A1AT Gene Research Articles

Inherited Chronic Obstructive Pulmonary Disease: New Selective-Sequencing Workup for α1-Antitrypsin Deficiency Identifies 2 Previously Unidentified Null Alleles

alpha(1)-Antitrypsin (alpha(1)AT) deficiency predisposes individuals to chronic obstructive pulmonary disease (COPD) and/or liver disease. Phenotyping of the protein by isoelectric focusing is often used to characterize alpha(1)AT deficiency, but this method may lead to misdiagnosis (e.g., by missing null alleles). We evaluated a workup that included direct sequencing of the relevant parts of the gene encoding alpha(1)AT, SERPINA1 [serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1], for patients with alpha(1)AT concentrations < or =1.0 g/L. During a 5-year period, we identified 66 patients with alpha(1)AT concentrations < or =1.0 g/L and amplified and sequenced exons 2, 3, and 5 of the alpha(1)AT gene in these patients. To ensure that no relevant genotypes were missed, we sequenced the same exons in 48 individuals with alpha(1)AT concentrations between 1.0 and 1.5 g/L. Sequence analysis revealed 18 patients with combinations of disease-associated alpha(1)AT alleles: 8 homozygous for the deficient Z allele and 10 compound heterozygotes for various deficient or null alleles. We identified and named 2 new null alleles, Q0(soest) (Thr(102)-->delA, which produces a TGA stop signal at codon 112) and Q0(amersfoort) (Tyr(160)-->stop). No relevant disease-associated allele combinations were missed at a 1.0-g/L threshold. Up to 22% of the alleles in disease-associated alpha(1)AT allele combinations may be missed by conventional methods. Genotyping by direct sequencing of samples from patients with alpha(1)AT concentrations < or =1.0 g/L detected these alleles and identified 2 new null alleles.

The Locus Control Region Activates Serpin Gene Expression through Recruitment of Liver-Specific Transcription Factors and RNA Polymerase II

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 comprises 11 serpin genes, many of which are expressed specifically in hepatic cells. Previous studies identified a locus control region (LCR) upstream of the human alpha1-antitrypsin (alpha1AT) gene that is required for gene activation, chromatin remodeling, and histone acetylation throughout the proximal serpin subcluster. Here we show that the LCR interacts with multiple liver-specific transcription factors, including hepatocyte nuclear factor 3beta (HNF-3beta), HNF-6alpha, CCAAT/enhancer binding protein alpha (C/EBPalpha), and C/EBPbeta. RNA polymerase II is also recruited to the locus through the LCR. Nongenic transcription at both the LCR and an upstream regulatory region was detected, but the deletion of the LCR abolished transcription at both sites. The deletion of HNF-3 and HNF-6 binding sites within the LCR reduced histone acetylation at both the LCR and the upstream regulatory region and decreased the transcription of the alpha1AT, corticosteroid binding globulin, and protein Z-dependent protease inhibitor genes. These results suggest that the LCR activates genes in the proximal serpin subcluster by recruiting liver-specific transcription factors and components of the general transcription machinery to regulatory regions upstream of the alpha1AT gene.

Alpha-1-Antitrypsin Deficiency: Current Concepts

Since the condition was first described four decades ago, alpha-1-antitrypsin (A1AT) deficiency has served as a model for other disease processes. A1AT is the archetypal serpin designed to ensnare proteases, a process that involves significant conformational change within the molecule. Mutations in the A1AT gene lead to misfolding of the protein and accumulation within the endoplasmic reticulum of hepatocytes resulting in two different pathologic processes. First, the accumulation of mutant A1AT protein has a directly toxic effect on the liver, resulting in hepatitis and cirrhosis. Second, the resultant decrease in circulating A1AT results in protease-antiprotease imbalance at the lung surface and emphysema ensues. A1AT deficiency therefore can be seen as two distinct disease processes: a conformational disease of the liver and a protease-antiprotease imbalance of the lung. This two-stage model of disease in A1AT deficiency is elegant in its simplicity and goes a long way to explaining the clinical manifestations that occur in patients with the condition. However, some aspects of the disease are not readily explained. Recent findings suggest that there is more to the lung damage in A1AT deficiency than simple proteolytic insult and that the presence of the mutant protein itself is proinflammatory and may indeed cause chronic injury to the cells that produce it. This review discusses some of the emerging concepts in alpha-1-antitrypsin research and outlines the implications these new ideas may have for treatment of this condition.

