Automated protein sequence pattern handling and PROSITE searching

The protein sequence searching program Scrutineer has been modifiedto search for targets from a file. We are distributing a reformattedfile of PROSITES which can be read by Scrutineer. In addition,Scrutineer still accepts targets typed in interactively butcan now write them out in the format required as input. Sincethe input format is the same as the output format, target managementand re-use is simple.     

Molecular cloning and sequence analysis of prion protein gene in Xiji donkey in China

Prion diseases are a group of human and animal neurodegenerative disorders caused by the deposition of an abnormal isoform prion protein (PrP^Sc) encoded by a single copy prion protein gene (PRNP). Prion disease has been reported in many herbivores but not in Equus and the species barrier might be playing a role in resistance of these species to the disease. Therefore, analysis of genotype of prion protein (PrP) in these species may help understand the transmission of the disease. Xiji donkey is a rare species of Equus not widely reared in Ningxia, China, for service, food and medicine, but its PRNP has not been studied. Based on the reported PrP sequence in GenBank we designed primers and amplified, cloned and sequenced the PRNP of Xiji donkey. The sequence analysis showed that the Xiji donkey PRNP was consisted of an open reading frame of 768 nucleotides encoding 256 amino acids. Amino acid residues unique to donkey as compared with some Equus animals, mink, cow, sheep, human, dog, sika deer, rabbit and hamster were identified. The results showed that the amino acid sequence of Xiji donkey PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of other Equus species in this study. Amino acid sequence analysis showed high identity within species and close relation to the PRNP of sika deer, sheep, dog, camel, cow, mink, rabbit and hamster with 83.1–99.7% identity. The results provided the PRNP data for an additional Equus species, which should be useful to the study of the prion disease pathogenesis, resistance and cross species transmission.     

HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment

Sequence-based protein function and structure prediction depends crucially on sequence-search sensitivity and accuracy of the resulting sequence alignments. We present an open-source, general-purpose tool that represents both query and database sequences by profile hidden Markov models (HMMs): 'HMM-HMM-based lightning-fast iterative sequence search' (HHblits; http://toolkit.genzentrum.lmu.de/hhblits/). Compared to the sequence-search tool PSI-BLAST, HHblits is faster owing to its discretized-profile prefilter, has 50-100% higher sensitivity and generates more accurate alignments.     

Comparative sequence analysis of the mRNAs coding for mouse and rat whey protein

Whey acidic protein (WAP) is a major milk protein found in mouse and rat. Cloned WAP cDNAs from both species have been sequenced and the respective protein sequences have been deduced. Mouse and rat WAP (134 and 137 amino acids respectively) are acidic, cysteine rich proteins which contain a N-terminal signal peptide of 19 amino acids. Most of the cysteines are located in two clusters containing six cysteine residues each, arranged in an identical pattern. Comparison of the mouse and rat WAPs show that the signal peptide and the first cysteine domain are conserved to a greater extent than the rest of the protein. This result is reflected in the nucleotide sequence homology, where the regions coding for the signal peptide and cysteine domain I are the only regions where the rate of replacement substitution is lower than the rate of silent substitution. The 3' non-coding regions show a 91% conservation which is half the substitution rate for the coding region. This low rate of sequence divergence in the 3' non-translated region of the mRNA may indicate a functional importance for this region.     

Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.     

Increased coverage obtained by combination of methods for protein sequence database searching

MOTIVATION: Sequence alignment methods that compare two sequences (pairwise methods) are important tools for the detection of biological sequence relationships. In genome annotation, multiple methods are often run and agreement between methods taken as confirmation. In this paper, we assess the advantages of combining search methods by comparing seven pairwise alignment methods, including three local dynamic programming algorithms (PRSS, SSEARCH and SCANPS), two global dynamic programming algorithms (GSRCH and AMPS) and two heuristic approximations (BLAST and FASTA), individually and by pairwise intersection and union of their result lists at equal p-value cut-offs. RESULTS: When applied singly, the dynamic programming methods SCANPS and SSEARCH gave significantly better coverage (p=0.01) compared to AMPS, GSRCH, PRSS, BLAST and FASTA. Results ranked by BLAST p-values gave significantly better coverage compared to ranking by BLAST e-values. Of 56 combinations of eight methods considered, 19 gave significant increases in coverage at low error compared to the parent methods at an equal p-value cutoff. The union of results by BLAST (p-value) and FASTA at an equal p-value cutoff gave significantly better coverage than either method individually. The best overall performance was obtained from the intersection of the results from SSEARCH and the GSRCH62 global alignment method. At an error level of five false positives, this combination found 444 true positives, a significant 12.4% increase over SSEARCH applied alone.     

