Biomedical subjects

P Roepstorff

Publications and source records attributed to P Roepstorff.

At least 109 records · Page 6Linked to original sources

PubMed1998

Partial amino acid sequence of gamma-46 gliadin.

We have determined the partial amino acid sequence (207 amino acids) of gamma-46 gliadin isolated from wheat cultivar Hardi. The molecular mass of the protein (Mr) estimated by electrospray mass spectrometry is 35191.3. The number of cysteine residues in gamma-46 gliadin was determined as a mass difference of the protein before and after reduction and alkylation with 4-vinylpyridine. It was shown that the protein has no free SH-groups, and all cysteine residues are involved in the formation of four disulfide bonds. The partial structure of gamma-46 gliadin was determined by N-terminal sequencing and sequencing of tryptic and chymotryptic peptides. The tryptic peptides were obtained by enzymatic hydrolysis of the protein, which was preliminarily reduced and immobilized at free SH-groups on thiopropyl-Sepharose 6B. The chymotryptic peptides were isolated by limited digestion of the native protein. The positions of cysteine residues, as well as surrounding amino acid sequences, are conserved in gamma-46 gliadin; this is typical of gliadins.

Amino Acid Sequence↗

PubMed1997

Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3.

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.

Amino Acid Sequence↗

PubMed1997

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNA.

The determination of RNA sequences using base- specific enzymatic cleavages is a well established method. Different synthetic RNA molecules were analyzed for uniformity of degradation by RNase T1, U2, A and PhyM under reaction conditions compatible with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), to identify the positions of G, A and pyrimidine residues. In order to get a complete set of fragments derived from cleavage at every phosphodiester bond, the samples were also subjected to a limited alkaline hydrolysis. Additionally, the 5'-terminus fragments of a 49mer RNA transcript were isolated by way of 5'-biotinylation and streptavidin-coated magnetic beads (Dynal), followed by a RNase U2digestion. MALDI-MS of the generated fragments is presented as an efficient technique for a direct read out of the nucleotide sequence.

Animals↗

PubMed1997

Immunohistochemical study of MUC5AC expression in human gastric carcinomas using a novel monoclonal antibody.

In order to investigate the expression of MUC5AC mucin in normal gastric mucosa and gastric carcinomas, we produced 3 monoclonal antibodies (MAbs) using a MUC5AC synthetic peptide. The immunohistochemical study was performed using one of these MAbs (CLH2) which reacted with the different designs of peptides based on the MUC5AC tandem repeat and with native and deglycosylated mucin extracted from gastric tissues. CLH2 immunoreactivity was restricted to foveolar and mucopeptic neck cells in normal gastric mucosa. No reactivity was observed in type-I intestinal metaplasia. Out of 66 gastric carcinomas, 42 (63.6%) expressed MUC5AC. Most diffuse carcinomas were positive (83.3%), whereas only 59.3% of intestinal and 40.0% of atypical carcinomas expressed MUC5AC (p < 0.05). Gastric carcinomas with mixed pattern showed immunoreactivity in diffuse areas and decreased immunoreactivity in intestinal areas. Every early gastric carcinoma expressed MUC5AC, in contrast to 58.6% of advanced carcinomas (p < 0.05). A trend toward decreased immunoreactivity was observed in deep areas of advanced carcinomas in comparison with the respective superficial areas. Taking together the specific staining of foveolar and mucopeptic neck cells and the absence of immunoreactivity in intestinal metaplasia, we conclude that MUC5AC expression may be used as a marker of gastric differentiation. This assumption is further supported by the finding of MUC5AC immunoreactivity in most diffuse carcinomas, which usually display morphologic and histochemical signs of gastric differentiation. The expression of MUC5AC in early gastric carcinomas, regardless of their histologic type, suggests that all gastric carcinomas retain at least some cells with a gastric phenotype during the first steps of neoplastic development.

Amino Acid Sequence↗

PubMed1997

Matrix-assisted laser desorption/ionization mass spectrometric peptide mapping of the neural cell adhesion protein neurolin purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis or acidic precipitation.

