Comparison Of Amino Acid Composition Research Articles

Predicting Proteolysis in Complex Proteomes Using Deep Learning.

Both protease- and reactive oxygen species (ROS)-mediated proteolysis are thought to be key effectors of tissue remodeling. We have previously shown that comparison of amino acid composition can predict the differential susceptibilities of proteins to photo-oxidation. However, predicting protein susceptibility to endogenous proteases remains challenging. Here, we aim to develop bioinformatics tools to (i) predict cleavage site locations (and hence putative protein susceptibilities) and (ii) compare the predicted vulnerabilities of skin proteins to protease- and ROS-mediated proteolysis. The first goal of this study was to experimentally evaluate the ability of existing protease cleavage site prediction models (PROSPER and DeepCleave) to identify experimentally determined MMP9 cleavage sites in two purified proteins and in a complex human dermal fibroblast-derived extracellular matrix (ECM) proteome. We subsequently developed deep bidirectional recurrent neural network (BRNN) models to predict cleavage sites for 14 tissue proteases. The predictions of the new models were tested against experimental datasets and combined with amino acid composition analysis (to predict ultraviolet radiation (UVR)/ROS susceptibility) in a new web app: the Manchester proteome susceptibility calculator (MPSC). The BRNN models performed better in predicting cleavage sites in native dermal ECM proteins than existing models (DeepCleave and PROSPER), and application of MPSC to the skin proteome suggests that: compared with the elastic fiber network, fibrillar collagens may be susceptible primarily to protease-mediated proteolysis. We also identify additional putative targets of oxidative damage (dermatopontin, fibulins and defensins) and protease action (laminins and nidogen). MPSC has the potential to identify potential targets of proteolysis in disparate tissues and disease states.

Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction

Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.

Favorable and unfavorable amino acid residues in water-soluble and transmembrane proteins

We analyzed the amino acid residues present in the water-soluble and transmembrane proteins of 6 thermophilic and 6 mesophilic species of the domains Archaea and Eubacteria, and characterized them as favorable or unfavorable. The characterization was performed by comparing the observed number of each amino acid residue to the expected number calculated from the percentage of nucleotides present in each gene. Amino acids that were more or less abundant than expected were considered as favorable or unfavorable, respectively. Comparisons of amino acid compositions indicated that the water-soluble proteins were rich in charged residues such as Glu, Asp, Lys, and His, whereas hydrophobic residues such as Trp, Phe, and Leu were abundant in transmembrane proteins. Interestingly, our results found that although the Trp residue was abundant in transmembrane proteins, it was not defined as favorable by our calculations, indicating that increased numbers of a particular amino acid does not necessary indicate it is a favorable residue. Amino acids with high G + C content such as Ala, Gly, and Pro were frequently observed as favorable in species with low G + C content. Comparatively, amino acids with low G + C content such as Phe, Tyr, Lys, Ile, and Met were frequently observed as favorable in species with high G + C content. These are the examples to increase the supply of amino acids than expected. Amino acids with neutral G + C content, i.e., Glu and Asp were favorable in water-soluble proteins from all species analyzed, and Cys was unfavorable both in water-soluble and transmembrane proteins. These results indicate that amino acid compositions are essentially determined by the nucleotide sequence of the genes, and the amino acid content is altered by a deviation from expectation.

In silico characterization of thermostable lipases

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.

Comparative genetic mutation frequencies based on amino acid composition differences

Genetic variation inferred from large-scale amino acid composition comparisons among genomes and chromosomes of several species, Saccharomyces cerevisiae, Drosophila melanogaster, Ceanorhabditis elegans, H. sapiens, is shown to be correlated (highest, r 2 = 0.9855, p < 0.01) with reported mutation rates for various genes in these species. This study, based largely on pseudogene data, helps to establish reference mutation frequencies that are likely to be representative of overall genome mutation rates in each of the species examined, and provides further insight into heterogeneity of mutation rates among genomes.

