Biomedical subjects

M Nimtz

Publications and source records attributed to M Nimtz.

At least 73 records · Page 4Linked to original sources

PubMed1995

7-O-acetyl-GD3 in human T-lymphocytes is detected by a specific T-cell-activating monoclonal antibody.

The monoclonal antibody U5, which is a potent inducer of proliferation in human T-cells, was found to bind to an alkali-sensitive derivative of ganglioside GD3. Using immunochemical and spectroscopic methods, the structure of the U5 antigen was determined as 7-O-acetyl-GD3. The antibody U5 did not react with 9-O-acetyl-GD3 and bound severalfold more stronger to 7-O-acetyl-GD3 than to GD3. U5 is the first antibody known to detect preferentially 7-O-acetyl-GD3. Flow cytometric analysis showed that each major class of human leukocytes contained a significant fraction of cells binding the U5 antibody.

Animals↗

PubMed1995

Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions.

The N-glycosylation patterns of a genetically engineered human interleukin-2 variant glycoprotein (IL-Mu6), produced by BHK-21 cells from long-term suspension and microcarrier cultures in the presence and absence of fetal calf serum were compared. IL-Mu6 was used as a model protein in studying the effect of different controlled cell culture conditions on the expression of N-glycans in recombinant glycoproteins. IL-Mu6 contains a single amino acid substitution (Glu100<==>Asn) generating a potential N-glycosylation recognition site (Asn100-Xxx-Thr/Ser) in addition to the natural O-glycosylation at position Thr3. Parallel cell cultivations were carried out in two continuously perfused 2.5-liter stirred bioreactors under defined culture conditions. Major differences were found in the glycoprotein products obtained during these different cultivation conditions. Serum-free cultures resulted in a higher level of terminal sialylation and proximal alpha 1-6 fucosylation. The ratio of O- to N-glycans as well as the amount of nonglycosylated product and the antennarity of N-linked carbohydrates in the model protein exhibited major differences depending on the presence or absence of serum, the condition of growth and the cultivation procedure.

Amino Acid Sequence↗

PubMed1995

Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)-branch of diantennary oligosaccharides from secreted recombinant beta-trace protein.

The human beta-trace protein has been cloned and has been expressed for the first time in a mammalian host cell line. Stable BHK-21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT-beta TP or pAB3-1 which contain the cDNAs encoding the human secretory glycoproteins beta-trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase (ST6N) gene. The beta-trace protein was purified by immunoaffinity chromatography and N-linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural beta-trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions in a ratio of 1:5. The monosialylated oligosaccharides of beta-trace protein coexpressed with human ST6N were found to contain NeuAc in alpha 2,6- or alpha 2,3-linkage in the same ratio. From 1H-NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both alpha 2,3- and alpha 2,6-linkage to Gal beta(1-4)GlcNAc-R, whereas 40% of the molecules of this fraction contained NeuAc in only alpha 2,3-linkage to Gal(beta 1-4)GlcNAc-R. The alpha 2,6-linked NeuAc was shown to be attached preferentially to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human-type glycosylated secretory recombinant polypeptides.

Animals↗

PubMed1995

Identification and structural characterization of a mannose-6-phosphate containing oligomannosidic N-glycan from human erythropoietin secreted by recombinant BHK-21 cells.

A sialidase resistant mono-charged N-glycan was isolated from glycosylation site I (Asn-24) of recombinant human erythropoietin expressed from baby hamster kidney (BHK-21) cells and constituted approximately 2-4% of the oligosaccharide material at this glycosylation site. Mass spectrometry and both 1- and 2-dimensional NMR techniques revealed a high mannose type structure (Man6) with a phospho-diesterbridged N-acetylglucosamine as follows: [formula: see text]

Animals↗

PubMed1995

'Brain-type' N-glycosylation of asialo-transferrin from human cerebrospinal fluid.

