The rate of bulk flow from the endoplasmic reticulum to the cell surface

Tripeptides containing the acceptor sequence for Asn-linked glycosylation (Asn-X-Ser/Thr) were added to CHO and HepG2 cells. The tripeptides were glycosylated in the ER and then secreted into the medium, via the Golgi complex in which the oligosaccharide chains were processed. The half-time for secretion, approximately 10 min, was faster than that of known proteins transported through the same pathway. Since much evidence suggests that oligosaccharide chains are not signals for transport, it appears that no signal is necessary for rapid and efficient transport from the ER to the Golgi, or from the Golgi to the cell surface. Rather, it appears that proteins retained as permanent residents en route through the ER-Golgi transport pathway must contain specific retention signals.     

Signal peptidase and oligosaccharyltransferase interact in a sequential and dependent manner within the endoplasmic reticulum

We demonstrate that the signal peptides of prepro-alpha-factor and preinvertase must be cleaved before Asn-X-Ser/Thr acceptor tripeptides located near the signal peptides of these precursors can be efficiently glycosylated within the endoplasmic reticulum of the yeast Saccharomyces cerevisiae. The data support a model whereby the interaction of a signal peptide with the membrane prevents an acceptor tripeptide juxtaposed to the signal peptide from accessing the oligosaccharyltransferase active site until the signal peptide is cleaved.     

N-glycosylation at noncanonical Asn-X-Cys sequences in plant cells

The vesicular transport pathway in plant cells is often used for higher accumulation of recombinant proteins. In the endoplasmic reticulum, which acts as a gateway to the vesicular transport pathway, N-glycosylation occurs on specific Asn residues. This N-glycosylation in recombinant proteins must be carefully regulated as it can impact their enzymatic activity, half lives in serum when injected, structural stability, etc. In eukaryotic cells, including plant cells, N-glycans were found to be attached to Asn residues in Asn-X-Ser/Thr (X ≠ Pro) sequences. However, recently, N-glycosylations at noncanonical Asn-X-Cys sequences have been found in mammals and yeast. Our laboratory has discovered that N-glycans are attached to Asn residues at Asn-Thr-Cys sequences of double-repeated B subunit of Shiga toxin 2e produced in plant cells, the first reported case of N-glycosylation at a noncanonical Asn-X-Cys sequence in plant cells.     

Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity.

Asparagine-linked N-glycosylation is an essential protein modification occurring in all eukaryotic cells. The central step is the co-translational transfer of the core oligosaccharide assembled on the lipid carrier dolichol phosphate to selected Asn-X-Ser/Thr residues of nascent polypeptide chains in the endoplasmic reticulum. This reaction is catalyzed by the enzyme N-oligosaccharyl transferase. In yeast, Wbp1p is an essential component of this enzyme. Using a high copy number suppression approach, the SWP1 gene was isolated as an allele specific suppressor of a wbp1 mutation. Swp1p is a 30 kDa type I transmembrane protein and essential for cell viability. Similar to Wbp1p, depletion of Swp1p results in reduced N-oligosaccharyl transferase activity in vivo and in vitro. Wbp1p and Swp1p can be chemically cross-linked, suggesting that both proteins are essential constituents of the N-oligosaccharyl transferase complex.     

Sequence specificity, statistical potentials, and three-dimensional structure prediction with self-correcting distance geometry calculations of beta-sheet formation in proteins

A statistical analysis of a representative data set of 169 known protein structures was used to analyze the specificity of residue interactions between spatial neighboring strands in beta-sheets. Pairwise potentials were derived from the frequency of residue pairs in nearest contact, second nearest and third nearest contacts across neighboring beta-strands compared to the expected frequency of residue pairs in a random model. A pseudo-energy function based on these statistical pairwise potentials recognized native beta-sheets among possible alternative pairings. The native pairing was found within the three lowest energies in 73% of the cases in the training data set and in 63% of beta-sheets in a test data set of 67 proteins, which were not part of the training set. The energy function was also used to detect tripeptides, which occur frequently in beta-sheets of native proteins. The majority of native partners of tripeptides were distributed in a low energy range. Self-correcting distance geometry (SECODG) calculations using distance constraints sets derived from possible low energy pairing of beta-strands uniquely identified the native pairing of the beta-sheet in pancreatic trypsin inhibitor (BPTI). These results will be useful for predicting the structure of proteins from their amino acid sequence as well as for the design of proteins containing beta-sheets.     

