Two-domain structure of the td intron-encoded endonuclease I-TevI correlates with the two-domain configuration of the homing site.

I-TevI, the T4 td intron-encoded endonuclease, catalyzes the first step in intron homing by making a double-strand break in the intronless allele within a sequence designated the homing site. The 28 kDa enzyme, which interacts with the homing site over a span of 37 bp, binds as a monomer, contacting two domains of the substrate. In this study, limited proteolysis experiments indicate that I-TevI consists of two domains that behave as discrete physical entities as judged by a number of functional and structural criteria. Overexpression clones for each domain were constructed and the proteins were purified. The carboxy-terminal domain has DNA-binding activity coincident with the primary binding region of the homing site and binds with the same affinity as the full-length enzyme. The isolated amino-terminal domain, contains the conserved GIY-YIG motif, consistent with its being the catalytic domain. Furthermore, site-directed mutagenesis of a conserved arginine residue within the extended motif rendered the full-length protein catalytically inactive, although DNA-binding was maintained. This is the first evidence that the GIY-YIG motif is important for catalytic activity. An enzyme with an N-terminal catalytic domain and a C-terminal DNA-binding domain connected by a flexible linker is in accord with the bipartite structure of the homing site.

High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli.

The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.

Alpha-fetoprotein derived synthetic peptides: assay of an estrogen-modifying regulatory segment.

This study describes the estrogen bioassay of a synthetic peptide fashioned after an amino acid sequence from human alpha-fetoprotein (HAFP). The synthetic peptide (P149), modeled after a portion of the estrogen binding pocket of rat/human AFP chimeras, was produced via F-MOC solid phase chemistry. Bioassay of P149 in the estrogen-sensitive immature rodent uterus demonstrated an anti-estrogenic (40-50% inhibitory) activity in the 23 h but not the 3-4 h uterine response. In contrast to purified HAFP, incubation of the peptide with estrogen was not a prerequisite for inhibitory activity. The estrogen-dependent increase in uterine thrombin and tissue factor, as determined by an enzymatic esterase assay, was inhibited by 30% in rat uterine cytosols. In an in vitro bioassay of estrogen-induced focus formation in MCF-7 human breast cancer cultures, focus development was inhibited by 70% following peptide exposure. The mechanism of the AFP-derived peptide inhibition of estrogen-dependent growth remains to be determined.

An overall perspective of X-ray damage to a DNA oligomer mediated by reactive oxygen species.

The products produced by X irradiation of an oxygenated aqueous solution containing d(CpApTpG) were analyzed by NMR spectroscopy and mass spectrometry. Thirteen different base modifications were detected, including a novel product formed by the addition of oxygen to guanine. Seven different strand break products were identified, including strands having 5'-phosphoryl groups, 3'-phosphoryl groups and groups having 3'-phosphoglycolates as termini. The products produced in largest yield contained base modifications: Pyrimidine bases degraded to a formamido moiety, the 8-oxo-7,8-dihydroguanine (8-oxoguanine) lesion, and double base lesions in which both the 8-oxo-7,8-dihydroguanine lesion and a formamido remnant are present.

Novel, specific O-glycosylation of secreted Flavobacterium meningosepticum proteins. Asp-Ser and Asp-Thr-Thr consensus sites.

A new type of O-linked oligosaccharide has been discovered on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum, including Endo F2 (three sites), Endo F3 (one site), and a P40 protease (one site). The oligosaccharide moiety is covalently attached via a mannose residue to a serine or threonine at consensus sites corresponding to Asp-Ser* or Asp-Thr*-Thr. Preliminary characterization by mass spectroscopy revealed an oligosaccharide of 1244 Da at each of the proposed glycosylation sites. Collision-associated dissociation analysis showed a characteristic daughter ion series of m/z 218, 394, and 556, indicative of a common Flavobacterium oligosaccharide. Compositional analysis demonstrated an unusual profile of monosaccharides, including hexoses, methylated hexoses, and uronic acid derivatives.

Protein sequencing by tandem mass spectrometry.

Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.

Molecular cloning and sequence analysis of flavastacin: an O-glycosylated prokaryotic zinc metalloendopeptidase.

