Tyr-129 is important to the peptide ligand affinity and selectivity of human endothelin type A receptor.

Molecular modeling and protein engineering techniques have been used to study residues within G-protein-coupled receptors that are potentially important to ligand binding and selectivity. In this study, Tyr-129 located in transmembrane domain 2 of the human endothelin (ET) type A receptor A (hETA) was targeted on the basis of differences between the hETA and type B receptor (hETB) sequences and the position of the residue on ET receptor models built using the coordinates of bacteriorhodopsin. Replacement of Tyr-129 of hETA by alanine, glutamine, asparagine, histidine, lysine, serine, or phenylalanine results in receptor variants with enhanced ET-3 and sarafotoxin 6C affinities but with unchanged ET-1 and ET-2 affinities. Except for Tyr-129-->Phe hETA, these hETA variants have two to three orders of magnitude lower binding affinity for the ETA-selective antagonist BQ123. Replacement of His-150, the residue in hETB that is analogous in sequence to Tyr-129 of hETA, by either tyrosine or alanine does not affect the affinity of peptide ligands. These results indicate that although transmembrane domain 2 is important in ligand selectivity for hETA, it does not play a significant role in the lack of ligand selectivity shown by hETB. Chimeric receptors have been constructed that further support these conclusions and indicate that at least two hETA regions contribute to ligand selectivity. Additionally, the data support an overlap in the binding site in hETA of agonists ET-3 and sarafotoxin 6C with that of the antagonist BQ123.

Molecular cloning and characterization of the major endothelin receptor subtype in porcine cerebellum.

Endothelin receptors (ETRs) display subtype heterogeneity and are widely distributed throughout the tissues of the periphery and central nervous system. In order to gain further insight into the potential molecular differences of ETRs, we initiated molecular cloning of ETR genes by screening for the appearance of 125I-ET-1 binding activity in COS cells transfected with pools of a porcine cerebellum cDNA expression library. Two independent clones (pPCETR 1.1 and pPCETR 5.6) were identified and isolated by repeated rounds of pool enrichment and COS cell expression. DNA sequence analysis of pPCET 1.1 and pPCET 5.6 indicated that both clones have the same nucleotide sequence; the deduced amino acid sequence indicated that the porcine cerebellum ETR is 443 residues in length and consists of seven potential transmembrane domains, with homology to members of the GTP-binding protein-coupled receptor superfamily. Northern analysis indicated a single mRNA species of about 5 kilobases, which is expressed significantly in cerebellum, lung, kidney, and pituitary. Expression of functional receptor was demonstrated by endothelin-1 (ET-1)-mediated Ca2+ mobilization in COS cells transfected with pPCETR 1.1 (COS/ETR 1.1) and ET-1-mediated electrophysiological responses in Xenopus oocytes injected with RNA derived from pPCETR 1.1. Quantitative comparison of saturation binding of 125I-ET-1 to either porcine cerebellum or COS/ETR 1.1 membranes indicated an identical apparent dissociation constant. The relative efficacy of ET-related peptides to compete for binding of 125I-ET-1 to receptor from porcine cerebellum and COS/ETR 1.1 indicated that both preparations encode a nonselective or ETBR subtype. Chemical cross-linking of 125I-ET-1 to receptor derived from cerebellum or COS/ETR 1 revealed two bands, with apparent molecular masses of 47 and 35 kDa. These data demonstrate that the pPCETR 1.1 encodes the major ETR subtype in the porcine cerebellum.

High-level production of fully active human alpha 1-antitrypsin in Escherichia coli.