Asialo-transferrin from human cerebrospinal fluid was purified to homogeneity. Investigation of the structural characteristics of its oligosaccharides support our hypothesis of 'brain-type' glycosylation of intrathecally synthesized cerebrospinal fluid proteins. For carbohydrate structural analysis, high-pH anion-exchange chromatography, methylation analysis, liquid secondary ion- and matrix-assisted laser desorption/ ionization mass spectrometry of the permethylated derivatives were used. The major structure turned out to be a complex-type agalactodiantennary oligosaccharide with bisecting N-acetylglucosamine and proximal fucose. Analysis of a second transferrin preparation containing both asialo- and sialo-transferrin revealed another major glycan species derived from the sialylated transferrin variant which is galactosylated and lacks bisecting N-acetylglucosamine and fucose.

Amino Acid Sequence↗

PubMed1995

Triterpenoid saponins from the aerial parts of Aster bellidiastrum.

Four major triterpenoid saponins were isolated from the aerial parts of Aster bellidiastrum. The structures were elucidated from their NMR and mass spectral data, and from derivatization. One is a new compound with the structure 3-O-beta-D-glucopyranosyl-2 beta,3 beta,16 alpha,23-tetrahydroxyolean-12-en-28- oic acid 28-O-beta-D-xylopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->2)-beta- D-fucopyranoside, while the three others have been previously identified in extracts from various Bellis species.

Carbohydrate Sequence↗

PubMed1995

Structure elucidation of a glycoside of 2 beta, 3 beta, 23-trihydroxy-16-oxoolean-12-en-28-oic acid from Bellis bernardii using mass spectrometry for the sugar sequence determination.

The structure of a novel saponin, possessing an unusual aglycone named bellisonic acid and isolated from whole plants of Bellis bernardii, has been elucidated by modern ms and nmr techniques as 3-O-alpha-L-rhamnopyranosyl-2 beta, 3 beta, 23-trihydroxy-16-oxoolean-12-en-28-oic acid 28-alpha-L-rhamnopyranosyl(1-->3)-O-beta-D-xylopyranosyl(1-->4)-alpha-L- rhamnopyranosyl(1-->2)-beta-D-fucopyranoside [1]. Ms procedures of general applicability allowed unambiguous sugar sequence determination from very small amounts of material.

Carbohydrate Conformation↗

PubMed1994

A strategy for the mapping of N-glycans by high-performance capillary electrophoresis.

We have evaluated high-performance capillary electrophoresis (HPCE) with respect to its suitability for use in establishing a carbohydrate-mapping database that would enable a carbohydrate structural analysis by mere comparison of migration times. The suitability of HPCE for carbohydrate structural assignments was ascertained by validation experiments. The migration times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation of usually less than 0.20%, requiring only femtomoles of N-glycan per injection for reliable measurements. By including mesityl oxide and sialic acid as internal standards and a triple-correction method, HPCE fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan-mapping database was established using a newly developed and optimized buffer system containing 1,5-diaminopentane as an organic modifier. Approximately 80 different sialylated N-glycans of known structure, which have thus far been measured and characterized, have been entered into our Lotus 1-2-3 mapping database. The database for structural determinations was tested using the N-linked carbohydrates released from recombinant human urinary erythropoietin (baby hamster kidney) by PNGase F treatment and from bovine serum fetuin and alpha 1-acid glycoprotein by automated and manual (large-scale) hydrazinolysis, respectively. The efficiency of the database and of the triple-correction method was further confirmed by HPCE measurements performed in a different laboratory and by a different analyst who used the HPCE system of a different manufacturer.

Electrophoresis↗

PubMed1994

Identification of the mouse helper T lymphocyte differentiation antigen 3G11 as the ganglioside IV3(NeuAc)2-GgOse4Cer.

The 3G11 antigen, originally defined by the monoclonal antibody SM3G11, is expressed to different extents by distinct murine CD4+ T helper cell subsets. This antigen was isolated from a 3G11+ murine thymoma by lipid extraction. Upon further purification 3G11 antigen was enriched in the disialoganglioside fraction and was finally purified by HPLC. Compositional and methylation analysis, liquid secondary ion mass spectroscopy (LSI-MS) and comparison with an authentic sample resulted in identification of the 3G11 antigen as the disialoganglioside IV3(NeuAc)2-GgOse4Cer.

