Residues in Torpedo californica acetylcholinesterase necessary for processing to a glycosyl phosphatidylinositol-anchored form.

Acetylcholinesterase from Torpedo californica (TcAChE) can be found as a glycosyl phosphatidylinositol (GPI)-anchored, membrane associated form. The C-terminal amino-acid sequence of the precursor protein resembles the signal peptide sequence found in proteins and enzymes destined for GPI-modification. Characteristics of such a signal peptide are a relatively polar stretch of amino acids, separating a cleavage- and modification-site (omega-site) residue from a hydrophobic C-terminus. We have introduced mutations, both at putative omega-sites and in the hydrophobic region, and analysed their effects on GPI-anchoring of TcAChE. Our results show that substitution of all three Ser residues in the region Ser542-Ser544 prevents GPI-modification and membrane anchoring. Individual substitution of each of these residues resulted in no or only a minor effect on the modification. We therefore conclude that more than one residue within this sequence can be utilised as the omega-site. Our analyses of double substitutions indicated that Ser543 and Ser544 are the preferred residues for GPI-modification. Moreover, the hydrophobic region is shown to be essential for GPI-anchoring of TcAChE.

Optimising the signal peptide for glycosyl phosphatidylinositol modification of human acetylcholinesterase using mutational analysis and peptide-quantitative structure-activity relationships.

Glycosyl phosphatidylinositol (GPI)-modified proteins have a C-terminal signal peptide (GPIsp) that mediates the addition of a GPI-anchor to an amino acid residue at the cleavage and modification site (omega-site). Within the GPIsp, a stretch of hydrophilic amino acid residues are found which constitutes the spacer region that separates the omega-site residue from a hydrophobic C-terminus. Deletions and insertions into the spacer region of human acetylcholinesterase (AChE) show that the length of this spacer region is very important for efficient GPI-modification. Surprisingly, the natural length of the spacer region in human AChE was not optimal for the highest degree of GPI modification. The importance of the two adjacent residues downstream of the omega-site, the omega+1 and omega+2 residues, was investigated by peptide-quantitative structure-activity relationships (Peptide-QSAR). A model was made that predicts the efficiency of the GPI modification when these residues are substituted with others, and suggests important features for these residues. The most preferred omega+1 and omega+2 residues, predicted by the model, in combination with an ideal spacer length resulted in an optimised GPIsp. This mutant protein is more efficiently GPI-modified than any mutant AChE tested thus far.

Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase.

The three-dimensional crystal structure of the glycosyl phosphatidylinositol (GPI)-modified form of Torpedo acetylcholinesterase reveals the participation of Arg-44 and Glu-92 in a salt bridge and a hydrogen bond between Asp-93 and Tyr-96. To investigate the biological significance of these interactions, we have made amino acid replacements in this form of AChE: R44E, R44K, E92Q, E92L, D93N, and D93V. None of the introduced mutations affected the production of the acetylcholinesterase polypeptide significantly. However, the mutations introduced at position 92, as well as the D93V and R44E mutations, resulted in a total loss of surface located, active acetylcholinesterase. Replacement of Asp-93 with Asn resulted in a reduced amount of active enzyme. This mutant enzyme was indistinguishable from the wild-type enzyme regarding catalytic constants, but was more sensitive to thermal inactivation. The results show that the salt bridge and hydrogen bond involving residues Arg-44, Glu-92, and Asp-93 have important structural roles and are needed for correct folding, required for transport to the cell surface of TcAChE. The GPI-modified form of acetylcholinesterase is a disulfide bonded dimer. Cys-537 is shown to be required for the formation of the intersubunit disulfide bond in the dimer. Replacement with Ser resulted in the production of an enzyme, that migrates as a monomer upon non-reducing SDS-PAGE and has a lower stability compared to the dimeric wild-type enzyme.

Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli.

During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene.

Cloning and sequencing of a cDNA encoding human milk beta-casein.

A cDNA of 1065 bp encoding the human milk beta-casein was cloned and sequenced using a synthetic oligodeoxyribonucleotide probe and a human mammary gland library. The nucleotide (nt) sequence contained an open reading frame sufficient to encode the entire amino-acid (aa) sequence of a beta-casein precursor protein consisting of 210 aa and a signal peptide of 15 aa. The nt sequence shows 45-62% homology to those of bovine, ovine, rat, and mouse beta-caseins. The highly phosphorylated site, which is responsible for the calcium-binding capacity of beta-casein, the signal peptide, and a sequence encoding for an inhibitor to the angiotensin-converting enzyme seem highly conserved among the beta-caseins with known sequences.

Gc X and Gc Y revealed by immunofixation electrophoresis.

Immunofixation electrophoresis is used to define two variants in the Gc system: Gc X and Gc Y. Gc X has one band with a mobility between Gc 1-1 and Gc 2-2 while Gc Y has two bands migrating faster than the cathodal band of Gc 1.

Distribution of alpha-1-antitrypsin phenotypes in Sweden.

The distribution of phenotypes of alpha-1-antitrypsin (Pi) in 1,062 unrelated Swedes was determined by isoelectric focusing with carrier ampholytes. The frequencies calculated were: PiM1 = 0.6940, PiM2 = 0.1384, PiM3 = 0.1139, PiZ = 0.0231, PiS = 0.0245, PiF = 0.0038, Pivar = 0.0024. A mother-child material consisting of 194 pairs is also presented.

Five new Gc variants detected by isoelectric focusing in agarose gel.

Five new genetically determined Gc variants were observed by isoelectric focusing. Seven rare variants 1A4, 1Cl, 1C3, 1C9, 1C11, 2A2, and 2A5 were also found in the material comprising Danish and Swedish paternity cases. All the variants were further analysed by electrophoresis in agarose gel. Two of the new variants had double bands of which the anodal one was susceptible to neuraminidase treatment (Gc 1C13 and 1C14). The three other new variants appeared as a single band, which was unaffected by neuraminidase treatment (Gc 2A9, 2C5, and 2C6). The Gc Ar variant originally detected by electrophoresis was reexamined by isoelectric focusing and named 2C4.

Implementation and dissemination of methods for prevention of alcohol problems in primary health care: a feasibility study.

Secondary prevention of alcohol problems in health care has been proved efficacious in many studies, yet its implementation remains scarce, and its effectiveness in regular health care remains unknown. This article reports results from a feasibility study of dissemination of alcohol prevention methods in primary health care in Stockholm. Initial interviews with general practitioners (GPs) and district health nurses indicated that few raised the issue of alcohol with patients, made notes about alcohol in patient charts or found working with alcohol issues rewarding. The impact of a training session, where a project nurse visited all willing GPs and nurses, was limited. Although the uptake of the prevention package was high, follow-up at 3 months indicated that little use was made of the materials. Specifically, screening rates were low. In the future, secondary prevention of alcohol problems will require better adaptation to the realities of primary care.

Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1.

The effect of the copy number of plasmid R1drd-19 on cell division of Escherichia coli K-12 was studied in populations growing as steady-state cultures at different growth rates, the growth rate being varied by use of different carbon sources. The plasmid copy number was also varied by using copy mutants of the R-factor. The mean cell size was larger in populations carrying an R-factor than in R-factorless populations, an effect that was more pronounced at low growth rates and in populations carrying R-factor copy mutants. The increased cell size was due to formation of elongated cells in a fraction of the population and to an increase in the diameter of all cells. The majority of the cells divided at a normal cell length, but the presence of an R-factor caused some cells to elongate, probably by the uncoupling of chromosome replication and cell division. This can be explained as a competition between the chromosome and plasmid replicons for some replication factor(s), presumably acting on both initiation and elongation of replication. The formation of elongated cells was a reversible process, but occasionally some of the elongated cells reached lengths 20 times that of newborn cells. If cell division did not occur at the normal cell size, the septum was not formed until the cell size was four times that of a newborn cell. When an elongated cell divided, it usually formed a polar septum, thus producing a newborn cell of normal cell length. The ability of plasmid-containing cells to omit one cell division but to retain the capacity of dividing one mass doubling later is compatible with a mechanical model for septum formation and cell division.

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii.

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.

Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli.

The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.

Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase.

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been isolated and the nucleotide sequence determined. The cDNA has a very high G+C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approximately 50%) with the final two-thirds of the sequences of all known eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergences are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved from the CuZn SODs before the evolution of fungi and plants.

Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product.

A complementary DNA clone from human placenta, encoding human extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1), has recently been isolated and characterized. An expression plasmid, based on the EC-SOD complementary DNA, was transfected into Chinese hamster ovary cells (CHO-K1). The transfected cells secreted human EC-SOD to the culture medium. The secreted recombinant (r) EC-SOD was isolated in high yield with a three-step procedure beginning with immobilized monoclonal anti-EC-SOD antibodies. The properties of the rEC-SOD were compared with native (n) EC-SOD isolated from human umbilical cords. The specific activities and amino-terminal amino acid sequences were identical. The amino acid compositions were virtually identical and very similar to the composition deduced from the complementary DNA sequence. Both rEC-SOD and nEC-SOD contained 4 Cu and 4 Zn atoms per molecule, and the presence of Zn in EC-SOD is thus now established. The rEC-SOD produced is type C, since its affinity for heparin-Sepharose was identical to that of nEC-SOD type C. Both enzymes bound to concanavalin A, lentil lectin, and wheat germ lectin and are thus glycoproteins. rEC-SOD and nEC-SOD seem to have the same subunit structure and composition as analyzed by polyacrylamide gel electrophoresis and gel chromatography.

Autoimmunity and ALS: studies on antibodies to acetylcholinesterase in sera.

The involvement of the immune system in the pathogenesis of amyotrophic lateral sclerosis is controversial. It has been suggested that ALS patients develop specific antibodies against acetylcholinesterase (AChE) and that these antibodies by retrograde axonal transport may be the cause of death of the spinal motor neurons. It has also been argued that these antibodies elicit hemolysis of erythrocytes. However, using recombinant human AChE as antigen in ELISA and Western blot analysis, we have been unable to find any evidence for the existence of specific AChE antibodies in ALS patients.

The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide.

The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.

Group specific component and susceptibility to HIV infection and progression to AIDS.

The distribution of phenotypes of the group specific component (Gc) was examined in 85 AIDS patients and in 40 couples, each consisting of one HIV seropositive patient and one seronegative sexual partner. Phenotype and allele frequencies in these groups did not differ significantly from those in a Swedish control population. Our observations did not indicate any involvement of the Gc system in susceptibility to HIV infection or progression to AIDS.

A non-glycosylated extracellular superoxide dismutase variant.

The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.

Sequencing of the Francisella tularensis strain Schu 4 genome reveals the shikimate and purine metabolic pathways, targets for the construction of a rationally attenuated auxotrophic vaccine.

Francisella tularensis is the etiological agent of tularemia, a serious disease in several Northern hemisphere countries. The organism has fastidious growth requirements and is very poorly understood at the genetic and molecular levels. Given the lack of data on this organism, we undertook the sample sequencing of its genome. A random library of DNA fragments from a highly virulent strain (Schu 4) of F. tularensis was constructed and the nucleotide sequences of 13,904 cloned fragments were determined and assembled into 353 contigs. A total of 1.83 Mb of nucleotide sequence was obtained that had a G+C content of 33.2%. Genes located on plasmids pOM1 and pNFL10, which had been previously isolated from low virulence strains of F. tularensis, were absent but all of the other known F. tularensis genes were represented in the assembled data. F. tularensis Schu4 was able to grow in the absence of aromatic amino acids and orthologues of genes which could encode enzymes in the shikimate pathway in other bacteria were identified in the assembled data. Genes that could encode all of the enzymes in the purine biosynthetic and most of the en- zymes in the purine salvage pathways were also identified. This data will be used to develop defined rationally attenuated mutants of F. tularensis, which could be used as replacements for the existing genetically undefined live vaccine strain.