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PubMed2006

[Construction and transfection of eucaryotic expression recombinant vector containing truncated region of UL83 gene of human cytomegalovirus and it's sheltered effect as DNA vaccine].

To construct eucaryotic expression recombinant vector containing vivo truncated region of UL83 gene of human cytomegalovirus, realize its steady expression in Hep-2 cell, and study sheltered effect of the eucaryotic expression recombinant vector as DNA vaccine. A vivo truncated UL83 gene fragment encoding for truncated HCMV pp65 was obtained by PCR from human cytomegalovirus AD169 stock genome. By gene recombinant ways, the truncated UL83 gene fragment was cloned into eucaryotic expression vector pEGFP-C1 with reported gene coding GFP to construct recombinant vector pEGFP-C1-UL83. The recombinant vector pEGFP-C1-UL83 was tested by different methods including PCR, restriction digestion and gene sequencing. Test results showed the recombinant vector was constructed successfully. After pEGFP-C1-UL83 was transfected into Hep-2 cell by lipofectin mediation, expression of GFP and truncated pp65 fusion protein in Hep-2 cell was observed at different time points by fluorescence microscope. Results showed that quantity of fusion protein expression was the highest at 36h point. Then, Hep-2 cell was cultured selectively by RPMI-1640 containing G418 (200 microg/mL) to obtain a new cell stock of expressing truncated UL83 Gene fragment steadily. RT-PCR and Western blot results showed the truncated fragment of UL83 gene could be expressed steadily in Hep-2 cell. The result showed a new cell stock of expressing Tpp65 was established. This cell stock could be useful in some HCMV research fields, for example, it could be a tool in study of pp65 and HCMV infection, and it could provide a platform for the research into the therapy of HCMV infection. Immune sheltered effect of pEGFP-C1-UL83 as DNA vaccine was studied in vivo of HCMV congenital infection mouse model. The mouse model was immunized solely by pEGFP-C1-UL83, and was immunized jointly by pEGFP-C1-UL83 and its expression product. When the mouse was pregnant and brought to bed, differential antibody of anti-HCMV pp65 was tested by indirect ELISA in mother mouse, the infectious virus was separated with the method of virus separation, and pp65 antigen was checked up by indirect immunofluorescence staining in fetal mouse. Results showed differential antibody of anti-HCMV pp65 was produced in mouse model. Tilter of the antibody was from 1:2.51 to 1:50.79. Results of virus separation and pp65 checkup of fetal mouse brain tissue were negative. So the conclusion can be reached that pEGFP-C1-UL83 as DNA vaccine in vivo has sheltered effect which can prevent HCMV vertical transmission from mother mouse to her fetus.

Animals↗
PubMed1994

Contribution of the C-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases. C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin.

In this study, we have used high resolution gel-filtration chromatography and measurements of Ki to compare the capacity of full-length native stromelysin, C-terminal truncated stromelysin (Phe100-Pro273), and matrilysin (the only metalloproteinase spontaneously lacking a C-terminal hemopexin-like domain) to bind to the tissue inhibitor of metalloproteinases (TIMP). While prostromelysin failed to bind TIMP, active stromelysin bound to the inhibitor avidly, exhibiting an affinity for TIMP (Ki = 8.3 x 10(-10) M) essentially identical to that of active interstitial collagenase as determined by competition experiments. C-terminal truncated stromelysin also formed a higher M(r) complex with TIMP which survived gel filtration. However, when truncated stromelysin was forced to compete with its full-length parent molecule for limiting amounts of TIMP, the full-length enzyme preferentially bound to the inhibitor. Indeed, binding studies indicated a Ki of 5.95 x 10(-9) M for the truncated variant's interaction with TIMP, only 14% as tight as that of full-length stromelysin. We also examined the interaction between TIMP and matrilysin, the only metalloproteinase which naturally lacks a C-terminal domain. Promatrilysin failed to bind the inhibitor. However, active matrilysin readily bound TIMP, forming a complex that resisted separation by gel filtration. When active matrilysin was forced to compete with truncated stromelysin for limiting amounts of TIMP, both enzymes appeared to complex the inhibitor with nearly equivalent efficacy. Indeed, active matrilysin exhibited a Ki for TIMP of 4.5 x 10(-9) M, essentially identical to that of truncated stromelysin. These data indicate that, as is true for collagenase, the C-terminal domain of stromelysin contributes significantly to its capacity to bind the physiologic inhibitor, TIMP. Furthermore, since stromelysin readily processes in vitro to a C-terminal truncated form, this enzyme species, as well as the full-length metalloproteinase matrilysin, may resist inhibition by TIMP in areas of active inflammation in vivo.