Comparison of amino acid compositions of peptides eluted from HLA-B27 molecules of healthy individuals and patients with ankylosing spondylitis

HLA-B27 is a relative risk factor for ankylosing spondylitis (AS) and is present in about 10% in European populations but in 95% of AS patients. Various data suggest that the HLA-B27 molecule itself could be the strongest risk factor, but there is no explanation for this association. To define differential antigen presenting features of HLA-B27 in healthy individuals and AS patients, a question that cannot be addressed by biochemical studies on cell lines, the HLA-B27 protein was purified from peripheral blood lymphocytes of AS patients and healthy controls and pool sequencing of the bound peptides was performed. Results show that peptides are rich in proline (Pro) and the content of arginine (Arg) is much lower in comparison with sequences listed in the register of peptides eluted from cell cultures. Statistically significant differences were detected in frequencies of a subset of amino acids, predominantly at positions in the middle of the peptides. The frequency of Glu was increased and Gln was decreased in peptides from AS patients. Detailed analysis of purity of the immunoisolated HLA molecules excluded that the peptides might originate from any co-purified HLA molecules other than B27. We conclude that statistically significant increase in the Glu/Gln ratio of peptides from AS patients, consistent with increased deamidation in vivo, may account for differential antigenicity of HLA-B27 in patients. Source protein(s) of deamidated peptides remain unknown.

On the Informational Content of Overlapping Genes in Prokaryotic and Eukaryotic Viruses

. In genetic language a peculiar arrangement of biological information is provided by overlapping genes in which the same region of DNA can code for functionally unrelated messages. In this work, the informational content of overlapping genes belonging to prokaryotic and eukaryotic viruses was analyzed. Using information theory indices, we identified in the regions of overlap a first pattern, exhibiting a more uniform base composition and more severe constraints in base ordering with respect to the nonoverlapping regions. This pattern was found to be peculiar to coliphage, avian hepatitis B virus, human lentivirus, and plant luteovirus families. A second pattern, characterized by the occurrence of similar compositional constraints in both types of coding regions, was found to be limited to plant tymoviruses. At the level of codon usage, a low degree of correlation between overlapping and nonoverlapping coding regions characterized the first pattern, whereas a close link was found in tymoviruses, indicating a fine adaptation of the overlapping frame to the original codon choice of the virus. As a result of codon usage correlation analysis, deductions concerning the origin and evolution of several overlapping frames were also proposed. Comparison of amino acid composition revealed an increased frequency of amino acid residues with a high level of degeneracy (arginine, leucine, and serine) in the proteins encoded by overlapping genes; this peculiar feature of overlapping genes can be viewed as a way with which they may expand their coding ability and gain new, specialized functions.

Characterisation and purification of ribulose‐bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium, Haloferax mediterranei

The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.

Comparison of amino-acid compositions and E 4/E 6 ratios of soil and water humic substances fractions obtained by polyacrylamide gel electrophoresis

Groundwater fulvic acids (GWFA), river fulvic acids (RFA), soil humic acids (SHA), and soil fulvic acids (SFA) were fractionated by electrophoresis in 10% polyacrylamide gel in the presence of denaturing agents. The humic substances (HS) from each source separated into several discrete fractions: SHA divided into five fractions; SFA and GWFA divided into four fractions; and RFA divided into three fractions. Identical fractions had similar electrophoretic mobilities independent of the HS sources. Amino-acid compositions and E 4/E 6 ratios of the HS and their fractions were studied. While HS and their fractions from different sources differed greatly in E 4/E 6 ratios, they had remarkably similar amino-acid compositions; differences in neutral and basic amino-acid compositions were not significant.

Interactions between extracellular Borrelia burgdorferi proteins and non-Borrelia-directed immunoglobulin M antibodies

Previous work showed that outer surface protein A (OspA) and OspB of Borrelia burgdorferi may occur within an extracellular multiprotein complex, which was resolved by electrophoresis as an 83-kDa major extracellular protein band. To characterize the 83-kDa band, we sequenced the N terminus of the predominant peptide in the band and examined the interaction between the associated proteins. Peptide sequence and amino acid composition comparisons showed identity with the heavy chain of immunoglobulin M (IgM). Reduction sensitivity experiments and the recognition of the band by antibodies specific for rabbit mu chain indicated that the multiprotein complex contained pentameric IgM. Immunoelectron microscopy showed that anti-mu chain antibodies and monoclonal antibodies to OspA and OspB bound to extracellular amorphous material surrounding cells. Furthermore, the Osps coprecipitated with either nonspecific polyclonal rabbit IgM antibodies or with murine monoclonal anti-human serum albumin IgM antibodies, using insoluble anti-mu chain antibody conjugates. Although the apparent 83-kDa complex was stable under conditions of chelation and concentrated salts, it was disrupted by treatment with neuraminidase. These results indicate that extracellular B. burgdorferi proteins, including OspA and OspB, interact with IgM. The association is apparently not a classic antibody-antigen interaction but may result from other mechanisms.

