The structure of a trisialyl di-antennaryN-type glycopeptide obtained from rat plasma hemopexin

Vliegenthart, Johannes F.G.; Bernard, Nicole F.; Engler, R; Strecker, Gérard; Montreuil, Jean; Halbeek, Herman van

doi:10.1007/bf01213726

Cited by 33 publications

(21 citation statements)

References 24 publications

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“…Diagnostic chemical shifts of 'structural reporter' protons were a-D-GlcNAc H-I (6 = 5.207, J,,* 2.1 Hz): P-D-GIcNAc H-1 (6 = 4.725, J,,* 7.8 Hz); P-D-Gal H-1 (in the a compound, 6 = 4.478, J,,* 7.9 Hz); P-D-Gal H-1 (in the / 3 compound, 6 = 4.475, I',* 7.7 Hz); and N-acetyl (CH,, 6 = 2.044). No evidence was seen for another potential product, GalpPl-3GlcNAc, a linkage which has been reported in rat plasma glycoproteins (Yoshima et al, 1981 ;Bernard et al, 1984).…”

Section: Identification Of the Product Of Galactosyltransferase Catalmentioning

confidence: 87%

Proteins of the Golgi apparatus

Bendiak

Ward

Simpson

1993

European Journal of Biochemistry

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The Golgi marker enzyme, UDP‐galactose: N‐acetylglucosamine β1‐4galactosyltransferase (β1‐4GalT) was purified 44300‐fold in its intact, membrane‐bound form from rat liver membranes. The protein was isolated from detergent extracts as a high‐Mr form, having a Stokes radius approximating a globular protein of Mr 440000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an Mr near 51000, and does not have intermolecular disulfide crosslinks. N‐terminal sequencing of the enzyme demonstrated that it contains an N‐terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5′ end of the mRNA [Russo, R. N, Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324–3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N‐terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low‐Mr forms which were fully catalytically active and which, upon sequencing, were missing a 66‐amino‐acid stretch from the N‐terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with Mr near 50000. The N‐terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non‐protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

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Section: Identification Of the Product Of Galactosyltransferase Catalmentioning

confidence: 87%

Proteins of the Golgi apparatus

Bendiak

Ward

Simpson

1993

European Journal of Biochemistry

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“…3)Gal/?(l-+3)-GlcNAc/? ( 1 +) structural element [30]. The relative broadness of the NeuAca(2+ 6) H-3ax and H-3eq signals is most probably caused by heterogeneity of the peptide moiety.…”

Section: Structure Determination Of Compound Amentioning

confidence: 95%

The structure of sialyl-glycopeptides of the O-glycosidic type, isolated from sialidosis (mucolipidosis I) urine

et al. 1984

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Sialyl-glycopeptides containing an 0-glycosidically linked tetrasaccharide chain were obtained from the urine of a patient suffering from mucolipidosis I. Isolation of these compounds was achieved by gel filtration, ion-exchange chromatography and preparative paper chromatography. Their structures were determined by a combination of carbohydrate and amino acid analysis, dansylation, periodate oxidation, methylation studies, enzymatic hydrolysis and 'H-NMR spectroscopy, to be as follows:wherein R = peptide linked through -Thr-, -Ser-, -Ser-Thr-or -Thr-Ser-.The finding of these glycopeptides in urine shows that mucolipidosis I is characterized by a general "glycoproteinspecific" sialidase deficiency. The possibility of the existence of a human endo-a-N-acetylgalactosaminidase is discussed.Mucolipidosis I (MLP-I, one of the forms of sialidosis) is an inherited metabolic disease characterized clinically by the presence of a cherry-red spot on the fundus, by elective overload of the Kupffer cells and by a variety of neurological syndroms. Chemical studies of this disease showed accumulation of abnormal amounts of sialic-acid-containing compounds in fibroblasts and leukocytes of the patients, due to a deficiency of sialidase activity in these cells. By consequence, an enhanced excretion of sialic-acid-containing oligosaccharides was observed in the urine of MLP-I patients; these oligosaccharides originate from the impaired catabolism of N-glycosidic glycoprotein carbohydrate chains [I -41.However, the sialidase deficiency in MLP-I does not only affect the catabolism of the N-glycosidic type of glycoprotein oligosaccharides but, as previously reported by us [5] and others [6], also the excretion of the 0-glycosidic type of glycoprotein carbohydrates is found increased about tenfold in MLP-I urine, as compared to normal. Structural studies on the excreted sialic-acid-containing compounds of the 0-glycosidic type have been scarce until now; however, the occurrence of a Abbreviations. MLP-I, mucolipidosis I; 'H-NMR, proton nuclear magnetic resonance; NeuAc, N-acetylneuraminic acid ; Gal, galactose; CalNAc, N-acetylgalactosamine; 2,4,6-Me3-Gal-ol, 2,4,6-tri-Ornethyl-l,3,5-tri-O-acetyl-galactitol; 2,3,4,6-Me4-Gal-ol, 2,3,4,6-tetra-O-methyl-1,5-di-O-acetyl-galactitol; 4-Me-GalNAc(Me)-ol, 2-deoxy -2 -N -methylace tamido -4-mono -0 -methyl -1,3,5,6 -te tra-0 -acetyl-galactitol; 4,6-Me2-GalNAc(Me)-01, 2-deoxy-2-N-methylacetamido-4,6-di-O-methyl-l,3,5-tri-O-acetyl-galactitol.Enzymes. a-Neuraminidase (EC 3.2.1.18); P-D-galactosidase (EC 3.2.1.23).glyco-aminoacid constituted of NeuAc, GalNAc and Ser has been reported [7].The present work describes the structure determination of some sialyl-glycopeptides of the 0-glycosidic type isolated from the urine of a patient suffering from MLP-I. The results provide further insight into the biochemical consequences of the enzyme deficiency. A preliminary account of the outcome of the structural studies has been presented [S]. MATERIALS AND METHODS MaterialsUrine of a patient (P.P.) with MLP-...