A new zinc metalloendopeptidase that cleaves peptides on the amino-terminal side of aspartic acid was isolated from the cultural filtrate of Flavobacterium meningosepticum. The gene for this new enzyme was cloned into pBluescript, and the complete nucleotide sequence was determined. Over 40% of the deduced amino acid sequence was verified independently by direct protein microsequencing. The most important structural features of this new enzyme include (i) the presence of an unusual O-linked oligosaccharide of unknown function located at a unique consensus site near the C-terminus and (ii) a characteristic extended zinc-binding site and corresponding Met-turn that places this metalloendopeptidase in the astacin family. This is the first example of a prokaryotic enzyme related to the eukaryotic astacin group; it is being designated hereafter as flavastacin.

Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: a single gene encodes the alpha and beta subunits.

A full-length insert for the Flavobacterium meningosepticum N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500-bp HindIII fragment and cloned into the plasmid vector pBluescript. DNA sequencing revealed an open reading frame of 1020 nucleotides encoding a putative 45-amino-acid leader sequence and a deduced precursor polypeptide of 295 amino acids. In F. meningosepticum this precursor polypeptide undergoes proteolytic processing by an as yet unknown mechanism to generate an alpha-subunit and a beta-subunit, which constitute the active form of the heterodimeric mature glycosylasparaginase. The Flavobacterium glycosylasparaginase gene was expressed in Escherichia coli and found to be enzymatically active. The recombinant enzyme was purified from crude lysates and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of the typical alpha- and beta-subunits. The recombinant beta-subunit cross-reacted to antibody specific for the rat liver beta-subunit, and Edman analysis demonstrated that its amino-terminus corresponded exactly to that of the mature native glycosylasparagine beta-subunit. A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 30% structural identity and 60% overall similarity between the prokaryotic and eukaryotic forms of the enzyme. Even more striking was the conservation of the amino acid sequence in both proteins where the post-translational cleavage to generate the active enzyme occurs. Our data demonstrate that deglycosylation of asparagine-linked glycans via hydrolysis of the AspNHGlcNAc linkage is an important reaction which has been preserved during evolution.

Mass spectrometry and amino acid sequencing of two cadmium-binding metallothionein isoforms from the terrestrial gastropod Arianta arbustorum.

1. Two cadmium-binding metallothionein (Mt) isoforms, called Mta and Mtb, were isolated from terrestrial snails (Arianta arbustorum), using various chromatographic techniques, such as gel-permeation chromatography and reversed-phase HPLC. The purified proteins were S-methylated and cleaved by means of different enzymes (trypsin, endoproteinase Glu-C, and endoproteinase Asp-N). Amino acid sequences were determined by automated Edman degradation and collision-induced dissociation (CID) tandem MS. According to their primary structures, both isoforms should be attributed to class-I Mts. 2. The two forms are structurally identical, differing only by one amino acid exchange in position 60 of the peptide chain. Both isoproteins consist of 66 amino acids, 18 of which are cysteine residues. Most of the cysteine residues are arranged in seven Cys-Xaa-Cys motifs. Mta and Mtb possess an N-terminal acetylated-serine residue and contain a short N-terminal motif which shows a high degree of similarity with the N-termini of histones H4 and H2A. 3. A comparison of Mta and Mtb with other invertebrate Mts shows a very high degree of sequence similarity with a cadmium-binding Mt from Helix pomatia, a species that is closely related to Arianta arbustorum. Moreover, Mta and Mtb, as expected, also exhibit structural similarities with Mts from other molluscan species, such as mussels and oysters. It is suggested that Mta and Mtb represent two allelic isoforms, reflecting the genetic polymorphism of Mt in Arianta arbustorum.

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization.

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]+ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thiols with pyrrolylated peptides.

Identification of disulfide bridges in a cardiotoxic peptide by electrospray ionization.

A new method was developed for subjecting a peptide to a specified number of Edman degradation cycles on an automated polypeptide sequencer and desorbing the residual peptide for further investigations. The procedure was applied in combination with electrospray ionization mass spectrometry to identify the four disulfide bridges present in a small tightly bound peptide. The task was accomplished using only a few nanomoles of the intact peptide.