Neurolin is a cell surface protein involved in the neural regeneration and neogenesis of the central nervous system of goldfish. Its theoretical molecular mass, based on the amino acid sequence translated from the cDNA, is 58 kDa, but in SDS-PAGE it shows an apparent MW of 86 kDa. Neurolin is stated to be a glycoprotein and it contains five potential N- and 96 potential O-glycosylation sites. The complete characterization of the primary structure and initial investigations on the postulated glycosylation of neurolin, immunopurified from goldfish brains, are described. The protein was either digested in situ in the sodium dodecyl sulfate polyacrylamide gel matrix or digested after trichloroacetic acid precipitation. Trypsin and endoprotease Glu-C were used as proteases and matrix-assisted laser desorption/ionization mass spectrometry was applied for direct peptide mapping analysis of the proteolytic mixtures. Various sample preparation techniques were performed and the mass spectra were recorded in both positive- and negative-ion modes.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed1997

Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry.

The site-specific glycan heterogeneity of human urinary erythropoietin was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Owing to the small amount of protein available, a strategy combining optimal sensitivity and specificity was used. Erythropoietin was reduced, S-alkylated and digested with endoproteinase Lys C. The peptides were separated by reversed-phase high-performance liquid chromatography and the molecular masses of the peptides determined by MALDI-MS. The peptides were identified by comparing the experimental masses with the masses predicted from the cDNA derived amino acid sequence. Glycopeptides were identified from the mass spectra based on the peak pattern caused by the glycan heterogeneity. They were further characterized after treatment with neuraminidase and endoproteases. All N-glycosylation sites exhibited fucose-containing complex-type glycans. The N-glycosylation sites at Asn38 and Asn83 are mainly occupied by tetraantennary glycans, whereas Asn24 is occupied by a mixture of bi-, tri- and tetraantennary glycans. A molecular mass glycoprofile for each glycosylation site was established based on the relative peak intensities observed in the MALDI mass spectra of the desialylated glycopeptides.

Alkylation↗

PubMed1997

Proteome analysis of Saccharomyces cerevisiae: a methodological outline.

Proteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function. This is achieved through the combination of two technologies: protein separation and selection by two-dimensional gel electrophoresis, and protein identification and characterization by mass spectrometry. This methodological outline sketches the strengths and weaknesses of the two central technologies used, and provides both practical tips and the theoretical background for their utilization. One application of these technologies is illustrated by the characterization of genes, revealed by sequencing, but which have no--or only weak homology--to any other known genes. Other applications, for example the identification of protein markers for particular human diseases, are only referred to. The aim of the article is thus to provide the basis for a sound understanding of the full potential and limitations of proteome analysis.

Databases, Factual↗

PubMed1997

Overexpression and characterization of Aspergillus awamori wild-type and mutant glucoamylase secreted by the methylotrophic yeast Pichia pastoris: comparison with wild-type recombinant glucoamylase produced using Saccharomyces cerevisiae and Aspergillus niger as hosts.

Glucoamylase from Aspergillus niger (identical to Aspergillus awamori glucoamylase) is an industrially important, multidomain N- and O-glycosylated starch-hydrolase. To produce protein-engineered glucoamylase, heterologous expression is established in the methylotrophic yeast Pichia pastoris. Using the vector pHIL-D2, the cDNA encoding A. awamori glucoamylase is inserted in the yeast genome downstream of the 5' AOX1 promoter to replace the AOX1 gene. Induction by 0.75% methanol for 48 h led to synthesis of secreted glucoamylase to give around 0.4 g/liter, as directed by the A. awamori signal sequence. Recombinant glucoamylase produced in P. pastoris, Saccharomyces cerevisiae, or A. niger displayed similar catalytic properties, thiol content, and isoelectric point. Glucoamylase from P. pastoris, however, has higher thermostability than the enzymes from S. cerevisiae, A. niger, or a commercial preparation of A. niger glucoamylase. The average Mr determined by matrix-assisted laser desorption ionization mass spectrometry of these enzymes is thus 82,327, 83,869, 82,839, and 80,370, respectively, and neutral sugar analysis shows the differences to be due to variation in the extent of glycosylation. Compared to wild-type, single-residue mutation generally reduced the amount of secreted glucoamylase in S. cerevisiae and A. niger. In P. pastoris, however, the Cys320 --> Ala/Glu400 --> Cys double mutant is produced at 0.3 g/liter, or 75% of wild-type glucoamylase, while the corresponding single mutants have been produced at l and 20% of the wild-type level in S. cerevisiae and A. niger, respectively.