The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro.

Asparagine-linked N-glycosylation is a highly conserved and functionally important modification of proteins in eukaryotic cells. The central step in this process is a cotranslational transfer of lipid-linked core oligosaccharides to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains, catalysed by the enzyme N-oligosaccharyl transferase. In this report we show that the essential yeast protein WBP1 (te Heesen et al., 1991) is required for N-oligosaccharyl transferase in vivo and in vitro. Depletion of WBP1 correlates with a defect in transferring core oligosaccharides to carboxypeptidase Y and proteinase A in vivo. In addition, in vitro N-glycosylation of the acceptor peptide Tyr-Asn-Leu-Thr-Ser-Val using microsomal membranes from WBP1 depleted cells is reduced as compared with membranes from wild-type cells. We propose that WBP1 is an essential component of the oligosaccharyl transferase in yeast.     

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.     

Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helveticus.

The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpTLH) was cloned with the aid of the inverse PCR technique and used to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coli E1772. Functional expression of the peptide transporter was shown by the uptake of prolyl-[14C] alanine in whole cells and membrane vesicles. Peptide transport via DtpT in membrane vesicles is driven by the proton motive force. The system has specificity for di- and tripeptides but not for amino acids or tetrapeptides. The dtpTLH gene consists of 1,491 bp, which translates into a 497-amino-acid polypeptide. DtpTLH shows 34% identity to the di- and tripeptide transport protein of Lactococcus lactis and is also homologous to various peptide transporters of eukaryotic origin, but the similarity between these proteins is confined mainly to the N-terminal halves.     

Origin of the duplicated ribonuclease gene in guinea-pig: Comparison of the amino acid sequences with those of two close relatives: Capybara and cuis ribonuclease

The amino acid sequences of the pancreatic ribonuclease from capybara (Hydrochoerus hydrochaeris) and cuis (Galea musteloides) were determined. Both species belong to the same superfamily of the hystricomorph rodents as the guinea-pig. In guinea-pig pancreas two ribonucleases are present as a result of a recent gene duplication, but in capybara and cuis pancreas only one single ribonuclease has been found. A most parsimonious tree of ribonucleases indicates that the gene duplication leading to both guinea-pig ribonucleases occurred before the divergence of guinea-pig from capybara and cuis. This would mean that changes in expression of the ribonuclease genes have occurred in these taxa. Cuis and capybara ribonuclease have no Asn-X-Ser/Thr sequences and are carbohydrate-free proteins. Capybara ribonuclease has leucine at position 114, a position occupied by proline in the cis-configuration in bovine pancreatic ribonuclease.     

Effect of some fragments of peptide hormones on the content of biogenic monoamines from mouse brain]

The effect of some tripeptides, which are fragments of peptide hormones, and their analogs on the content of biogenic monoamines (BM) from albino mice brain was studied. It was found that thyroliberin, melanostatin and the C-terminal tripeptide of gonadoliberin activate the dophaminergic (DA-ergic) system in the forebrain of mice treated with reserpine or haloperidol, whereas the C-terminal tripeptide of gastrin acts as a synergic blocker of the DA receptors. The N-terminal tripeptides (with and without the amido group) do not affect the content of BM. No effect of the tripeptides was observed in intact animals. It is assumed that the agonistic or antagonistic effect of the tripeptides on BM is due to certain structural peculiarities of the tripeptides, e.g. the presence of the C-terminal amido group and their endogenous nature.     

