Rare phenotypes of alpha-1 antitrypsin: study of a case of the M1X variant

Alpha-1 antitrypsin is the major component responsible for the normal alpha 1 band in human serum. Some genetic variants giving double alpha-1 band, may be associated with pathological process. In the course of a systematic screening of blood donors a double-band alpha-1 pattern was observed in a serum, due to the heterozygous expression of a genetic variant of the PI system. A possible clinical significance of the variant was investigated by characterizing it. The very rare allotype PI*X was identified and its frequency in the population of french blood donors was estimated around to one for 10,000.     

Partition of whey milk proteins in aqueous two-phase systems of polyethylene glycol-phosphate as a starting point to isolate proteins expressed in transgenic milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha lactoalbumin and beta lactoglobulin) and alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000; 1500 and 3350)-potassium phosphate was analysed. Bovine serum albumin and alpha lactoalbumin concentrated in the polyethyleneglycol rich phase with a partition coefficient of 10.0 and 27.0, respectively, while beta lactoglubulin and alpha-1 antitrypsin showed affinity for the phosphate-rich phase with a partition coefficient of 0.07 and 0.01, respectively. An increase of medium pH induced an increase of the partition coefficient of these proteins while the increase in polyethyleneglycol molecular mass induced the opposite behaviour. The system polyethyleneglycol 1500-pH 6.3 showed the best capacity for recovering the alpha-1 antitrypsin with a yield of 80% and a purification factor between 1.5 and 1.8 from an artificial mixture of the milk whey proteins and alpha-1 antitrypsin. The method appears to be suitable as a starting point to isolate proteins expressed in transgenic milk.     

Tris reversibly inhibits secretion of albumin and alpha-1-antitrypsin at different sites

We have investigated the effect of the weak base Tris on the processing and secretion of albumin and alpha-1 antitrypsin by hepatocytes in culture. We show that the secretion of both proteins is 90% inhibited by 30 mM Tris. The post-synthetic processing of both proteins is inhibited to the same extent. These effects are completely reversible. Cell fractionation indicates that albumin accumulates in the Golgi, whereas alpha-1 antitrypsin fails to leave the endoplasmic reticulum.     

Features of the milk whey protein partitioning in polyethyleneglycol-sodium citrate aqueous two-phase systems with the goal of isolating human alpha-1 antitrypsin expressed in bovine milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha-lactoalbumin and beta-lactoglobulin) and human alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000, 1450 and 3350)-sodium citrate was analysed at pH 5.2, 6.2 and 8.2. Alpha lactoalbumin concentrated in the polyethyleneglycol rich-phase, while beta-lactoglobulin, bovine serum albumin and alpha-1 antitrypsin showed affinity for the citrate rich-phase. In aqueous two-phase systems of high medium pH and high polyethyleneglycol molecular mass the protein partitioning equilibrium is displaced to the citrate rich-phase. The polyethyleneglycol 1450-pH 5.2 system with a top/bottom phase-volume ratio of 3 showed to have the best capability of recovering the alpha-1 antitrypsin from a mixture prepared with natural milk whey and human alpha-1 antitrypsin. The recovery of this protein in the bottom phase was of 90% and the purity of the obtained product was of 98%. The method appears to be suitable as a starting point to isolate other human proteins expressed in transgenic bovine milk.     

A novel model and molecular therapy for Z alpha-1 antitrypsin deficiency

Animal models that closely resemble human disease can present a challenge. Particularly so in alpha-1 antitrypsin deficiency (α(1)ATD), as the mouse alpha-1 antitrypsin (α(1)AT) cluster encodes five highly related genes compared with the one in humans. The mouse PI2 homologue is closest to the α(1)AT human gene. We have changed the equivalent mouse site that results in the Z variant in man (Glu342Lys) and made both the "M" and "Z" mouse PI2 α(1)AT proteins. We have tested the ability of a small-molecular-weight compound CG to alleviate polymerisation of these mouse α(1)AT proteins as it has been shown to reduce aggregates of Z α(1)AT in man. We found that (1) CG specifically reduces the formation of polymers of recombinant mouse "Z" protein but not "M" protein; (2) whereas there is significantly more α(1)AT secreted from Chinese Hamster Ovary cells transfected with the mouse "M" α(1)AT gene than with the "Z" (20.8?±?3.9 and 6.7?±?3.6, respectively; P?相似文献   

The role of bronchial epithelial cells in the pathogenesis of COPD in Z-alpha-1 antitrypsin deficiency

Background

Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown.We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients.

