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Biomedical subjects

P Roepstorff

Publications and source records attributed to P Roepstorff.

At least 91 records · Page 5Linked to original sources
PubMed1998

Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat.

A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.

Amino Acid Sequence↗
PubMed1998

Combining MALDI mass spectrometry and biomolecular interaction analysis using a biomolecular interaction analysis instrument.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been combined with biomolecular interaction analysis (BIA) in a Biacore instrument. A method has been developed for the recovery of the affinity-bound molecules from the sensor chip in a few microliters ready for mass spectrometric analysis. The procedure is illustrated with two molecular systems which exemplify antibody-antigen and DNA-protein interactions. In both cases, femtomole quantities of the affinity-bound proteins were eluted and subsequently detected by MALDI-MS. Whereas the Biacore analysis yields the surface concentration of protein bound to the sensor chip, identity of the bound compounds is revealed in the second step by accurate molecular mass determination. Combining the information of the two analyses allows calculation of the total surface molar concentration of affinity-bound molecules.

Antigen-Antibody Reactions↗
PubMed1998

DNA sequence analysis by MALDI mass spectrometry.

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.

Animals↗
PubMed1998

Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells.

Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III containing Asn233 was quantitatively recovered without carbohydrate. The remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosylation on Asn52 (domain I) and Asn172 (domain II) is dominated by the smaller biantennary complex-type oligosaccharides, while Asn162 (domain II) and Asn200 (domain III) predominantly carry tri- and tetraantennary complex-type oligosaccharides. The carbohydrate moiety on Asn52 in uPAR domain I could be selectively removed by N-glycanase treatment under nondenaturing conditions. This susceptibility was abrogated when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of carbohydrate on domain I exhibited altered binding kinetics toward uPA (a 4-6-fold increase in Kd) as assessed by real time biomolecular interaction analysis.

Amino Acid Sequence↗
PubMed1998

Restoration of catalytic activity beyond wild-type level in glucoamylase from Aspergillus awamori by oxidation of the Glu400-->Cys catalytic-base mutant to cysteinesulfinic acid.

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

Alkylation↗
PubMed1998

Hereditary cystatin C amyloid angiopathy: monitoring the presence of the Leu-68-->Gln cystatin C variant in cerebrospinal fluids and monocyte cultures by MS.

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant condition in which the patients suffer at an early age from repeated cerebral haemorrhages. The development of HCCAA is directly linked to a Leu-68-->Gln (L68Q) mutation in the cystatin C protein sequence. The concentration of cystatin C in cerebrospinal fluid (CSF) of HCCAA patients is markedly diminished and cultivated monocytes from affected individuals accumulate cystatin C. The goal of this work was to characterize cystatin C isolated from CSF and monocyte cultures originating from healthy persons and HCCAA patients with respect to the L68Q mutation. Cystatin C was isolated by carboxymethylpapain affinity chromatography. Proteins from CSF and monocyte cultures that bound specifically to the carboxymethylated papain column were resolved by reverse-phase HPLC chromatography and tryptic peptides were subsequently analysed by matrix-assisted laser desorption ionization MS. No evidence for mutated cystatin C protein was found in CSF samples from healthy subjects or HCCAA patients, but approx. 60% of the protein was found to be hydroxylated on Pro-3. No evidence was found for secretion of mutated cystatin C from HCCAA monocytes. However, we obtained evidence for the presence of mutated cystatin C in HCCAA monocytes. These results support the conclusion that the mutated cystatin C is retained in association with the monocytes and not secreted. An increased intracellular concentration would presumably promote the aggregation and denaturation of the mutated cystatin C, leading to the formation of amyloid fibrils and cell death.

Amino Acid Sequence↗
PubMed1998

Posttranslational modifications of human inter-alpha-inhibitor: identification of glycans and disulfide bridges in heavy chains 1 and 2.

