The Streptococcus pneumoniae beta-galactosidase is a surface protein.

The beta-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous beta-galactosidase activity. The results are consistent with beta-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.

Identification of lysosomal and Golgi localization signals in GAP and ARF domains of ARF domain protein 1.

ADP ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin and phospholipase D and are critical components of vesicular trafficking pathways. ARF domain protein 1 (ARD1), a member of the ARF superfamily, contains a 46-kDa amino-terminal extension, which acts as a GTPase-activating protein (GAP) with activity towards its ARF domain. When overexpressed, ARD1 was associated with lysosomes and the Golgi apparatus. In agreement with this finding, lysosomal and Golgi membranes isolated from human liver by immunoaffinity contained native ARD1. ARD1, expressed as a green fluorescent fusion protein, was initially associated with the Golgi network and subsequently appeared on lysosomes, suggesting that ARD1 might undergo vectorial transport between the two organelles. Here we show by microscopic colocalization that GAP and ARF domains determine lysosomal and Golgi localization, respectively, consistent with the presence of more than one signal motif. Using truncated ARD1 molecules, expressed as green fluorescent fusion proteins, it was found that the signal for lysosomal localization was present in residues 301 to 402 of the GAP domain. Site-specific mutagenesis demonstrated that the sequence (369)KXXXQ(373) in the GAP domain was responsible for lysosomal localization. Association of ARD1 with the Golgi apparatus required tyrosine-based motifs. A green fluorescent fusion protein containing the QKQQQQF motif was partially associated with lysosomes, suggesting that this motif contains the information sufficient for lysosomal targeting. These results suggest that ARD1 is a multidomain protein with ARF and GAP regions, which contain Golgi and lysosomal localization signals, respectively, that could function in vesicular trafficking.

A Bayesian system integrating expression data with sequence patterns for localizing proteins: comprehensive application to the yeast genome.

We develop a probabilistic system for predicting the subcellular localization of proteins and estimating the relative population of the various compartments in yeast. Our system employs a Bayesian approach, updating a protein's probability of being in a compartment, based on a diverse range of 30 features. These range from specific motifs (e.g. signal sequences or the HDEL motif) to overall properties of a sequence (e.g. surface composition or isoelectric point) to whole-genome data (e.g. absolute mRNA expression levels or their fluctuations). The strength of our approach is the easy integration of many features, particularly the whole-genome expression data. We construct a training and testing set of approximately 1300 yeast proteins with an experimentally known localization from merging, filtering, and standardizing the annotation in the MIPS, Swiss-Prot and YPD databases, and we achieve 75 % accuracy on individual protein predictions using this dataset. Moreover, we are able to estimate the relative protein population of the various compartments without requiring a definite localization for every protein. This approach, which is based on an analogy to formalism in quantum mechanics, gives better accuracy in determining relative compartment populations than that obtained by simply tallying the localization predictions for individual proteins (on the yeast proteins with known localization, 92% versus 74%). Our training and testing also highlights which of the 30 features are informative and which are redundant (19 being particularly useful). After developing our system, we apply it to the 4700 yeast proteins with currently unknown localization and estimate the relative population of the various compartments in the entire yeast genome. An unbiased prior is essential to this extrapolated estimate; for this, we use the MIPS localization catalogue, and adapt recent results on the localization of yeast proteins obtained by Snyder and colleagues using a minitransposon system. Our final localizations for all approximately 6000 proteins in the yeast genome are available over the web at: http://bioinfo.mbb.yale. edu/genome/localize.

The production and biological assessment of cervine interferon gamma.

Cervine interferon gamma (IFN-gamma) was cloned and expressed using an Escherichia coli expression system pET-32. The expressed protein contained a 6 histidine purification tag and an 11 kDa thioredoxin fusion partner 5' to the IFN-gamma molecule. The ability of IFN-gamma to inhibit the killing of Madin-Darby bovine kidney cells by Semliki forest virus was used as a measure of the bioactivity of the recombinant cervine IFN-gamma (rIFN-gamma). It was shown that the presence of the thioredoxin fusion partner 5' to the IFN-gamma molecule did not affect its biological activity. As in the mouse model, it was shown that cervine rIFN-gamma was able to down-regulate the transcription of interleukin 10 mRNA while up-regulating the transcription of interleukin 12 mRNA in lipopolysaccharide-sensitized, peripheral blood mononuclear cells. A prototype ELISA was tested for its ability to detect both recombinant and native IFN-gamma. The ELISA was able to detect rIFN-gamma at concentrations greater than 100 pg/ml. It was also used to detect native IFN-gamma produced by peripheral blood lymphocytes from Mycobacterium bovis infected or vaccinated deer after in vitro restimulation with antigen. The rIFN-gamma and the cervine IFN-gamma specific ELISA provide valuable tools with which to study important zoonotic infections in farmed and wild deer.

Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism.

The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery. It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid. The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes. The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide. A minimal Tat system requires a copy of tatA and a copy of tatC. The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer. The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes. The topology of evolution trees suggests a bacterial origin of the Tat system. In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway. However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances. Tat systems show a substrate-Tat component specificity and a species specificity. The pore size of the Tat channel is estimated as being between 5 and 9 nm. Operational models of the Tat system are proposed.

Secretion of cytoplasmic and nuclear proteins from animal cells using novel secretion modules.

Production of recombinant proteins that are not secreted outside the producing cells usually requires purification steps that can result in significant yield reductions and loss of biological activity. Using insect cells as a model system to devise the means for secreting recombinant proteins that are not normally destined for secretion outside the producing cells, we initially examined the ability of an insect-specific signal peptide sequence to direct secretion of two intracellular proteins (the cytoplasmic enzyme chloramphenicol acetyl transferase [CAT] and the nuclear protein Bombyx mori chorion factor 1 [BmCF1]) expressed in transfected silkmoth cells. Although this signal sequence functioned efficiently as a chimera with normally secreted proteins, it failed to secrete CAT and BmCF1, suggesting that additional signals are required for passage of these polypeptides through the secretion pathway. For this reason, we also generated a secretion module consisting of the secreted protein juvenile hormone esterase (JHE), a spacer region containing a histidine tag and an endopeptidase cleavage site, to which coding sequences of choice can be cloned as C-terminal extensions. In C-terminal fusions with the CAT and BmCF1 open reading frames, the N-terminal JHE moiety was able to provide all the signals necessary for secretion of CAT and BmCF1 into the extracellular environment. The histidine tag present in the spacer region allowed purification of fusion proteins by metal affinity chromatography under nondenaturing conditions, and the enteropeptidase cleavage site was recognized and cleaved by the cognate protease causing the release of the intracellular proteins from the secretion module. We also show that another secreted protein, human granulocyte-macrophage colony stimulating factor (GM-CSF) can substitute for JHE in the secretion module and that these secretion modules can function in mammalian cells.

Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59.

CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.

Na(+)/H(+) exchanger NHE3 has 11 membrane spanning domains and a cleaved signal peptide: topology analysis using in vitro transcription/translation.

The transmembrane topology of Na(+)/H(+) exchanger NHE3 has been studied using in vitro transcription/translation of two types of fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NHE3 membrane spanning domains (msds). These vectors encode N-terminal 101 (HKM0) or 139 (HKM1) amino acids of the H,K-ATPase alpha-subunit, a linker region and a reporter sequence containing five N-linked glycosylation consensus sites in the C-terminal 177 amino acids of the H,K-ATPase beta-subunit. The glycosylation status of the reporter sequence was used as a marker for the analysis of signal anchor and stop transfer properties of each putative msd in both the HKM0 and the HKM1 vectors. The linker region of the vectors was replaced by sequences that contain putative msds of NHE3 individually or in pairs. In vitro transcription/translation was performed using [(35)S]methionine in a reticulocyte lysate system +/- microsomes, and the translation products were identified by autoradiography following separation using SDS-PAGE. We propose a revised NHE3 topology model, which contains a cleaved signal peptide followed by 11 msds, including extracellular orientation of the N-terminus and intracellular orientation of the C-terminus. The presence of a cleavable signal peptide in NHE3 was demonstrated by its cleavage from NHE3 during translational processing of full-length and truncated NHE3 in the presence of microsomes. Of 11 putative msds, six (msds 1, 2, 4, 7, 10, and 11) acted as both signal anchor and stop transfer sequences, while five (msds 3, 5, 6, 8, and 9) had signal anchor activities when tested alone. Of the latter, 3, 5, 6, and 9 were shown to act as stop transfer sequences after C-terminal extension. The actual membrane orientation of each sequential transmembrane segment of NHE3 was deduced from the membrane location of the N- and C-termini of NHE3. The regions between putative msds 8 and 9 and between msds 10 and 11, which correspond to the fourth and fifth extracellular loops, did not act as msds when tested alone. However, the extension of the fifth extracellular loop with adjacent putative msds showed some membrane-associated properties suggesting that the fifth extracellular loop might be acting as a "P-loop"-like structure.

Structures of type 2 peroxisomal targeting signals in two trypanosomatid aldolases.

Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These "PTS2 dimers", which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.

Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.

A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed. Using N-terminal sequence information only, it discriminates between proteins destined for the mitochondrion, the chloroplast, the secretory pathway, and "other" localizations with a success rate of 85% (plant) or 90% (non-plant) on redundancy-reduced test sets. From a TargetP analysis of the recently sequenced Arabidopsis thaliana chromosomes 2 and 4 and the Ensembl Homo sapiens protein set, we estimate that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%. TargetP also predicts cleavage sites with levels of correctly predicted sites ranging from approximately 40% to 50% (chloroplastic and mitochondrial presequences) to above 70% (secretory signal peptides). TargetP is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.

Targeting to rhoptry organelles of Toxoplasma gondii involves evolutionarily conserved mechanisms.

Intracellular parasites of the phylum Apicomplexa contain specialized rhoptry secretory organelles that have a crucial function in host-cell invasion and establishment of the parasitophorous vacuole. Here we show that localization of the Toxoplasma gondii rhoptry protein ROP2 is dependent on a YEQL sequence in the cytoplasmic tail that binds to micro-chain subunits of T. gondii and mammalian adaptors, and conforms to the YXXstraight phi mammalian sorting motif. Chimaeric reporters, containing the transmembrane domains and cytoplasmic tails of the low-density lipoprotein receptor and of Lamp-1, are sorted to the Golgi or the trans-Golgi network (TGN), and partially to apical microneme organelles of the parasite, respectively. Targeting of these reporters is mediated by YXXstraight phi- and NPXY-type signals. This is the first demonstration of tyrosine-dependent sorting in protozoan parasites, indicating that T. gondii proteins may be targeted to, and involved in biogenesis of, morphologically unique organelles through the use of evolutionarily conserved signals and machinery.

Extracytoplasmic proteins of Mycobacterium tuberculosis - mature secreted proteins often start with aspartic acid and proline.

A surrogate expression system, based on fusions to the phoA bacterial reporter gene, was used to identify Mycobacterium tuberculosis genes that encode exported proteins and the promoter regions required for their expression in the heterologous host Mycobacterium smegmatis. To assess these results in the context of the complete M. tuberculosis genome sequence, the corresponding genes were identified and computational algorithms were employed to identify signal peptide (SP), transmembrane domain and membrane lipoprotein attachment motifs. This information was used to predict the subset of M. tuberculosis genes that encode exported proteins. Of the 34 genes identified by the phoA method, 22 were classified to encode potential soluble secreted proteins. Among these, 14 genes may encode novel secreted proteins. Six of the remaining 12 genes were predicted to encode membrane lipoproteins and an additional six to encode integral membrane proteins. Published observations of proteins proven to be secreted into M. tuberculosis culture filtrates were reviewed to further characterize the mycobacterial SP motif. It was concluded that mycobacterial SPs are comparable in size to Gram-positive SPs, but certain features are different. In particular, arginine was the predominant N-terminally positively charged amino acid in contrast to lysine in the Gram-positives. The hydrophobic transmembrane segment of the SP was dominated by alanine, in contrast to leucine. At the C-terminal end of the SPs, the (-3, -1) rule (AXA motif) holds, with alanine as the dominant amino acid in both positions, being most dominant in the (-1) position. A high proportion of mature sequences start with aspartic acid in the (+1) position and proline in the (+2) position - the DP motif. The authors propose that the DP sequence serves as a sorting signal, following translocation and cleavage by signal peptidase I. Alternatively, the DP motif may be part of the recognition site for the signal peptidase.

