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At least 19 recordsLinked to original sources
PubMed1989

Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342.

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

Amino Acid Sequence↗
PubMed1989

Intragenic revertants of yeast invertase variants with secretion-defective leader sequences.

Several secretion-defective variants of invertase from Saccharomyces cerevisiae were generated by replacement of the wild-type signal sequence codons with DNA fragments with random sequences. Strains encoding these proteins failed to grow on medium containing sucrose as the sole source of carbon. The invertase that was made in these strains was found to fractionate with soluble, cytoplasmic proteins, and indirect immunofluorescence confirmed that the mutant invertase was located throughout the cytoplasm. To define the defects in the secretion-defective leader sequences, we selected revertants by requiring growth on sucrose. Surprisingly, most of the reversion events consisted of point changes and duplications in the upstream noncoding portion of the gene. Each of these changes introduced several hydrophobic residues into the nonfunctional leader sequences, suggesting that the defective random leader peptides might simply lack adequate hydrophobicity to be effective signal peptides.

Amino Acid Sequence↗
PubMed1989

Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects.

Defects in platelet cytoplasmic Ca++ mobilization have been postulated but not well demonstrated in patients with inherited platelet secretion defects. We describe studies in a 42-year-old white woman, referred for evaluation of easy bruising, and her 23-year-old son. In both subjects, aggregation and 14C-serotonin secretion responses in platelet-rich plasma (PRP) to adenosine diphosphate (ADP), epinephrine, platelet activating factor (PAF), arachidonic acid (AA), U46619, and ionophore A23187 were markedly impaired. Platelet ADP and adenosine triphosphate (ATP), contents and thromboxane synthesis induced by thrombin and AA were normal. In quin2-loaded platelets, the basal intracellular Ca++ concentration, [Ca++]i, was normal; however, peak [Ca++]i measured in the presence of 1 mmol/L external Ca++ was consistently diminished following activation with ADP (25 mumol/L), PAF (20 mumol/L), collagen (5 micrograms/mL), U46619 (1 mumol/L), and thrombin (0.05 to 0.5 U/mL). In aequorin-loaded platelets, the peak [Ca++]i studied following thrombin (0.05 and 0.5 U/mL) stimulation was diminished. Myosin light chain phosphorylation following thrombin (0.05 to 0.5 U/mL) stimulation was comparable with that in the normal controls, while with ADP (25 mumol/L) it was more strikingly impaired in the propositus. We provide direct evidence that at least in some patients with inherited platelet secretion defects, agonist-induced Ca++ mobilization is impaired. This may be related to defects in phospholipase C activation. These patients provide a unique opportunity to obtain new insights into Ca++ mobilization in platelets.

Adenine Nucleotides↗
PubMedDate not supplied

Acquired secretion defect in platelets after cryopreservation in dimethyl sulfoxide.

Despite the use of preservatives, platelets are severely damaged during cryopreservation and, following freezing, function poorly in a number of in vitro tests. We report here that cryopreserved platelets show diminished aggregation in response to collagen. This may be a consequence of a secretion defect as evidenced by a 20 to 30 percent loss of dense- and alpha-granule content (p less than 0.05) and an impaired secretion mechanism. Analysis of adenine nucleotides confirmed the defect in dense granule adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content (storage pool), but in addition revealed a 50 percent fall in cytosolic ATP (metabolic pool). In contrast, the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + adenosine monophosphate), was normal. We concluded that platelet cryopreservation leads to a secretion defect, probably as a result of activation during freezing and thawing procedures.

Adenosine Diphosphate↗
PubMed1988

Platelet-associated immunoglobulins G, A and M are secreted during platelet activation: normal levels but defective secretion in grey platelet syndrome.

Platelet-associated (PA) IgG is known to be released from normal human platelets when they are stimulated by aggregating agents. We have studied whether PA-IgA and PA-IgM are also secreted during platelet activation or during blood collection and processing and whether their levels are related to those in serum. Processing of platelets from normal donors in the presence of secretion inhibitors prostaglandin E1 (PGE1) and theophylline increased levels of both surface and total PA-immunoglobulins (Ig) in intact and lysed platelets respectively, with increases being significant for surface PA-IgA and PA-IgM and total PA-IgM. About 50% of total PA-IgM, 40% PA-IgA and 20% PA-IgG was detectable on intact platelets. All three PA-Ig and PA-albumin were secreted in response to thrombin and this release was inhibited by PGE1. The platelet:serum ratio of each Ig and albumin were similar. In grey platelets deficient in alpha-granules, PA-Ig and PA-albumin levels were raised per platelet but when increased platelet size was taken into account PA-Ig were normal and PA-albumin just below normal. Although thrombin caused release of most of the small amounts of beta-thromboglobulin present, PA-Ig and PA-albumin were not released. This suggests that PA-Ig and albumin from plasma may enter a pool of secretory proteins in normal platelets, but in grey platelets they remain in some other site.