High Prevalence of α‐1‐Antitrypsin Heterozygosity in Children With Chronic Liver Disease

Alpha-1-antitrypsin (A1AT) deficiency is the most common genetic cause of liver disease in children; however, the role of polymorphic heterogeneity in the A1AT gene as a modifier of other forms of pediatric liver disease is not clear. We hypothesized that non-M A1AT allele variants are more common in children with chronic liver disease than in the general population. A retrospective, single-center study was performed in which A1AT phenotypes were obtained by reviewing charts of children with chronic liver disease. Chi-square analysis was used to compare allele frequencies in the population of children with liver disease with published epidemiologic data and to compare allele frequencies among disease subgroups. The frequency of A1AT Z and other alleles was increased in children with chronic liver disease (n = 241) when compared with the published reference database (P < 0.001). This increase remained significant when the population was divided into disease subsets: biliary atresia (n = 67) and other liver disease (n = 174) (P < 0.001 for both). Among children with biliary atresia referred for liver transplant evaluation, the presence of a non-M allele was associated with a lower mean age at transplant listing than the MM phenotype (235 vs 779 days, P = 0.036) and more frequent loss of native liver by 24 months of age (90% vs 65%, P = 0.04). A1AT non-M alleles are more frequent in children with chronic liver disease than in the general population. We speculate that these non-M alleles may act as genetic modifiers in pediatric liver disease in general and modulate disease progression in children with biliary atresia in particular.

An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene

In an attempt to produce transgenic cloned cows secreting alpha 1-antitrypsin (α1-AT) protein into milk, bovine cumulus cells were transfected with a plasmid containing an α1-AT gene and green fluorescent protein (GFP) reporter gene using Fugene 6 ® as a lipid carrier. The GFP-expressing cells were selected and transferred into enucleated bovine oocytes. Couplets were fused, chemically activated and cultured. Developmental competence was monitored and the number of inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts were counted after differential staining. The percentage of blastocysts was lower ( P < 0.05) in transgenic cloned embryos compared to non-transgenic cloned embryos (23% versus 35%). No difference in the numbers of ICM and TE cells between the two groups of embryos was observed. One or two GFP-expressing blastocysts were transferred into the uterus of each recipient cow. Out of 49 recipient cows, three pregnancies were detected by non-return estrus and rectal palpation. However, the pregnancies failed to maintain to term; two fetuses were aborted at Day 60 and 150, respectively, and one fetus at Day 240. The genomic DNA from the aborted fetus was amplified by polymerase chain reaction (PCR) to investigate integration of the transgene in the fetus. The expected PCR product was sequenced and was identical to the sequence of α1-AT transgene. In conclusion, the present study demonstrated that developmental competence of cloned embryos derived from transgenic donor cells was lower than embryos derived from non-transfected donor cells. Although we failed to obtain a viable transgenic cloned calf, integration of α1-AT gene into the fetus presents the possibility of producing transgenic cloned cows by somatic cell nuclear transfer.

155. Non-Viral Gene Therapy for Type 1 Diabetes by Electrotransfer of Insulin and Glucokinase Genes to Skeletal Muscle

Alpha1-antitrypsin (α1-AT) deficiency is caused by mutations in the α1-AT gene, a protease inhibitor, and can result in emphysema and/or liver disease. The lung disease is caused by a decrease in secreted α1-AT enzyme, a loss-of-function which results in tissue damage from unchecked leukocyte proteases. In contrast, the liver disease occurs by a gain-of-function, whereby mutant protein polymers are retained in the endoplasmic reticulum of hepatocytes. In persuit of an alternative to the only current treatment for the liver disease, to transplant the organ, we are investigating a genetic therapy that would combine the technologies of RNA interference (RNAi) with the Sleeping Beauty transposon system. The RNAi pathway consists of endogenous enzymes that destroy mRNAs selectively in response to small RNAs that are complimentary in sequence. Small inhibitory RNAs (siRNAs), which can be transcribed from plasmid constructs introduced into cells, have been shown to mediate RNAi in vitro and in vivo. Moreover, siRNAs can mediate long-term repression of a target gene if they are constitutively expressed from the genome. The Sleeping Beauty (SB) transposon system can integrate DNA transgenes into the genome for long-term expression in vitro and in vivo, and is being developed as a novel, non-viral gene therapy tool. A SB transposon vector was constructed to express siRNAs that target human α1-AT mRNA. The results demonstrate that siRNA transposons can be used to reduce wildtype and mutant α1-AT expression in cultured cells, whereas the house-keeping gene GAPDH was not affected. Next, we plan to test these siRNA transposons in vivo, in the transgenic mouse model of α1-AT liver disease. This gene therapy model combines two technologies, RNAi and the SB transposon, for a potential new therapeutic for the liver disease associated with mutant α1-AT.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

Cell-specific involvement of HNF-1beta in alpha(1)-antitrypsin gene expression in human respiratory epithelial cells.