Methods

Experiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of < 0.05 was considered to be significant.

Results

Alpha-1 antitrypsin polymers were found at a higher concentration in the culture medium of ex vivo bronchial epithelial cells from Z-variant homozygotes, compared with M-variant homozygotes (P < 0.01), and detected in the bronchial epithelial cells and submucosa of patient biopsies. Oncostatin M significantly increased the expression of alpha-1 antitrypsin mRNA and protein (P < 0.05), and the presence of Z-variant polymers in ex vivo cells (P < 0.01).

Conclusions

Polymers of Z-alpha-1 antitrypsin form in bronchial epithelial cells, suggesting that these cells may be involved in the pathogenesis of lung emphysema and in bronchial epithelial cell dysfunction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0112-3) contains supplementary material, which is available to authorized users.     

Polymorphisms of hemochromatosis, and alpha-1 antitrypsin genes in Egyptian HCV patients with and without hepatocellular carcinoma

Hereditary hemochromatosis and alpha-1antitrypsin deficiency are genetic diseases characterized by endoplasmic reticulum (ER) stress with subsequent development of liver disease. Our aim was to estimate the frequency of hemochromatosis gene (HFE) mutant alleles (C282Y and H63D) and alpha-1 antitrypsin S/Z variants among Egyptian HCV cirrhotic patients and in hepatocellular carcinoma patients and to evaluate their effects on disease progression. HFE and alpha-1 antitrypsin polymorphisms were characterized in 200 Egyptian patients with HCV infection (100 patients complicated with cirrhosis, 100 patients with HCC) and 100 healthy subjects who had no history of any malignancy. The frequencies of HD genotype of H63D mutation were significantly increased in HCC patients compared to control group and to cirrhosis group. Also, the frequencies of DD genotype were significantly increased In HCC group compared to control group and to cirrhosis group. Our results suggested that Carriers of the D allele of H63D mutation were significantly more likely to develop HCC.     

Polymorphism of alpha-1 antitrypsin in dogs

• 1.1. A genetically determined polymorphism of alpha-1 antitrypsin is demonstrated in dog serum by isoelectric focusing in a pH range of 3.5–5.0, followed by direct immunoblotting using a specific antiserum.
• 2.2. Alpha 1 antitrypsin focuses as two major bands at isoelectric points of 4.60 and 4.64 or 4.67 and 4.7 in presumed homozygous animals. Heterozygotes show both sets of bands.
• 3.3. The results of seven crosses with 33 offspring are best explained by two codominant alleles, Pi^M and Pi^S at a single locus designated as Pi for proteinase inhibitor.
• 4.4. The concentration of alpha-1 antitrypsin in serum of healthy dogs was 2.65 ± 0.42 mg/ml and 2.19 ± 0.38 mg/ml in females andv males respectively.
• 5.5. The higher concentration in female dogs suggests that estrogens may influence the serum level of alpha-1 antitrypsin.

     

PCR-Based Screening for the Most Prevalent Alpha 1 Antitrypsin Deficiency Mutations (PI S, Z, and Mmalton) in COPD Patients from Eastern Tunisia

It is generally agreed that the protease inhibitor (PI) alleles PI*S (Val264Glu) and PI*Z (Lys342Glu) are the most common alpha 1 antitrypsin deficiency variants worldwide, but the PI*Mmalton allele (ΔPhe52) prevails over these variants in some Mediterranean regions. In eastern Tunisia (Mahdia), we screened 100 subjects with chronic obstructive pulmonary disease for these variants. The PI*S and PI*Z alleles were genotyped by the previously described SexAI/Hpγ99I RFLP–PCR. We provide here a new method for PI*Mmalton genotyping using mismatched RFLP–PCR. These methods are suitable for routine clinical application and can easily be reproduced by several laboratories, since they do not require extensive optimization, unlike the previously described bidirectional allele-specific amplification PCR for PI*Mmalton genotyping. Our results were in agreement with previous reports from central Tunisia (Kairouan), suggesting that the PI*Mmalton mutation is the most frequent alpha 1 antitrypsin deficiency-related mutation in Tunisia.     