Binding Sites↗
PubMed1993

Origin of a soluble truncated transferrin receptor.

It has recently become evident that elevation of reticulocytes in the circulation of several species, including humans, leads to the formation of a noncellular transferrin receptor (TFR). In humans, the majority of the released receptor is in truncated form (Shih et al: J Biol Chem 265:19077, 1990). In other species (sheep, rat, chicken) the receptor is associated with a vesicle (exosome) and is full length (Johnstone et al: J Cell Physiol 147:27, 1991). In this report we show that in sheep reticulocytes incubated in vitro, the majority (approximately 75%) of the released receptor is of native size and is exosome associated. A fraction (approximately 25%) is a truncated form of approximately 80 Kd corresponding to the exofacial domain of the TFR. Herein we also address the question of whether the truncated receptor originates by proteolytic cleavage directly from the cell surface or by cleavage from exosomes. Using surface 125I-labeled sheep reticulocytes as the experimental model, we show that during in vitro maturation, 125I-TFR of native size appears in exosomes before the soluble, truncated, exofacial domain of the receptor is detected in the medium. Because cleavage and release of the exofacial domain would likely leave the truncated cytoplasmic and transmembrane domains in the originating membrane (plasma membranes or exosomes), both fractions were probed with antibodies specifically generated against the cytoplasmic domain of the receptor. Only exosomes, not plasma membranes, show the presence of a approximately 17-Kd peptide recognized by the antibody to the cytoplasmic domain of the transferrin receptor. Thus, it is concluded that the truncated, soluble receptor originates from exosomes in sheep. A 17-Kd cytoplasmic domain of the TFR was also detected in exosomes from the reticulocytes of an anemic man, suggesting that the truncated receptor in man may also originate from exosomes. Using in vitro cultures of surface 125I-labeled sheep reticulocytes, it is concluded that exosome formation is the principal route for maturation-associated loss of the TFR. A similar conclusion was made earlier (Johnstone et al: J Cell Physiol 147:27, 1991) for the nucleoside transporter of maturing sheep reticulocytes.

Animals↗
PubMed1996

Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation.

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c-derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid-specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.

Animals↗
PubMed1998

The hyperresponsiveness of cells expressing truncated erythropoietin receptors is contingent on insulin-like growth factor-1 in fetal calf serum.

We demonstrate herein that the well documented hyperresponsiveness to erythropoietin (Epo) of Ba/F3 cells expressing C-terminal truncated erythropoietin receptors (EpoRs) is contingent on these cells being in fetal calf serum (FCS). In the absence of FCS, their Epo-induced proliferation is far poorer than Ba/F3 cells expressing wild-type (WT) EpoRs. This hyporesponsiveness in the absence of serum is also seen in DA-3 cells expressing these truncated EpoRs. In fact, long-term proliferation studies performed in the absence of serum show that even at saturating concentrations of Epo, Ba/F3 cells expressing these truncated receptors die via apoptosis, while cells bearing WT EpoRs do not, and this programmed cell death correlates with an inability of Epo-stimulated Ba/F3 cells expressing truncated EpoRs to induce the tyrosine phosphorylation of MAPK and the activation of p70(S6K). Using neutralizing antibodies to insulin-like growth factor (IGF)-1, we show that a major non-Epo factor in FCS that contributes to the hyperresponsive phenotype of Ba/F3 cells expressing truncated EpoRs is IGF-1. Our results suggest that the Epo-hypersensitivity of truncated EpoR expressing Ba/F3 cells is due to the combined effects of these EpoRs not possessing a binding site for the negative regulator, SHP-1, and the triggering of proliferation-inducing/apoptosis-inhibiting cascades, lost through EpoR truncation, by IGF-1.

