Biomedical subjects

H Ahorn

Publications and source records attributed to H Ahorn.

32 records · Page 2Linked to original sources

PubMed1995

[Relation between structure and anti-viral activity in interferon system].

The interferons (IFN) were discovered in 1957 as biological agents interfering with viral replication. IFNs were initially classified according to their sources as leukocyte, fibroblast and immune IFNs. Both leukocyte and fibroblast IFNs are designated as Type I IFNs and immune IFN as a Type II IFN. There are almost twenty related IFNs which belong to the group of Type I IFN. IFN-alpha 2 and IFN-omega 1 are described as members of the large group of Type I IFN. Both natural IFN-alpha 2 and IFN-omega 1 are isolated from human leukocytes. IFN-alpha 2 carries an O-linked carbohydrate moiety, whereas IFN-omega 1 has an N-linked complex oligosaccharide. Recombinant IFN-alpha 2 expressed in E coli. and Sf-9 cells have been characterized. Moreover, recombinant IFN-omega 1 expressed in CHO cells and Sf-9 cells have also been characterized. In the present study, the recombinant human IFN-alpha 2 and IFN-omega 1 were analyzed for their anti-viral activity compared with native products.

Animals↗

PubMed1994

Structural and functional characterization of the interaction between 2',3'-dialdehyde guanine nucleotide analogues and the stimulatory G protein alpha-subunit.

We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombinant alpha-subunit of the stimulatory G protein, rGs alpha-s. oGTP and oGTP gamma S bind to rGs alpha-s in a quasi-irreversible manner via formation of a Schiff's base, which can be reduced with borhydrid resulting in covalent incorporation of [alpha-32P]oGTP and [35S]oGTP gamma S into rGs alpha-s. When bound to rGs alpha-s, oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rGs alpha. Thus, oGTP and oGTP gamma S act as irreversible G protein antagonist and agonist, respectively, and represent a pair of nucleotide analogues suitable as functional and structural tools. Cleavage of covalently labeled rGs alpha-s with cyanogen bromide generates several labeled fragments. Labeled fragments were assigned to the G1 and G4 region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragment was identified by amino-terminal sequencing and corresponded to the helix alpha A in the recently determined crystal structure of the transducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663). In the oGDP-liganded conformation, incorporation occurs predominantly into the G1-fragment, while [35S]oGTP gamma S labels the additional fragments to a similar extent indicating tight packing around the guanine nucleotide binding pocket in the active conformation. Furthermore, rGs alpha-s contains a single acid cleavable bond (Asp317-Pro318), such that formic acid releases a carboxyl-terminal fragment from [alpha-32P]oGTP- and [35S]oGTP gamma S-liganded rGs alpha-s. This fragment contains a single lysine residue (Lys324) which is only labeled by [35S]oGTP gamma S. Lys324 is unique to Gs alpha and lies within its effector binding region. Hence, during the switch from the inactive to the active state, this region undergoes a major conformational change that moves it closer to the nucleotide binding pocket.

Adenylyl Cyclases↗

PubMed1994

Characterization of cytosolic malic enzyme in human tumor cells.

Cytosolic NADP(+)-dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E. coli. Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human 'normal cell' ME and NADP(+)-ME from tumor cells were identical. Thus, NADP(+)-ME found in tumor cells does not constitute a tumor-specific isoform as suggested by other studies but is identical to the 'housekeeping protein' predominantly expressed in human liver and white adipose tissue.

Adipose Tissue↗

PubMed1994

Periplasmic expression of human interferon-alpha 2c in Escherichia coli results in a correctly folded molecule.

Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine.

Alkaline Phosphatase↗

PubMed1994

Isolation and functional reconstitution of a phosphate binding protein of the cyanobacterium Anacystis nidulans induced during phosphate-limited growth.

Adaptation of the blue-green algae Anacystis nidulans to phosphate-deficient growth leads to the expression of two membrane proteins, which appear as major constituents after separation by gel electrophoresis. One of these proteins, referred to as high affinity phosphate binding protein, has been isolated and its function reconstituted in liposomes. Partial sequencing showed no significant homologies to other proteins. The binding capacity of the proteoliposomes could be inhibited by arsenate but not by sulfhydryl reagents. Scatchard plot analyses of phosphate binding to reconstituted proteoliposomes suggested the existence of two different binding sites, one with a dissociation constant below micromolar and the other in the micromolar range.

Amino Acid Sequence↗

PubMed1994

2A proteinases of coxsackie- and rhinovirus cleave peptides derived from eIF-4 gamma via a common recognition motif.