Air pollution causes atherosclerosis through inducing pathological changes in the blood (a new perspective)

Atherosclerosis has been correlated with air pollution. However, the air pollution's atherogenic mechanism is not clear yet. This hypothesis proposes an atherogenic mechanism for air pollution and claims that the air pollution changes the physiological properties of the blood and then these changes promote the formation of atherosclerotic plaques. The first atherogenic effect of air pollution is hypothesized to be the inactivation of alpha-1 antitrypsin molecules in plasma via oxidation. In the next step, oxidized alpha-1 antitrypsin attaches LDL and forms a complex in the blood stream of lungs. This complex goes through intima with the action of a mediator and when in the vascular wall it is catabolised 4 times more effectively than LDL alone. The result is the formation of foamy cells that characterize atherosclerosis. In this hypothesis, it is also reasoned that the mediator of the complex passage through the arterial walls is elastase. At the end, this hypothesis argues on the bases of its documents that the mean distribution of atherosclerosis in the body obeys a pattern of decreased intensity centrifugally from lungs.     

Endoplasmic reticulum stress--a double edged sword for Z alpha-1 antitrypsin deficiency hepatoxicity

Several diverse disorders, including the liver disorder Z alpha-1 antitrypsin deficiency as well as cystic fibrosis, Alzheimer's, and Parkinson's disease arise from the same general disease mechanism and are now categorized under the term “conformational diseases”, characterized by abnormal folding and subsequent aggregation of an underlying protein. In recent years, several important research advances in the cell biology of aggregation-prone mutant proteins and pathobiological mechanisms of liver disease in general have proven paramount to our understanding of Z alpha-1 antitrypsin deficiency. This liver disease underlines the principle mechanisms of conformational disorders contained within the four pillars of endoplasmic reticulum stress: (1) protein degradation, (2) endoplasmic overload response, (3) unfolded protein response and (4) cellular death pathway. This four-stage model of Z alpha-1 antitrypsin hepatoxicity is elegant in its simplicity and helps explain the clinical manifestations of this condition. Endoplasmic reticulum stress responses have evolved to be protective, however when they are ineffective, toxic damage occurs demonstrating how these responses can be described as a double edged sword. In this context, one of the most perplexing problems in modern biology is to understand how the cell “chooses” between adaptation and demise in response to stress. When one pathway becomes predominant, a delicate balance is perturbed and either an adaptive or a lethal response ensues. Understanding how the endoplasmic reticulum stress signals potentially play a role in directing a clinical outcome may lead to better prospects of more rational approaches to investigation and therapy for this liver disease.     

Rapid Genotyping of Alpha 1 Antitrypsin Deletion Mutation (PI*Mmalton) Using Bi-directional PCR Allele-specific Amplification

Alpha 1 antitrypsin deficiency (AATD) is a well recognized genetic risk factor for pulmonary disease and less common liver disease. The two most common deficiency alleles worldwide PI*S and PI*Z can be easily detected using several molecular methods. However, there are at least 30 other AATD variants, which are only detectable by alpha 1 antitrypsin (AAT) gene sequencing and, therefore, seem to be more under-recognized than the PI*S and PI*Z alleles. PI*Mmalton is the most frequent AATD variant in different regions of the Southern Mediterranean basin countries, where its prevalence seems to prevail over PI*S and PI*Z. In this work, we report the development of a simple PCR-based analysis designed for the detection of the PI*Mmalton deficiency alleles using two specific primers. A one-tube reaction enables the distinction between the different genotypes. This reliable, easy, fast, and low-cost technique might be useful for laboratories involved in the study of AATD-related diseases, especially those of the Southern Mediterranean basin area with modest budget or where sophisticated equipment is not available. This will allow larger targeted screening for PI*Mmalton in order to better understand this mutation epidemiology and its origin.     

