Comparison of sequence homology of poly(A) and non-poly(A) containing 34S RNA of AKR murine leukemia virus.

AKR MuLV 70S RNA was separated on Poly(U)-Sepharose into poly(A) and non-poly(A) containing 34S subunits. The ratio of the two fractions was 2:1, respectively. Both fractions were hybridized to AKR MuLV [³H]cDNA, and the hybrids were assayed by nuclease S₁ and cesium sulfate centrifugation. The poly(A) and non-poly(A) subunits hybridized to [³H]cDNA to the same extent (80%), with identical $\text{CO}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values; and the hybrids of both fractions had identical T_m values (81°C in 0.15 M NaCl). These results demonstrate that the poly(A) and non-poly(A) containing subunits of the AKR genome have identical or very similar base sequences in the heteropolymeric regions.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.     

Purification and partial sequence analysis of murine B cell growth factor II (interleukin 5)

Murine B cell growth factor II (BCGF-II/interleukin 5) was purified from the conditioned media of the helper T cell line D10 . G4 . 1. The purification scheme consisted of sequential batch adsorption onto trimethylsilyl-controlled pore glass beads, high pressure ion exchange chromatography, and reverse phase high pressure liquid chromatography. The purified BCGF-II had a relative molecular weight of 45,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Identical analysis of BCGF-II under reducing conditions yielded a m.w. of 22,500, suggesting that native BCGF-II exists as a homodimer. The NH2-terminal amino acid sequence of the purified lymphokine was determined by automated Edman degradation. A single amino acid sequence of 24 residues was obtained that, upon comparison, was contained within the cDNA pSP6K-mTRF23 recently described as encoding murine BCGF-II/T cell-replacing factor. The NH2-terminal methionine in mature BCGF-II is found at position 21 of the amino acid sequence predicted from the cDNA pSP6K-mTRF23. This finding supports the contention of Kinashi et al. (Kinashi, T., N. Harada, E. Severinson, T. Tanabe, P. Sideras, M. Konishi, C. Azuma, A. Tominaga, S. Bergstedt-Lindqvist, M. Takahashi, F. Matsuda, Y. Yaoita, K. Takatsu, and T. Honjo. 1986. Nature 324:70) that amino acids 1-20 serve as the signal sequence for the BCGF-II gene. The ability of BCGF-II to stimulate the proliferation of the B cell lymphoma BCL1 was used to assess the potency of the lymphokine. BCGF-II at 13.5 pM induced 50% of the maximal proliferative response in the BCL1 cells; concentrations as low as 2 pM were still effective in stimulating the growth of the cells. Assuming that the amount of BCGF-II necessary to mount a 50% response in the BCL1 assay is defined as one unit of activity, then the purified BCGF-II has a specific activity of 16.5 U/ng of protein.     

Biochemical characterization of the murine S100A9 (MRP14) protein suggests that it is functionally equivalent to its human counterpart despite its low degree of sequence homology.

Due to the low degree of sequence similarity it has been speculated that murine and human S100A9 (MRP14), an inflammatory marker protein belonging to the S100 protein family, may have different cellular functions in mouse and man. The present study was undertaken to investigate the murine S100A9 protein (mS100A9) biochemically. We demonstrate that in murine peripheral CD11b+ cells up to 20% of the protein of the cytosolic fraction consists of mS100A9 and that several minor mS100A9 isoforms are present. Cell fractionation experiments with CD11b+ murine leukocytes showed that mS100A9 is found in the cytosol as well as in the insoluble fraction. Transient expression of a green fluorescence protein-mS100A9 fusion in mammalian cells revealed that mS100A9 is localized in neither the nucleus nor the vesicles. Recombinantly expressed murine S100A9 interacts in vitro with murine and human S100A8 in an in vitro glutathione S-transferase pull-down assay. Homodimerization was not observed. For further biochemical analysis the myeloid 32D cell line is presented as a suitable model, to study murine myeloid expressed S100 proteins. Both murine S100A9 and its dimerization partner mS100A8 are expressed at the onset of granulocyte-colony stimulating factor induced myeloid differentiation. Substantial amounts of this complex are constitutively secreted by granulocytic 32D cells into the medium. In summary, these data suggest, that the human and murine S100A9 may share a higher degree of functional homology than of sequence similarity.     

Arachidonate 15-lipoxygenase (omega-6 lipoxygenase) from human leukocytes. Purification and structural homology to other mammalian lipoxygenases

The enzyme responsible for 15-lipoxygenation of arachidonic acid was purified to homogeneity from human eosinophil-enriched leukocytes using a combination of ammonium sulfate precipitation, hydrophobic interaction chromatography, and high pressure liquid chromatography on hydroxyapatite and cation-exchange columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein revealed a single major band (apparent Mr 70,000). Amino acid sequence analysis yielded a single N-terminal sequence. Comparison of the N-terminal 15 residues reveals 71% sequence identity to the rabbit reticulocyte lipoxygenase and 36% sequence identity to the rat basophilic leukemia 5-lipoxygenase. In contrast, sequence identity to the soybean lipoxygenase-1 is not observed. These results demonstrate that human 15-lipoxygenase can be isolated from eosinophil-enriched leukocytes and is accessible for direct sequence analysis. Furthermore, we present initial evidence that the mammalian lipoxygenases constitute an homologous family of enzymes. The availability of homogeneous human 15-lipoxygenase will play a key role in elucidating other relationships in this family of enzymes.     

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE)

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.     