The cleavage specificities of the 2A proteinases from coxsackievirus B4 (CVB4) and human rhinovirus 2 (HRV2) on oligopeptide substrates have been determined. Comparison of the specificity of CVB4 2A proteinase with that of HRV2 2A proteinase allowed cleavable peptides to be designed using the common motif IIe/Leu-X-Thr-X*Gly; little resemblance to the viral cleavage site remained. The data also allowed the prediction of three possible cleavage sites for 2A proteinases on eIF-4 gamma; two peptides derived from these sequences were cleaved by both 2A proteinases. One of these peptides corresponds to the cleavage site for 2A proteinases mapped on eIF-4 gamma [B. J. Lamphear et al. (1993) J. Biol. Chem. 268, 19200-19203]. This supports the hypothesis that cleavage of eIF-4 gamma by picornaviral 2A proteinases occurs directly.

Amino Acid Sequence↗

PubMed1993

Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen--Cas s 1.

Pollens from trees of the order Fagales (e.g. birch, alder, hazel, and hornbeam) all contain one major allergen--the main cause for tree pollen allergy. So far the major allergens from birch (Bet v 1), alder (Aln g 1), hazel (Cor a 1), and hornbeam (Car b 1) have been characterized, showing high sequence similarity with each other (1-4). We present the molecular and immunologic characterization of Cas s 1, the major allergen from the European chestnut (Castanea sativa). From aqueous pollen extracts from European chestnut a protein was purified to homogeneity and was subjected to further investigation. The protein revealed a Mr of 22 kDa and was shown to represent the major allergen of the European chestnut (immunoblotting, histamine release) and designated Cas s 1. Despite a marked difference in Mr, Cas s 1 shows significant amino acid sequence similarity at the N-terminus and is antigenically closely related to the major birch pollen allergen Bet v 1 (17 kDa), as shown by binding to the anti-Bet v 1 monoclonal antibody BIP-1 and by IgE-inhibition tests using recombinant Bet v 1.

Allergens↗

PubMed1993

Expression of human interferon omega 1 in Sf9 cells. No evidence for complex-type N-linked glycosylation or sialylation.

Human interferon omega 1 (IFN-omega 1) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. Half of the protein purified by immunoaffinity chromatography was shown to be N-glycosylated at the same site as the natural IFN-omega 1. The degree of glycosylation was independent of the expression rate. While natural IFN-omega 1 was shown to carry complex-type oligosaccharides [Adolf, G. R., Maurer-Fogy, I., Kalsner, I. & Cantell, K. (1990) J. Biol. Chem. 265, 9290-9295], the insect cell produced protein which was demonstrated by lectin blot, mass spectroscopy and HPLC analysis to contain only the core oligosaccharide. Two different structures, (Man)2(GlcNAc)2[Fuc] and (Man)3(GlcNAc)2[Fuc] were identified. The fucosylation was identified to be (alpha 1-6)-linked to the core saccharide. Sialic acid residues were clearly absent. IFN-omega 1 expressed in S. frugiperda cells was shown to be partially truncated at the C-terminus by nine residues; its antiviral activity when glycosylated was significantly lower than the activity of IFN-omega 1 produced by Sendai-virus-stimulated leukocytes. Circular dichroism and fluorescence spectroscopy did not reveal any structural differences between glycosylated and nonglycosylated IFN-omega 1. This implies the importance of a complex-type glycosylation for the maximal biological activity of human IFN-omega 1.

Animals↗

PubMed1993

Expression of human interferon-alpha 2 in Sf9 cells. Characterization of O-linked glycosylation and protein heterogeneities.

Human interferon alpha 2 (IFN-alpha 2) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. The protein purified by immunoaffinity chromatography exhibited biological activity identical to that of leukocyte-derived 'natural' IFN-alpha 2. However, the protein was found to be heterogeneously glycosylated, partially truncated by proteolysis and partially lacking a disulfide bridge. The major product was shown to be O-glycosylated at the same position as natural human IFN-alpha 2. Enzymatic cleavage, reverse-phase HPLC peptide mapping and plasma-desorption mass spectroscopy analysis revealed the presence of two types of O-linked carbohydrates. The major O-linked carbohydrate was found to be the disaccharide galactosyl(beta 1-3)-N-acetylgalactosamine, the minor component the monosaccharide N-acetylgalactosamine. No evidence for sialylation was found. The non-glycosylated species representing about 40% of the total material were shown to partially lack the C-terminal three amino acids. In addition an unglycosylated, reduction-sensitive dimer was observed. This was formed due to the lack of the N-terminal cysteine normally forming an intramolecular disulfide bridge. Furthermore, a minor species was identified which contains Cys1 and Cys98 in a modified form, thereby hindering the formation of a disulfide bridge between these two residues.

Amino Acid Sequence↗

PubMed1993

Identification of the phosphorylation site on human erythrocyte band 7 integral membrane protein: implications for a monotopic protein structure.

Band 7 membrane protein was metabolically labelled with [32P]phosphate in the presence of cAMP, isolated and digested, the labelled peptides were purified and sequenced. Ser-9 was identified as the only phosphorylation site. This proves that the N-terminal region is located at the cytoplasmic side of the membrane and implies a monotopic rather than the predicted bitopic structure.