Hepatocyte-specific gene expression from integrated lentiviral vectors

BACKGROUND: For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors. METHODS: HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines. RESULTS: Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes. CONCLUSIONS: Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.     

Synthesis of antithrombin III and alpha-1-antitrypsin by the perfused rat liver.

Livers isolated from control or turpentine-injected rats were perfused for 3 h with human red cells suspended in Krebs-Henseleit solution containing bovine serum albumin, dextran, glucose, heparin, cortisol, insulin, a mixture of 20 amino acids and [3H]leucine. Changes in the concentrations of antithrombin III and alpha-1-antitrypsin were evaluated by rocket immunoelectrophoresis using specific antisera, and incorporation of the 3H radioactivity into the total protein, albumin, antitrhombin III and alpha-1-antitrypsin in the perfusate was measured. The results indicate that both antithrombin III and alpha-1-antitrypsin are synthesized in the liver. Local inflammation induced in the liver donors moderately stimulated the synthesis of alpha-1-antitrypsin but it affected only marginally that of antithrombin III.     

A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress

Endoplasmic reticulum (ER) stress is increasingly recognized as an important mechanism in a wide range of diseases including cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's and Alzheimer's disease. Therefore, there is an increased need for reliable and quantitative markers for detection of ER stress in human tissues and cells. Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause ER stress, which leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of X-box binding protein-1 (XBP1) mRNA, which is frequently used as a marker for ER stress. In most studies, the splicing of the XBP1 mRNA is visualized by gel electrophoresis which is laborious and difficult to quantify. In the present study, we have developed and validated a quantitative real-time RT-PCR method to detect the spliced form of XBP1 mRNA.     

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex

1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse- chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1- antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1- antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha- mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N- linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.     

Primary hepatic malignant fibrous histiocytoma: a case report and review of the literature

Primary malignant fibrous histiocytoma (MFH) of the liver remains extremely rare with only several cases having been reported in literature. We report a case of hepatic MFH in a 53-year-old man who presented with upper abdominal pain, and weight loss for one month. Ultrasound and computed tomography (CT) scan showed a large mass with fine tumor vessels over the left lobe of the liver. Histopathological findings indicated a mesenchymal tumor consisting of spindle cells in storiform pattern intermingled with histiocyte-like cells and giant cells. Immunohistochemically, most tumor cells expressed vimentin, alpha-1 anti-chymotrypsin, alpha-1 antitrypsin and CD68. Morphological and immunohistochemical findings support that the tumor should be classified as a primary malignant fibrous histiocytoma. The literatures is briefly reviewed.     

Does the PI polymorphism alone control alpha-1-antitrypsin expression?

Whether genetic factors other than the protease-inhibitor (PI) polymorphism itself contribute to variation in alpha-1-antitrypsin is of both theoretical and practical interest. We have measured the quantity of alpha-1-antitrypsin (by an immunoturbidometric assay) and its activity (by assaying elastase inhibitory capacity [EIC]) in 583 individuals from 114 twin kinships who were also typed for PI by isoelectric focusing. Models of variation were fitted directly to the raw observations by a maximum-likelihood method. Specification of phenotypic means led to highly significant improvements in fit over models including only individual environment variance and additive genetic variance. The 29 phenotype means could also be described as the appropriate additive combinations of the 12 allelic effects. Only small improvements in fit could then be obtained by addition of polygenic components of variance. We conclude that nearly all genetic variation in alpha-1-antitrypsin quantity and activity can be explained by detectable variation at the PI locus and that this variance is largely additive. Bivariate analysis of alpha-1-antitrypsin and EIC revealed marginal evidence for differences in specific activities of molecules coded by different PI alleles. The correlation between environmental deviations for the two measures was only .63, which may reflect, in part, the rather low reliability of the assays and account for the modest heritabilities (less than .5) of the two measures. An intriguing finding was the presence of significant differences in E1 variance for different PI types, suggesting that different phenotypes have differing capacities to react to environmental challenges.     

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.     

Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression.

We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets [H. Mori et. al. Life Sci., 741-747 44 (1989)]. This method involves the measurement of the accumulation of [3H]-inositol-1-phosphate (IP-1) as an index of PI hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine (10 microM and 100 microM) in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression.