Recombinant interferon gamma for the therapy of immunological and oncological diseases.
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Escherichia coli↗
Biomedical subjects
I Maurer-Fogy
Publications and source records attributed to I Maurer-Fogy.
[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↗
Purification, cDNA cloning and heterologous expression of the human mitochondrial NADP(+)-dependent malic enzyme.
Mitochondrial NADP(+)-dependent malic enzyme (ME; EC 1.1.1.39) has been purified to homogeneity and characterized kinetically from bovine heart. Partial amino acid sequence information allowed amplification of a specific bovine cDNA, which was used to isolate a full-length human cDNA of this isoform of ME. The cDNA is 1930 bp long and codes for a protein of 604 amino acids. Comparison of the amino acid sequence of this isoform with published sequences of other human ME isoforms shows stretches of homology interrupted by larger regions with significant differences. The human protein has been expressed in Escherichia coli, and the recombinant human protein has the same kinetic properties as the corresponding protein purified from bovine heart. Northern blot analysis showed a strong tissue-specific transcription with a predominantly high expression-rate in organs with a low division-rate.
Amino Acid Sequence↗
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↗
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↗
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↗
Aggregation of phospholipid vesicles by a chimeric protein with the N-terminus of annexin I and the core of annexin V.
A chimeric protein was produced with the N-terminal domain (amino acids 1-45) of annexin I and the core of annexin V (amino acids 19-320). This protein, annexin IN-VC, has a similar Ca2+ requirement for binding to phospholipid bilayers of 20% phosphatidylserine (PS)/80% phosphatidylcholine (PC) as annexin V. In contrast to annexin V, this protein has a strong potency to aggregate phospholipid vesicles as is shown by turbidimetric measurements and cryo-electron microscopy. Ellipsometry was employed to study quantitatively the phenomenon of phospholipid vesicle adhesion to annexin IN-VC bound to a planar phospholipid bilayer. The amount of phospholipid vesicles bound by annexin IN-VC on the planar bilayer is proportional to its surface coverage and can be inhibited by coadsorption of annexin V on the planar bilayer or by shielding the phospholipid surface of the vesicles with blood coagulation factor Va. Annexin IN-VC, like annexin V, does not bind to pure PC bilayers, but its adsorption on anionic phospholipid bilayers brings about the capacity to bind pure PC vesicles. This suggests that annexin IN-VC generates or exposes after binding to anionic phospholipids another phospholipid binding site, that differs from the annexin V phospholipid binding site. Collectively, the data suggest that two-dimensional cluster formation of annexin IN-VC on a bilayer with anionic phospholipids is involved in vesicle adherence.
Adsorption↗
Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase.
The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.
Amino Acid Sequence↗
Natural human tumor necrosis factor beta (lymphotoxin). Variable O-glycosylation at Thr7, proteolytic processing, and allelic variation.
Natural human tumor necrosis factor beta (TNF-beta) purified from supernatants of a human B-lymphoblastoid cell line was found to be heterogeneous in molecular mass, with seven components resolved by gel electrophoresis. All components are N-glycosylated at Asn62; N-glycosylation does not contribute to heterogeneity. In addition, part of the molecules are O-glycosylated at Thr7; O-glycosylation is heterogeneous due to variable decoration with neuraminic acid. The four lower molecular mass forms are derived from the full-length protein by trypsin-like proteolytic cleavage in the N-proximal region; these clipped molecules lack O-linked carbohydrates. Two allelic variants differing in amino acid position 26 (threonine/asparagine) were identified.
Alleles↗
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↗
Identification of functionally active fragments of staphylococcal enterotoxin B.
It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N-terminal sequences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N-terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin. The toxin's C-terminal fragment possesses an ability to activate calmodulin-dependent enzymes and is probably the toxicogenic part of the enterotoxin.
Amino Acid Sequence↗
Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.
Methylation of mammalian DNA can lead to repression of transcription and alteration of chromatin structure. Recent evidence suggests that both effects are the result of an interaction between the methylated sites and methyl-CpG-binding proteins (MeCPs). MeCP1 has previously been detected in crude nuclear extracts. Here we report the identification, purification, and cDNA cloning of a novel MeCP called MeCP2. Unlike MeCP1, the new protein is able to bind to DNA that contains a single methyl-CpG pair. By staining with an antibody, we show that the distribution of MeCP2 along the chromosomes parallels that of methyl-CpG. In mouse, for example, MeCP2 is concentrated in pericentromeric heterochromatin, which contains a large fraction (about 40%) of all genomic 5-methylcytosine.
Amino Acid Sequence↗
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↗
Constitutive production of interferon-alpha 2 by a human B-lymphoblastoid cell line.
Human B-lymphoblastoid cell lines immortalized by Epstein-Barr virus constitutively produce low levels of acid-stable interferon (IFN) that differs in composition from IFN produced by the same cell type after virus induction. We use three approaches to study the constitutive IFN produced by the lymphoblastoid cell line, LuKII: (i) antiviral bioassays with a neutralizing monoclonal antibody; (ii) amino-terminal sequence analysis of the single protein obtained after purification of the IFN by immunoaffinity chromatography and reverse-phase HPLC; (iii) sequence analysis of cDNA clones obtained by screening a LuKII library with an IFN-alpha 2 probe. Our data show that more than 90% of the constitutive IFN results from expression of a single gene coding for IFN-alpha 2. The significance of these results is discussed.
Amino Acid Sequence↗
Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis.
BSAP has been identified previously as a transcription factor that is expressed at early, but not late, stages of B-cell differentiation. Biochemical purification and cDNA cloning has now revealed that BSAP belongs to the family of paired domain proteins. BSAP is encoded by the Pax-5 gene and has been highly conserved between human and mouse. An intact paired domain was shown to be both necessary and sufficient for DNA binding of BSAP. Binding studies with several BSAP recognition sequences demonstrated that the sequence specificity of BSAP differs from that of the distantly related paired domain protein Pax-1. During embryogenesis, the BSAP gene is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other Pax genes in the developing central nervous system (CNS). Later, the expression of the BSAP gene shifts to the fetal liver where it correlates with the onset of B lymphopoiesis. BSAP expression persists in B lymphocytes and is also seen in the testis of the adult mouse. All of this evidence indicates that the transcription factor BSAP may not only play an important role in B-cell differentiation but also in neural development and spermatogenesis.
Amino Acid Sequence↗
Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.
RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen.
Amino Acid Sequence↗
Cloning and nucleotide sequence of cDNA encoding human erythrocyte band 7 integral membrane protein.
cDNA clones encoding the human erythrocyte band 7 membrane protein were isolated by immunoscreening from bone marrow and HeLa cell lambda gt 11 cDNA libraries, and their nucleotide sequences were determined. HeLa- and bone marrow cell-derived sequences were identical, except for one nucleotide; the deduced sequence of 287 amino acids was confirmed by sequence identity with peptides of the erythroid protein. Structure analysis assigned band 7 protein to the type Ib transmembrane proteins.
Amino Acid Sequence↗
Human interferon omega 1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein.
A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.
Amino Acid Sequence↗