A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability.

A cell-associated pullulanase (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55,000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr- Ala- Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75-80 degrees C and at 80-85 degrees C, respectively. The enzyme was stable up to 90 degrees C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae----B. acidopullulyticus----B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.

Cloning, sequencing and expression of cDNA for chick liver haem oxygenase. Comparison of avian and mammalian cDNAs and deduced proteins

A cDNA from a chick liver library that encodes for haem oxygenase has been cloned and sequenced. Positive clones were identified with monospecific antibodies to the purified enzyme from chick liver and a cDNA of rat haem oxygenase-1. The length of the cDNA is 1258 bases. An open reading frame of 888 bases was identified by comparison of nucleotide and amino acid sequences with those previously identified for haem oxygenase of mammalian or avian origin. The protein corresponding to this fragment of DNA is composed of 296 amino acid residues and has a molecular mass of 33,509 Da, which is similar to that previously estimated for haem oxygenase purified from chick liver. Unequivocal identification of this clone as that complementary to haem oxygenase was provided by (a) comparison of amino acid compositions and partial sequences with those previously established for the purified enzyme, (b) comparison with nucleotide and amino acid sequences for haem oxygenase from rat and human sources and (c) expression in Escherichia coli with production of high levels of mRNA, protein and haem oxygenase activity after exposure of the transfected bacteria to isopropyl beta-D-thiogalactopyranoside (IPTG). Overall, the similarity of chick haem oxygenase to rat and human haem oxygenase (nucleotides 66% and amino acids 62%) is moderately high. The region between proline-129 and alanine-157 is identical in all three enzymes, including histidine-135, which is proposed to play a key role in binding the substrate haem at the active centre of the enzyme. Northern blots also show that treatment of chicks with CdCl2, a potent inducer of haem oxygenase, results in increases in 1.65-1.70 kb mRNA, which hybridizes selectively to the full-length cDNA or to a synthetic 24-base oligonucleotide with sequence identical to that of a portion of the haem oxygenase cDNA. These results suggest that Cd-dependent induction of haem oxygenase is due to increased transcription of the gene or stabilization of its message.

Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol

The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with [3H]ethanolamine and myo-[3H]inositol. 5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.

Dye-sensitized Photooxidation of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Histidine Residue Oxidized.

The neutral protease of Bacillus subtilis var. amylosacchariticus was photooxidized in the presence of methylene blue, by which treatment the enzyme was rapidly inactivated. The inactive enzyme was digested with endoproteinase Asp-N, the resultant peptides were separated by HPLC, and their amino acid sequences were compared with those obtained from the unmodified enzyme. Of four peptides that contained histidine residues, only the recovery of one peptide was found to be decreased by the photooxidation with the appearance of a new peptide. Comparisons of amino acid compositions and sequences between these two peptides showed that the latter peptide lacked His228 of the former one, indicating that His228 was photooxidized. This result suggests that His228 is involved in the catalytic reaction of the neutral protease or interaction with substrates.

Isolation and characterization of the major and the minor phosvitin from eight species (three genera) of salmonid fishes: Genus specific nature of the amino acid composition of the major phosvitin, and novel features of the chemical composition of the minor phosvitins

1. 1. Three classes of phosvitin molecules were isolated from the eggs of eight species (three genera) of salmonid fishes and chemically characterized. 2. 2. Comparison of amino acid composition of the major phosvitin among the eight fish species revealed marked genus and species specific differences, indicating possibility of making use of these molecules in chemotaxonomy of fishes. 3. 3. The minor phosvitins (L and S) are characterized by the absence of carbohydrate prosthetic groups and lower phosphorus content than the major phosvitin. 4. 4. The minor phosvitins can be considered to be the ancestral molecules of the major phosvitin.