Alprostadil↗
PubMed1989

Interleukin 2 and soluble interleukin 2-receptor secretion defect in vitro in newly diagnosed type I diabetic patients.

In this study, we investigated whether an interleukin 2 (IL-2) secretion defect by peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with phytohemagglutinin (PHA-M) occurs in either newly diagnosed or long-standing type I (insulin-dependent) diabetic patients and whether it is accompanied by a dysregulation of soluble IL-2-receptor (IL-2RS) production. PBMC cultures (2.5 x 10(6) cells), unstimulated or stimulated with PHA-M (25 micrograms/ml), from 20 type I diabetic patients (10 with time since onset less than 3 mo and 10 with long-term diabetes of less than 3 yr) and 10 control subjects were studied for the production of IL-2 and IL-2RS in their respective supernatants. No difference was found in IL-2 production in unstimulated cultures of type I patients compared with control subjects, although a significant decrease from PHA-M-stimulated cultures were seen (newly diagnosed, 1.7 +/- 0.3 ng/2.5 x 10(6) cells; long-standing, 2.2 +/- 0.3 ng/2.5 x 10(6) cells; P less than .001 and P less than .05, respectively) compared with control subjects (3.6 +/- 0.4 ng/2.5 x 10(6) cells). In regard to the production of IL-2RS, no difference exists for unstimulated cultures, whereas, after PHA-M stimulation, both newly diagnosed and long-term-diabetic patients showed a decrease in the IL-2RS levels (318 +/- 50 and 331 +/- 62 U/2.5 x 10(6) cells; P less than .02 and P less than .05, respectively) compared with normal subjects (463 +/- 34.2 U/2.5 x 10(6) cells). Thymus-activated cell phenotypes confirmed the T-lymphocyte activation after a 48-h culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1988

Repair of the secretion defect in the Z form of alpha 1-antitrypsin by addition of a second mutation.

Homozygous inheritance of the Z-type mutant form of the alpha 1-antitrypsin (alpha 1AT) gene results in the most common form of alpha 1AT deficiency, a human hereditary disease associated with a high risk for the development of emphysema and an increased incidence of neonatal hepatitis. The alpha 1AT-synthesizing cells of individuals with the Z gene have normal alpha 1AT messenger RNA levels, but alpha 1AT secretion is markedly reduced secondary to accumulation of newly synthesized alpha 1AT in the rough endoplasmic reticulum. Crystallographic analysis of alpha 1AT predicts that in normal alpha 1AT, a negatively charged Glu342 is adjacent to positively charged Lys290. Thus the Glu342----Lys342 Z mutation caused the loss of a normal salt bridge, resulting in the intracellular aggregation of the Z molecule. The prediction was made that a second mutation in the alpha 1AT genet that changed the positively charged Lys290 to a negatively charged Glu290 would correct the secretion defect. When the second mutation was added to the Z-type complementary DNA, the resulting gene directed the synthesis and secretion of amounts of alpha 1AT similar to that directed by the normal alpha 1AT complementary DNA in an in vitro eukaryotic expression system. This suggests the possibility that a human hereditary disease can be corrected by inserting an additional mutation in the same gene.

Animals↗
PubMed1986

Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis.

We devised a new selection for conditionally lethal suppressors of secA mutants. This selection allows the isolation of both temperature-sensitive and cold-sensitive suppressor mutations, whereas previous studies were limited to nonlethal or cold-sensitive suppressor mutations. Two temperature-sensitive suppressor mutations lie in genes required for protein synthesis: asnS, the gene for the asparaginyl-tRNA synthetase, and divE, which encodes the tRNASer1. A previously characterized mutation in alaS, the gene for the alanyl-tRNA synthetase, suppresses the growth and secretion defects of a secA mutant. Although the primary effects of these suppressor mutations are different, it is likely that they cause suppression of secA mutations by altering the rate of protein synthesis, since the protein synthesis inhibitors, chloramphenicol and tetracycline, also suppress secA mutations. Chloramphenicol also suppresses the growth defect of certain other sec mutants. We postulate that the impaired secretory capacity of sec mutants can be offset by decreasing the rate of elongation of secreted proteins or by decreasing the total amount of secreted proteins per cell. The results indicate that our initial goal to identify cellular secretory components as suppressors of secA mutations might be difficult to achieve because of a high frequency of nonspecific suppressors that alter protein synthesis. Unexpectedly, the suppressor approach provides a direct genetic selection for mutants in protein synthesis.