The human alpha-1-antitrypsin (A1AT) gene expressed in Escherichia coli as a full-length, non-fusion gene product accumulates to a relatively low level approaching less than or equal to 0.1% of total cellular protein. In contrast, deletion of the first 5, 10 or 15 codons leads to production of truncated A1AT derivatives at levels between 10 and 30% of total cellular protein. The protein with the largest truncation was insoluble and inactive following solubilization by chaotropic agents. In contrast, the two derivatives with the smaller truncations were found to be soluble, and exhibit identical specific activities in both trypsin and elastase inhibition assays to authentic human A1AT. The expression of the full-length A1AT was also optimized by making silent third position mutations within its first 15 codons. These mutations were chosen to optimize codon usage and minimize the possibility of RNA secondary structure formation in this region. Via this approach, expression of full-length, authentic, fully active A1AT was increased at least 20-fold to 2% of total cellular protein. Optimal expression was obtained using as few as three silent mutations in the first five codons, confirming the importance of this 5'-terminal region as had been defined by our deletion mutants. Both the full-length derivatives as well as the small N-terminal deletion derivative can be readily purified from bacterial extracts in fully active form suitable for the examination of their potential therapeutic application.

Selection of mutations that increase alpha 1-antitrypsin gene expression in Escherichia coli.

The gene encoding human alpha-1-antitrypsin (A1AT), when cloned and expressed as a full-length, non-fusion gene product in Escherichia coli, accumulates to levels up to 0.1% of total cellular protein. Truncation of the gene at its 5' end or synthesis as a fusion protein increases expression up to 200-fold. Extensive mutagenesis in vitro within this same 5'-terminal region aimed at improving codon usage and disrupting potential secondary structure increased expression only 10 to 20-fold. We have developed a translational fusion system for selecting mutations and applied it to the study of A1AT expression in E. coli. With this methodology, we have obtained single base-pair mutations having up to a 20-fold effect on A1AT expression. When we combined these multiple single base-pair mutations, we achieve up to a 200-fold increase in A1AT expression. The resulting gene product is of authentic size (394 amino acid residues) and contains two amino acid substitutions (Asn in place of Asp) in codons 2 and 6. This protein is primarily in the soluble fraction of the E. coli lysate and has identical activity to A1AT purified from human sera. The methodology used to generate these mutations may be generally applicable to the study of genes that do not express well in E. coli initially, and provides an alternative to secondary structure analysis in the redesign of such genes.

Characterization of a crude selective PolI transcription system from Tetrahymena pyriformis.

A crude in vitro transcription system which selectively transcribes DNA fragments containing the promoter region of the Tetrahymena pyriformis rRNA gene has been prepared from T. pyriformis. The system requires both an S100 fraction of lysed isolated macronuclei and an S100 extract of whole cells. When a HhaI-HindIII fragment of the promoter containing plasmid pEN 19-1 is employed as a template, transcription yields two major products of about 560 (A) and 510 (B) bases in length. The analysis of the transcription products of truncated templates showed that RNA A is a runoff transcript and RNA B is produced by nucleolytic cleavage of RNA A at a site about 50 nucleotides to the left of the HindIII cleavage site. S1 nuclease mapping was used to demonstrate that the 5' end of RNA A is identical with that predicted for a transcript which was initiated at the same site on the gene as the in vivo 35S rRNA precursor. Transcription is dependent upon the addition of promoter containing DNA, is inhibited by 1 microgram/mL actinomycin D, and is insensitive to 200 micrograms/mL alpha-amanitin. Transcription is dependent upon the salt levels in the assay exhibiting activity peaks at 58 mM KCl, 28 mM (NH4)2SO4, and 3 mM MgCl2. Several minor transcription start sites to the left of the major initiation site become active at high salt, yielding several minor longer transcripts. High salt also inhibits the RNA cleavage activity, reducing the levels of RNA B produced.

Structure of the Tetrahymena pyriformis rRNA gene. Nucleotide sequence of the transcription initiation region.

Portions of rRNA gene repeat from Tetrahymena pyriformis were cloned in pBR322, and a subclone containing the transcription initiation site was isolated. The promoter region was identified by S1 nuclease mapping of a portion of the HindIIIB fragment of the rRNA gene, using the 35 S rRNA primary transcript as a probe. The nucleotide sequence of the initiation site and the 5'-flanking region reveals three sets of highly conserved repeat sequences. A model of the promoter region is presented in which the rDNA is wrapped around a series of histone octamers. In this model the families of repeat sequences are brought in close proximity and suggest several possible mechanisms for the regulation of rRNA synthesis in T. pyriformis.