A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosidases

A p-nitrophenyl-α-d-glucopyranoside-hydrolysing oligo-1,6-glucosidase (dextrin 6-α-d-glucanohydrolase, EC 3.2.1.10) of Bacillus sp. KP 1071 capable of growing at 30°–66°C was purified to homogeneity. The molecular weight was estimated to be 62,000. The amino-terminal amino acid was methionine. The enzyme shared its antigenic groups in part with its homologous counterpart from Bacillus thermoglucosidasius KP 1006 (obligate thermophile), but did not at all with any one of oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus flavocaldarius KP 1288 (extreme thermophile). A comparison of amino acid composition showed that the proline content increased greatly in a linearity with the rise in thermostability in the order, mesophile → facultative thermophile → KP 1071 → obligate thermophile → extreme thermophile enzymes.

Purification of cysteine proteinases from adult Schistosoma mansoni

Proteolytic activity against hemoglobin and low molecular weight synthetic substrates has been previously found in homogenates and excretion/secretion products of adult Schistosoma mansoni worms. This activity is stimulated in the presence of thiol compounds and is maximally active at acidic pH. To characterize further this proteolytic activity, lyophilized adult worms were extracted, and proteinases were isolated and purified. From extracts prepared in 0.2 m citrate buffer, pH 4.9, two proteinase species were purified to homogeneity by centrifugation, gel filtration, dialysis, and chromato-focusing chromatography. The proteinases, designated SMw32 and SMw28, have apparent molecular weights (SDS-PAGE) of 31,700 ± 1400 and 27,800 ± 1700, respectively. Both are thiol-dependent, acidic endopeptidases that cleave hemoglobin and a synthetic substrate, CBZ-arg-arg-AFC. A statistical comparison of amino acid compositions reveals that the proteinases are highly related.

Identification and characterization of Thy-1 homologues from bovine thymocytes

Two forms of Thy-1 homologues of apparent mol. wt of 25,000 (designated BTp25) and 45,000 (designated BTp45) were isolated from bovine thymocyte membrane by solubilization, affinity chromatography with Con A, and preparative SDS-PAGE. Both forms reacted with a rabbit antiserum to murine Thy-1 in an enzyme-linked immunosorbent assay (ELISA). BTp45 is most likely a dimer of BTp25, since the two are indistinguishable in their amino acid compositions. Comparison of amino acid compositions of BTp25 and BTp45 to that of rodent and human Thy-1 by the S Δ Q index revealed significant relatedness among these molecules. BTp25 and BTp45 demonstrate more structural homology to rodent Thy-1 than to human Thy-1. Detailed chemical analyses indicate that bovine Thy-1 homologues contain neutral sugars and fatty acids covalently bound to the polypeptide chain; therefore, they are lipoglycoproteins.

Comparison of Amino Acid Compositions Suggest There May Be Sequence Similarities Between Bacterial Cytochromes b557.5 and Eukaryotic Ferritins

Comparison of animo acid compositions by the procedure of Cornish-Bowden [ Meth. Enzymol. 91, 60–75 (1983)] suggests that eukaryotic ferritins and bacterial cytochromes b 557.5, also known as bacterioferritin, have similar amino acid sequences. The comparison test also indicates that eukaryotic cytochrome b 5 and bacterioferritin may be related.

Human T-cell leukemia virus type II: Primary structure analysis of the major internal protein, p24 and the nucleic acid binding protein, p15

The 24,000-molecular-weight major internal protein (p24) and the 15,000-molecular-weight nucleic acid binding protein (p15) of human T-cell leukemia virus type II (HTLV-II) were subjected to amino acid composition and amino-terminal amino acid sequence analysis. A comparison of amino acid composition of p24 and p15 of HTLV-II with those of the analogous proteins of HTLV-I revealed that these two proteins share overall similarity. Further, alignment of the amino-terminal amino acid sequence for the first 27 residues of p24 and 34 residues of p15 from HTLV-II showed extensive sequence homology with analogous proteins of HTLV-I. These data suggest that although disease associated with HTLV-I is malignant T-cell leukemia and that associated with HTLV-II is a relatively benign variant of hairy-cell leukemia, HTLV-I and HTLV-II are closely related to each other, at least in their gag-gene-encoded sequences.