Detailed structural analysis of a novel, specific O-linked glycan from the prokaryote Flavobacterium meningosepticum.

In the preceding paper, preliminary analysis revealed a new type of O-linked oligosaccharide of 1244 Da at each of two proposed glycosylation sites on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum (Plummer, T. H., Jr., Tarentino, A. L., and Hauer, C. R. (1995) J. Biol. Chem. 270, 13192-13196). In this report we detail the linkage, sequence, and branching of this unusual heptasaccharide by electrospray (ES) ionization mass spectrometry (MS), and collision-induced dissociation (CID). The proposed structure was supported by a combination of isotopic labeling, composition and methylation analysis, and the preparation of several chemical analogs and derivatives with each product evaluated by MS and CID. The singly branched structure contained seven residues, including three different uronyl analogs: a methylated rhamnose and mannose, a glucose, and a reducing terminal mannose. Only pyranose ring forms were detected ((2-OMe)Man1-4GlcNAcU1-4GlcU1-4Glc1-4(2-OMe)G lcU-4 [(2-OMe)Rham1-2]Man).

Analysis of CA(2+)-binding S100 proteins in human heart by HPLC-electrospray mass spectrometry.

Calcium dependent processes are often impaired in myocardial dysfunctions and calcium-binding proteins might be involved as mediators of Ca2+ signals. Here we present a method to assess a footprint of the calcium-binding proteins of the S100 family in human heart. The S100 proteins are purified through calcium dependent affinity chromatography and reverse phase high-performance liquid chromatography and are analyzed by electrospray-ionization mass spectrometry (ESI-MS) and liquid secondary ionization/tandem quadrupole mass spectrometry (LS-MS/MS). In human heart we identified S100 alpha, CACY, and CAPL as the most abundant S100 proteins and showed that they occur as monomers and homodimers.

6-pyruvoyl tetrahydropterin synthase from salmon liver: amino acid sequence analysis by tandem mass spectrometry.

The most frequent variant of atypical phenylketonuria, an inborn error of metabolism, is characterized by a low activity of the 6-pyruvoyl tetrahydropterin synthase. We purified and characterized this enzyme from salmon liver known to contain high levels. After digestion, peptides were sequenced by tandem mass spectrometry and/or automated Edman microsequence analysis. Both a free amine terminus and an N-acetylated amine terminus were found, indicating the presence of two isoforms. The peptide sequences determined here have a high degree of homology with the protein sequence deduced from cDNA for rat 6-pyruvoyl tetrahydropterin synthase (1), however, the amine termini of these proteins differ significantly.

Identification and characterization of mycobacterial proteins differentially expressed under standing and shaking culture conditions, including Rv2623 from a novel class of putative ATP-binding proteins.

The environmental signals that affect gene regulation in Mycobacterium tuberculosis remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including acr (also known as hspX) (alpha-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling of M. bovis BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified M. tuberculosis Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in M. tuberculosis's response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.

Molecular cloning, primary structure, and properties of a new glycoamidase from the fungus Aspergillus tubigensis.

A new glycoamidase, peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubigensis. The enzyme was purified to homogeneity, and the DNA sequence was determined by cloning in Escherichia coli. Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/or electrospray ionization-mass spectrometry of protease fragments of native PNGase At. This glycoamidase contains 12 potential asparagine-linked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides. PNGase At consists of two non-identical glycosylated subunits that are derived from a single polypeptide gene precursor. Evidence is presented suggesting that autocatalysis is involved in subunit formation. PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad range of glycopeptides, including those with core-linked alpha1-->6 or alpha1-->3 fucose, under conditions not favorable with existing glycoamidases.

Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry.

A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N6-[14C]benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-His-Asp-Ala-Asp-Glu.

Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A.

A cadmium-binding metallothionein has been purified from metal-exposed Roman snails (Helix pomatia) using gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography. The S-methylated protein was digested with trypsin and the endoproteinases Asp-N, Glu-C and Arg-C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N-terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N-terminal amino acid was an acetylated serine residue. Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys-Xaa-Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N-terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N-terminal sequences of histones H2A and H4 which may be important for regulatory functions.