Trend of amino acid composition of proteins of different taxa

Archaea, bacteria and eukaryotes represent the main kingdoms of life. Is there any trend for amino acid compositions of proteins found in full genomes of species of different kingdoms? What is the percentage of totally unstructured proteins in various proteomes? We obtained amino acid frequencies for different taxa using 195 known proteomes and all annotated sequences from the Swiss-Prot data base. Investigation of the two data bases (proteomes and Swiss-Prot) shows that the amino acid compositions of proteins differ substantially for different kingdoms of life, and this difference is larger between different proteomes than between different kingdoms of life. Our data demonstrate that there is a surprisingly small selection for the amino acid composition of proteins for higher organisms (eukaryotes) and their viruses in comparison with the "random" frequency following from a uniform usage of codons of the universal genetic code. On the contrary, lower organisms (bacteria and especially archaea) demonstrate an enhanced selection of amino acids. Moreover, according to our estimates, 12%, 3% and 2% of the proteins in eukaryotic, bacterial and archaean proteomes are totally disordered, and long (> 41 residues) disordered segments are found to occur in 16% of arhaean, 20% of eubacterial and 43% of eukaryotic proteins for 19 archaean, 159 bacterial and 17 eukaryotic proteomes, respectively. A correlation between amino acid compositions of proteins of various taxa, show that the highest correlation is observed between eukaryotes and their viruses (the correlation coefficient is 0.98), and bacteria and their viruses (the correlation coefficient is 0.96), while correlation between eukaryotes and archaea is 0.85 only.     

Characteristic sequential residue environment of amino acids in proteins

The occurrence of all di- and tripeptide segments of proteins was counted in a large data base containing about 119 000 residues. It was found that the abundance of the amino acids does not determine the frequency of the various di- and tripeptide segments. In addition, the frequency of the various tripeptides cannot be predicted from the observed pair-frequency values. The pair-frequency distribution of amino acids is highly asymmetrical, pairs formed from identical residues are generally preferred and amino acids cannot be clustered on the basis of their first neighbour preferences. These data indicate the existence of general short range regularities in the primary structure of proteins. The consequences of these short range regularities were studied by comparing Chou-Fasman parameters with analogous parameters determined from the results of conformational energy calculations of single amino acids. This comparison shows that Chou-Fasman parameters carry significant information about the environment of each amino acid. The success of the Chou-Fasman's prediction and the properties of the pair and triplet distribution of the amino acid residues suggest that every amino acid has a characteristic sequential residue environment in proteins. The observed preferences could be invoked, for example, in protein design or in the study of the evolutionary relationship of proteins.     

Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit

The key step of N-glycosylation of proteins in the endoplasmic reticulum is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). It transfers the lipid-linked core-oligosaccharide to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains. Biochemical and genetic approaches have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1p, Swp1p, Stt3p, Ost1p, Ost2p, Ost4p, Ost5p, Ostp3 and Ost6p. By blue native polyacrylamide electrophoresis we show that yeast OST consists of two isoforms with distinct functions differing only in the presence of the two related Ost3 and Ost6p proteins. The OST6-complex was found to be important for cell wall integrity and temperature stress. Ost3p and Ost6p are not essential for OST activity, and can in part displace each other in the complex when overexpressed, suggesting a dynamic regulation of the complex formation.     

Bacterial proteins fold faster than eukaryotic proteins with simple folding kinetics

Protein domain frequency and distribution among kingdoms was statistically analyzed using the SCOP structural database. It appeared that among chosen protein domains with the best resolution, eukaryotic proteins more often belong to α-helical and β-structural proteins, while proteins of bacterial origin belong to α/β structural class. Statistical analysis of folding rates of 73 proteins with known experimental data revealed that bacterial proteins with simple kinetics (23 proteins) exhibit a higher folding rate compared to eukaryotic proteins with simple folding kinetics (27 proteins). Analysis of protein domain amino acid composition showed that the frequency of amino acid residues in proteins of eukaryotic and bacterial origin is different for proteins with simple and complex folding kinetics.     

Carbohydrate in pancreatic ribonucleases.

A survey of the presence and compositions of carbohydrate chains attached to pancreatic ribonucleases is given. Carbohydrate chains may occur at asparagine residues in Asn-X-Ser/Thr sequences at four exposed sites of the molecule (positions 21, 34, 62 and 76). These sites form part of highly variant sequences in pancreatic ribonucleases with the consequence that the enzymes from very closely related species may differ in the presence or absence of carbohydrate. In a number of ribonucleases Asn-X-Ser/Thr sequences occur which carry carbohydrate in only a part of the molecules. The occurrence of Asn-X-Ser/Thr sequences entirely without any carbohydrate also has been demonstrated. Compositions of the carbohydrate moieties of ribonucleases from cow, sheep, pig, whales, giraffe, okapi, moose, horse, coypu, chinchilla, and guinea-pig are presented. Striking differences in complexity have been found, both between chains attached to the same site in different species (cow and giraffe), between chains attached to different sites of the same enzyme in one-species (pig) and even between chains attached to the same site in a single species (chinchilla).     