Animals↗
PubMed1999

Defective internalization and sustained activation of truncated granulocyte colony-stimulating factor receptor found in severe congenital neutropenia/acute myeloid leukemia.

Acquired mutations truncating the C-terminal domain of the granulocyte colony-stimulating factor receptor (G-CSF-R) are found in about 20% of severe congenital neutropenia (SCN) patients, with this cohort of patients predisposed to acute myeloid leukemia (AML). In myeloid cells, such mutations act in a dominant-negative manner leading to hyperproliferation and lack of differentiation in response to G-CSF. However, why these truncated receptors are dominant in function over wild-type receptors has remained unclear. We report that ligand-induced internalization of truncated G-CSF-R is severely impaired compared with the wild-type receptor, which results in sustained activation of STAT proteins. Strikingly, in cells coexpressing both truncated and wild-type forms, the truncated receptors acted dominantly with regard to both internalization and sustained activation. Site-directed mutagenesis of the C-terminus showed that receptor tyrosines in this region were dispensable for internalization, whereas a di-leucine-containing motif in Box B3 played some role. However, loss of the di-leucine motif was not the critical determinant of the sustained activation status of truncated receptors. These data suggest that defective internalization, leading to extended receptor activation, is a major cause of the dominant hyperproliferative effect of truncated G-CSF receptors, which is only partially due to the loss of a di-leucine motif present in the Box B3 region of the full-length receptor.

Animals↗
PubMed2001

Truncated forms of the recombinant Escherichia coli ADP-glucose pyrophosphorylase: the importance of the N-terminal region for allosteric activation and inhibition.

Truncated forms of Escherichia coli ADPglucose pyrophosphorylase were constructed using recombinant DNA techniques. A truncated form of the enzyme having the first 11 amino acid residues from the N-terminus and 2 amino acid residues from the C-terminus deleted was found to be highly active in absence of activator. A 1.6-fold activation by 1.5 mM fructose 1,6 bis-phosphate was observed for the truncated enzyme as compared to the 30-fold activation seen for the intact enzyme. Inhibition of the truncated enzyme by AMP was less than that seen with the intact enzyme. Similar properties were displayed by an enzyme truncated only at the N-terminal. Conversely, the C-terminal truncated enzyme shortened by 2 amino acid residues at the C-terminus is as sensitive as the intact enzyme to activation and inhibition. These results suggest that the N-terminal region is required for allosteric regulation of the enzyme.

Adenosine Monophosphate↗
PubMed1993

Bacterial synthesis of truncated forms of the human vitamin D receptor and characterization of anti-receptor monoclonal antibodies.

We biosynthesized full-length (amino acids 1-427) and truncated human 1,25-dihydroxyvitamin D3 receptor proteins that encompassed only the putative DNA binding domain (amino acids 1-112) or the DNA binding domain and parts of the sterol binding domain (amino acids 1-193 and 1-328) in a bacterial expression system. We also prepared monoclonal antibodies against the full-length vitamin D receptor. The binding properties of the monoclonal antibodies were characterized by their ability to bind to full-length and truncated vitamin D receptor protein constructs. Seven of twelve monoclonal antibodies recognized the full-length receptor protein. These antibodies bound to truncated hVDR proteins with decreasing affinities as successive truncations were made from the carboxy-terminal end of the receptor protein. The five remaining monoclonal antibodies recognized the full-length and truncated receptor proteins with equally low affinities. Truncated forms of the vitamin D receptor and region-specific antibodies will be useful in assessing the properties of the receptor.

Antibodies, Monoclonal↗
PubMed1999

Truncation of betaA3/A1-crystallin during aging of the bovine lens; possible implications for lens optical quality.