Animals↗

PubMed1994

Structure and physical properties of the extracellular polysaccharide PS-P4 produced by Sphingomonas paucimobilis P4 (DSM 6418).

A new strain, Sphingomonas paucimobilis P4 (DSM 6418), was found during a screening programme for exopolysaccharide-producing bacteria. The highly viscous fermentation broth yields a polysaccharide (up to 10 kg/m3), named PS-P4, and shows thixotropic flow behaviour. In the presence of phosphate ions, PS-P4 forms aqueous gels after heating and cooling at alkaline pH. After isolation and purification of the exopolysaccharide, structural analysis by 1D and 2D 1H NMR spectroscopy and mass spectrometry was performed. The deacylated exopolysaccharide has the following repeating trisaccharide structure:-->4)-beta-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D- Glcp(1-->Additionally, the presence of ester-bound acetic acid, D-glyceric acid, and (R)-3-hydroxybutyric acid in the native polysaccharide was demonstrated.

Carbohydrate Sequence↗

PubMed1994

Betacyanins from bracts of Bougainvillea glabra.

Betacyanins from the bracts of Bougainvillea glabra were isolated and characterized by a combination of spectroscopic techniques (DAD-HPLC, NMR, LC-MS, GC-MS, electrospray MS, tandem MS) as gomphrenin I (betanidin 6-O-beta-glucoside) and various derivatives of bougainvillein-v (betanidin 6-O-beta-sophoroside), i.e. mono- and diglucosylsophorosides which are acylated with 4-coumaric and caffeic acid (mono- and diesters). Besides the betacyanins, B. glabra bracts accumulated large amounts of flavonols (kaempferol and quercetin conjugates) reaching ratios of flavonol to betacyanin of 1:1.

Betalains↗

PubMed1994

Carbohydrate structures of beta-trace protein from human cerebrospinal fluid: evidence for "brain-type" N-glycosylation.

The carbohydrate structures of beta-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N-glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N-acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N-acetylglucosamine residues of nearly all structures were found to be alpha 1,6-fucosylated. Peripheral fucose (about 20% alpha 1,3-linked to N-acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N-acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N-acetylglucosamine only were present. At least 20 different N-glycans were identified. Analysis of the site-specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N-acetylglucosamine, as well as terminal N-acetylglucosamine and galactose residues, and significant amounts of N-acetylneuraminic acid in alpha 2,3 linkage), this protein can be classified as "brain-type" glycosylated.

Acetylglucosamine↗

PubMed1993

Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications.

Human interleukin 2 (IL-2) and human IL-2 mutant proteins, with artificially introduced N-glycosylation or O-glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodoptera frugiperda) using recombinant baculovirus vectors. Only approximately 25% of the total recombinant IL-2 protein synthesized by Sf21 cells was secreted into the culture medium. Significant N-terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). Alanine and proline were absent in the major truncated forms; the first 3-5 amino acids were also absent in a small proportion of the purified proteins. The introduction of potential artificial O-glycosylation peptide sequences (..GGKAPTPPPK..), to the C-terminus or between positions 80 and 81 of the IL-2 polypeptide chain, resulted in the secretion of unglycosylated and O-glycosylated variant forms. Fast atom bombardment mass spectrometry, compositional analysis and methylation analysis, of the tryptic glycopeptide APTPPPK, revealed the presence of either GalNAc or the disaccharide Gal(beta 1-3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual IL-2 mutant protein. The Gal(beta 1-3)GalNAc protein forms could be partially altered in vitro to mammalian-type glycoforms by porcine liver beta-galactoside alpha-2,3-sialyltransferase in the presence of CMP-N-acetylneuraminic acid. An IL-2 mutant form, with an 11-amino-acid peptide of human interferon-beta at position 4, which includes its only N-glycosylation site, had exclusively truncated proximally fucosylated oligomannosidic glycans; Man3GlcNAc[Fuc(alpha 1-6)]GlcNAc or Man2GlcNAc[Fuc(alpha 1-6)]GlcNAc structures, in a ratio of 3:1, were detected in the secreted proteins. No evidence was obtained for the presence of secreted proteins with complex oligosaccharide chains, irrespective of the cell-culture conditions used or the harvesting time, for infected cells with recombinant baculovirus constructs.

Amino Acid Sequence↗

PubMed1993

Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells.

The native structures of the Asn-linked oligosaccharides and the O-glycans at Ser126 of human erythropoietin expressed from recombinant BHK cells have been elucidated. Enzymatically released N-glycans were studied by methylation analyses, fast-atom-bombardment mass spectrometry as well as one- and two-dimensional 1H-NMR spectrometry at 600 MHz. Many (82.7%) were found to be tetraantennary N-acetyllactosamine-type (22.8% with one, 3.6% with two and 0.4% with three N-acetyllactosamine repeats) being tetrasialylated (41%), trisialylated (29.6%) and disialylated (12.2%). A few (9.7%; 4.1% 2,4-branched, 5.6%, 2,6-branched) of the chains were triantennary (5.4% trisialyl, 4.3% disialyl) and 4.6% were of the disialyl diantennary type. Almost all of the innermost GlcNAc residues were alpha 1-6 fucosylated and NeuAc was exclusively alpha 2-3 linked to Gal beta 1-4GlcNAc-R; 60% of the protein was found to be O-glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Gal beta 1-3GalNAc core type. Glycosylation patterns at individual Asn-Xaa-Thr/Ser sites were determined by analytical high-pH anion-exchange chromatography with pulsed amperometric detection. Only tetraantennary chains with 0-3 N-acetyllactosamine repeats were detected at Asn38 and Asn83, while almost all of the di- and triantennary oligosaccharides were attached to Asn24. Batch analysis of different preparations of recombinant erythropoietin revealed the high reproducibility of the production procedure. Structures containing terminal GalNAc-GlcNAc were detected in small amounts in a few batches.

Amino Acid Sequence↗

PubMed1993

Purification and chemical characterization of beta-trace protein from human cerebrospinal fluid: its identification as prostaglandin D synthase.

beta-Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23-29 kDa was determined for the polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of beta-trace protein as prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (Thr instead of Ser) was detected at amino acid position 154 of the beta-trace polypeptide chain in the corresponding tryptic peptide. The two N-glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29 and Asn56 bear exclusively complex-type oligosaccharide structures (partially sialylated with alpha 2-3- and/or alpha 2-6-linked N-acetylneuraminic acid) that are almost quantitatively alpha 1-6 fucosylated at the proximal N-acetylglucosamine; approximately 70% of these molecules contain a bisecting N-acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.

Amino Acid Sequence↗

PubMed1992

A strategy for the mapping of N-glycans by high-pH anion-exchange chromatography with pulsed amperometric detection.

We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of less than 0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients "S" and "A" for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of less than +/- 0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of less than +/- 1%, three of them even with an accuracy of less than 0.1%, providing the reliability of the established HPAE-PAD mapping database.

Carbohydrate Conformation↗

PubMed1991

NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin.

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]

Amino Acid Sequence↗

PubMed1990

Carbohydrate structures of a human tissue plasminogen activator variant expressed in recombinant Chinese hamster ovary cells.

The carbohydrate structures of a genetically engineered human tissue plasminogen activator variant bearing a single N-glycosylation site at Asn 448 are reported. After isolation of the tryptic glycopeptide and liberation of the N-linked carbohydrates by polypeptide:N-glycosidase F, 6 major oligosaccharide fractions were separated by HPLC on NH2-bonded phase. Their structures were determined by compositional and methylation analyses combined with fast atom bombardment mass spectrometry. Seventy percent of the carbohydrates were of the biantennary complex type with fucose at the proximal GlcNAc and zero, one or two alpha 2-3 linked NeuAc. The remainder were triantennary structures with one, two or three NeuAc.

Animals↗