Inter-alpha-inhibitor (IalphaI) is a serine proteinase inhibitor found in high concentrations in human plasma. The protein is composed of a light inhibitory chain called bikunin and two heavy chains of unknown function. The three polypeptide chains are covalently assembled via a carbohydrate cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., & Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751]. The aim of this study was to complete the primary structure by characterizing additional covalent posttranslational modifications of the heavy chains. Analysis revealed three N-linked oligosaccharides located on Asn251 and Asn554 of heavy chain 1 and on Asn64 of heavy chain 2: all these were complex biantennary structures composed of (Asn)-GlcNAc2-Man-(Man-GlcNAc-Gal-SA)2. In addition, the IalphaI heavy chains carried several O-linked glycans located on Thr619 of heavy chain 1 and a cluster of four O-linked oligosaccharides on Thr612, Ser619, Thr621, and Thr637 of heavy chain 2. The oligosaccharides were short (Ser/Thr)-GalNAc-Gal-SA trisaccharides. The IalphaI heavy chains contain nine Cys residues, of which eight are involved in disulfide bridges. The unpaired Cys residue residing on heavy chain 1, Cys26, appears to be modified by dihexosylation. The other Cys residues exclusively form intrachain disulfide bridges. In heavy chain 1 the two disulfide bonds are formed between Cys210 and Cys213 and between Cys234 and Cys506, and in heavy chain 2, between Cys207 and Cys210 and between Cys596 and Cys597. Interestingly, three of these four disulfides are formed between Cys residues that are either adjacent or only two amino acid residues apart.

Alpha-Globulins↗
PubMed1998

Characterization of enzymatic pectin digests by matrix-assisted laser desorption/ionization mass spectrometry.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the characterization of partially methyl-esterified enzymatic pectin digests is described. The sensitivities of several matrices, positive and negative ion modes and desalting techniques for these acidic oligosaccharides were compared. The most favorable results were obtained with a thin-layer preparation of a mixture of 2,4,6-trihydroxyacetophenone and nitrocellulose in the negative ion mode. Results are presented demonstrating the sensitive characterization of separated and unseparated high-ester pectin digests obtained after complete digestion using Aspergillus niger pectin lyase and the analysis of digests after chemical modification. In the case of unseparated digests, the analysis of methylation patterns is demonstrated. Oligomers with a degree of polymerization up to 40 were detected after enrichment of large oligomers by propan-2-ol precipitation.

1-Propanol↗
PubMed1998

Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: strategies and applications.

The entire genomic DNA sequences of a number of prokaryotic and eukaryotic species are now available and many more, including the human genome, will be completed in the near future. The state-of-life of a cell at any given time, however, is defined by its protein composition, i.e., its proteome. Gel electrophoresis, mass spectrometry, and bioinformatics will be important tools for protein and proteome analysis in the post-genome era. Protein identification from electrophoretic gels by mass spectrometric peptide mapping or peptide sequencing combined with sequence database searching is established and has been applied to numerous biological systems. We describe current strategies and selected applications in molecular and cell biology. The next challenges are detailed structure/function analyses, which include studying the molecular composition of multiprotein complexes and characterization of secondary modifications of proteins. The advantages and limitations of a number of mass spectrometry-based strategies designed for microcharacterization of low amounts of protein from electrophoretic gels are discussed and illustrated by examples.

Amino Acid Sequence↗
PubMed1998

Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification.

Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross-references demonstrated that there is no obvious pattern by which the mobility of a protein in one gel system can be used to predict its mobility in the other. Thus, as laboratories adopt the immobilized pH gradient-based 2-D gel systems, the only reliable means of translating the data gained with the carrier ampholyte-based gel system is to positively identify the proteins in both 2-D systems.

Ampholyte Mixtures↗
PubMed1998

Revised primary structures of rat pituitary gamma-lipotrophin and beta-endorphin.

In-depth investigations, by high performance liquid chromatographic purification, radio-immunoassay, mass spectrometry, tandem mass spectrometry, Edman sequencing and limited C-terminal ladder sequencing, were prompted by mass spectrometric charting experiments which suggested that the amino acid sequences for rat gamma-lipotrophin and beta-endorphin require revision. The results for gamma-lipotrophin identify a histidine for glutamine substitution at position 12, and heterogeneity in the expressed protein presumably due to partial dehydration. Partial dehydration for acidic joining peptide, previously reported by Toney et al was corroborated. The results for beta-endorphin confirm the presence of alanine at position 26 and provide no evidence for the expression of multiple forms of the hormone.

