C-terminal Sequence Of Protein Research Articles

The C-Terminal RNLL Sequence of the Plasma Retinol-Binding Protein Is Not Responsible for Its Intracellular Retention

Anin vitromodel system using COS cells that transiently express human plasma retinol binding protein has been set up in which we are able to mimic the retinol dependent secretion of this protein observed in hepatocytes. In the absence of its ligand, plasma retinol binding protein is retained in the endoplasmic reticulum. It contains a C-terminal sequence, RNLL, that could function as a cryptic KDEL motif and thus be responsible for its retention in the endoplasmic reticulum. The model system has been used to test a mutant lacking these four last amino acids for retention and retinol induced secretion. The results obtained show that although plasma retinol binding protein is retained in the endoplasmic reticulum, the RNLL sequence does not seem to be responsible for its retention.

Structure-Function Analysis of the Adherence-Binding Domain on the Pilin of Pseudomonas aeruginosa Strains PAK and KB7

The pili of Pseudomonas aeruginosa mediate bacterial binding to human epithelial cell surfaces. We have previously shown that a 17-residue synthetic peptide, KCTSDQDEQFIPKGCSK, corresponding to the C-terminal sequence of the PAK pilin protein (residues 128-144) contains the adherence binding domain. Another pilin strain, KB7, has been cloned and sequenced [Paranchych et al. (1990) in Pseudomonas Biotransformations, Pathogenesis and Evolving Biotechnology, pp 343-351, American Society for Microbiology, Washington, DC]. The C-terminal 17-residue sequence of the KB7 pilin is SCATTVDAKFRPNGCTD, which is semiconserved as compared to the PAK sequence. In this study, the interactions between the A549 human lung carcinoma cells and the two P. aeruginosa pilin strains were elucidated using a single alanine replacement analysis on the C-terminal 17-residue synthetic peptide of the pilins. The ability of these peptide analogs to inhibit the binding of the biotinylated PAK pili to A549 cells was assessed. Six PAK amino acid side chains (Ser131, Gln136, Ile138, Pro139, Gly141, and Lys144) and nine KB7 side chains (Ala130, Thr131, Thr132, Val133, Asp134, Ala135, Lys136, Arg138, and Pro139) were found to be important in mediating the pilus adhesin binding to A549 cells. In addition, a flexible peptide analog with both cysteine residues replaced by alanine failed to inhibit the binding of PAK pili to A549 cells. This suggests that the interactions between the pilin ligand and the A549 cell surface receptors are dependent on the conformation mediated by the disulfide bridge (Cys129 and Cys142). The residues considered to contribute to bacterial adherence are referred to as the "adhesintope". Four PAK and three KB7 side chains were located in a structurally more rigid region of the disulfide-bridged peptide as revealed by two-dimensional NMR studies [McInnes et al. (1993) Biochemistry 32, 13432-13440]. The structural aspects of the pilin-receptor interactions related to the mapped adhesintope sequences are discussed. The dissimilarities between the PAK and KB7 adhesintopes may suggest that compensatory mutations could occur among different pilin strains so as to allow the pilin adhesins to interact with the same receptor.

The Gene Structure of Tissue Inhibitor of Metalloproteinases (TIMP)-3 and Its Inhibitory Activities Define the Distinct TIMP Gene Family

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a critical role in extracellular matrix homeostasis. We have previously cloned human and mouse TIMP-3 cDNAs and mapped their chromosomal loci (Apte, S. S., Mattei, M-G., and Olsen, B. R. (1994) Genomics 19, 86-90; Apte, S. S., Hayashi, K., Seldin, M. F., Mattei, M-G., Hayashi, M., and Olsen, B. R. (1994) Dev. Dynam. 200, 177-197); the identification of TIMP3 mutations in Sorsby's fundus dystrophy has underscored the functional importance of TIMP-3. We now report that TIMP-3 is encoded by five exons spanning over 30 kilobase pairs of mouse genomic DNA. In the attribution of protein domains to specific exons, as well as exon structures, the Timp-3 and Timp-1 genes are similar, confirming the common evolutionary origin of the TIMPs and defining a distinct gene family. We have expressed human and mouse TIMP-3 in mouse NSO myeloma cells. In each case, an N-glycosylated 27-kDa protein was generated, that, like TIMP-1 and TIMP-2, inhibited collagenase-1, stromelysin-1, and gelatinases A and B. TIMP-3 and TIMP-1 inhibition were quantitatively similar, implying that all TIMPs are equally efficient in MMP inhibition. Instead, differential regulation of the TIMP genes or divergent C-terminal protein sequences may underlie distinct biological functions for each TIMP.

Importance of the C terminus of the hepatitis B virus precore protein in secretion of HBe antigen.