During aging, extensive modifications of eye lens proteins take place, which may contribute to the development of cataract. Truncation of the accessible extensions of beta-crystallins has been suggested to be an important factor in this process. We therefore studied the truncations of bovine betaA3- and betaA1-crystallin in more detail. These proteins are identical except for the length of their N-terminal extension, 30 and 13 residues, respectively. The water-soluble and -insoluble proteins from cortex and nucleus of bovine lenses of different ages were separated by 2D-gel electrophoresis and immuno-blotted with an antiserum against betaA3. Two major truncation products were detected, which by sequence analysis were found to correspond to betaA3 having lost 11 or 22 amino acids. betaA3(-11) was only observed in the nucleus, whereas betaA3(-22) was present both in cortex and nucleus. We argue, therefore, that each of these two products is produced by a separate proteolytic enzyme. betaA3(-22) can originate by cleavage of betaA3, betaA1 and betaA3(-11). Truncation of betaA3 occurs more readily than that of betaA1, while betaA3(-11) disappears at an intermediate rate. It appears that the longer the N-terminal extension, the easier proteolysis takes place. Truncated proteins are not necessarily prone to end up in the water-insoluble fractions; other modifications leading to charge changes are more likely to be responsible for insolubilization. Truncation of the extensions of beta-crystallins could be a functional rather than a harmful process during aging of the lens; by modulating protein repulsion, it may help to maintain the protein concentration gradient that is necessary for the optical quality of the lens.

Aging↗
PubMed1993

Truncated GLP-1 (proglucagon 78-107-amide) inhibits gastric and pancreatic functions in man.

We studied the effect of intravenous infusion of synthetic truncated GLP-1 (proglucagon 78-107-amide) on fasting and postprandial gastric acid secretion, gastric emptying, and pancreatic secretion of trypsin and lipase in eight normal volunteers using marker dilution and aspiration technique. The infusion resulted in a plasma concentration of 110 +/- 14 pmol/liter (mean +/- SEM). Truncated GLP-1 significantly inhibited postprandial acid secretion by 43 +/- 11% in spite of unchanged plasma gastrin concentration. Gastric emptying rate decreased significantly; 50% emptying time increased from 16 +/- 2 min to 30 +/- 5 min. Postprandial trypsin and lipase outputs were significantly inhibited by 47 +/- 17% and 40 +/- 9% during truncated GLP-1 infusion. Pancreatic enzyme output was linearly correlated to gastric emptying, and truncated GLP-1 did not affect this relationship, suggesting that the effect on pancreatic secretion was secondary to the effect on gastric emptying. Postprandial insulin and glucagon concentrations were similar with and without truncated GLP-1 infusion in spite of significantly lower blood glucose levels (5.2 +/- 0.2 versus 3.7 +/- 0.3), indicating that GLP-1 stimulated insulin secretion and inhibited glucagon secretion. In conclusion, our results suggest that truncated GLP-1 act as a physiological inhibitor of gastric and pancreatic functions in man.

Adult↗
PubMed2006

Effect of truncated-ApoE4 overexpression on tau phosphorylation in cultured N2a cells.

The carboxyl-terminal amino acids 272-299-truncated apoE4 (delta272-299) is the main fragments of apoE4 hydrolysate in neurons. The effects of truncated-ApoE4 (delta272-299) overexpression on tau phosphorylation in cultured N2a cells were investigated. The truncated-apoE4 (delta272-299) cDNA was subcloned into pEGFP-c3 to form recombinant pEGFP-T-apoE4. pEGFP-c3, pEGFP-T-apoE4 and pEGFP-apoE4 were transfected into N2a cells respectively by lipofectamine 2000 method. After 24--48 h, tau phosphorylation was detected by Western blot assay and glycogen synthase kinase-3 (GSK-3) activity by using GSK-3 activity assay. The results showed that the overexpression of both full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau at Ser202 sites and the activation of GSK-3 as compared with untransfected cells, most significantly in the cells transfected with pEGFP-T-apoE4 (P < 0.05). It was concluded that in vitro overexpression of truncated-ApoE4 (delta272-299) can result in tau hyperphosphorylation in N2a cells by activating GSK-3, suggesting truncated-ApoE4 (delta272-299) might contribute the pathogenesis of Alzheimer disease.

