectroscopy.     

Structural Analysis of N-Glycans of Storage Glycoproteins in Soybean (Glycine max. L) Seed

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz ¹H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man₃Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 3.4%).     

Studies on the sugar chains of interferon-gamma from human peripheral-blood lymphocytes

Sugar chains of interferon-gamma (IFN-gamma) from human peripheral-blood lymphocytes (PBL) were liberated by hydrazinolysis. After N-acetylation, the reducing end residues of the sugar chains were tagged with 2-aminopyridine and the pyridylamino (PA-) derivatives were purified by gel filtration and reversed-phase HPLC. Five major PA-sugar chains were obtained. The structure of each PA-sugar chain was estimated by comparing its elution times on anion exchange, reversed-phase, and size-fractionation HPLC with those of PA-sugar chains of IFN-gamma from the human myelomonocyte cell line HBL-38 (Yamamoto, S. et al. (1989) J. Biochem. 105, 547-555) as standards, and also by comparison of their elution times after partial desialylation. The results showed that IFN-gamma (PBL) contained mono- and disialo-biantennary structures with 0 or 1 mol of fucose residue, as found for IFN-gamma (HBL-38), but the N-acetylneuraminyl alpha 2-6 linkage was dominant in IFN-gamma (PBL), unlike IFN-gamma (HBL-38), which contains both N-acetylneuraminyl alpha 2-3 and alpha 2-6 linkages.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).     

Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains.

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.     

Structures of sugar chains of ricin D

The toxic lectin, ricin D, contains mannose, fucose, xylose, and N-acetylglucosamine as sugar components. Sugar chains are linked to Asn-10 of the A-chain, and to Asn-95 and Asn-135 of the B-chain (Funatsu, G. et al. (1978) Agric. Biol. Chem. 42, 501-503; Araki, T. & Funatsu, G. (1985) FEBS Lett. 191, 121-124). Asparagine-linked sugar chains of each glycopeptide from ricin D were liberated by hydrazinolysis followed by N-acetylation. The reducing end residues of the sugar chains were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives obtained were purified by gel-filtration and reversed-phase HPLC. Eight main PA-sugar chains were obtained from three glycopeptides and the structures of these sugar chains were determined by component analysis, stepwise exoglycosidase digestions, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. The results show that oligomannose type sugar chains (Man6-7GlcNAc2) are linked to Asn-95; Man5-7 GlcNAc2 and M4X (structure, see below) to Asn-135 of the B-chain, and M3FX and M3X to Asn-10 of the A-chain. (Formula: see text).     

Structural analysis of N-linked sugar chains of human blood clotting factor IX

The structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.     

Application of 2-aminopyridine fluorescence labeling to glycosaminoglycans

A pyridylamination method was applied to glycosaminoglycans and the characteristics of the resulting pyridylamino glycosaminoglycans were examined. First, glycosaminoglycan chains, which uniformly possess a xylose residue at their reducing termini, were liberated from proteoglycan by successive digestion with protease and endo-beta-xylosidase. Then the glycosaminoglycan chains were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride for 15 h according to the method of Hase, S. et al. [J. Biochem. 95, 197-203 (1984)]. The pyridylamination reaction caused neither depolymerization, de-N-acetylation, nor de-N- or de-O-sulfation. The pyridylamino glycosaminoglycan chains had an intact linkage region (GlcA-Gal-Gal-Xyl) between the carbohydrate chain and the peptide core of the proteoglycan. These pyridylamino glycosaminoglycans should be useful as substrates for endo-type glycosidases that act on glycosaminoglycan chains and as markers for studies of glycosaminoglycan metabolism.     

Release of O-linked sugar chains from glycoproteins with anhydrous hydrazine and pyridylamination of the sugar chains with improved reaction conditions.

A method for preparation of pyridylamino (PA-) derivatives of O-linked sugar chains from glycoproteins was developed. A glycopeptide containing O-linked Gal beta 1-3GalNAc was prepared from fetuin and treated with anhydrous hydrazine followed by N-acetylation of free amino groups. Sugar chains released were pyridylaminated with improved reaction conditions and excess reagents were removed by gel filtration. Gal beta 1-3GalNAc-PA obtained together with PA-Gal as a by-product was quantified by HPLC. Conditions for the hydrazine treatment were investigated and the treatment at 40 degrees C for 350 h gave the best results for releasing O-linked sugar chains. The total yield of Gal beta 1-3GalNAc-PA from the glycopeptide was 53% under the established conditions and that of PA-Gal was 18%. The present method was applied to a glycoprotein, and the expected PA-O-linked sugar chains were obtained. Under these conditions, N-linked sugar chains were also released.     

Structural Elucidation of N-Linked Sugar Chains of Storage Glycoproteins in Mature Pea (Pisum sativum) Seeds by Ion-spray Tandem Mass Spectrometry (IS-MS/MS)

The structures of N-linked sugar chains of the storage glycoproteins in mature pea seeds have been estimated. Nine pyridylaminated (PA-) N-linked sugar chains were derived and purified from the hydrazinolysate of the storage glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping, considering the data of sugar composition analysis or sequential exoglycosidase digestions. The deduced structures were further analyzed by IS-MS/MS analysis. Every relevant fragment ion derived from all PA-sugar chains could be assigned on the basis of deduced structures. The estimated nine structures fell into two categories; the first was a typical oligomannose type (Man_8-3GlcNAc₂; 77.7%) which can be hydrolyzed by endo-β-N-acetylglucosaminidase PS [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 228–232 (1996)], the second was a xylose-containing type (Man_4-3Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 22.3%). Among these structures, Man₈GlcNAc₂ (19.7%), Man₆GlcNAc₂ (24.7%), and Man₃Fuc₁Xyl₁GlcNAc₂ (18.8%) were the dominant structures.