Combinatorial control of prion protein biogenesis by the signal sequence and transmembrane domain

The prion protein (PrP) is synthesized in three topologic forms at the endoplasmic reticulum. (sec)PrP is fully translocated into the endoplasmic reticulum lumen, whereas (Ntm)PrP and (Ctm)PrP are single-spanning membrane proteins of opposite orientation. Increased generation of (Ctm)PrP in either transgenic mice or humans is associated with the development of neurodegenerative disease. To study the mechanisms by which PrP can achieve three topologic outcomes, we analyzed the translocation of proteins containing mutations introduced into either the N-terminal signal sequence or potential transmembrane domain (TMD) of PrP. Although mutations in either domain were found to affect PrP topogenesis, they did so in qualitatively different ways. In addition to its traditional role in mediating protein targeting, the signal was found to play a surprising role in determining orientation of the PrP N terminus. By contrast, the TMD was found to influence membrane integration. Analysis of various signal and TMD double mutants demonstrated that the topologic consequence of TMD action was directly dependent on the previous, signal-mediated step. Together, these results reveal that PrP topogenesis is controlled at two discrete steps during its translocation and provide a framework for understanding how these steps act coordinately to determine the final topology achieved by PrP.     

Mass accuracy and sequence requirements for protein database searching.

To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein matches was examined by varying the mass error allowed in the search over a wide range (2-500 ppm). For the "clean" data obtained from apomyoglobin, very few masses were needed to return valid protein matches, and the mass error allowed in the search had little effect up to 500 ppm. However, in the case of alpha-lactalbumin more mass values were needed, and low mass errors increased the search specificity. Mass errors below 30 ppm were particularly useful in eliminating false protein matches when few mass values were used in the search. Collision-induced dissociation of an unassigned peak in the alpha-lactalbumin digest provided sufficient data to unambiguously identify the peak as a fragment from alpha-lactalbumin and eliminate a large number of spurious proteins found in the peptide mass search. The results show that even with a relatively high mass error (0.8 Da for mass differences between singly charged product ions), collision-induced dissociation can help identify proteins in cases where unfavorable digest conditions or modifications render digest peaks unidentifiable by a simple mass mapping search.     

A comparative analysis of rapid methods for purification and refolding of recombinant bovine prion protein

Bacterially-produced recombinant prion protein (rPrP) is a frequently used model system for the study of the properties of wild-type and mutant prion proteins by biochemical and biophysical approaches. A range of approaches have been developed for the purification and refolding of untagged rPrP expressed as inclusion bodies in Escherichia coli, including refolding by dialysis and simultaneous on-column purification and refolding. In order to perform a higher-throughput analysis of different rPrP proteins, an approach that produces highly pure rPrP with a minimum of purification steps and a high yield per liter of induced bacterial culture is desired. Here, we directly compare purification approaches for untagged bovine rPrP as adapted to rapid, small-scale formats useful for higher-throughput studies. An analysis of protein yield, purity, oxidation, and refolding revealed significant differences between preparative methods as adapted to the small-scale format, and based on these findings we provide recommendations for future purifications. We also describe the utility of a sensitive commercial kit for thiol analysis of these preparations, the pH dependence of dimer formation during refolding of bovine rPrP, and bovine rPrP binding to cobalt affinity resin.     

MacPattern: protein pattern searching on the Apple Macintosh

A program is described for rapid detection of protein sequencepatterns on the Apple Macintosh which makes full use of theinformation contained in the PROSITE protein pattern database.     

Comparison of methods for searching protein sequence databases.