Amino Acid Sequence↗
PubMed1998

Characterization of exoskeletal proteins from the American lobster, Homarus americanus.

Proteins from the calcified exoskeleton of the lobster, Homarus americanus, were extracted and separated by two-dimensional gel-electrophoresis. Electroblotting the proteins onto polyvinylidene difluoride (PVDF) membranes followed by sequence determination gave 16 N-terminal amino-acid sequences and revealed that further eight proteins were N-terminally blocked. The relative molecular mass, M(r), was obtained for most of the electrophoretically separated proteins by means of matrix-assisted laser desorption mass spectrometry (MALDIMS) after electroelution from Coomassie-stained two-dimensional polyacrylamide gels. Eleven proteins were purified from extracts of the exoskeleton by low pressure ion exchange chromatography and reversed-phase high performance chromatography, and their sequences were determined by combined use of Edman degradation and mass spectrometry. Good agreement was obtained between the M(r)-values measured by mass spectrometry and those calculated from the sequences. Five of the sequenced proteins contain two copies of a previously observed 18-residue sequence motif, while a couple of the remaining sequences show similarity to sequences of exoskeletal proteins from shrimps and spiders. Only limited similarity to insect cuticular proteins was observed.

Amino Acid Sequence↗
PubMed1998

Development and characterization of an antibody directed to an alpha-N-acetyl-D-galactosamine glycosylated MUC2 peptide.

In an attempt to raise anti-Tn antibodies, an alpha-N-acetyl-D-galactosamine glycosylated peptide based on the tandem repeat of the intestinal mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMVTPTPTPTC) was glycosylated in vitro using concentrated alpha-N-acetylgalactosaminyltransferases activity from porcine submaxillary glands which resulted in the incorporation of 8-9 mol of Ga/NAc. Rabbits and mice developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not show reactivity with the unglycosylated MUC2 peptide or with other GalNAc glycosylated peptides. A mouse monoclonal antibody (PMH1) representative of the observed immune response was generated and its immunohistological reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the staining was not restricted to the supranuclear area but extended to the entire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glycoforms of MUC2. The present work presents a useful approach for development of anti-mucin antibodies directed to different glycoforms of individual mucins.

Animals↗
PubMed1998

Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.

The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

Amino Acid Sequence↗
PubMed1998

Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition.

Coeliac disease probably results from a T-cell response to wheat gliadin and is associated to HLA-DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut-lesion-derived DQ2-restricted T cells from coeliac disease patients were used to identify an epitope within a purified gamma-type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T-cell recognition. Other gut-derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut-derived DQ2-restricted T cells isolated from several patients. Several DQ2-restricted T-cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.

Amides↗
PubMed1998

Characterization of intramolecular disulfide bonds and secondary modifications of the glycoprotein from viral hemorrhagic septicemia virus, a fish rhabdovirus.

Viral hemorrhagic septicemia virus (VHSV) infections cause high losses in cultured rainbow trout in Europe. Attempts to produce a recombinant vaccine based on the transmembrane glycoprotein (G protein) have indicated that proper folding is important for the antigenicity and immunogenicity of the protein. The present study was initiated to identify the disulfide bonds and other structural aspects relevant to vaccine design. The N-terminal amino acid residue was identified as being a pyroglutamic acid, corresponding to Gln21 of the primary transcript. Peptides from endoproteinase-degraded G protein were analyzed by mass spectrometry before and after chemical reduction, and six disulfide bonds were identified: Cys29-Cys339, Cys44-Cys295, Cys90-Cys132, Cys172-Cys177, Cys195-Cys265, and Cys231-Cys236. Mass spectrometric analysis in combination with glycosidases allowed characterization of the glycan structure of the G protein. Three of four predicted N-linked oligosaccharides were found to be predominantly biantennary complex-type structures. Furthermore, an O-linked glycan near the N terminus was identified. Alignment of the VHSV G protein with five other rhabdovirus G proteins indicates that eight cysteine residues are situated at conserved positions. This finding suggests that there might be some common disulfide bonding pattern among the six rhabdoviruses.

Amino Acid Sequence↗