Formation and properties of S-protein complex with S-peptide-containing fusion protein

A fusion protein (FP) comprised of the RNase A S-peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S-protein at a stoichiometric ratio 1:1 with Kdiss = 5.0 x 10(-7) M. The S-protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis. The fusion protein can be determined specifically and quantitatively in the presence of S-protein by RNase activity assays. The possibility of effective purification of S-peptide-containing proteins by affinity chromatography on an S-protein-Sepharose column has been demonstrated.     

Binding characteristics of Escherichia coli enterotoxin b (STb) to the pig jejunum and partial characterization of the molecule involved

Escherichia coli heat-stable enterotoxin b (STb) causes severe diarrhoea in weaning piglets. STb most probably has to bind to intestinal epithelial cells in order to achieve its effect. Using biotinylated biologically active STb, we developed a semi-quantitative binding assay using indirect fluorescence microscopy. We demonstrated the attachment of the biotinylated toxin to microvilli of the pig jejunum. However, binding was abolished when biotinylated STb was either boiled or treated with 2-mercaptoethanol, treatments known to abolish biological activity. Different characteristics of STb attachment to the pig small intestine were determined. The reaction was rapid and reached maximum intensity after approximately 10 min. The binding was pH dependent showing an optimum at pH 5.8. Incubation at either 4 degrees C, 25 degrees C or 37 degrees C did not affect the binding. No competition was observed with non-biotinylated STb. However, preincubation of biotinylated STb with streptavidin conjugated to horseradish peroxidase completely abolished the binding. Pig tissues other than jejunum demonstrated binding towards STb including duodenum, ileum, caecum, colon, liver, lung, spleen and kidney. The molecule involved was then partially characterized. Metaperiodate treatment of the jejunum sections abrogated binding but protease treatment had no effect. Enzymatic treatments of jejunal sections demonstrated that N- and O-glycosidases, and several exoglycosidases did not affect binding, whereas reduced binding was observed with ceramide glycanase and alpha-glucosidase, and was completely abolished following neuraminidase treatment. Overall, our results suggest that in vitro STb binding was rapid, pH dependent, temperature independent, not restricted to jejunum and involves a molecule that seems to be composed of a ceramide moiety, terminal neuraminic acid and/or alpha-linked terminal glucose residue(s).     

Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q and H13A, at 2.0 A resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.     

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.     

DNA "melting" proteins. I. Effects of bovine pancreatic ribonuclease binding on the conformation and stability of DNA

Bovine pancreatic ribonuclease is a DNA "melting" protein, since it binds with greater overall affinity to the single-stranded than to the double-stranded form of natural and synthetic deoxyribose-containing polynucleotides. As such, the DNA-RNase system provides a simple model for the more complex and biologically relevant melting protein-nucleic acid systems. Aspects of the DNA-RNase interactions which are related to the quantitative assessment of this system as a melting protein model are investigated here. A boundary sedimentation velocity technique is used to measure thermodynamic parameters of the interaction; association constants (Kh and Kc) and site sizes (nh and nc) are determined for the interaction of ribonuclease with native (double helical) and denatured (random coil) DNA. It is shown that log Kh and log Kc are linear functions of log [Na+], binding decreasing with increasing Na+ concentration, with Kh about 2 orders of magnitude smaller than Kc at the ionic strengths studied, nh and nc are approximately 8 and approximately 11 nucleotide residues, respectively, indicating that potential binding sites overlap. Binding to both forms of DNA is non-cooperative. It is shown by CD and ultraviolet spectroscopy that the binding of RNase to single- and double-stranded DNA perturbs the conformations of these polynucleotide conformations very little relative to the unliganded structures. Hydrodynamic methods are used to show that RNase binds to native DNA without altering the overall solution structure of the latter; however conditons which permit binding to, and stabilization of, transiently exposed single-stranded sequences result in a collapse of the stiff native DNA structure. We demonstrate by melting transition studies that ribonuclease does bring about an equilibrium destabilization of native DNA and poly [d(A-T)] and, by applying a ligand-perturbed helic in equilibrium coil theory developed by McGhee (McGhee, J.D. (1976) Biopolymers 15, 1345-1375), it is shown that the extent of the observed destabilization is in semiquantitative accord with expectations based on the measured affinity constants and site sizes for RNase binding to both DNA conformations. Spectral methods are used to show that the relative stability of native DNA sequences of varying base composition is the same in the presence and absence of ribonuclease, strongly arguing that this "melting" ligand "traps" single-stranded sequences transiently exposed by thermal fluctuations. RNase also undergoes an order in equilibrium disorder conformational transition as a function of temperature (the denatured form of RNase stabilizes native DNA, while native RNase destabilizes the native double helix), and the coupled equilibria involved in these interacting conformational changes are interpreted and discussed as possible models of genome regulatory interactions.     

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.     

The scapular manipulation method for reducing anterior shoulder dislocations

Poly[2'-O-(2,4-dinitrophenyl)]poly(A)[DNP-poly(A)] has been found to be a potent inhibitor in solution for RNases A, B, S, T1, T2 and H as well as phosphodiesterases I and II. Kinetic measurements with RNase B and RNase T1 showed DNP-poly(A) to be a reversible competitive inhibitor with K1 equal to 1.03 and 1.05 microM, respectively. Data on the quenching of fluorescence of RNase T1 by DNP-poly(A) indicate the existence of more than one RNase-binding site in each DNP-poly(A) molecule. By attaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column was prepared for selective removal of RNases from aqueous solutions by simple filtration. It was found that a 1000-fold reduction in RNase concentration can be obtained by passing either 7.0 microM or 7.0 nM RNase A solution through a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solution and subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedly with no significant decrease in RNase-binding affinity and capacity. By titration of the derivatized beads with RNase, the first dissociation constant (Kd) and binding capacity for the bound enzyme can be determined. The (Kd) was found to be 0.66 microM for RNase B and 0.48 microM for RNase T1; the corresponding binding capacities were found to be 21.0 x (10)-8 and 9.6 x (10)-8 mol/g, respectively.     