Aspergillus niger↗

PubMed1997

Mass spectrometry in protein studies from genome to function.

The demands for highly sensitive and specific analytical techniques in biochemistry, molecular biology and biotechnology are met by new developments in mass spectrometry. Femto- to attomole sensitivity and mass accuracy in a low parts per million range can now be routinely obtained. Mass spectrometry, already accepted for studies of protein secondary modifications, must, in the future, be expected to be an important tool in protein studies on all levels, ranging from proteome analysis to studies of protein higher order structures and protein interaction.

Biotechnology↗

PubMed1997

Cuticular proteins from the giant cockroach, Blaberus craniifer.

The extractable proteins from selected cuticular regions of nymphs and adults of the cockroach, Blaberus craniifer, have been compared by two-dimensional gel-electrophoresis. Only minor differences in protein patterns were observed when nymphal and adult pre-ecdysial cuticles (presumptive exocuticle) were compared, whereas the pattern obtained from nymphal mid-instar cuticle (mainly endocuticle) differed markedly from that obtained from mature adult cuticle. The pattern obtained from nymphal mid-instar cuticle depended upon the specific cuticular region analysed, but the differences within a stage were, to a large extent, quantitative and not qualitative. Seven nymphal endocuticular proteins have been purified to near homogeneity, and the complete amino acid sequence has been determined for three of them. One of the proteins, Bc-NCP1, contains a 16-residue motif repeated three times and containing a disulphide bridge. Protein Bc-NCP2 has a twice repeated motif in common with a pupal protein from Bombyx mori, and Bc-NCP4 contains a twice-repeated sequence of nine residues and is moreover characterized by an unusual high content of valine (22.0%). None of the protein sequences shows significant similarities to the sequences determined for locus endocuticular proteins, except that they all have pyroglutamate as the N-terminal residue.

Amino Acid Sequence↗

PubMed1997

Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor.

Complete amino acid sequences have been determined for six larval-pupal cuticular proteins from Tenebrio molitor. The sequenced proteins are major components in both larval and pupal cuticle, and both basic and slightly acidic proteins are represented. The proteins show pronounced similarities to some of the proteins sequenced from other insect cuticles. Three slightly acidic larval-pupal Tenebrio cuticular proteins contain a 66-residue central, hydrophilic region, resembling regions in cuticular proteins from insect species of four different orders (Coleoptera, Diptera, Lepidoptera and Orthoptera), and three basic proteins from larval-pupal Tenebrio cuticle have a 51-residue hydrophilic region in common with two proteins from cuticle of pharate adult locusts (Locusta migratoria). The Tenebrio larval-pupal cuticular proteins are also similar to locust adult cuticular proteins, by frequent occurrence of the short sequence motif Ala-Ala-Pro-Ala/Val. The pronounced sequence similarities between cuticular proteins from different insect orders indicate that the conserved regions are functionally important.

Amino Acid Sequence↗

PubMed1997

Determination of isoforms, N-linked glycan structure and disulfide bond linkages of the major cat allergen Fel d1 by a mass spectrometric approach.

The domestic cat (Felis domesticus) is an important source of indoor allergens, the major allergen being Fel d1 (formerly cat allergen 1). Fel d1 is responsible for cat allergy and has also been established to cause cat-induced asthma. The allergen is a 38 kDa dimer composed of two 19 kDa subunits. Each 19 kDa subunit comprises two disulfide linked polypeptide chains, a light alpha-chain and a heavy beta-chain containing an N-linked oligosaccharide. In this study a variety of endoproteinase digestions of the native allergen in combination with HPLC and matrix-assisted laser desorption mass spectrometry was used to determine the position of the disulfide bridges and to demonstrate that the peptide chains are linked in an anti parallel way. Enzymatic digestion of the reduced and alkylated peptides located the N-glycan to Asn33. Moreover, Fel d1 is found to be partially truncated and to exist in several isoforms. Sequential degradation of the glycosylated peptide with specific glycosidases monitored by mass spectrometry, shows that the glycan is a heterogeneous triantennary complex type structure. The heterogeneity is caused by terminal sialic acid and a fucose residue attached to a beta-galactose residue.

Alkylation↗

PubMed1996

Biosynthesis and N-glycosylation of human interferon-gamma. Asn25 and Asn97 differ markedly in how efficiently they are glycosylated and in their oligosaccharide composition.

