The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical.

Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals. Removal of the signals is carried out by specific, membrane-bound processing peptidases. In this report we show that the reaction specificities of these three peptidases are essentially identical. Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase. Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E. coli enzymes generates the correct mature amino terminus. Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase. No evidence of incorrect processing was obtained. Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.     

Fibroblast cellular and plasma fibronectins are similar but not identical

Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.     

Requirements for glycosylphosphatidylinositol attachment are similar but not identical in mammalian cells and parasitic protozoa

The general features of the glycosylphosphatidylinositol (GPI) signal have been conserved in evolution. To test whether the requirements for GPI attachment are indeed the same in mammalian cells and parasitic protozoa, we expressed the prototype GPI-linked protein of Trypanosoma brucei, the variant surface glycoprotein (VSG), in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI linked. This impaired processing is not caused by the VSG ectodomain, since replacement of the VSG GPI signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Furthermore, whereas fusion of the DAF GPI signal to the COOH terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous hGH fusion using the VSG GPI signal does not, indicating that the VSG GPI signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI signals and fusing them to the COOH terminus of hGH, we show that of the two critical elements that comprise the GPI-signal--the cleavage/attachment site and the COOH terminal hydrophobic domain--the former is responsible for the impaired activity of the VSG GPI signal in COS cells. To confirm this, we show that the VSG GPI signal can be converted to a viable signal for mammalian cells by altering the amino acid configuration at the cleavage/attachment site. We also show that when fused to the COOH terminus of hGH, the putative GPI signal from the malaria circumsporozoite (CS) protein produces low levels of GPI- anchored hGH, suggesting that the CS protein is indeed GPI linked, but that the CS protein GPI signal, like the VSG-signal, functions poorly in COS cells. The finding that the requirements for GPI attachment are similar but not identical in parasitic protozoa and mammalian cells may allow for the development of selective inhibitors of GPI-anchoring that might prove useful as antiparasite therapeutics.     

TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast.

The Saccharomyces cerevisiae genes TOR1 and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TOR1 gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TOR1 is not essential, but a TOR1 TOR2 double disruption uniquely confers a cell cycle (G1) arrest as does exposure to rapamycin; disruption of TOR2 alone is lethal but does not cause a cell cycle arrest. TOR1-TOR2 and TOR2-TOR1 hybrids indicate that carboxy-terminal domains of TOR1 and TOR2 containing a lipid kinase sequence motif are interchangeable and therefore functionally equivalent; the other portions of TOR1 and TOR2 are not interchangeable. The TOR1-1 and TOR2-1 mutations, which confer rapamycin resistance, alter the same potential protein kinase C site in the respective protein's lipid kinase domain. Thus, TOR1 and TOR2 are likely similar but not identical, rapamycin-sensitive PI kinases possibly regulated by phosphorylation. TOR1 and TOR2 may be components of a novel signal transduction pathway controlling progression through G1.     

Rhizobium etli CE3 bacteroid lipopolysaccharides are structurally similar but not identical to those produced by cultured CE3 bacteria

Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin.     

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.     

Cholecystokinin-58 and cholecystokinin-8 produce similar but not identical activations of myenteric plexus and dorsal vagal complex

The enteric nervous system (ENS: myenteric and submucosal plexuses) of the gastrointestinal tract may have a role in the reduction of food intake by cholecystokinin (CCK). Exogenous cholecystokinin-8 (CCK-8) activates the myenteric plexus and the feeding control areas of the dorsal vagal complex (DVC) of the brainstem. An increasing number of reports, however, have shown that CCK-58 is the sole or the major circulating form of CCK in rat, human and dog, and that it is qualitatively different from CCK-8 in evoking various gastrointestinal physiological responses (e.g., contraction of the gallbladder and exocrine pancreatic secretion). In the current report, we compared the abilities of exogenous CCK-58 to activate the myenteric plexus and the dorsal vagal complex with those of exogenous CCK-8 by quantifying Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation). We report that CCK-58 (1, 3, and 5 nmol/kg) increased Fos-LI in the myenteric plexus (p<0.001) and in the DVC (p<0.001) compared to the saline vehicle. The highest dose of CCK-58 increased Fos-LI more than an equimolar dose of CCK-8 in the myenteric plexus and the area postrema. Thus, CCK-8 and CCK-58 produce the same qualitative pattern of activation of central and peripheral neurons, but do not provoke identical quantitative patterns at higher doses. The different patterns produced by the two peptides at higher doses, in areas open to the circulation (myenteric plexus and area postrema) may reflect endocrine actions not observed at lower doses.     

The two Xenopus Gbx2 genes exhibit similar, but not identical expression patterns and can affect head formation.

Gbx2 homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. Two Gbx2 genes were identified in Xenopus laevis, differing in 13 amino acids, including a change in the homeodomain. Xgbx2a is activated earlier during gastrulation and reaches higher levels of expression while Xgbx2b is expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns.     

Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical.

Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli. The Ntg1 and Ntg2 proteins were overexpressed in E.coli and purified to apparent homogeneity. The substrate specificity of Ntg1 and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas chromatography/isotope-dilution mass spectrometry. The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(dG-dC). Excision was measured as a function of enzyme concentration and time. Furthermore, kinetic parameters were determined for each lesion. The results show that kinetic constants varied among the different lesions for the same enzyme. We also investigated the capacity of the Ntg1 and Ntg2 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases. The results show that the Ntg1 protein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine. Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua/Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenine, thymine or cytosine. In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases. These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells.     

The biological activities of gro beta and IL-8 on human neutrophils are overlapping but not identical.

Gro beta and IL-8 are two members of the small induced secreted (SIS) cytokine family (C-X-C subgroup) with proinflammatory activities on neutrophils. In order to assess whether or not the interaction with their receptors results in similar biological actions, we compared the two cytokines in five different bioassays. Gro beta showed similar biological activities as IL-8 in tests of chemotaxis, induction of the respiratory burst, and induction of interleukin 6 (IL-6) production. However, for two other biological activities: augmentation of the expression of CD11b on the cell surface and rapid elevation of the intracellular calcium concentration, maximal effects required 100 times more gro beta than IL-8. Taken together, these results suggest that the stimulation of the IL-8 or gro beta receptor evokes three similar responses, but that only the activation of the IL-8 receptor and not that of gro beta results in elevated CD11b expression and calcium mobilization in human neutrophils.     

Sterol transfer between cyclodextrin and membranes: similar but not identical mechanism to NPC2-mediated cholesterol transfer

Niemann--Pick C disease is an inherited disorder in which cholesterol and other lipids accumulate in the late endosomal/lysosomal compartment. Recently, cyclodextrins (CD) have been shown to reduce symptoms and extend lifespan in animal models of the disease. In the present studies we examined the mechanism of sterol transport by CD using in vitro model systems and fluorescence spectroscopy and NPC2-deficient fibroblasts. We demonstrate that cholesterol transport from the lysosomal cholesterol-binding protein NPC2 to CD occurs via aqueous diffusional transfer and is very slow; the rate-limiting step appears to be dissociation of cholesterol from NPC2, suggesting that specific interactions between NPC2 and CD do not occur. In contrast, the transfer rate of the fluorescent cholesterol analogue dehydroergosterol (DHE) from CD to phospholipid membranes is very rapid and is directly proportional to the acceptor membrane concentration, as is DHE transfer from membranes to CD. Moreover, CD dramatically increases the rate of sterol transfer between membranes, with rates that can approach those mediated by NPC2. The results suggest that sterol transfer from CD to membranes occurs by a collisional transfer mechanism involving direct interaction of CD with membranes, similar to that shown previously for NPC2. For CD, however, absolute rates are slower compared to NPC2 for a given concentration, and the lysosomal phospholipid lysobisphosphatidic acid (LBPA) does not stimulate rates of sterol transfer between membranes and CD. As expected from the apparent absence of interaction between CD and NPC2, the addition of CD to NPC2-deficient fibroblasts rapidly rescued the cholesterol accumulation phenotype. Thus, the recent observations of CD efficacy in mouse models of NPC disease are likely the result of CD enhancement of cholesterol transport between membranes, with rapid sterol transfer occurring during CD--membrane interactions.     

Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificities

The genome of Arabidopsis thaliana contains six putative cell-wall type invertase genes (AtcwINV1-6). Heterologous expression of AtcwINV1, 3 and 6 cDNAs in Pichia pastoris revealed that the enzymes encoded by AtcwINV3 and 6 did not show invertase activity. Instead, AtcwINV3 is a 6-FEH and AtcwINV6 is a fructan exohydrolase (FEH) that can degrade both inulin and levan-type fructans. For AtcwINV6 it is proposed to use the term (6&1) FEH. In contrast, AtcwINV1 is a typical invertase. FEH activity was also detected in crude extracts of different parts of Arabidopsis. To verify that the FEH activity of AtcwINV3 and 6 were not artefacts of the heterologous expression system, the protein corresponding to AtcwINV3 was isolated from whole Arabidopsis plants and indeed showed only 6-FEH activity and no invertase activity. Although no fructans can be detected in Arabidopsis plants, it is shown that kestoses (trimers) can be synthesized in crude leaf extracts. The putative physiological significance of FEH in so-called non-fructan plants is discussed.     

Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity.

Interactions between similar but not identical (homeologous) DNA sequences play an important biological role in the evolution of genes and genomes. To gain insight into the underlying molecular mechanism(s) of genetic recombination, we have studied inter- and intramolecular homeologous recombination in S. cerevisiae during transformation. We found that homeologous DNAs recombine efficiently. Hybrid sequences were obtained between two mammalian cytochrome P450 cDNAs, sharing 73% identity, and between the yeast ARG4 gene and its human homeologous cDNA, sharing 52% identity. Sequencing data showed that the preferred recombination events are those corresponding to the overall alignment of the DNA sequences and that the junctions are within stretches of identity of variable length (2-21 nt). We suggest that these events occur by a conventional homologous recombination mechanism.     