Animals↗
PubMed1995

Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.

Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IAk's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-Ak molecules. Ak beta TM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-Ak conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the Ak beta chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the Ak beta chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to assess a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the Ak alpha Cy domains is proposed to prevent complete masking of the ER retention sequence of the Ii chain.

Animals↗
PubMed1990

Truncated sinc slice excitation for 31P spectroscopic imaging.

The shortest possible delay (Td) between slice selection and data acquisition is important for producing high quality 31P spectra. In single slice multivoxel spectroscopic imaging, conventional excitation using sinc-shaped rf pulses within typical gradient limitations can have values of Td that lead to significant spectral distortion and loss of signal. Truncated sinc excitation, which ends the excitation close to the center of the main rf lobe has been suggested for MR angiographic applications to produce short values of Td. In this work, the slice profiles, spectral signal-to-noise ratio (SNR) and spectral distortions are compared using the minimum delay achievable on a commercial MRI system for conventional 'sinc' rf excitation and truncated sinc excitation. Slice profiles are calculated using the Bloch equations and measured with a phantom. SNR and spectral distortions are evaluated from whole slice spectra on a human volunteer. On an MRI system with 1 G/cm gradients (0.5 msec risetime), for a 2.5-cm slice at 31P frequencies, conventional excitation can be adjusted to achieve Td = 2.5 msec while truncated sinc excitation yields Td = 1.5 msec. The truncated sinc excitation's shorter value of Td leads to much smaller spectral distortions, but its slice profile has "dispersive tails" which increase as more of the rf is truncated. Slice profile corrected SNR for the beta-ATP peak of 31P on a human volunteer is equivalent for both sequences while, qualitatively, in the PDE region the truncated sinc approach has improved SNR.

Brain↗
PubMed2005

Impairment of the ubiquitin-proteasome system by truncated cardiac myosin binding protein C mutants.

OBJECTIVE: Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHC patients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin-proteasome system (UPS). METHODS AND RESULTS: By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHC patients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70+/-4% and 11+/-5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS. CONCLUSION: Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC.

Adenoviridae↗
PubMed1995

Truncated K+ channel DNA sequences specifically suppress lymphocyte K+ channel gene expression.

We have constructed a series of deletion mutants of Kv1.3, a Shaker-like, voltage-gated K+ channel, and examined the ability of these truncated mutants to form channels and to specifically suppress full-length Kv1.3 currents. These constructs were expressed heterologously in both Xenopus oocytes and a mouse cytotoxic T cell line. Our results show that a truncated mutant Kv1.3 must contain both the amino terminus and the first transmembrane-spanning segment, S1, to suppress full-length Kv1.3 currents. Amino-terminal-truncated DNA sequences from one subfamily suppress K+ channel expression of members of only the same subfamily. The first 141 amino acids of the amino-terminal of Kv1.3 are not necessary for channel formation. Deletion of these amino acids yields a current identical to that of full-length Kv1.3, except that it cannot be suppressed by a truncated Kv1.3 containing the amino terminus and S1. To test the ability of truncated Kv1.3 to suppress endogenous K+ currents, we constructed a plasmid that contained both truncated Kv1.3 and a selection marker gene (mouse CD4). Although constitutively expressed K+ currents in Jurkat (a human T cell leukemia line) and GH3 (an anterior pituitary cell line) cells cannot be suppressed by this double-gene plasmid, stimulated (up-regulated) Shaker-like K+ currents in GH3 cells can be suppressed.

Animals↗
PubMed2002

Alternative splicing of the GABA(A) receptor alpha 4 subunit creates a severely truncated mRNA.