On the origin of eukaryotic cytoskeleton.

The origin of eukaryote-specific cytoskeletal proteins is an issue which is closely related to the origin of the domain Eukarya. As nearly all of these proteins are not found in prokaryotes, the prokaryotic origin of eukaryotic cytoskeletal network suggested by most models is questionable. Eukaryotic cytoskeletal proteins might descend from subpopulations of pre-cells co-existing with Bacteria and Archaea prior to the origin of eukaryotes. The pre-karyote (the host for a-proteobacterial ancestors of mitochondria) might have already possessed eukaryotic-like cytoskeleton. A possible role for viruses in the origin of eukaryotic cytoskeletal proteins is discussed. Viruses parasitizing on pre-cells and/or on the pre-karyote might have themselves used several eukaryotic-like cytoskeletal proteins for segregation and packing of their genomes.     

Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering

In N-glycosylated glycoproteins, carbohydrate is attached to Asn in the sequence Asn-X-Ser/Thr, where X denotes any amino acid. However, the presence of this consensus peptide does not always lead to glycosylation. We have compiled an extensive collection of glycosylated and non-glycosylated Asn-X-Thr/Ser sites and present a statistical study based on this data set. Our results indicate that non-glycosylated sites tend to be found more frequently towards the C termini of glycoproteins, and that proline residues in positions X and Y in the consensus Asn-X-Thr/Ser-Y strongly reduce the likelihood of N-linked glycosylation. Beyond this, there are no obvious local sequence features that seem to correlate with the absence or presence of N-linked glycosylation. These findings are discussed in terms of the prediction and engineering of glycosylation sites in secretory proteins.     

Identification of novel plant peroxisomal targeting signals by a combination of machine learning methods and in vivo subcellular targeting analyses

In the postgenomic era, accurate prediction tools are essential for identification of the proteomes of cell organelles. Prediction methods have been developed for peroxisome-targeted proteins in animals and fungi but are missing specifically for plants. For development of a predictor for plant proteins carrying peroxisome targeting signals type 1 (PTS1), we assembled more than 2500 homologous plant sequences, mainly from EST databases. We applied a discriminative machine learning approach to derive two different prediction methods, both of which showed high prediction accuracy and recognized specific targeting-enhancing patterns in the regions upstream of the PTS1 tripeptides. Upon application of these methods to the Arabidopsis thaliana genome, 392 gene models were predicted to be peroxisome targeted. These predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal. The prediction methods were able to correctly infer novel PTS1 tripeptides, which even included novel residues. Twenty-three newly predicted PTS1 tripeptides were experimentally confirmed, and a high variability of the plant PTS1 motif was discovered. These prediction methods will be instrumental in identifying low-abundance and stress-inducible peroxisomal proteins and defining the entire peroxisomal proteome of Arabidopsis and agronomically important crop plants.     

Simple sequences are rare in the Protein Data Bank

A simple sequence is abundant in the proteins that have been sequenced to date. But unusual protein features, such as a simple sequence, are not present in the same high frequency within structural databases. A subset of these simple sequences, a group with a highly repetitive nature has been shown to be abundant in eukaryotes but not in prokaryotes. In this study, an examination of the eukaryotic proteins in the Protein Data Bank (PDB) has revealed a large deficiency of low complexity, highly repetitive protein repeats. Through simulated databases of similar samples of eukaryotic proteins taken from the National Center for Biotechnology Information (NCBI) database, it is shown that the PDB contains a significantly less highly repetitive, simple sequence than artificial databases of similar composition randomly derived from NCBI. When the structural data for those few PDB sequences that did contain a highly repetitive simple sequence is examined in detail, it is found that in most cases the tertiary structure is unknown for the regions consisting of a simple sequence. This lack of a simple sequence both in the PDB database and in the structural information suggests that this type of simple sequence may produce disordered structures that make structural characterization difficult.