Analysis of N‐acetyl‐4‐O‐acetylneuraminic‐acid‐containing. N‐linked carbohydrate chains released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F

Damm, Jan B. L.; Voshol, Hans; Hård, Karl; Kamerling, Johannis P.; Vliegenthart, Johannes F.G.

doi:10.1111/j.1432-1033.1989.tb14620.x

Cited by 33 publications

(25 citation statements)

References 23 publications

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“…The 'H-NMR analysis of the mixture of glycopeptides isolated from the pronase digest of Tf2a confirms the presence of NeuSAc and Neu(4,5)Ac2 [23]. Similar 'H-NMR results were obtained for the mixture of glycopeptides isolated from the pronase digest of Tf 4b indicating that in both glyeopeptides Neu5Ac and Neu(4,5)Ac2 are found in the ratio 2: 1.…”

Section: Determination Ofglycan Primary Structuresupporting

confidence: 70%

“…Occurrence of this type of sialic acid in horse serum glycoproteins is not new since, in 1968, Pepper [28] has found in horse serum the four following types of sialic acids : NeuSAc, NeuSGc, Ncu(4,5)Ac, and N-glycolyl-4-0-acetylneuraminic acid in the relative ratio 35: 5 : 50: 10, respectively. The presence of Neu(4.5)Ac2 has been determined in horse a,-acid glycoprotein [29], in a,-macroglobulin [30] and in fibrinogen [23]. Concerning the presence of Neu(4,5)Ac2 in horse serotransferrin variants, we have found that it is limited to two of the three analysed variants of Tf 0.…”

Section: Djscussionmentioning

confidence: 76%

“…The NAc resonances from GlcNAc at positions 5 and 5' involved in the sialylated antennae were assigned at 2.110 ppm and 2.048 ppm, respectively, according to Damm et al [23]. The NAc signal of GlcNAc at position 5 also resonates at a lower field than that …”

Section: Determination Ofglycan Primary Structurementioning

confidence: 99%

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Primary structure of horse serotransferrin glycans

Coddeville

Stratil

Wieruszeski

et al. 1989

European Journal of Biochemistry

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Three serotransferrin variants Tf 2al Tf4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants T1'4b and Tf 5b contain two glycans. The structure of all of the glycans has been cstablished by combining methylation analysis, mass spectrometry and 400-MHz 'H-NMR spectroscopy. From the obtained rcsults, it appears that the two glycans of Tf 5b variant are, like in human serotransferrin, of the Nacetyllactosaminic biantennary type, fully sialylated by two residues of N-acetylneuraminic acid (NeuSAc ; glycan In contrast, in addition to this structure, two N-acetyllactosaminic biaritennary isomeric structures named type 11-A and type 11-B sialylated by one Neu5hc residue and one N-acetyl-4-0-acetylneuraminic acid [Neu(4,5)Ac2] residue located either at Gal6 or 6' and one N-acetyllactosaminic biantennary structure (named type 111) sialylated by two residues of Neu(4,5)Ac2, were identified in variants Tf 2a and Tf 4b.These results demonstrate that in an homozygous preparation of horse scrotransferrin Tf 0, the heterogeneity is dependent, on the one hand, on the nature of the neuraminic acid substituting a N-acetyllactosaminic biantennary structure and, on the other hand, on the number of glycans bound to the polypeptide chain.Moreover, the differences which exist in the molecular mass of 77.5 kDa, 80 kDa and 82 kDa for serotransferrin variants Tf 2a, Tf 4b and Tf Sb, respectively, are not completely explained by the structure and the number of the glycans suggesting that the three variants should also differ in their polypeptide chain. . In order to define the molecular basis of this heterogeneity, homozygous serotransferrin Tf 0 was submitted to chromatographyAn a DEAE-Sephadex A-SO column and eight fractions were collected [5]. Threc of them (fractions 11, IV and VI, corresponding to serotransferrin variants named 'rf 2a, Tf 4b and Tf 5b, respectively) were found to be homogeneous by starch gel electrophoresis. Previous results obtained by Strati1 ct al. [S] have shown that the two main serotransferrin variants Tf 2a and Tf 4b possess a similar amino acid composition but differ in their molecular mass; 75.2 kDa and 80.5 kDa, respectively. and in their carbohydrate compoCorrespondence to G.

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Section: Determination Ofglycan Primary Structuresupporting

confidence: 70%

Section: Djscussionmentioning

confidence: 76%

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Primary structure of horse serotransferrin glycans

Coddeville

Stratil

Wieruszeski

et al. 1989

European Journal of Biochemistry

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“…The N-linked carbohydrate chains were released enzymically from the glycoprotein with PNGase-F [28]. Briefly, a solution of 46 mg K,tu-PA-I in 17 ml 25 mM sodium acetate pH 5.0, diluted to 23.3 ml, containing 30 mM Hepes, 50 m M EDTA, 1 % (by vol.)…”

Section: Liberation Of the N-linked Carbohydrate Chainsmentioning

confidence: 99%

“…The molar ratio of oligosaccharides was determined on basis of absorbance at 205 nm (HPLC on Lichrosorb-NH,) and at 214 nm (FPLC on Mono Q) [28].…”