The hepatitis B virus (HBV) e antigen (HBeAg) is a 15 kDa soluble antigen derived from a precursor protein (precore protein) by two processing events, cleavage of the N-terminal signal peptide and cleavage of the C-terminal 34 amino acids. So far, the role of the C-terminal sequences in secretion has not been analysed in full. In this study deletion of the last 60 amino acids was found to abrogate HBeAg secretion whereas deletions of the last 10, 25 or 39 amino acids decreased its secretion rate. These data demonstrate that C-terminal precore protein sequences are crucial for HBe secretion and determine its secretion rate.

A Chemical Method for the C-Terminal Sequence Analysis of Proteins

During the past several years there have been increased interest and substantial progress toward the development of a routine automated method for the C-terminal sequence analysis of peptides and proteins. Such a procedure would complement current methodology for N-terminal sequence determination and would be especially valuable for the sequence determination of proteins with blocked N-termini and for the detection of post-translational processing at the C-terminus of natural and recombinant proteins. Of the chemical methods proposed, the method based on the derivatization of the carboxy-terminal amino acid to a thiohydantoin has been the most widely studied and appears to offer the most promise. Recent work from several laboratories has solved many of the limitations of the thiocyanate approach. This work, coupled with the availability of modern analytical instrumentation, has made the prospects for a routine automated method for C-terminal sequence analysis feasible. This article discusses some of the most recent advances in the chemistry, describes detailed procedures for automation of the chemistry on our new compact protein sequencer and the prototypic G1009A Hewlett-Packard C-Terminal protein sequencing system, and presents several examples of the application of this automated approach to the C-terminal sequencing of peptide and protein samples.

Affinity of Synthetic Peptides for the HSV-2 Ribonucleotide Reductase R1 Subunit Measured with an Iodinated Photoaffinity Peptide

The herpes simplex virus (HSV) ribonucleotide reductase comprises two nonidentical subunits, R1 and R2, which associate to form the active holoenzyme. A sensitive binding assay was developed to measure the affinity of inhibitory peptides for the HSV R1 subunit. The assay involved the use of a photoreactive radioligand [4′-azido-Phe328,3′,5′-125I-Tyr329] HSV R2-(328-337), an analogue of the decapeptide Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu which corresponds to the C-terminal sequence (328-337) of the HSV R2 protein. As the radioligand binds covalently to the HSV R1 subunit upon uv irradiation, the affinity of peptide inhibitors can be easily determined by measuring their ability to compete with this highly specific binding. The method, which did not require any pure preparation of R1, was tested at 25 and 4°C and showed a significant increase in the affinity of the peptide inhibitors at 4°C. The relative affinity of these peptides was in agreement with their relative potency to inhibit reductase activity. The affinity of R2 subunit for R1 was also determined, and an IC50 of 0.05 μM was measured. Altogether, this assay represents a precise and reliable tool with which to study more potent HSV ribonucleotide reductase peptide inhibitors, and the method could be applied to the study of other protein-protein and peptide-protein interactions.

C-Terminal Sequencing of Peptides and Proteins by Successive Degradation with Heptafluorobutyric Anhydride Vapor

Abstract A chemical C-terminal sequencing method of proteins, involving cyanogen bromide cleavage of proteins, selective isolation of the C-terminal peptide and the C-terminal successive degradation with heptafluorobutyric anhydride vapor is presented. FAB/MS analysis of the C-terminal truncated peptide mixture clearly indicated the C-terminal sequence of a protein.

Structure and expression of a 72-kDa regulatory subunit of protein phosphatase 2A. Evidence for different size forms produced by alternative splicing

The trimeric form of protein phosphatase 2A consisting of 36-, 65-, and 72-kDa subunits (previously termed polycation-stimulated protein phosphatase M) was purified from rabbit skeletal muscle. Amino acid sequence data of the 72-kDa regulatory subunit (termed PR72) were used to isolate cDNAs from human heart and fetal brain libraries and libraries derived from WI-38 and MCF-7 cells. The clones isolated from the heart cDNA library revealed an open reading frame encoding a protein with a predicted molecular mass of 62 kDa. All the peptides sequenced from the protein matched with the sequence predicted from the cDNA. However, in vitro transcription and translation from this cDNA yielded a protein with an apparent molecular mass of 72 kDa on sodium dodecyl sulfate-polyacrylamide gels. From brain we isolated cDNA clones spanning an open reading frame encoding a 130-kDa protein (termed PR130). The apparent molecular mass of the protein produced by in vitro transcription and translation was 130 kDa. This protein has exactly the same deduced C-terminal protein sequence as the PR72 subunit from amino acids 45 to 527 but has an N-terminal extension of 665 amino acids. It is likely, therefore, that these two proteins arise from the same gene by alternative splicing. In human tissues several transcripts were detected by Northern analysis generated probably by the use of different polyadenylation signals and alternative splicing. High levels of the PR72 mRNAs were detected in heart and muscle, while lower levels of PR130 transcripts were found in heart, brain, placenta, lung, muscle, and kidney.

Sequencing of peptides and proteins from the carboxy terminus

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with {NCS} − anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.

Purification and characterization of two serine carboxypeptidases from Aspergillus niger and their use in C-terminal sequencing of proteins and peptide synthesis.

