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PubMed1995

Truncated profibrillin of a Marfan patient is of apparent similar size as fibrillin: intracellular retention leads to over-N-glycosylation.

We studied profibrillin-1 (proFib) synthesis and microfibril formation in cultured fibroblasts from an individual with severe Marfan syndrome harboring a premature stop codon (W2756ter) in one FBN1 allele. Rotary shadowing analysis of extracellular matrix produced by these cells revealed the presence of only a very few intact microfibrils which showed marked disorganisation within the interbeaded domains. Metabolic pulse-chase studies identified intracellularly a population of truncated proFib molecules which were secreted more slowly than the normal proFib derived from the normal allele. Culture media contained strikingly reduced amounts of wild-type proFib in comparison to fibrillin (Fib). Our findings imply that (1) the truncated proFib is secreted and disturbs microfibril assembly; (2) the mutation is probably close to a putative cleavage site in the proFib C terminus necessary for the conversion of proFib to Fib; (3) the truncated proFib is over-N-glycosylated due to intracellular retention rather than incomplete cleavage of proFib with persistence of N-glycosylated sites; (4) not all potential N-glycosylation sites in proFib seem to be normally used, since we could produce over-N-glycosylated proFib in normal cells by brefeldin A mediated intracellular captivation and subsequent appearance of over-glycosylated Fib in culture medium upon removal of the compound. It is conceivable that post-translational over-modification might be important for modulating the phenotype of FBN1 mutations in Marfan syndrome.

Actin Cytoskeleton↗
PubMed2000

Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation.

N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We have determined the crystal structure of the NAD(+) complex of a truncated form of the enzyme, GFORDelta, in which the first 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, refined at 2.7 A resolution (R(cryst)=24.1%, R(free)=28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of the N-terminal arm also greatly increases the solvent exposure of the cofactor; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORDelta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity.

Amino Acid Sequence↗
PubMed2001

The structure of truncated recombinant human bile salt-stimulated lipase reveals bile salt-independent conformational flexibility at the active-site loop and provides insights into heparin binding.

Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of BSSL on bile acids for activity with water-insoluble substrates differentiates it from other lipases. We have determined the crystal structure of a truncated variant of human BSSL (residues 1-5.8) and refined it at 2.60 A resolution, to an R-factor of 0.238 and R(free) of 0.275. This variant lacks the C-terminal alpha-helix and tandem C-terminal repeat region of native BSSL, but retains full catalytic activity. A short loop (residues 115-126) capable of occluding the active-site (the active site loop) is highly mobile and exists in two conformations, the most predominant of which leaves the active-site open for interactions with substrate. The bile salt analogue 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid (CHAPS) was present in the crystallisation medium, but was not observed bound to the enzyme. However, the structure reveals a sulfonate group from the buffer piperizine ethane sulfonic acid (PIPES), making interactions with Arg63 and His115. His115 is part of the active-site loop, indicating that the loop could participate in the binding of a sulphate group from either the glycosaminoglycan heparin (known to bind BSSL) or a bile acid such as deoxycholate. Opening of the 115-126 active-site loop may be cooperatively linked to a sulphate anion binding at this site. The helix bundle domain of BSSL (residues 319-398) exhibits weak electron density and high temperature factors, indicating considerable structural mobility. This domain contains an unusual Asp:Glu pair buried in a hydrophobic pocket between helices alpha(H) and alpha(K) that may be functionally important. We have also solved the structure of full-length glycosylated human BSSL at 4.1 A resolution, using the refined coordinates of the truncated molecule as a search model. This structure reveals the position of the C-terminal helix, missing in the truncated variant, and also shows the active-site loop to be in a closed conformation.

Animals↗
PubMed1998

Rapid RT-PCR-based protein truncation test in the screening for 5' located mutations of the APC gene.

Although in vitro protein synthesis is a rapid method to screen for translational stops in the adenomatous polyposis coli (APC) gene, truncating mutations at the 5' most end are at risk of being overseen due to their small size. The authors describe a reverse transcriptase-polymerase chain reaction (RT-PCR)-based protein truncation test specifically designed for detecting truncated polypeptide chains of less than 10 kDa. Using this detection system, three novel germline mutations in familial adenomatous polyposis (FAP) patients were identified, i.e. a Gly101 Ter non-sense mutation in exon 3, an exon 4 splice acceptor mutation and a 555delC deletion in exon 5. Morever, a patient manifesting congenital hypertrophy of the retinal pigmented epithelium (CHPRE) was detected with an Arg232Ter mutation in exon 6. This is, to the authors' knowledge, the fourth exception to the rule that FAP patients manifesting CHRPE harbour genetic alternations downstream from APC exon 9. Hence, an alternative hotspot for non-sense mutations associated with CHRPE appears to encompass the codons 215, 216 and 232. Patients reported in this study, exhibited relatively mild clinical symptoms with respect to the age of onset of malignancy (> 50 years of age) and the number of polyps (70-100 adenomas). However, manifestation of severe duodenal adenomatosis was independent of the attenuated colorectal FAP phenotype.