The synergistic action of hepatocyte nuclear factor (HNF)-1alpha and HNF-4 plays an important role in expression of the alpha(1)-antitrypsin (alpha(1)-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the alpha(1)-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that alpha(1)-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1alpha and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that alpha(1)-AT gene expression in H441 cells is predominantly driven by HNF-1beta, even though HNF-1beta has no effect on alpha(1)-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of alpha(1)-AT and HNF-1beta was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on alpha(1)-AT gene expression in H441 cells, even when it is cotransfected with HNF-1beta or HNF-1alpha. HNF-3 by itself has little effect on alpha(1)-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1beta in alpha(1)-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.

The HNF-4/HNF-1alpha transactivation cascade regulates gene activity and chromatin structure of the human serine protease inhibitor gene cluster at 14q32.1.

Hepatocyte-specific expression of the alpha1-antitrypsin (alpha1AT) gene requires the activities of two liver-enriched transactivators, hepatocyte nuclear factors 1alpha and 4 (HNF-1alpha and HNF-4). The alpha1AT gene maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene encoding corticosteroid-binding globulin (CBG), and the chromatin organization of this approximately 130-kb region, as defined by DNase I-hypersensitive sites, has been described. Microcell transfer of human chromosome 14 from fibroblasts to rat hepatoma cells results in activation of alpha1AT and CBG transcription and chromatin reorganization of the entire locus. To assess the roles of HNF-1alpha and HNF-4 in gene activation and chromatin remodeling, we transferred human chromosome 14 from fibroblasts to rat hepatoma cell variants that are deficient in expression of HNF-1alpha and HNF-4. The variant cells failed to activate either alpha1AT or CBG transcription, and chromatin remodeling failed to occur. However, alpha1AT and CBG transcription could be rescued by transfecting the cells with expression plasmids encoding HNF-1alpha or HNF-4. In these transfectants, the chromatin structure of the entire alpha1AT/CBG locus was reorganized to an expressing cell-typical state. Thus, HNF-1alpha and HNF-4 control both chromatin structure and gene activity of two cell-specific genes within the serpin gene cluster at 14q32.1.

Ribozyme-mediated specific gene replacement of the α1-antitrypsin gene in human hepatoma cells

Backgroundl Aims: Some of the mutant forms of cellular proteins not only lose their function, but also cause diseases by their toxic effects. One of the challenging tasks in the field of gene therapy will be “gene replacement” accomplished by inhibiting mutant gene expression and providing normal function of the same gene, simultaneously. Although lung involvement in α1-antrypsin (α1-AT) deficiency is caused by the lack of α1-AT function, the liver involvement is due to the accumulation of the mutated α1-AT protein. Therefore, one possible approach to prevent and treat the disease manifestations of α1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning α1-AT protein in the liver. Methods: For the inhibition of α1-AT gene expression, panels of α1-AT-specific hammerhead ribozymes designed to target different GUC sites in the α1-AT mRNA were evaluated in a human hepatoma cell-line, transduced with retroviral vectors which express ribozymes under the control of a human tRNA promoter. A bi-functional vector was also constructed, which contained a functional α1-AT ribozyme and was combined with a modified α1-AT gene, whose product was engineered to be resistant to the specific α1-AT ribozyme. This construct was transduced into target hepatoma cells. Results: The transduced hepatoma cells showed the effective expression of modified α1-AT, under the conditions where the endogenous α1-AT gene expression was inhibited. Conclusion: This ribozyme-mediated, specific gene replacement is a first step in the gene therapy of α1-AT deficiency.

Regulation of alpha1-antitrypsin gene expression in human intestinal epithelial cell line caco-2 by HNF-1alpha and HNF-4.

There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha1-antitrypsin (alpha1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1alpha (HNF-1alpha), HNF-1beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha1-AT gene. Cotransfection studies showed that HNF-1alpha and HNF-4 had a synergistic effect on alpha1-AT gene expression. RNA blot analysis showed that HNF-1alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1alpha binding activity increase coordinately with alpha1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha1-AT gene expression. Together, these results provide evidence that HNF-1alpha and HNF-4 play a role in the mechanism by which the alpha1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.