A procedure was developed to prepare in large amounts two carboxypeptidases, CPD-I and CPD-II, from Aspergillus niger. They were each shown to be serine proteases and single-chain monomers with molecular masses of ca. 81 kDa and containing 22% carbohydrates. Amino acid analysis, carbohydrate determination, and N-terminal sequencing (20 to 25 residues) were performed on each enzyme. CPD-I showed sequence homologies with malt carboxypeptidase II, while the N terminus of CPD-II was different from that of any known serine carboxypeptidase. Like carboxypeptidase Y from Saccharomyces cerevisiae and carboxypeptidase III from malt, CPD-II contained a free sulfhydryl group that could play a role in catalysis. Both A. niger enzymes had pH optima of about 4 and were unstable above pH 7. Their specificities for substrate positions P1 and P'1 were characterized by use of, as substrates, a series of N-blocked amino acid esters and dipeptides. Both enzymes were specific for Arg, Lys, and Phe in P1. CPD-I preferred hydrophobic residues in P'1, while CPD-II was highly specific for Arg and Lys in this position. Each displayed an original specificity when P1 and P'1 were considered together. The specificities were also studied by analyzing the time course of the release of amino acids from eight different peptides of various lengths. CPD-I and CPD-II appeared to be quite suitable for C-terminal sequence studies as well as for the synthesis of peptide bonds. The latter was studied with two peptide esters as aminolysis substrates and a series of amino acid amides as nucleophiles.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional dissection of the HIV-1 Rev trans-activator—Derivation of a trans-dominant repressor of Rev function

Human immunodeficiency virus type 1 (HIV-1) encodes a nuclear trans-activator, termed Rev, that is required for the expression of the viral structural proteins and, hence, for viral replication. The Rev protein acts posttranscriptionally to induce the sequence-specific nuclear export of unspliced HIV-1 mRNA species that are otherwise excluded from the cell cytoplasm. We have used site-directed mutagenesis to identify two distinct regions of the HIV-1 Rev protein that are required for in vivo biological activity. The larger and more N-terminal of these two regions includes, but extends beyond, an arginine-rich sequence element required for nuclear localization. Mutation of a second, more C-terminal Rev protein sequence element was found to yield defective Rev proteins that act as trans-dominant inhibitors of Rev function. These Rev mutants are shown to inhibit HIV-1 replication when expressed in transfected cells and may have potential application in the treatment of HIV-1 related disease.

The computer-assisted carboxypeptidase method for C-terminal sequencing of proteins and polypeptides

On further study of the computer-assisted carboxypeptidase method for sequencing the C-terminals of proteins or peptides, two computer programs—DPS and CPA were successfully developed on VAX-11/780 computer and tested with some synthetic peptides and the degradation fragment of the natural protein as model substrates. The C-terminal sequence of CB-3, one of the CNBr degradation fragments of trichosanthin, had first been determined by this method as: -Ser-Ala-Ser-Ala-Ala-Leu-Hse-OH, which was later confirmed by other ways of sequencing. This method is not only able to extend the C-terminal sequencing up to 7 amino acid residues, but also useful for determining the C-terminal sequence with repeating amino acid residues.

Sequence homology between barley endosperm protein Z and protease inhibitors of the α 1-antitrypsin family

Six cDNA clones encoding parts of protein Z, a major barley endosperm albumin, have been identified. Nucleotide and amino acid sequencing have established a 180 residues long C-terminal amino acid sequence of protein Z as well as two minor amino acid sequences (14 and 7 residues). These sequences show that barley protein Z is homologous with human α 1-antitrypsin, human otj-antichymotrypsin, human antithrombin III, mouse contrapsin and chicken ovalbumin (26–32% of the 180 residues in the C-terminal sequence in identical positions). The sequence homology and specific cleavage of protein Z at a bond corresponding to the reactive site of the inhibitors indicate a possible inhibitory function. Inhibition of microbial or pancreatic serine proteases could, however, not be associated with protein Z.

A membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3–9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3–9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3–9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3–9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3–9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex.

The structure of tobacco necrosis virus: II. Terminal amino acid residues of the protein subunit

The amino end group of the protein of tobacco necrosis virus (TNV), unlike those of most other plant viruses, was found to be unsubstituted. Reactions with fluorodinitrobenzene and 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl) resulted in the identification of alanine as the amino terminal residue. Leucine aminopeptidase, at slightly alkaline pH, released up to 62% of the theoretical value of alanine from the whole virus. The TNV coat protein N-terminus is therefore identical to that of its satellite and the RNA-containing bacteriophages. Kinetic studies with carboxypeptidase suggested that the C-terminal sequence of tobacco necrosis virus coat protein is …Ser·Val·Val·Met. Quantitative end-group analysis was in agreement with the earlier suggested molecular weight of the protein subunit as 33,300. The number of tryptic peptides separated on fingerprints was also consistent with these data.

C-terminal amino-acid sequence of tobacco mosaic virus protein

C-terminal amino-acid sequence of tobacco mosaic virus protein