PubMed Health⌕ Search

Biomedical subjects

R A Kastelein

Publications and source records attributed to R A Kastelein.

51 records · Page 3Linked to original sources
PubMed1995

Involvement of the fourth alpha-helix of mouse granulocyte-macrophage colony-stimulating factor in binding to the alpha-subunit of the receptor complex.

Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions for residues located within alpha-helix D were examined for biological activity and receptor binding properties. Alanine substitutions of the surface exposed positions indicated that several residues contribute to the ligand-receptor interface. Position K108 and particularly D102 appeared to dominate the binding epitope recognized by mGM-R alpha. Several amino acid substitutions were made for K108 which reduced binding with concommitant losses in bioactivity. Substitutions for D102 resulted in binding affinities less than 0.1% that of the wild-type mGM-CSF and bioactivity decreased to 1.0%. Comparative analysis using high and low affinity binding conditions indicated that mGM-R beta binding was unaffected by these mutations.

Amino Acids↗
PubMed1992

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.

Animals↗
PubMed1992

Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors. Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity. In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding. Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A). Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved. The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.

Alanine↗
PubMed1991

Identification of critical amino acid residues in human and mouse granulocyte-macrophage colony-stimulating factor and their involvement in species specificity.

Segments critical to the activity of human granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified by scanning deletion analysis and compared with the critical regions previously identified in the homologous mouse GM-CSF protein. Three of the four critical regions thus identified are in equivalent positions in their respective polypeptides, while a fourth critical region of each is uniquely located. To investigate whether unique critical regions are responsible for the observed species specificity of human and mouse GM-CSF, all critical regions were substituted into their opposite homologue. This identified one specific, but different, critical region in each homologue that could not be replaced. Further characterization of the nature of the species specificity of these two proteins was accomplished by the generation of a series of human/mouse GM-CSF hybrids. Each hybrid protein was assayed for specific activity on human- and mouse GM-CSF-dependent cell lines. Significant differences in the specific activity of these hybrids was observed, suggesting that different segments of each molecule interact with their respective receptors. Based on these two approaches, individual amino acids were identified that could provide, at least in part, the interactions between these protein ligands and their respective receptors. These residues are Thr-78 and Met-80 in human GM-CSF and Asp-92, Thr-98, and Asp-102 in mouse GM-CSF.

Amino Acid Sequence↗
PubMed1991

The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors.

Transduction of the biological effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) requires the interaction of each cytokine with at least two cell surface receptor components, one of which is shared between these two cytokines. A strategy is presented that allowed us to identify receptor binding determinants in GM-CSF and IL-5. Mixed species (human and mouse) receptors were used to locate unique receptor binding domains on a series of human-mouse hybrid GM-CSF and IL-5 cytokines. Results show that the interaction of these two cytokines with the shared subunit of their high affinity receptor complexes is governed by a very small part of their peptide chains. The presence of a few key residues in the amino-terminal alpha-helix of each ligand is sufficient to confer specificity to the interaction. Comparison with other cytokines suggests that the amino-terminal helix of many of these proteins may contain the recognition element for the formation of high affinity binding sites with the alpha subunit of their multi-component receptors.

Amino Acid Sequence↗
PubMed1989

Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional three-amino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed.

Amino Acid Sequence↗
PubMed1988

Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli.

The lymphokine human interleukin 4 (IL-4) has been expressed from a plasmid in the cytoplasm of Escherichia coli. Advantage has been taken of insolubility of the human IL-4 in E. coli for rapid purification of this protein in only a few steps. We describe extraction and renaturation procedures which solubilize human IL-4 yielding biologically active protein. The protein was purified to homogeneity by one passage over a gel-filtration column. The refolded human IL-4 was characterized by N-terminal sequence analysis, amino acid analysis and bioassays. The refolded E. coli-derived human IL-4 has biological activity on T and B cells and binds to the human IL-4 receptor, comparable to mammalian expressed human IL-4, indicating that the protein is folded correctly.

Amino Acids↗
PubMed1987

Cloning and overexpression of the colicin E1 immunity gene.

A DNA fragment containing only the putative immunity gene-coding sequence was cloned under the control of the trp and lambda PL promoters, generating pRKA11 and pIPL, respectively. Escherichia coli hosts containing either construction were immune to colicin E1. Cells harboring both pIPL and pNT204, which encodes a temperature-sensitive cI repressor, were sensitive to colicin E1 at 30 degrees C, but became immune after 0.5 h of incubation at 42 degrees C. In addition, pRKA11 directed the synthesis of a 14.5-kDA protein in maxicells, identical to that found with the wild-type immunity gene. This evidence identifies unequivocally the coding sequence of the immunity gene as that extending from bases 1214 to 1552 [OKA, A., et al., Mol. Gen. Genet. 172, 151-159 (1979)]. The entire immunity gene operon was also cloned under the control of the tac promoter, generating pTCU2, which, upon induction with isopropyl beta-D-thiogalactopyranoside, produced the imm gene product in amounts sufficient to be visualized by autoradiography.