Alpha-1 antitrypsin deficiency alleles and severe cystic fibrosis lung disease

BACKGROUNDAlpha-1 antitrypsin (α1-AT) is the most abundant proteinase inhibitor within the lung. We have recently reported the surprising observation that cystic fibrosis patients with mild to moderate deficiency of α1-antitrypsin...

Clinical Features and Natural History of Severe α1-Antitrypsin Deficiency: Roger S. Mitchell Lecture

Since its first description by Laurell and Eriksson1 in 1963, α1-antitrypsin (α1-AT) deficiency has been studied extensively, with substantial strides made in understanding the cellular and molecular biology of this disorder. The α1-AT gene has been located on the long arm of chromosome 14 and the gene has been sequenced and cloned.2 The complete primary structure of the normal α1-AT molecule has been characterized, and the mechanism of impaired hepatic secretion of the most common severe deficiency variant, the Z-type protein, has been characterized as “loop-sheet polymerization.”3 In the context of this extensive basic understanding, it remains a paradox that current cellular and molecular understanding of α1-AT deficiency greatly outstrips current knowledge about clinical aspects of this disease.

Temporal and tissue-specific expression of the MET ORF driven by the complete transcriptional unit of human A1AT gene in transgenic mice

We inserted the sequence coding for the cytoplasmic portion of the human MET receptor into an 18-kb genomic fragment containing the entire human A1AT gene (encoding α-l-antitrypsin). Stringent control of gene expression, at the transcriptional, post-transcriptional and translational levels, was ensured by insertion of the MET open reading frame into A1AT, thus maintaining: (i) all the elements that confer tissue-specific transcription initiation, ( ii) all the sequences involved in transcript processing and ( iii) all the sequences which influence messenger stability and translational efficiency. The expression pattern of this vector in transgenic mice was identical to that of the human A1AT transgene, as well as to that of A1AT in humans with regard to both temporal and tissue-specific regulation.

Molecular mechanism of hepatocellular injury in alpha 1 antitrypsin deficiency.

Liver injury in PiZZ α1-antitrypsin (α1-AT) deficiency probably results from toxic effects of the abnormal α1-AT molecule accumulating within the ER of liver cells. However, only 12-15% of individuals with this same genotype develops liver disease. Therefore, we predicted that other genetic traits that determine the net intracellular accumulation of the mutant α1-AT molecule would also determine susceptibility to liver disease. To address this prediction, we transduced skin fibroblasts from PiZZ individuals with liver disease or without liver disease with amphotropic recombinant retroviral particles designed for constitutive expression of the mutant α1-AT Z gene. Human skin fibroblasts do not express the endogenous α1-AT gene but presumably express other genes involved in postsynthetic processing of secretory proteins. The results show that expression of human α1-AT M gene was conferred on each fibroblast cell line. Compared to the same cell line transduced with the wild-type α1-AT M gene, there was selective intracellular accumulation of the mutant α1-AT Z protein in each case. However, there was a marked delay in degradation of the mutant α1-AT Z protein after it accumulated in the fibroblasts from ZZ individuals with liver disease (“susceptible hosts”) as compared to those without liver disease (“protected hosts”). Appropriate disease controls showed that the lag in degradation in susceptible hosts is specific for the combination of PiZZ phenotype and liver disease. Biochemical characteristics of α1-AT Z degradation in the protected hosts were found to be similar to those of a common ER degradation pathway previously described in model experimental cell systems for T-cell receptor α subunits and asialoglycoprotein receptor subunits, therefore, raising the possibility that the lag in degradation in the susceptible host is a defect in this common ER degradation pathway. Thus, these data provide evidence that other genetic traits that affect the fate of the abnormal α1-AT Z molecule, at least in part, determine susceptibility to liver disease. These data also validate a system for elucidating the biochemical/genetic characteristics of these traits and for examining the relevance to human disease of pathways for protein déradation in the ER.

Disruption of the LF-A1 and LF-B1 binding sites in the human alpha-1-antitrypsin gene has a differential effect during development in transgenic mice.