Colchicine-binding sites of brain tubulins from an antarctic fish and from a mammal are functionally similar, but not identical: implications for microtubule assembly at low temperature.

The tubulins of Antarctic fishes possess adaptations that favor microtubule formation at low body temperatures (Detrich et al.: Biochemistry 28:10085-10093, 1989). To determine whether some of these adaptations may be present in a domain of tubulin that participates directly or indirectly in lateral contact between microtubule protofilaments, we have examined the energetics of the binding of colchicine, a drug thought to bind to such a site, to pure brain tubulins from an Antarctic fish (Notothenia gibberifrons) and from a mammal (the cow, Bos taurus). At temperatures between 0 and 20 degrees C, the affinity constants for colchicine binding to the fish tubulin were slightly smaller (1.5-2.6-fold) than those for bovine tubulin. van't Hoff analysis showed that the standard enthalpy changes for colchicine binding to the two tubulins were comparable (delta H degrees = +10.6 and +7.4 kcal mol-1 for piscine and bovine tubulins, respectively), as were the standard entropy changes (delta S degrees = +61.3 eu for N. gibberifrons tubulin, +51.2 eu for bovine tubulin). At saturating concentrations of the ligand, the maximal binding stoichiometry for each tubulin was approximately 1 mol colchicine/mol tubulin dimer. The data indicate that the colchicine-binding sites of the two tubulins are similar, but probably not identical, in structure. The apparent absence of major structural modifications at the colchicine site suggests that this region of tubulin is not involved in functional adaptation for low-temperature polymerization. Rather, the colchicine site of tubulin may have been conserved evolutionarily to serve in vivo as a receptor for endogenous molecules (i.e., "colchicine-like" molecules or MAPs) that regulate microtubule assembly.     

The bacteriophage T2 and T4 DNA-[N6-adenine] methyltransferase (Dam) sequence specificities are not identical.

Bacteriophages T2 and T4 encode DNA-[N6-adenine] methyltransferases (Dam) which differ from each other by only three amino acids. The canonical recognition sequence for these enzymes in both cytosine and 5-hydroxymethylcytosine-containing DNA is GATC; at a lower efficiency they also recognize some non-canonical sites in sequences derived from GAY (where Y is cytosine or thymine). We found that T4 Dam fails to methylate certain GATA and GATT sequences which are methylated by T2 Dam. This indicates that T2 Dam and T4 Dam do not have identical sequence specificities. We analyzed DNA sequence data files obtained from GenBank, containing about 30% of the T4 genome, to estimate the overall frequency of occurrence of GATC, as well as non-canonical sites derived from GAY. The observed N6methyladenine (m6A) content of T4 DNA, methylated exclusively at GATC (by Escherichia coli Dam), was found to be in good agreement with this estimate. Although GATC is fully methylated in virion DNA, only a small percentage of the non-canonical sequences are methylated.     

Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes.

Vipp1 (vesicle inducing protein in plastids 1) is found in cyanobacteria and chloroplasts where it is essential for thylakoid formation. Arabidopsis thaliana mutant plants with a reduction of Vipp1 to about 20% of wild type content become albinotic at an early stage. We propose that this drastic phenotype results from an inability of the remaining Vipp1 protein to assemble into a homo-oligomeric complex, indicating that oligomerization is a prerequisite for Vipp1 function. A Vipp1-ProteinA fusion protein, expressed in the Deltavipp1 mutant background, is able to reinstate oligomerization and restore photoautotrophic growth. Plants containing Vipp1-ProteinA in amounts comparable to Vipp1 in the wild type exhibit a wild type phenotype. However, plants with a reduced amount of Vipp1-ProteinA protein are growth-retarded and significantly paler than the wild type. This phenotype is caused by a decrease in thylakoid membrane content and a concomitant reduction in photosynthetic activity. To the extent that thylakoid membranes are made in these plants they are properly assembled with protein-pigment complexes and are photosynthetically active. This strongly supports a function of Vipp1 in basic thylakoid membrane formation and not in the functional assembly of thylakoid protein complexes. Intriguingly, electron microscopic analysis shows that chloroplasts in the mutant plants are not equally affected by the Vipp1 shortage. Indeed, a wide range of different stages of thylakoid development ranging from wild-type-like chloroplasts to plastids nearly devoid of thylakoids can be observed in organelles of one and the same cell.     

Class I molecules with similar peptide-binding specificities are the result of both common ancestry and convergent evolution

HLA class I molecules can be classified into supertypes associated with overlapping peptide-binding motifs and repertoires. Herein, overlaps in peptide-binding and T-cell recognition repertoires were demonstrated between mouse and human molecules. Since rodent and primate lineages separated before the current allelic variation of mouse and human class I molecules, these data demonstrate that supertypic specificities originated by convergent evolution. Phylogenetic and structural analyses demonstrated that convergent evolution also occurs amongst primates and within the human species, resulting from the selection of different pocket structures having similar specificity or independent repeated selection of the same pocket structure.