We have compared commonly used sequence comparison algorithms, scoring matrices, and gap penalties using a method that identifies statistically significant differences in performance. Search sensitivity with either the Smith-Waterman algorithm or FASTA is significantly improved by using modern scoring matrices, such as BLOSUM45-55, and optimized gap penalties instead of the conventional PAM250 matrix. More dramatic improvement can be obtained by scaling similarity scores by the logarithm of the length of the library sequence (In()-scaling). With the best modern scoring matrix (BLOSUM55 or JO93) and optimal gap penalties (-12 for the first residue in the gap and -2 for additional residues), Smith-Waterman and FASTA performed significantly better than BLASTP. With In()-scaling and optimal scoring matrices (BLOSUM45 or Gonnet92) and gap penalties (-12, -1), the rigorous Smith-Waterman algorithm performs better than either BLASTP and FASTA, although with the Gonnet92 matrix the difference with FASTA was not significant. Ln()-scaling performed better than normalization based on other simple functions of library sequence length. Ln()-scaling also performed better than scores based on normalized variance, but the differences were not statistically significant for the BLOSUM50 and Gonnet92 matrices. Optimal scoring matrices and gap penalties are reported for Smith-Waterman and FASTA, using conventional or In()-scaled similarity scores. Searches with no penalty for gap extension, or no penalty for gap opening, or an infinite penalty for gaps performed significantly worse than the best methods. Differences in performance between FASTA and Smith-Waterman were not significant when partial query sequences were used. However, the best performance with complete query sequences was obtained with the Smith-Waterman algorithm and In()-scaling.     

AGenDA: gene prediction by comparative sequence analysis

Comparative sequence analysis is a powerful approach to identify functional elements in genomic sequences. Herein, we describe AGenDA (Alignment-based GENe Detection Algorithm), a novel method for gene prediction that is based on long-range alignment of syntenic regions in eukaryotic genome sequences. Local sequence homologies identified by the DIALIGN program are searched for conserved splice signals to define potential protein-coding exons; these candidate exons are then used to assemble complete gene structures. The performance of our method was tested on a set of 105 human-mouse sequence pairs. These test runs showed that sensitivity and specificity of AGenDA are comparable with the best gene- prediction program that is currently available. However, since our method is based on a completely different type of input information, it can detect genes that are not detectable by standard methods and vice versa. Thus, our approach seems to be a useful addition to existing gene-prediction programs. Availability: DIALIGN is available through the Bielefeld Bioinformatics Server (BiBiServ) at http://bibiserv.techfak.uni-bielefeld.de/dialign/ The gene-prediction program AGenDA described in this paper will be available through the BiBiServ or MIPS web server at http://mips.gsf.de.     

Aberrant metal binding by prion protein in human prion disease

Human prion diseases are characterized by the conversion of the normal prion protein (PrP(C)) into a pathogenic isomer (PrP(Sc)). Distinct PrP(Sc) conformers are associated with different subtypes of prion diseases. PrP(C) binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrP(Sc) type, combined. Both zinc and manganese were undetectable in PrP(C) preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrP(Sc) type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrP(Sc) and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.     

Effective detection of remote homologues by searching in sequence dataset of a protein domain fold

Profile matching methods are commonly used in searches in protein sequence databases to detect evolutionary relationships. We describe here a sensitive protocol, which detects remote similarities by searching in a specialized database of sequences belonging to a fold. We have assessed this protocol by exploring the relationships we detect among sequences known to belong to specific folds. We find that searches within sequences adopting a fold are more effective in detecting remote similarities and evolutionary connections than searches in a database of all sequences. We also discuss the implications of using this strategy to link sequence and structure space.     

Generating recombinant C-terminal prion protein fragments of exact native sequence

Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aβ1-40 versus Aβ1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or β-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease step. This technique consistently produced sufficient yields (～10 mg/L) of high purity peptides (>95%) equating to C1 and C2 of exact native primary sequence in the α-helical conformation suitable for biological and biophysical investigations.     

Understanding the improved sensitivity of spectral library searching over sequence database searching in proteomics data analysis

Spectral library searching has been recently proposed as an alternative to sequence database searching for peptide identification from MS/MS. We performed a systematic comparison between spectral library searching and sequence database searching using a wide variety of data to better demonstrate, and understand, the superior sensitivity of the former observed in preliminary studies. By decoupling the effect of search space, we demonstrated that the success of spectral library searching is primarily attributable to the use of real library spectra for matching, without which the sensitivity advantage largely disappears. We further determined the extent to which the use of real peak intensities and non-canonical fragments, both under-utilized information in sequence database searching, contributes to the sensitivity advantage. Lastly, we showed that spectral library searching is disproportionately more successful in identifying low-quality spectra, and complex spectra of higher- charged precursors, both important frontiers in peptide sequencing. Our results answered important outstanding questions about this promising yet unproven method using well-controlled computational experiments and sound statistical approaches.     