Previous work in transfected cell lines and in nuclear extracts has led to the identification of two cis-acting elements important for transcription of the human alpha-1-antitrypsin (A1AT) gene, which bind to two liver specific trans-acting factors, LF-A1 and LF-B1. Mutations EM3 and PM1, which abolish the binding of LF-A1 and LF-B1 respectively, drastically reduce transcription activity of the A1AT gene in vitro and in cell culture. The same mutants have now been introduced in a larger DNA context and their effect has been tested in transgenic mice. A stretch of DNA was constructed which carries two transcriptional units: 18 kb of the human retinol binding protein (RBP) gene, driving the expression of the bacterial chloramphenicol acetyl transferase, linked to 17.5 kb containing the entire A1AT coding sequence with additional 5' and 3' flanking sequences. Transcription from the RBP promoter was shown to predominate in liver, and could be used as an internal marker of 'active copy number'. Mutations in the A1AT gene promoter were introduced by homologous recombination in bacterial cells. The results show that base pair substitutions in the binding site for LF-A1 and LF-B1 drastically reduce transcription in non-hepatic adult tissues, yolk sac, and fetal liver, whereas only LF-B1 binding site mutations have a marked, albeit variable, effect in adult liver.

Effect of Pseudomonas Elastase on Human Mononuclear Phagocyte α1-Antitrypsin Expression

The net balance of neutrophil elastase and its inhibitor, alpha 1-antitrypsin (alpha 1-AT), is a critical determinant of connective tissue turnover during homeostasis and in disease states. In addition to liver-derived alpha 1-AT, which translocates from blood to tissues, this elastase-alpha 1-AT balance is maintained by expression of alpha 1-AT at the local tissue level in resident mononuclear phagocytes. Our previous studies have shown that this elastase-alpha 1-AT balance is also tightly controlled at a cellular level in that addition of exogenous neutrophil elastase (serpine-type elastase) to cultured mononuclear phagocytes is associated with an increase in expression of the alpha 1-AT gene. Subsequent studies have demonstrated that this novel regulatory loop involves interaction between exogenous neutrophil elastase and endogenous alpha 1-AT inducing a structural rearrangement in the alpha 1-AT molecule and exposing highly conserved conformation-specific domain of alpha 1-AT, which can then be recognized by a specific cell surface receptor, the serpine-enzyme complex receptor. In the following study, we examined the effect of a bacterial metalloelastase, Pseudomonas aeruginosa elastase, on expression of alpha 1-AT in human mononuclear phagocytes. We show that pseudomonas elastase inactivates monocyte-derived alpha 1-AT by limited proteolysis but, in so doing, alpha 1-AT becomes recognized by the serpine-enzyme complex receptor and mediates an increase in de novo synthesis of alpha 1-AT in these cells. However, the concentrations of pseudomonas elastase needed to proteolytically inactivate alpha 1-AT in monocyte culture fluid are higher than those required for inactivation of purified plasma alpha 1-AT.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the sequence of the normal alpha-1-antitrypsin M3 allele and function of the M3 protein.

Alpha-1-antitrypsin (alpha 1AT), a highly pleomorphic 52 kD glycoprotein, functions chiefly as the major inhibitor of neutrophil elastase. Of the known alpha 1AT variants, greater than 95% in the U.S. Caucasian population are those of the "normal" M family, including M1(Ala213), M1(Val213), M2 and M3, with M3 the least common of the group. Quantification of the functional capacity of the M3 protein as an inhibitor of neutrophil elastase demonstrated a Kassociation for neutrophil elastase of 10.1 +/- 1.5 x 10(6) M-1 s-1, a value comparable to the common normal M1(Val213) alpha 1AT. To define the nucleotide sequence of the M3 gene, the five coding exons of the alpha 1AT gene of an M3 homozygote were amplified by the polymerase chain reaction and cloned into the plasmid vector pUC19. Sequence analysis demonstrated that the alpha 1AT M3 gene differs from the alpha 1AT M1(Val213) gene by a single base substitution (Glu376 GAA----Asp376 GAC) and from the alpha 1AT M2 gene by a single base substitution (His101 CAT----Arg101 CGT). To establish the consistency of the alpha 1AT M3 genotype among individuals identified by isoelectric focusing of serum to have the M3 phenotype, analysis of genomic DNA of 16 individuals by means of allele-specific amplification revealed that residues 101 and 376 were Arg and Asp, respectively, in all M3 alleles, while residue 101 was His in all M2 alleles and residue 376 was Glu in all M1 alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum α1-Antitrypsin Deficiency Associated with the Common S-type (Glu264 → Val) Mutation Results from Intracellular Degradation of α1- Antitrypsin Prior to Secretion