Adenomatous Polyposis Coli Protein↗
PubMed2002

Chemical chaperones reduce aggregate formation and cell death caused by the truncated Machado-Joseph disease gene product with an expanded polyglutamine stretch.

Machado-Joseph disease/spinocerebellar ataxia-3 (MJD/SCA-3) is an inherited neurodegenerative disorder caused by expansion of the polyglutamine stretch in the MJD gene-encoded protein ataxin-3. The truncated form of mutated ataxin-3 causes aggregation and cell death in vitro and in vivo. Abnormal conformation and misfolding of the pathological protein are assumed critical to pathogenesis. To test this hypothesis, we transfected BHK-21 and Neuro2a cells transiently with N-terminal truncated ataxin-3 with an expanded polyglutamine stretch. We then studied the effects of organic solvent dimethyl sulfoxide (DMSO), cellular osmolytes glycerol, and trimethylamine N-oxide (TMAO) on aggregate formation and cell death. These reagents stabilize proteins in their native conformation and are called chemical chaperones based on their influence on protein folding. Aggregate formation and cytotoxicity induced by truncated expanded ataxin-3 were reduced by exposing cells to these chemical chaperones. Our results indicate the potentially useful therapeutic strategy of the chemical chaperones in preventing cell death in MJD.

Acetylcysteine↗
PubMed1997

Expression, purification, and characterization of the two human primase subunits and truncated complexes from Escherichia coli.

Eukaryotic DNA replication is primed by small RNA primers synthesized by the two-subunit primase complex, p58 and p49, where the p49 subunit contains the catalytic activity. The cDNA's for these two human DNA primase subunits were amplified, sequenced, and overexpressed in Escherichia coli. Specific assays for initiation revealed that although the smaller subunit contains catalytic function, initiation requires the presence of the larger subunit. A two-plasmid system was developed for the coexpression of both subunits in E. coli. This system was exploited to express and study truncations of the larger, human p58 subunit in order to investigate its role in primer formation. Analysis of the complexes formed between the truncated human p58 subunits and the native human p49 subunit revealed that protein-protein contacts between these two subunits occur over several regions of the human p58 subunit. Of four primase complexes containing different truncated p58 subunits only one complex supported initiation as measured by the formation of dinucleotides. All complexes supported the extension of oligoA-primed polydT, suggesting that the intrinsic RNA polymerase activity residing in the smaller subunit was not affected. These results suggest that several regions of the human p58 subunit are in contact with the human p49 subunit during the initiation of primer synthesis.

Cloning, Molecular↗
PubMed1999

Recombinant and truncated tetanus neurotoxin light chain: cloning, expression, purification, and proteolytic activity.

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.

Cloning, Molecular↗
PubMed1993

Topology of the major capsid protein P3 of bacteriophage PRD1: analysis using monoclonal antibodies and C-terminally truncated proteins.

Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid-protein vesicle containing the viral dsDNA genome. In this study we characterize a panel of monoclonal antibodies (MAb) against P3. The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules. Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions. Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies. Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein. The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid. MAbs 3N81 and 3R2 recognize epitopes in the region of amino acids 159-168, which is part of the first predicted beta-barrel structure of P3. The third antigenic region is in the second predicted beta-barrel, between amino acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map. The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17. Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation.

Antibodies, Monoclonal↗
PubMed1994

Presence of a truncated form of the Sendai virus P protein in a long-term persistent infection: implications for the maintenance of the persistent state.

In this report we have monitored viral gene expression, both at the RNA and protein level, after the establishment of a long-term persistent infection of Sendai virus. The persistent infection was initially established by infecting BHK cells with a viral stock containing a short (1.4 kb) copy-back DI (DIH4). After over 120 weeks in culture this short copy-back DI had been replaced by two large deletion DIs (approximately 7 and 12 kb) from which was expressed an N-terminally truncated form of the P protein. The mRNA for this protein was detected in cells and the deletion within the P gene was mapped by PCR cloning and sequencing of intracellular nucleocapsid RNA. This truncated P protein (derived by deleting the N-terminal half of the cloned Pwt gene) has already been shown to function as a dominant negative for DI replication when driven by cloned viral genes. Cloning and expression of the truncated P from the long-term persistent infection revealed that this protein had retained the dominant negative phenotype. The presence of such a protein would severely depress viral gene expression and may therefore play an important role in the maintenance of persistence.