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“…N-Glycans containing type-1 lactosamine antennae have been found in bovine prothrombin [35], blood coagulation factors IX and X [36, 371, fibrinogen [38] and fetuin [34,, in rat plasma hemopexin [44], in mouse monoclonal IgM and IgG antibodies [45,461, in recombinant erythropoietin from baby hamster kidney cells as well as human urinary erythropoietin [47l and in the mouse specific cross-reacting antigen-2 from human meconium [48]. Corresponding oligosaccharide structures were found to comprise di-, tri-and tetraantennary species with one or two type-1 chains.…”

Section: Discussionmentioning

confidence: 99%

Carbohydrate structure of a thrombin‐like serine protease from Agkistrodon rhodostoma

Pfeiffer

Dąbrowski

et al. 1992

European Journal of Biochemistry

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The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-~-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary-ion mass spectrometry and 'H-NMR spectroscopy.The results reveal that this snake venom glycoprotein contains partially truncated di-, tri-and tetraantennary complex type N-glycans carrying Fuc(a1-6) residues at the innermost Nacetylglucosamine and solely (a2-3)-linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Galj3GlcNAcp lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAca3GalNAc~4GlcNAc~ antenna exclusively linked to C-2 of Man(a1-3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein-N-glycan structures.The venom of the Malayan pit viper Agkistrodon rhodostoma contains a thrombin-like serine protease termed ancrod which acts specifically on fibrinogen. In contrast to thrombin, however, this enzyme splits off only fibrinopeptide A leaving the BD-chain of the molecule intact. Furthermore, it does not activate factor XI11 [l, 21. Consequently, ancrodgenerated clots are not cross-linked and are more susceptible to plasmin degradation resulting in a rapid clearance from the microcirculation. When administered to humans, the enzyme acts as an anticoagulant and lowers the plasma viscosity by reducing the fibrinogen concentration. Therefore, purified ancrod is used for therapeutic defibrinogenation in patients suffering from various vascular diseases. Repeated administration of this agent, however, results in a decrease of its pharmacological activity due to the induction of an antibody response, thus limiting its clinical applicability.Ancrod is a glycoprotein with a relative molecular mass of about 35000 comprising about 36% (by mass) carbohydrate [2, 31. Since there is evidence that the carbohydrate constituents of this enzyme might be involved in the emergence of the so-called resistance to ancrod [4], we have carried out a detailed analysis of its glycosylation. In this contribution, we describe the structure analysis of the oligosaccharide constituents present by exoglycosidase studies, methylation analysis, liquid secondary-ion mass spectrometry and 'H-NMR.

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The structure of a trisialyl di-antennaryN-type glycopeptide obtained from rat plasma hemopexin

Cited by 33 publications

References 24 publications

Proteins of the Golgi apparatus

Proteins of the Golgi apparatus

The structure of sialyl-glycopeptides of the O-glycosidic type, isolated from sialidosis (mucolipidosis I) urine

Carbohydrate structure of a thrombin‐like serine protease from Agkistrodon rhodostoma

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