Bacteriocin Plasmids↗
PubMed1987

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

Amino Acid Sequence↗
PubMed1985

Translational interference at overlapping reading frames in prokaryotic messenger RNA.

In overlapping reading frames of prokaryotic mRNA, the ribosome-binding site (RBS) of the downstream cistron is part of the coding sequence of the upstream message. We have examined whether the rate of translation in Escherichia coli can be sufficiently high to preclude the use of an RBS in initiation of protein synthesis when it is part of an actively decoded reading frame. The two sets of gene overlap present in the RNA phage MS2 are used as a model system. We find that translation of an upstream cistron can fully block initiation of protein synthesis at the overlapping RBS of the downstream cistron. Nonsense mutations in the upstream gene restore the translation of the downstream gene.

Binding Sites↗
PubMed1983

tuf gene dosage effects on the intracellular concentration of EF-TuB.

In this paper we have studied the effect of raising the intracellular EF-Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF-Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal. We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (PL) of phage lambda. Transcription from PL can be repressed at low temperature by a temperature-sensitive repressor and activated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of PL. Cells carrying either plasmid were shown to contain an almost doubled amount of EF-Tu at temperatures from 28 degrees C to 37 degrees C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF-Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37 degrees C to 43 degrees C. The multicopy plasmid pTuB1 described by Miyajima et al. [FEBS Lett. 102, 207-210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid-borne tufB. Transformation with either plasmid raised the intracellular EF-Tu concentration by 30-60% depending on the nutritional conditions. Suppression of tufB expression was observed when the intracellular level of EF-Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF-Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.

Chromosomes, Bacterial↗
PubMed1983

Effect of the sequences upstream from the ribosome-binding site on the yield of protein from the cloned gene for phage MS2 coat protein.

The translational efficiency of the coat protein gene of phage MS2 has been examined in vivo with respect to neighbouring sequences. The cloned MS2 DNA has been gradually shortened starting at the 5' or 3' terminus, and its effect on coat protein synthesis monitored. Removal of the 3'-terminal sequences had little influence. In contrast, the gradual removal of the 5'-terminal region profoundly reduces translation. Long before the ribosomal binding site (RBS) of the coat protein (CP) gene is reached, the yield of CP has dropped by one order of magnitude. Functional half-lives of the various messengers were found not to be significantly different. Available evidence indicates that the secondary structure of the RBS in native and shortened MS2 RNA is identical. We infer that important determinants for ribosome recognition lie 5' to the RBS region of the MS2 RNA coat gene.

Base Sequence↗
PubMed1978

Non-ribosomal nucleotide sequences in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA in yeast.

The topography and the length of the non-ribosomal sequences present in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA, from the yeast Saccharomyces carlsbergensis were determined by analyzing the nucleotide sequences of the products obtained after complete digestion of 7-S RNA with RNase T1. The results show that 7-S RNA contains approximately 150 non-ribosomal nucleotides. The majority (90%) of the 7-S RNA molecules was found to have the same 5'-terminal pentadecanucleotide sequence as mature 5.8-S rRNA. The remaining 10% exhibited 5'-terminal sequences identical to those of 5.9-S RNA, which has the same primary structure as 5.8-S rRNA except for a slight extension at the 5' end [Rubin, G.M. (1974) Eur. J. Biochem. 41, 197--202]. These data show that the non-ribosomal nucleotides present in 7-S RNA are all located 3'-distal to the mature 5.8-S rRNA sequence. Moreover, it can be concluded that 5.9-S RNA is a stable rRNA rather than a precursor of 5.8-S rRNA. The 3'-terminal sequence of 5.8-S rRNA (U-C-A-U-U-UOH) is recovered in a much longer oligonucleotide in the T1 RNase digest of 7-S RNA having the sequence U-C-A-U-U-U-(C-C-U-U-C-U-C)-A-A-A-C-A-(U-U-C-U)-Gp. The sequences enclosed in brackets are likely to be correct but could not be established with absolute certainty. The arrow indicates the bond cleaved during processing. The octanucleotide sequence -A-A-A-C-A-U-U-C- located near the cleavage site shows a remarkable similarity to the 5'-terminal octanucleotide sequence of 7-S RNA (-A-A-A-C-U-U-U-C-). We suggest that these sequences may be involved in determining the specificity of the cleavages resulting in the formation of the two termini of 5.8-S rRNA.

Base Sequence↗