Amino Acid Sequence↗

PubMed1993

A role for the transcription factors Mbp1 and Swi4 in progression from G1 to S phase.

In budding yeast genes that encode G1 cyclins and proteins involved in DNA synthesis are transcriptionally activated in late G1. A transcription factor, called SBF, is composed of Swi4 and Swi6 proteins and activates transcription of G1 cyclin genes. A different, but related, complex called MBF binds to MCB elements (Mlu I cell cycle box) found in the promoter of most DNA synthesis genes. MBF contains Swi6 and a 120-kilodalton protein (p120). MBF was purified and the gene encoding p120 (termed MBP1) was cloned. A deletion of MBP1 was not lethal but led to deregulated expression of DNA synthesis genes, indicating a direct regulatory role for MBF in MCB-driven transcription. Mbp1 is related to Swi4. Strains deleted for both MBP1 and SWI4 were inviable, demonstrating that transcriptional activation by MBF and SBF has an important role in the transition from G1 to S phase.

Amino Acid Sequence↗

PubMed1992

Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A.

Proteinase 2A of human rhinovirus serotype 2 (HRV2 2A) was expressed in Escherichia coli and partially purified; the preparation was used to study various enzymatic parameters. Using a 16-amino acid peptide representing the native cleavage region of HRV2 2A, an apparent Km value of 5.4 x 10(-4) mol/liter was determined. A minimum of 9 amino acids (comprising residues P8 to P1') was necessary for cleavage to occur. Proteolysis of substituted peptides was highly tolerant toward changes at P1, P2', and P3' but an absolute requirement for glycine P1' and a high preference for threonine P2 was found. Furthermore, HRV2 2A only cleaved peptide substrates derived from other rhinovirus serotypes and poliovirus that possessed P2 Thr and P1' Gly. Thus, the sequence Thr-X-Gly may form the basis of the cellular cleavage site processed by rhinoviral 2As during viral replication. Studies with various inhibitors support the hypothesis that HRV2 2A belongs to a new class of cysteine proteinases.

Amino Acid Sequence↗

PubMed1992

Comparison of the carbohydrate moieties of recombinant soluble Fc epsilon receptor (sFc epsilon RII/sCD23) expressed in Saccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells.

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFc epsilon RII/sCD23) was produced in Saccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFc epsilon RII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(alpha 2-3)Gal(beta 1-3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only alpha-mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.

Amino Acid Sequence↗

PubMed1991

Natural human interferon-alpha 2 is O-glycosylated.

Natural human interferon alpha 2 (IFN-alpha 2) was isolated from a preparation of partially purified human leucocyte IFN by monoclonal-antibody immunoaffinity chromatography. The purified protein had a specific activity of 1.5 x 10(8) i.u./mg; it was estimated to constitute 10-20% of the total antiviral activity of leucocyte IFN. N-Terminal amino-acid-sequence analysis identified the subspecies IFN-alpha 2b and/or IFN-alpha 2c, whereas IFN-alpha 2a was not detectable. The structure of natural IFN-alpha 2 was found to differ from that of its recombinant (Escherichia coli-derived) equivalent. First, reverse-phase h.p.l.c. showed that natural IFN-alpha 2 was significantly more hydrophilic then expected. Secondly, the apparent molecular mass of the natural protein determined by SDS/PAGE was higher than that of recombinant IFN-alpha 2; incubation under mild alkaline conditions known to eliminate O-linked carbohydrates resulted in a reduction of the apparent molecular mass to that of the recombinant protein. On sequence analysis of proteolytic peptides, Thr-106 was found to be modified. These results suggested that Thr-106 of natural IFN-alpha 2 carries O-linked carbohydrates. Reverse-phase h.p.l.c. as well as SDS/PAGE of natural IFN-alpha 2 showed that glycosylation is heterogeneous. For characterization of the carbohydrate moieties, the protein was treated with neuraminidase and/or O-glycanase and analysed by gel electrophoresis; in addition, glycopeptides obtained by proteinase digestion and separated by h.p.l.c. were characterized by sequence analysis and m.s. Further information on the composition of the glycans was obtained by monosaccharide analysis. The results indicate that natural IFN-alpha 2 contains the disaccharide galactosyl-N-acetylgalactosamine (Gal-GalNAc) linked to Thr-106. In part of the molecules, this core carbohydrate carries (alpha-)N-acetylneuraminic acid, whereas a disaccharide, probably N-acetyl-lactosamine, is bound to Gal-GalNAc in another proportion of the protein. Further glycosylation isomers are present in small amounts. As IFN-alpha 2 is the only IFN-alpha species with a threonine residue at position 106, it may represent the only O-glycosylated human IFN-alpha protein.

Amino Acid Sequence↗