Germline repertoire of the immunoglobulin V(H)3 family in rhesus monkeys.

To facilitate molecular studies of antibody responses in rhesus monkeys (Macaca mulatta), we cloned and sequenced germline segments from its largest and most diverse immunoglobulin heavy-chain gene family, V(H)3. Using a PCR-based approach, we characterized 29 sequences, 20 with open reading frames (ORFs) and 9 pseudogenes. The leader sequences, introns, exons, and recombination signal sequences of M. mulatta V(H)3 gene segments are not strictly identical to those of humans, but the mature coding regions demonstrate, on average, greater than 90% sequence similarity. Although the framework regions are more highly conserved, the complementarity-determining regions (CDRs) also show strong similarities, and their predicted three-dimensional structures resemble those of their human homologues. In one instance, homologous macaque and human CDR1 sequences were 100% identical at the nucleotide level, and some CDR2s shared nucleotide identity as high as 96.5%. However, some rhesus V(H)3 ORFs have unusual structural features, including atypical CDR lengths and uncommon amino acids at structurally crucial positions. The similarity of rhesus and human V(H)3 homologues reinforces the notion that humoral immunity in this nonhuman primate species is an appropriate system for modeling human antibody responses.

The long signal peptide isoform and its alternative processing direct the intracellular trafficking of interleukin-15.

Two isoforms of interleukin (IL)-15 exist: one with a short and another with a long signal peptide (LSP). Experiments using combinations of the LSP and mature proteins IL-2, IL-15, and green fluorescent protein revealed complex pathways of intracellular trafficking. In one pathway, the LSP was unprocessed, and IL-15 was not glycosylated, remained in the cytoplasm, and was degraded. The second trafficking pathway involved endoplasmic reticulum entry, N-linked glycosylation, and alternative partial LSP processing. The third pathway involved endoplasmic reticulum entry, followed by glycosylation, complete processing, and ultimately secretion. The complex intracellular trafficking patterns of LSP-IL-15 with its impediments to secretion as well as impediments to translation may be required due to the potency of IL-15 as an inflammatory cytokine. In terms of a more positive role, we propose that intracellular infection may relieve the burdens on translation and intracellular trafficking to yield effective IL-15 expression.

Inefficient function of the signal sequence of PTHrP for targeting into the secretory pathway.

Parathyroid hormone-related peptide (PTHrP) is not only secreted out of cells, but also targeted to the nucleoli due to a nucleolar targeting signal (NTS). We assessed the molecular mechanism underlying the dual targeting of PTHrP by constructing a series of truncated forms of rat PTHrP cDNA and expressing them in CHO cells. Immunostaining was observed in both the Golgi apparatus and nucleoli in the same cell expressing PTHrP with the N-terminal full-length signal sequence. When PTHrP molecules were translated from CUGs downstream of the AUG-initiator codon in the signal sequences, potential alternative initiators of the translation, they were exclusively localized in the nucleoli. In contrast, when a construct containing only the ATG-initiator codon was expressed, PTHrP was found to localize in both the nucleolus and the Golgi apparatus. No nucleolar staining of PTHrP was observed in the CHO cells transfected with PTH/PTHrP receptors even after incubating with a conditioned medium containing PTHrP, ruling out a possibility that PTHrP is, once secreted, internalized via receptor-mediated endocytosis and subsequently conveyed to nucleoli. Compatible with these morphological observations, a preproform of PTHrP was found in the cells expressing PTHrP in addition to proPTHrP, indicative of molecules along the secretory pathway. These results strongly indicate that the signal sequence of PTHrP is not sufficient to direct all the newly synthesized molecules across the endoplasmic reticulum, resulting in part of it being delivered to the nucleoli due to the NTS.

Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems.

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).

A novel double-headed proteinaceous inhibitor for metalloproteinase and serine proteinase.

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.

Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization.

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.