Bacterial Proteins↗
PubMed1987

X-linked hypogammaglobulinaemia with a late secretion defect. A family study.

Three boy cousins suffering from x-linked hypogammaglobulinaemia have been described. Their disease is quite different from the classical x-linked hypogammaglobulinaemia. We present data from 15 family members of three families. The three mothers are sisters and 3 boys from 5 are suffering from immunodeficiency. HLA A, B and DR typing and in vitro diagnostics of immune functions of all family members were carried out. In the patients we could demonstrate a well functioning T cell system which seems to be regular for T cell help on PMW driven B cell maturation into cytoplasmic immunoglobulin positive (cIg+) cells of all immunoglobulin classes but we had no evidence for IgG secreting cells in a plaque forming cell (PFC) assay. The registration of spontaneous suppressor phenomena should be taken into account. If patients undergo an gamma-globulin therapy the changed suppressor effects come back to normal values but the secretion defect is unchanged. The three healthy mothers express one identical haplotype (A2 B8 DR3) which we could demonstrate in the three patients but also in one healthy boy.

Agammaglobulinemia↗
PubMed1986

The interleukin 2 secretion defect in vitro in systemic lupus erythematosus is reversible in rested cultured T cells.

Interleukin 2 (IL 2) secretion in response to mitogenic stimulation in vitro is strongly reduced in circulating T lymphocytes from patients with SLE. It is still not clear how this abnormality relates to the B cell hyperactivity in the disease. Some investigators proposed that an intrinsic T helper cell defect could lead to suppressor cell dysfunction and autoimmunity. Others have found that in fact increased suppressor cell activity can cause IL 2 hyposecretion. In the present study we report that the IL 2 secretion in response to PHA plus PMA by T cells from patients with SLE, which initially was decreased by a factor of 10 as compared with the IL 2 secretion in blood donor T cells, was restored when the T cells were rested for 2 to 3 days in culture before stimulation. IL 2 hyposecretion in SLE T cells and the kinetics of normalization in culture were not changed by the addition of normal adherent cells during the stimulation with PHA/PMA, occurred in the absence of significant cell death or proliferation or change of the T4:T8 cell ratio during the resting culture, were not due to a maturation of immature T6-positive cells (less than 1.5% T6 cells in SLE T cells), and also occurred in T8-depleted T4 cells alone. Furthermore, a normalization of IL 2 secretion took place in the presence of either SLE serum or normal serum, and the addition of fresh autologous T cells to 3-day-cultured SLE T cells did not cause suppression of IL 2 secretion. These data show that some rapidly reversible defect occurs in circulating T helper cells in SLE. That this could reflect an exhaustion of T helper cells that have been activated in vivo is discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

Alleles↗
PubMed1988

Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants.

SecA protein synthesis levels were elevated 10- to 20-fold when protein secretion was blocked in secA, secD, and secY mutants or in a malE-lacZ fusion-containing strain but not in a secB null mutant. An active secB gene product was not required to derepress secA, since SecA levels were elevated during protein export blocks in secB secY and secB malE-lacZ double mutants.

Bacterial Proteins↗
PubMed1990

Platelets acquire a secretion defect after high-dose chemotherapy.

Patients receiving high-dose chemotherapy (HDC) and autologous bone marrow transplantation (ABMT) may experience life-threatening hemorrhagic myocarditis. The authors investigated whether HDC was associated with an acquired platelet defect. Platelet aggregation and release were evaluated after HDC in ten patients with either metastatic breast carcinoma or melanoma. Platelets underwent shape change and a primary wave of aggregation. High-dose chemotherapy was associated with the inhibition of secondary aggregation of platelets induced by adenosine diphosphate (ADP), arachidonic acid, prostaglandin H2 (PGH2) analog (U44619), and collagen. Although electron microscopic study of the platelets revealed normal morphologic features with an adequate number of dense bodies and alpha-granules, release of adenosine triphosphate (ATP) from dense granules was less than 20% of normal. The acquired platelet defect occurred before development of thrombocytopenia. Aggregation of platelets from normal volunteers was not inhibited by either the addition of the chemotherapeutic agents, chemotherapy metabolites, or the patients' sera. In conclusion, HDC induces an acquired abnormality in platelet secretion and aggregation which may contribute to the development of hemorrhagic complications after ABMT.

Adenosine Triphosphate↗
PubMed1990

Recovery of Staub Effect and amelioration of insulin secretion defects after glyburide treatment in non-insulin-dependent diabetes.

Staub Effect or improved glucose disposal after repetitive glucose loads does not occur in untreated diabetes. In non-insulin dependent diabetes (NIDDM) there is impaired insulin response to intravenous (i.v.) glucose injection, especially in early insulin release (EIR) and the lesser known post EIR suppression of insulin levels below basal, or acute insulin decrement (AID). To test the ability of a second generation sulfonylurea, glyburide, to affect glucose primed glucose disposal and insulin secretory patterns, sixteen NIDDM male subjects received three hourly intravenous glucose loads while untreated and again after six months of glyburide therapy. After treatment there was a fall of fasting glycemia from 204 +/- 11 to 147 +/- 8 mg/dl (p less than 0.001), of all glucose levels during the i.v. glucose tolerance tests (p less than 0.025) and glycosylated hemoglobin from 8 +/- 0.3% to 7.6 +/- 0.3% (p less than 0.005). Before treatment i.v. glucose disposal (K value) changed very little after successive glucose challenges, but after glyburide all mean K values were higher, and glucose primed glucose disposals were faster after the second (K2) and third (K3) glucose injection than after the first (K1) (p less than 0.025 and p less than 0.01 respectively). In the untreated state, there was higher and significant EIR by the third glucose load, (p less than 0.025) while AID was clearly more pronounced after the second load (p less than 0.001). After glyburide treatment EIR was significantly higher than before in all loads, and mean AID was no longer demonstrable. Insulin summation (S) after successive i.v. loads maintained a stepwise increase both before and after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Glucose↗
PubMed1991

Isolation of a mutant of Chinese hamster ovary cells with defective secretion of a subset of secretory proteins.

The secretory pathway of mammalian cells involves a complex sequence of steps beginning with the translation and translocation of proteins across the rough endoplasmic reticulum membrane followed by their transport through a variety of intracellular compartments. Understanding the cellular mechanisms involved in protein transport would be facilitated by the application of somatic cell genetics techniques. Therefore, we have developed a procedure for the isolation of Chinese hamster ovary cell lines that are deficient in this pathway. Mutant (sec) clones that secrete into the medium a lesser amount of newly synthesized protein relative to wild type are detected in the screen. One of the sec cell lines, LEH6, has similar growth and protein synthetic rates as do wild-type cells. The extent of total protein secretion is 60-70% of wild-type with some proteins more reduced than others. Immunoprecipitation studies indicate that for LEH6 cells there is impaired secretion of fibronectin (three- to fourfold) and major excreted protein (two- to sixfold).

Animals↗
PubMed1985

Defective secretion of maltose- and ribose-binding proteins caused by a truncated periplasmic protein in Escherichia coli.

The secretion in Escherichia coli of a C-terminally truncated periplasmic enzyme from Salmonella typhimurium, the glpQ-encoded glycerolphosphate phosphodiesterase, was studied. Plasmid pRH100, carrying the truncated glpQ gene, directs the synthesis of a 30,000-molecular-weight (30 K) protein that is processed to a mature 27.5 K protein. (The mature wild-type protein is a 38 K protein.) The truncated protein is not released into the periplasm but remains membrane associated, although it becomes protease sensitive after conversion of cells to spheroplasts. The presence of pRH100 strongly reduces the amount of some other proteins in the periplasm, including the maltose- and ribose-binding proteins. The reduction does not occur at the level of transcription or early translation, as shown by lacZ fusions to the gene coding for the structural gene of the maltose-binding protein. Outer membrane proteins are not affected. A hydroxylamine-induced mutation in the sequence of glpQ corresponding to the mature polypeptide overcomes the inhibitory effect of pRH100. The mutated gene no longer directs the synthesis of the 30/27.5 K protein but directs that of a new 19 K protein which is not membrane bound. We propose that sorting signals in the mature GIpQ protein are necessary for effective translocation to the periplasm and that the C-terminal third of the protein is essential for release into the periplasm.

ATP-Binding Cassette Transporters↗
PubMed1990

Soluble interleukin-2 receptor secretion defect in vitro in HLA-B8, DR3 positive subjects.

Several studies have shown that HLA-B8,DR3 positive subjects may display T cell dysfunctions. Recently, a soluble form of the receptor for IL-2 (sIL-2R) has been demonstrated in human sera and in vitro-stimulated culture supernatant from human T lymphocytes. In the present paper we report sIL-2R serum levels and sIL-2R production from peripheral blood mononuclear cells in HLA-B8,DR3 positive subjects. We found that HLA-B8,DR3 positive subjects have the highest values of serum sIL-2R, but comparing the values of these subjects with those of negative ones no significant difference was observed. As regards the in vitro production of sIL-2R, no difference exists for unstimulated cultures, whereas after stimulation, the HLA-B8,DR3 positive subjects showed the lowest values compared with negative ones. It is noteworthy that these changes are observed in autoimmune diseases linked to this HLA phenotype.

Adolescent↗
PubMed1992

A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus.

The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.

Amino Acid Sequence↗