Section: Isolation Of the Liberated N-linked Carbohydrate Chainsmentioning

confidence: 99%

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K₂tu‐PA expressed in Chinese hamster ovary cells

Bergwerff

Oostrum

Asselbergs

et al. 1993

European Journal of Biochemistry

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A recombinant human plasminogen activator hybrid variant KJu-PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asnl2 (A chain, kringle-2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N-linked carbohydrate chains by peptide-N4-(N-acetyl-/3-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb-NH,, and analysed by 500-MHz 'H-NMR spectroscopy. The following types of carbohydrates occur : monosialylated diantennary (8 %), disialylated diantennary (45 %), disialylated tri-and tri'-antennary (1 %), trisialylated tri-and tri'-antennary (28 %), and tetrasialylated tetra-antennary (18 %) structures, all having fucose in a(l-g)-linkage at the Asn-bound Nacetylglucosamine. Sialic acid occurred exclusively in a(2-3)-linkage to galactose, and consisted of N-acetylneuraminic acid (94 %), N-glycolylneuraminic acid (3 %), and N-acetyl-9-O-acetylneuraminic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K,tu-PA were analysed. The oligosaccharides attached to Asnl2 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K,tu-PA with that of tissue-type plasminogen activator from different biological sources showed significant differences.Profiling studies on different K,tu-PA production batches demonstrated that the structures of Nlinked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.Plasminogen activators (PA) are serine proteases, that convert the inactive proenzyme plasminogen into the active enzyme plasmin, thereby functioning as important initiators of fibrinolysis. Two types of plasminogen activators can be distinguished, namely, the urinary-type (urokinase, u-PA) and the tissue-type (t-PA) [l]. Urokinase and recombinant t-PA are of clinical interest for the treatment of acute vascular diseases like myocardial infarction [2-41. To obtain artery reperfusion, high blood levels of PA are required, because of its rapid clearance by the liver. Such pharmaceutical use is associated with a variable degree of systemic fibrinolytic acCorrespondence to J. P. Kamerling, Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P. 0. Box 80.075, NL-3508 TB Utrecht, The Netherlands Abbreviations. PNGase-F, peptide-P-(N-acetyl-/%glucosaminyl)-asparagine amidase F; PA, plasminogen activator; u-PA, urinarytype plasminogen activator; t-PA, tissue-type plasminogen activator; CHO, Chinese hamster ovary ; NeuSAc, N-acetylneuraminic acid; NeuSGc, N-glycolylneuraminic acid ; Neu5,9Ac2, N-acetyl-9-O-acetylneuraminic acid; lD, one-dimensional ; 2D, two-dimensional; HOHAHA, homonuclear Hartmann-Hahn ; MLEV, composite pulse devised by Malcom Levitt; COSY, scalar shift correlated spectroscopy ; ROESY, rotating-frame nuclear Overhauser enhancement spectroscopy.Enzymes. Peptid...

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Sialic acid patterns in N‐linked carbohydrate chains. Structural analysis of the N‐acetyl‐/N‐glycolyl‐neuraminic‐acid‐containing N‐linked carbohydrate chains of bovine fibrinogen

Damm

Bergwerff

Hård

et al. 1989

Recl. Trav. Chim. Pays‐Bas

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Abstract. The N-linked carbohydrate chains of bovine fibrinogen have been released by peptide--N4-(N-acetyl-P-glucosaminyl)asparagine amidase-F, fractionated by FPLC and HPLC procedures and characterized by 500/600-MHz 'H NMR spectroscopy. Evidence is presented for the native sialic acid patterns, involving the occurrence of a 2 + 6-linked N-acetyl-and N-glycolylneuraminic acid residues in the terminal position of the di-antennary carbohydrate chains. Details are included of minor oligosaccharides containing NeuSAcu2 --t 3GalP1-+ 4/Pl -+ 3GlcNAc pl + structural elements, which have not been previously described in this glycoprotein.

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Analysis of N‐acetyl‐4‐O‐acetylneuraminic‐acid‐containing. N‐linked carbohydrate chains released by peptide‐N⁴‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F

Cited by 33 publications

References 23 publications

Primary structure of horse serotransferrin glycans

Primary structure of horse serotransferrin glycans

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K₂tu‐PA expressed in Chinese hamster ovary cells

Sialic acid patterns in N‐linked carbohydrate chains. Structural analysis of the N‐acetyl‐/N‐glycolyl‐neuraminic‐acid‐containing N‐linked carbohydrate chains of bovine fibrinogen

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Analysis of N‐acetyl‐4‐O‐acetylneuraminic‐acid‐containing. N‐linked carbohydrate chains released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F

Cited by 33 publications

References 23 publications

Primary structure of horse serotransferrin glycans

Primary structure of horse serotransferrin glycans

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells

Sialic acid patterns in N‐linked carbohydrate chains. Structural analysis of the N‐acetyl‐/N‐glycolyl‐neuraminic‐acid‐containing N‐linked carbohydrate chains of bovine fibrinogen

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Product

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Analysis of N‐acetyl‐4‐O‐acetylneuraminic‐acid‐containing. N‐linked carbohydrate chains released by peptide‐N⁴‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K₂tu‐PA expressed in Chinese hamster ovary cells