Chronic wasting disease of elk and deer and Creutzfeldt-Jakob disease: comparative analysis of the scrapie prion protein

Chronic wasting disease (CWD), a transmissible prion disease that affects elk and deer, poses new challenges to animal and human health. Although the transmission of CWD to humans has not been proven, it remains a possibility. If this were to occur, it is important to know whether the "acquired" human prion disease would show a phenotype including the scrapie prion protein (PrP(Sc)) features that differ from those associated with human sporadic prion disease. In this study, we have compared the pathological profiles and PrP(Sc) characteristics in brains of CWD-affected elk and deer with those in subjects with sporadic Creutzfeldt-Jakob disease (CJD), as well as CJD-affected subjects who might have been exposed to CWD, using histopathology, immunohistochemistry, immunoblotting, conformation stability assay, and N-terminal protein sequencing. Spongiform changes and intense PrP(Sc) staining were present in several brain regions of CWD-affected animals. Immunoblotting revealed three proteinase K (PK)-resistant bands in CWD, representing different glycoforms of PrP(Sc). The unglycosylated PK-resistant PrP(Sc) of CWD migrated at 21 kDa with an electrophoretic mobility similar to that of type 1 human PrP(Sc) present in sporadic CJD affecting subjects homozygous for methionine at codon 129 (sCJDMM1). N-terminal sequencing showed that the PK cleavage site of PrP(Sc) in CWD occurred at residues 82 and 78, similar to that of PrP(Sc) in sCJDMM1. Conformation stability assay also showed no significant difference between elk CWD PrP(Sc) and the PrP(Sc) species associated with sCJDMM1. However, there was a major difference in glycoform ratio of PrP(Sc) between CWD and sCJDMM1 affecting both subjects potentially exposed to CWD and non-exposed subjects. Moreover, PrP(Sc) of CWD exhibited a distinct constellation of glycoforms distinguishable from that of sCJDMM1 in two-dimensional immunoblots. These findings underline the importance of detailed PrP(Sc) characterization in trying to detect novel forms of acquired prion disease.     

Nucleotide sequence analysis of zein mRNAs from maize endosperm

A comparison of the DNA and protein sequences of a group of zein cDNA clones reveals that they share extensive sequence homology and probably originated from a common ancestral gene. A comparison of clones corresponding to Mr 22,000 polypeptides shows they are 92% homologous, while five clones corresponding to the Mr 19,000 zeins vary in homology from 75 to 95%. The clones corresponding to the Mr 22,000 proteins are 60-65% homologous to clones encoding the Mr 19,000 zein proteins. A clone corresponding to the Mr 15,000 zein has little homology to either the Mr 22,000 or 19,000 zeins. Clones corresponding to both the Mr 22,000 and 19,000 zeins have two putative polyadenylation signals. S1 nuclease mapping indicates that the first polyadenylation signal following the stop codon is utilized by the Mr 22,000 sequences, while primarily the second polyadenylation signal is utilized by the Mr 19,000 sequences.     

SARS transmission pattern in Singapore reassessed by viral sequence variation analysis

Background

Epidemiological investigations of infectious disease are mainly dependent on indirect contact information and only occasionally assisted by characterization of pathogen sequence variation from clinical isolates. Direct sequence analysis of the pathogen, particularly at a population level, is generally thought to be too cumbersome, technically difficult, and expensive. We present here a novel application of mass spectrometry (MS)–based technology in characterizing viral sequence variations that overcomes these problems, and we apply it retrospectively to the severe acute respiratory syndrome (SARS) outbreak in Singapore.

Methods and Findings

The success rate of the MS-based analysis for detecting SARS coronavirus (SARS-CoV) sequence variations was determined to be 95% with 75 copies of viral RNA per reaction, which is sufficient to directly analyze both clinical and cultured samples. Analysis of 13 SARS-CoV isolates from the different stages of the Singapore outbreak identified nine sequence variations that could define the molecular relationship between them and pointed to a new, previously unidentified, primary route of introduction of SARS-CoV into the Singapore population. Our direct determination of viral sequence variation from a clinical sample also clarified an unresolved epidemiological link regarding the acquisition of SARS in a German patient. We were also able to detect heterogeneous viral sequences in primary lung tissues, suggesting a possible coevolution of quasispecies of virus within a single host.

Conclusion

This study has further demonstrated the importance of improving clinical and epidemiological studies of pathogen transmission through the use of genetic analysis and has revealed the MS-based analysis to be a sensitive and accurate method for characterizing SARS-CoV genetic variations in clinical samples. We suggest that this approach should be used routinely during outbreaks of a wide variety of agents, in order to allow the most effective control.