GABA(A) receptors, important sites of drug action, are chloride channels composed of 5 subunits chosen from among 19 or more. Alternative splicing for alpha 5, alpha 6, and rho 1 subunits results in truncated proteins which appear to lack function. We report a similar, relatively common (about 20%) form of alternative splicing of the alpha 4 subunit mRNA in mice and humans which, remarkably, creates a severely truncated message containing only the first two and last coding exons, with a frameshift in between. The only apparent translation product includes a short piece (39 amino acids) of the N-terminus right after the signal peptide. The splicing was developmentally and regionally regulated; the highest proportions of truncated alpha 4 mRNA, about 40%, were observed in embryonic day 18 whole brain and adult cerebellum. The truncated mRNA, when coexpresssed in human embryonic kidney (HEK) 293 cells with the complete alpha 4 subunit and beta1 and gamma 2 S subunits, reduced observed GABA currents without kinetic alterations. No such effect of truncated alpha 4 was observed with alpha1 subunit-containing receptors. Thus, the truncated alpha 4 N-terminus may play a post-translational regulatory role in intracellular folding/glycosylation/assembly of the alpha 4 subunit.

Alternative Splicing↗
PubMed1996

Expression and characterization of a truncated, soluble, low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor mediates the clearance of apolipoprotein B- and E-containing lipoproteins from the bloodstream. In the current study, we characterized the binding properties of the amino terminus of the LDL receptor. We produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human LDL receptor. This truncated receptor protein (LDL-R354) was secreted from recombinant baculovirus-infected Spodoptera frugiperda (Sf-21) cells. Upon electrophoresis, LDL-R354 migrated with a mobility of 55,000. Treatment of cells with tunicamycin decreased the size of the truncated receptor, suggesting the presence of asparagine-linked carbohydrates. Nonreducing SDS-PAGE resulted in at least three discernible bands with M(r)s consistent with the truncated receptor existing as monomers and multimers, suggesting the possibility of intermolecular disulfide cross-linking. All forms of the truncated receptor bound LDL on a ligand blot in a calcium-dependent manner. The purified truncated receptor bound 125I-LDL with high affinity in a competitive binding assay (IC50 = 0.8 microgram/ml). LDL-R354 also bound calcium. This interaction was sensitive to the conformation of the ligand binding domain, as reduction of the disulfide bonds greatly decreased the affinity of the receptor for calcium. The ligand and calcium binding activities of this truncated receptor protein demonstrate that the ligand binding domain of the LDL receptor can fold into a functionally active protein.

Animals↗
PubMed1989

Stimulation of cation transport in mitochondria by gramicidin and truncated derivatives.

Gramicidin and the truncated derivatives desformylgramicidin (desfor) and des(formylvalyl)gramicidin (desval) stimulate monovalent cation transport in rat liver mitochondria. Cation fluxes were compared indirectly from the effect of cations on the membrane potential at steady state (state 4) or from the associated stimulation of electron transport. Rb+ transport was measured directly from the uptake of 86Rb. The truncated gramicidins show enhanced selectivity for K+ and Rb+ when compared to gramicidin. Moreover, the pattern of selectivity within the alkali cation series is altered, i.e., Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+ for desfor and desval as compared to Cs+ greater than Rb+ greater than K+ = Na+ greater than Li+ for gramicidin. The cation fluxes through the truncated derivatives are more strongly dependent on the cation concentration. The presence of high concentrations of permeating cation enhances the transport of other cations through the truncated derivative channels, suggesting that cations are required for stabilizing the channel structure. In high concentrations of KCl, desfor and desval are nearly as effective as gramicidin in collapsing the mitochondrial membrane potential, and, consequently, in the uncoupling of oxidative phosphorylation and enhancement of ATP hydrolysis. Preliminary experiments with liposomes show that 86Rb exchange is stimulated by desfor and desval almost to the same extent as gramicidin. These results strongly suggest that the truncated gramicidins form a novel conducting channel which differs from the gramicidin head-to-head, single-stranded beta 6.3-helical dimer ("channel") in its conductance characteristic and its structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