Animals↗
PubMed1995

Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form.

The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus. Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure. The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not. This conversion was dilution independent and thought to be intramolecular. Isolation of the approximately 2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxy-terminus of the proteinase. A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity. Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form. The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form.

Amino Acid Sequence↗
PubMed2001

Truncation of the human adenovirus type 5 L4 33-kDa protein: evidence for an essential role of the carboxy-terminus in the viral infectious cycle.

The subgroup C human adenovirus L4 33-kDa protein is a nuclear phosphoprotein that plays a direct, but dispensable, role in virion assembly. The r-strand open reading frame (ORF) for this protein lies opposite to the 5' end of the l-strand E2 early (E2E) transcription units. To facilitate studies of regulation of E2E transcription, we wished to construct a mutant virus in which the 33-kDa ORF was truncated to serve as a background into which specific E2E mutations could be introduced without also altering the 33-kDa protein. We constructed viral DNA (vDNA) containing within the 33-kDa ORF two tandem, premature stop codons that should prevent translation of the C-terminal 47 amino acids of the protein (Delta47). We report here the unanticipated lethality of such truncation of the L4 33-kDa protein. Viral DNA harboring the Delta47 mutations did not produce infectious virus when transfected into cultured cells. In contrast, infectious virus was recovered upon transfection of revertant vDNA, indicating that the Delta47 mutations were responsible for the observed phenotype. The Delta47 mutations did not affect E2E transcription or production of the E2 DNA-binding protein. Transfected Delta47 vDNA was replicated and directed the production of early and late viral proteins, including hexon protein in the trimer conformation. However, no virus particles of any kind were produced. We propose that truncation of the adenovirus 33-kDa protein results in a lethal, late block in the infectious cycle during the assembly of progeny virions and discuss the implications of this phenotype for the mechanism of virion assembly.

Adenoviruses, Human↗
PubMed1996

Purification and characterization of a truncated Bacillus subtilis alpha-amylase produced by Escherichia coli.

A Bacillus subtilis amylase gene was inserted into a plasmid which was transferred to Escherichia coli. During cloning, a 3' region encoding 171 carboxy-terminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of the B. subtilis complete alpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with a Km of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50 degrees C, and the purified enzyme was stable at temperatures up to 50 degrees C. While Hg2+, Fe3+ and Al+3 were effective in inhibiting the truncated enzyme, Mn2+ and Co2+ considerably enhanced the activity.

Bacillus subtilis↗
PubMed1996

Another CT sign of sinonasal polyposis: truncation of the bony middle turbinate.

In order to study the features of sinonasal polyposis (SNP) on CT, 100 consecutive coronal sinus CT examinations done for chronic inflammatory sinonasal disease were reviewed. The CT findings of the 27 fully documented SNPs were analyzed. All our SNPs were bilateral. There was a strong tendency for extensive involvement. Nasal polyps were seen in 22 of 27(81%); bony trabecular deossification in 23 of 27 (85%); widening of infundibulum in 26 of 27 (96%). We discovered a new sign "truncation of the bony middle turbinate", where the bulbous part of bony middle turbinate was missing, in 15 of 26 (58%) of SNP patients without a previous history of middle turbinectomy, 12 of 15(80%) were bilateral. The one SNP patient (1 of 27) with previous middle turbinectomy was not regarded to be real truncation. Truncation of the bony middle turbinate is a characteristic and easily recognizable ancillary sign, and is not seen in other patterns of sinusitis. Together with other features on coronal sinus CT, this adds diagnostic confidence in diagnosing sinonasal polyposis.

Adolescent↗
PubMed1990

Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.

Tomato plants were transformed with a chimaeric polygalacturonase (PG) gene, designed to produce a truncated PG transcript constitutively. In these plants expression of the endogenous PG gene was inhibited during ripening, resulting in a substantial reduction in PG mRNA and enzyme accumulation. This inhibition was comparable to that achieved previously using antisense genes. The expression of the truncated gene in ripe fruit was substantially lower than its expression in green fruit. Thus expression of both the endogenous and truncated genes is reduced in ripe fruit in which both are active. The implication of this observation is discussed in relation to the possible mechanism whereby sense constructs inhibit gene expression.

Blotting, Northern↗
PubMed1990

Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events.