DNAMAT: an efficient graphic matrix sequence homology algorithm and its application to structural analysis

We present a fast algorithm to produce a graphic matrix representationof sequence homology. The algorithm is based on lexicographicalordering of fragments. It preserves most of the options of asimple naive algorithm with a significant increase in speed.This algorithm was the basis for a program, called DNAMAT, thathas been extensively tested during the last three years at theWeizmann Institute of Science and has proven to be very useful.In addition we suggest a way to extend our approach to analysea series of related DNA or RNA sequences, in order to determinecertain common structural features. The analysis is done by‘summing’ a set of dot-matrices to produce an overallmatrix that displays structural elements common to most of thesequences. We give an example of this procedure by analysingtRNA sequences. Received on June 26, 1986; accepted on September 28, 1986     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.     

Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T. pallidum and Borrelia burgdorferi proteins

Abstract An Escherichia coli clone containing recombinant plasmid C19 was identified from a Treponema pallidum genomic DNA library by in situ immunoassay. E. coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa, designated TpN38(b). Pulse-chase and protein processing studies showed that TpN38(b) is synthesized with a cleavable amino-terminal signal peptide. A 2.0-kb fragment of pC19 containing the tpn38(b) gene was subcloned and sequenced. The tpn38(b) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa. The deduced amino acid sequence of TpN38(b) has homology with the T. pallidum TpN35 lipoprotein and the Borrelia burgdorferi BmpA, BmpB, BmpC, and BmpD proteins.     

Immunological evidence for structural homology between Drosophila melanogaster (S14), rabbit liver (S12), Saccharomyces cerevisiae (S25), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomal proteins.

Specific antibodies directed against Drosophila melanogaster acidic ribosomal protein S14 were used in a comparative study of eucaryotic and procaryotic ribosomes by immunoblotting and enzyme-linked immunosorbent assays. Common antigenic determinants and, thus, structural homology were found between D. melanogaster, Saccharomyces cerevisiae (S25), rabbit liver (S12), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomes.     

Primary sequence and structural analysis of sterol carrier protein 2 from rat liver: homology with immunoglobulins

Sterol carrier protein 2 (SCP2) is involved in the later steps of cholesterol biosynthesis and in the intracellular transport of cholesterol. In the present investigation, the amino acid sequence of SCP2 from rat liver has been determined. It is a single polypeptide chain with 122 amino acid residues. Secondary structure prediction indicates an amphipathic alpha-helix region for residues 21-34 and antiparallel beta-sheet structure for residues 35-95. A major finding is the significant homology which exists over approximately 80 residues between SCP2 and the variable domains of the heavy chain of immunoglobulin G.     

cDNA sequence analysis of CP94: rat lens fiber cell beaded-filament structural protein shows homology to cytokeratins.

To study the molecular structure of the gene responsible for a lens fiber cell beaded-filament structural protein of 94kDa (CP94), we isolated its specific cDNA from a rat lens cDNA library by use of anti-mouse CP94 antiserum. The expressed fusion protein kept the epitopes specific against anti-chick CP97 as well as anti-mouse CP94 antibody, and the size was estimated as 190-200kDa, indicating that the cDNA insert of the clone seemed to encode a polypeptide with 80-90kDa in appearance. Northern analysis indicated that CP94 mRNA is expressed only in the lens, and not in the brain, skin, heart, kidney, lung, and liver, and the size was estimated to 2.1-2.3kb. In a lens of inherited microphthalmic mouse, Elo, a trace amount of mRNA with the size closely similar to that of rat mRNA was observed. The entire compiled sequence (1,873bp) showed an open reading frame covering the sequence of 533 amino acids totalling 58,857Da. No sequence homologous to the entire CP94 was found among the entries of any nucleotide and amino acid sequence databases; but with respect to a limited amino acid sequence of N-side region of CP94, a significant homology with cytokeratins was found.     

Redox modifications of the C-terminal cysteine residue cause structural changes in S100A1 and S100B proteins

S100 is a family of small, acidic, calcium binding proteins involved in the control of a multitude of intra- and extracellular processes, including many pathologies. The application of the analytical methodology based on the combination of RP HPLC and ESI-MS allowed for the characterization of S-nitrosylation and S-glutathionylation in two representative S100 proteins: S100A1 and S100B. The GSNO related S-nitrosylation of the conserved C-terminal cysteine is strongly activated by the binding of Ca(II) to S100A1 and of Ca(II) and Zn(II) to S100B. This modification results in a global alteration of protein structure, as demonstrated by a variety of techniques. The presented results provide a mechanistic basis for further studies of the function of S100 proteins in the control of redox-based and metal-based signal transduction.     

Purification and sequence analysis of the atypical maltohexaose-forming alpha-amylase of the B. stearothermophilus US100

The maltohexaose-forming alpha-amylase, of B. stearothermophilus US100, was purified to homogeneity by a combination of osmotic shock, starch adsorption and anion exchange chromatography. This enzyme has a relative molecular mass of 59 kDa. The analysis of the nucleotide sequence, of the corresponding gene, allowed the identification of a single open reading frame encoding a 549 amino acid protein, exhibiting a large homology to the other B. stearothermophilus alpha-amylases. This homology reaches a maximum with those of DY-5 and DN1792 strains with respectively 3 and 4 aa different over 549. The relatively small differences, between Amy US100 and that of DN1792 strain, take in more importance since we have demonstrated that these enzymes differ essentially by their starch hydrolysis pattern.