Interferon-gamma (IFN-gamma) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N-glycosylation, and secretion of IFN-gamma in human CD8+ T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-gamma mRNA levels were found as early as 1 h after stimulation. Maximal IFN-gamma protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-gamma mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-gamma from T cells was very rapid, the secretion half-time being approximately 20-25 min. Inhibition of N-glycosylation by tunicamycin dramatically reduced the expression of IFN-gamma, but did not block its secretion. Natural IFN-gamma is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-gamma constructs lacking either one of the potential glycosylation sites suggested that Asn25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-gamma by glycosidase treatment followed by matrix-assisted-laser-desorption-ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Asn25 consisted of fucosylated, mainly complex-type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high-mannose structures. Our results emphasize the essential role of N-linked glycans in the biology of IFN-gamma and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.

Amino Acid Sequence↗

PubMed1996

Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Amino Acid Sequence↗

PubMed1996

Ligand-binding sites in human serum amyloid P component.

Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes. AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly and specifically to certain glycosaminoglycans that are components of amyloid deposits, AP may play an important role in the maintenance of amyloid. In the present work, we isolated and identified two proteolytic fragments of AP that are responsible for its heparin-binding activity. Neither fragment corresponds to published heparin-binding sequences. The structural requirements for activity of the peptides (amino acid residues 27-38 and 192-203 of AP) were examined by means of solid-phase inhibition assays with synthetic peptides. AP-(192-203)-peptide inhibits the Ca(2+)-dependent binding of AP to heparin with an IC50 of 25 microM, while the IC50 of AP-(27-38)-peptide and AP-(33-38)-peptide are 10 microM and 2 microM, respectively. The understanding of the structure and function of active AP peptides will be useful for development of amyloid-targeted diagnostics and therapeutics.

Amino Acid Sequence↗

PubMed1996

Does matrix-assisted laser desorption/ionization mass spectrometry allow analysis of carbohydrate heterogeneity in glycoproteins? A study of natural human interferon-gamma.

Interferon-gamma (IFN-gamma) is a dimeric, secretory glycoprotein produced by T-lymphocytes. The glycan micro-heterogeneity of natural human IFN-gamma was characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) combined with glycosidase digestion. The glycan structures at the two potential glycosylation sites, asparagine 25 and 97, differ in composition and heterogeneity. The glycan at Asn 25 consists of a mixture of hybrid structures and fucosylated complex bi-, tri- and tetra-antennary structures, whereas the glycan at Asn 97 is more heterogeneous and consists of a mixture of high mannose structures, hybrid structures and unfucosylated complex bi- and tri-antennary structures. The contribution to the observed glycan heterogeneity by prompt and metastable fragmentation was evaluated by treatments with different exoglycosidases and by comparison of linear, reflected and delayed extraction MALDI/TOF mass spectra. Heterogeneity observed with the matrices alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid and 2,4,6-trihydroxyacetophenone was compared. Most of the heterogeneity can be attributed to native structure diversity and only to a minor extent to mass spectrometric fragmentation such as fragmentational loss of sialic acid residues.

Carbohydrates↗

PubMed1996

Mass spectrometric characterization of glycosylated interferon-gamma variants separated by gel electrophoresis.

Glycosylated proteins in polyacrylamide gels were characterized by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and glycosidase digestion. Sodium dodecyl sulfate-polyacrylamide get electrophoresis (SDS-PAGE) of natural, human interferon-gamma (IFN-gamma) showed two glycosylated variants with apparent molecular masses of 20 and 24 kDa. MALDI-MS of the intact IFN-gamma, electroeluted from the two bands, confirmed that these correspond to IFN-gamma molecules glycosylated at one or both of the two potential glycosylation sites, respectively. The peptide map obtained by MALDI-MS after digestion in the gel covers 92% of the IFN-gamma sequence and revealed an N-terminal pyroglutamate residue and one oxidized methionine residue. One glycosylated peptide was detected after treatment of the peptide mixture with neuraminidase, and the carbohydrate structure partially elucidated by sequential glycosidase digestion monitored by MALDI-MS. A second glycosylated peptide, due to a very heterogeneous glycan structure, could only be observed after separation of the peptides by high performance liquid chromatography (HPLC).

Cells, Cultured↗

PubMed1996

Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris.

Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.

Amylases↗