During Agrobacterium tumefaciens infection, the T-DNA flanked by 24 bp imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions. Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome. When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (greater than 70%) in transcriptionally active regions of the plant genome. When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation. These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length. Deletion of the right border region prevents the detection of activated marker genes. Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process. In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 10(5) regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency.

Base Sequence↗
PubMed2002

Truncated (des-[27-31]) C-peptide is not a major secretory product of human islets.

AIMS/HYPOTHESIS: It has been suggested that C-peptide is bioactive and that such bioactivity is lost when the last five amino acids are removed. In rats, C-peptide is truncated in beta-cell granules leading to the loss of these last five residues and secretion of des-[27-31]-C-peptide. The aim of this study was to determine whether this truncated form of C-peptide was also a secretory product of human islets. METHODS: Plasma from healthy subjects, patients with Type II (non-insulin-dependent) diabetes mellitus or insulinoma and cord blood was analysed by HPLC and ELISA. This method allows for separation and quantification of intact C-peptide and des-[27-31]-C-peptide. Human islets were pulse-chased and secretion stimulated by a mixture of secretagogues. Radioactive products secreted to the medium were analysed by HPLC and the relative amount of intact and truncated C-peptide measured. RESULTS: The proportion of total C-peptide immunoreactivity comprised of des-[27-31]-C-peptide was 1.5% or less in all plasma samples, except for that from one patient with insulinoma where it was 4.2%. The proportion of radiolabelled des-[27-31]-C-peptide released from isolated islets was less than 1%. CONCLUSION/INTERPRETATION: In contrast to the situation in rats, des-[27-31]-C-peptide is not a major secretory product of human islets and its contribution to total circulating C-peptide is not increased in Type II diabetes or in patients with insulinoma.

Adult↗
PubMed1999

Experimental, extrapolated and truncated areas under the concentration-time curve in bioequivalence trials.

OBJECTIVE: The extrapolated area under the concentration-time curve (AUC0-infinity) for any drug is considered by operating guidelines as the primary parameter related to the extent of absorption in single-dose bioavailability and bioequivalence trials. Not more than 20% should be added to the experimental AUC (AUC0-t) in the extrapolating procedure. However, in certain specific cases, it is problematic and, in other cases, impossible to respect the above requirement. It was intended to demonstrate that truncated AUC or AUC0-t would be used in bioequivalence trials when the AUC cannot be extrapolated. METHODS: AUC0-t and truncated AUC were compared at various time intervals with AUC0-infinity in a series of 19 single-dose bioequivalence trials covering 15 different drugs, each carried out on 12-24 healthy volunteers. Point estimators and 90% confidence intervals in the 0.80-1.25 and 0.70-1.43 ranges were statistically processed using log-transformed parameters. RESULTS: In all the trials considered, overlapping point estimators and 90% confidence intervals were invariably obtained, regardless of the AUC used. CONCLUSION: The use of AUC0-t or truncated AUC may be considered an ancillary procedure in bioequivalence trials when AUC cannot be extrapolated. This occurs with most extended-release formulations, endogenous substances, and poorly absorbed drugs. It also occurs in trials with one or more poor metabolisers, with drugs possessing very long elimination half-lives, and with bioassays having problems of sensitivity that preclude sufficiently precise plasma concentration measurement over the required sampling period.

Anticarcinogenic Agents↗
PubMed2001

High-efficiency synthesis of oligosaccharides with a truncated beta-galactosidase from Bifidobacterium bifidum.

An exceptionally large beta-galactosidase, BIF3, with a subunit molecular mass of 188 kDa (1,752 amino acid residues) was recently isolated from Bifidobacterium bifidum DSM20215 [Møller et al. (2001) Appl Environ Microbiol 67:2276-2283]. The BIF3 polypeptide comprises a signal peptide followed by an N-terminal beta-galactosidase region and a C-terminal galactose-binding motif. We have investigated the functional importance of the C-terminal part of the BIF3 sequence by deletion mutagenesis and expression of truncated enzyme variants in Escherichia coli. Deletion of approximately 580 amino acid residues from the C-terminal end converted the enzyme from a normal, hydrolytic beta-galactosidase into a highly efficient, transgalactosylating enzyme. Quantitative analysis showed that the truncated beta-galactosidase utilised approximately 90% of the reacted lactose for the production of galacto-oligosaccharides, while hydrolysis constituted a 10% side reaction. This 9:1 ratio of transgalactosylation to hydrolysis was maintained at lactose concentrations ranging from 10% to 40%, implying that the truncated beta-galactosidase behaved as a "true" transgalactosylase even at low lactose concentrations.

Bifidobacterium↗