The S-type α1-antitrypsin (α1AT) deficiency allele differs from the normal M1(Val213) allele by a single amino acid substitution (Glu264 → Val). To evaluate the molecular pathophysiology responsible for the reduced serum levels of al AT associated with the S-type allele, α1AT gene expression was examined in blood monocytes, cells which normally produce α1AT, as well as murine fibroblasts modified by retroviral gene transfer to express the S-type and normal M-type human α1AT genes. Northern analysis and S1 protection analysis demonstrated that monocytes of M and S homozygotes both express 1.8-kilobase α1AT mRNA transcripts in comparable levels and similar in structure. Pulse-chase labeling studies demonstrated that both M and S monocytes synthesized and secreted a 52-kDa protein, but the S monocytes secreted significantly less. The cellular lysates of both M and S monocytes contained a newly synthesized 50-kDa precursor form of α1AT, but the S monocytes contained reduced amounts. Pulse-chase labeling in the presence of tunicamycin, an inhibitor of core oligosaccharide addition, demonstrated that S monocytes exhibited a selective inhibition of secretion of 45-kDa nonglycosylated α1AT not observed in M monocytes. Consistent with these observations, murine fibroblasts modified by retroviral gene transfer to contain an integrated human S-type α1AT cDNA demonstrated reduced secretion of α1AT compared with fibroblasts containing an integrated human M-type α1 AT cDNA and also reproduced the abnormality of α1AT biosynthesis observed with S-type monocytes. Furthermore, in the presence of leupeptin, an inhibitor of cellular proteinases, the S-type modified fibroblasts demonstrated a selective augmentation of human α1AT secretion not observed for the M-type. Together, these observations are consistent with the concept that the single A → T mutation of the S-type α1AT gene results in reduced cellular secretion of α1AT because the newly synthesized S-type α1AT protein is degraded intracellularly prior to secretion.

Molecular basis of alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder associated with a major reduction in serum A1AT levels. Clinically, A1AT deficiency is associated with emphysema in adults and, less commonly, liver disease in neonates. A1AT is a 52-kDa, 394-amino acid, single-chain glycoprotein normally present in serum at 150 to 350 mg/dl. The A1AT gene, composed of seven exons dispersed over 12 kb of chromosomal segment 14q31-32.3, is expressed in heptocytes and mononuclear phagocytes. The A1AT protein, a member of the class of protease inhibitor proteins known as serpins (serine protease inhibitors), is a globular molecule composed of nine alpha-helices and three beta-pleated sheets. The major function of A1AT is to inhibit neutrophil elastase; A1AT does so through an active site centered around Met358 contained within an external stressed loop on the surface of the molecule. A1AT is a highly pleomorphic protein with greater than 75 variants determined at the protein and/or gene level. These variants can be categorized into four groups according to their serum A1AT level and function: normal, deficient, dysfunctional, and absent. There are two important salt bridges within the A1AT molecule (Glu342—Lys290; Glu263—Lys367); a mutation in the A1AT gene causing disruption of either salt bridge causes distinct molecular pathology resulting in reduced serum A1AT levels. Clinically relevant variants can be distinguished by a combination of isoelectric focusing of serum, restriction fragment length analysis of genomic DNA, oligonucleotide probes, and direct sequencing of the variant A1AT genes. Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder associated with a major reduction in serum A1AT levels. Clinically, A1AT deficiency is associated with emphysema in adults and, less commonly, liver disease in neonates. A1AT is a 52-kDa, 394-amino acid, single-chain glycoprotein normally present in serum at 150 to 350 mg/dl. The A1AT gene, composed of seven exons dispersed over 12 kb of chromosomal segment 14q31-32.3, is expressed in heptocytes and mononuclear phagocytes. The A1AT protein, a member of the class of protease inhibitor proteins known as serpins (serine protease inhibitors), is a globular molecule composed of nine alpha-helices and three beta-pleated sheets. The major function of A1AT is to inhibit neutrophil elastase; A1AT does so through an active site centered around Met358 contained within an external stressed loop on the surface of the molecule. A1AT is a highly pleomorphic protein with greater than 75 variants determined at the protein and/or gene level. These variants can be categorized into four groups according to their serum A1AT level and function: normal, deficient, dysfunctional, and absent. There are two important salt bridges within the A1AT molecule (Glu342—Lys290; Glu263—Lys367); a mutation in the A1AT gene causing disruption of either salt bridge causes distinct molecular pathology resulting in reduced serum A1AT levels. Clinically relevant variants can be distinguished by a combination of isoelectric focusing of serum, restriction fragment length analysis of genomic DNA, oligonucleotide probes, and direct sequencing of the variant A1AT genes.