A coenzyme A-independent transacylase is linked to the formation of platelet-activating factor (PAF) by generating the lyso-PAF intermediate in the remodeling pathway

The remodeling pathway for the biosynthesis of platelet-activating factor (PAF) consists of the following reaction sequence: alkylacylglycerophosphocholine----lyso-PAF----PAF. Results presented in this article describe a novel transacylase activity that generates the lyso-PAF intermediate, which can then be acetylated to form PAF. Ethanolamine-containing lysoplasmalogens, 1-acyl-2-lyso-sn-glycero-3-phosphoethanolamine, alkyllysophosphoethanolamine, unlabeled lyso-PAF, 1-acyl-2-lyso-GPC, where GPC is sn-glycero-3-phosphocholine, and choline-containing lysoplasmalogens were all able to stimulate the formation of [3H]lyso-PAF from a [3H]alkylacyl-GPC precursor pool associated with HL-60 cell (granulocytic type) membranes. Other glycerolipids containing free hydroxyl groups (3-alkyl-2-lyso-sn-glycero-1-phosphocholine, lysophosphatidylserine, lysophosphatidylinositol, diacylglycerols, alkylglycerols, and monoacylglycerols), cholesterol, phosphatidylcholine, and phosphatidylethanolamine had no stimulatory effect on the release of [3H]lyso-PAF from the prelabeled membranes under identical incubation conditions. The observed transacylase reaction is directly coupled to PAF production, since the addition of a lysoethanolamine plasmalogen preparation to HL-60 membranes in the presence of [14C]acetyl-CoA stimulated PAF formation; under these conditions the lysoethanolamine plasmalogen was acylated. The transacylase responsible for the release of lyso-PAF from the membrane-associated alkylacyl-GPC was not affected by Ca2+, EGTA, or a known phospholipase A2 inhibitor, p-bromophenacyl bromide. The fact that the unnatural analog of lyso-PAF, lysophosphatidylserine, and lysophosphatidylinositol did not influence transacylase activity, whereas detergents such as deoxycholate and Triton X-100 inhibited the activity, demonstrated the observed stimulatory effects of the choline- and ethanolamine-containing lysophospholipids on the formation of [3H]lyso-PAF from [3H]alkylacyl-GPC were not due to any detergent property of these lysophospholipids. Thus, we conclude a CoA-independent transacylase (possessing phospholipase A2/acyltransferase activities) can be responsible for the formation of the lyso-PAF intermediate in the remodeling route of PAF biosynthesis.     

Isolation and characterization of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminases.

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.     

Mutations in the small subunit of cyanobacterial ribulose-bisphosphate carboxylase/oxygenase that modulate interactions with large subunits

In the cyanobacterium Anacystis nidulans (Synechococcus PCC6301), ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) is composed of eight large subunits and eight small subunits. There are three regions of the small subunit that contain amino acids that are conserved throughout evolution, from bacteria to higher plants. Since the function of the small subunit is not fully understood, site-directed mutagenesis was performed on highly conserved residues in the first and second conserved regions. Ser-16, Pro-19, Leu-21, and Tyr-54 were replaced by Asp-16, His-19, Glu-21, and Ser-54, respectively. Crude extracts containing the recombinant His-19 mutant enzyme indicated that there was little effect on either Rbu-P2 carboxylase activity or interactions between large and small subunits. However, the Asp-16, Glu-21, and Ser-54 mutations showed effects on Rbu-P2 carboxylase activity and the interaction between large and small subunits. The large and small subunits of the Asp-16, Glu-21, and Ser-54 enzymes were found to dissociate during nondenaturing gel electrophoresis or sucrose density gradient centrifugation. However, the dissociated small subunits remained functional and were capable of reconstituting Rbu-P2 carboxylase activity when added to large subunits. These results indicated that Ser-16, Leu-21, and Tyr-54 might play an important role in interactions between large and small subunits of the A. nidulans enzyme.     

Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis

The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to Asp57, Asp13, and Lys109 in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is Asp57, and in the three-dimensional structure of CheY the Asp57 carboxylate side chain is in close proximity to the beta-carboxylate of Asp13 and the epsilon-amin of Lys109. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution. Asn for Asp and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the Asp to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys109 mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys109 mutant protein cannot produce tumbly behavior. Thus, Lys109 is required for an event subsequent to phosphorylation. We propose that an interaction between the epsilon-amino of Lys109 and the phosphoryl group at Asp57 is essential for the conformational switch that leads to activation of CheY.     

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.     

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.     

Multiexon deletion in the procollagen III gene is associated with mild Ehlers-Danlos syndrome type IV

We have characterized a deletion of approximately 9 kilobases which spans from intron 33 to exon 48 of one pro-alpha 1 (III) collagen allele in a patient with Ehlers-Danlos syndrome type IV. The mutation results in the production of an in-frame species of mRNA which lacks the sequences corresponding to residues 595-1,008 of the triple-helical domain. Thus, half of the pro-alpha 1 (III) chains synthesized by the patient's fibroblasts are nearly 30% shorter than normal. The procollagen III molecules composed of either three normal length or three shortened chains are thermally stable and efficiently secreted. In contrast, the procollagen III molecules that contain one or two shortened chains are unstable and are not secreted. Failure to secrete unstable molecules and a residual functional role of the shortened but stable homotrimers may explain the somewhat milder phenotype of this individual compared with that of another Ehlers-Danlos type IV patient bearing a deletion of similar size in the amino-terminal portion of the alpha 1 (III) collagen chain.     

Mutants of initiator tRNA that function both as initiators and elongators

We describe the effect of mutations in the acceptor stem of Escherichia coli initiator tRNA on its function in vivo. The acceptor stem mutations were coupled to mutations in the anticodon sequence from CAU----CUA to allow functional studies on the mutant tRNAs in initiation and in elongation in vivo. We show that, with one exception, there is a good correlation between the kinetic parameters for formylation of the mutant tRNAs in vitro (preceding paper, Lee, C.P., Seong, B. L., and RajBhandary, U.L. (1991) J. Biol. Chem. 266, 18012-18017) and their activity in initiation in vivo. These results suggest an important role for formylation of initiator tRNA in its function in initiation, at least when it is aminoacylated with glutamine as is the case with the mutant tRNAs used here. Mutant tRNAs that have a base pair between nucleotides 1 and 72 at the top of the acceptor stem function as elongators, as analyzed by their ability to suppress an amber mutation in the E. coli beta-galactosidase gene. One of these mutants is also quite active in initiation. Thus, activities of a tRNA in initiation and elongation steps of protein synthesis are not mutually exclusive. Using a mRNA with two in frame UAG codons, we show that this mutant tRNA can both initiate protein synthesis from the upstream UAG and suppress the down-stream UAG. We discuss the potential use of tRNAs with such "dual" functions in tightly regulated expression of genes for proteins in E. coli.