The chlamydial EUO gene encodes a histone H1-specific protease

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.     

Cloning, subcellular localization and functional expression of human RNase HII

Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.     

Differential exposure of surface epitopes in the beta-strand region of LOOP1 of the yeast H+-ATPase during catalysis

The plasma membrane H+-ATPase of yeast assumes distinct conformational states during its catalytic cycle. To better understand structural changes in the LOOP1 domain, a catalytically important cytoplasmic loop segment linking transmembrane segments 2 and 3, surface epitopes were examined at different stages of catalysis. A polyclonal rabbit antibody was prepared to a fusion protein consisting of LOOP1 and the maltose binding protein. This antibody was affinity-purified to produce a LOOP1-specific fraction that could be used in competition enzyme-linked immunosorbent assays to assess surface exposure of the LOOP1 epitopes. It was found that in an E1 conformation stabilized with either adenosine 5'-(beta,gamma -imino)triphosphate (AMP-PNP) or ADP, less than 10% of the LOOP1 epitopes were accessible on native enzyme. However, when the enzyme was stabilized in an E2-state with ATP plus vanadate, approximately 40% of the surface epitopes on LOOP1 became accessible to antibody. The remaining 60% of the LOOP1 epitopes were fully occluded in the native enzyme and never showed surface exposure. Enzyme-linked immunosorbent assays utilizing fusion proteins consisting of LOOP1 subdomains demonstrated that all of the available epitopes were contained in the beta-strand region (Glu-195-- Val-267) of LOOP1. The epitopes that were differentially exposed during catalysis were included in regions upstream and downstream of the highly conserved TGES sequence. Our results suggest that during catalysis either the beta-strand region of LOOP1 or an interacting domain undergoes substantial structural rearrangement that facilitates epitope exposure.     

Reinventing the academic medical center

In this study we have demonstrated that the native rabbit sperm protein, Sp17, is a 22- to 24-kDa triplet of proteins in washed ejaculated rabbit spermatozoa and is unaffected by capacitation. However, during the acrosome reaction, Sp17 is processed from a 22- to 24-kDa triplet of proteins to a triplet of proteins at 17-19 kDa by the removal of amino acids from the C-terminal. Recombinant rabbit Sp17 (rRSp17) can also be proteolytically processed by acrosome-reacted spermatozoa in a similar manner. Protease inhibitors prevent the proteolytic processing of Sp17. Both forms of native Sp17 remain associated with acrosome-reacted spermatozoa and are solubilized by ionic detergents. Previously, sequence analysis of Sp17 revealed that Sp17 amino acids 108-137 were 52% identical to the calmodulin binding domain of neuromodulin and contained an IQ motif found in other calmodulin binding proteins. In this study, a truncated recombinant Sp17, rRSp17CB, which lacks amino acids 118-146, including the potential calmodulin binding site, was made. Recombinant rabbit Sp17, but not rRSp17CB, binds to calmodulin in the presence of Ca2+ or EDTA, under reduced or nonreduced conditions in biotinylated-calmodulin overlay assays. In DSS crosslinker experiments, calmodulin bound to rRSp17 in a 1:1 ratio but not to rRSp17CB. Additionally, biotinylated rRSp17 interacts with native sperm calmodulin. We propose that the processing of native Sp17, by removing a C-terminal fragment during the acrosome reaction, might be a mechanism to regulate the calmodulin binding activity of Sp17 and provide calmodulin at specific sites after the acrosome reaction.     

In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and alpha-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of alpha-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For alpha-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.     

Expression and possible role of muscle-type carnitine palmitoyltransferase I during sperm development in the rat

Because we had found whole testis from adult rats to be much richer in the messenger RNA for the muscle (M) than for the liver (L) form of mitochondrial carnitine palmitoyltransferase I (CPT I), we sought to determine which cell type(s) accounts for this expression pattern and how it might relate to reproductive function. Studies with immature (14-day-old) and adult animals included 1) Northern blot analysis of testis mRNA; 2) in situ hybridization with slices of testis; 3) enzyme assays for CPT I, CPT II, and carnitine acetyltransferase (CAT) in testicular germ cells and nongerm cells, together with measurement of the malonyl-coenzyme A (CoA) sensitivity and affinity for carnitine of CPT I; 4) labeling of testicular CPT I with [3H]etomoxir, a covalent inhibitor of the enzyme; and 5) the response of testicular and nontesticular CPT I to dietary etomoxir. The data established the following: 1) L-CPT I was the sole isoform detected in immature testis. 2) Expression of the M-CPT I gene was associated only with meiotic and postmeiotic germ cells. 3) Adult testis contains a mixture of the L- and M-CPT I enzymes, the L and M form dominating in extratubular cells and spermatids, respectively. Mature epididymal spermatozoa appear to be devoid of CPT I activity while possessing abundant levels of CPT II and CAT. 4) Five days of dietary etomoxir treatment at a dose that resulted in essentially complete inhibition of CPT I in liver, heart, skeletal muscle, and kidney was totally without effect on either the L- or M-type enzyme in the testis of mature rats. The data point to an important role for transient expression of M-CPT I, coupled with sustained activity of CAT, in the maturation and/or function of rat sperm. They also suggest that, at least in the case of one CPT I inhibitor (etomoxir), the testis is unusually resistant to the agent when given orally.     

Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.