Normal Alpha 1AT Research Articles

Alpha1-antitrypsin gene mutation hot spot associated with the formation of a retained and degraded null variant [corrected; erratum to be published

Null alpha1-antitrypsin (alpha1AT) alleles represent the end of a continuum of variants associated with profound alpha1AT deficiency and an increased risk of emphysema. This study characterizes the molecular basis of QOclayton, a new example of an alpha1AT null allele arising from a mutational hot spot in the alpha1AT gene. The QOclayton allele is identical to the normal M1(V213) alpha1AT allele except for an insertion of a cytosine. This insertion occurs in the alpha1AT sequence which normally has seven cytosines corresponding to amino acid residues 360 to 362. The QOclayton mutation is located in the same reiterated DNA sequence as the alpha1AT QObolton deletion mutation and the insertion mutation allele QOsaarbruecken. The QOclayton cytosine insertion causes a 3' frameshift and results in the formation of a termination codon at residue 376, the same consequence as the alpha1AT QOmattawa mutation (L353 T-insertion with a 3' frameshift). To determine the molecular mechanisms responsible for the absence of alpha1AT associated with the QOclayton gene, an in vitro model of QOclayton was established using Chinese hamster ovary cells (CHO) transfected with the QOclayton gene. These cells were evaluated for alpha1AT mRNA expression, protein synthesis and secretion. Although the QOclayton gene expresses a similar amount of alpha1AT mRNA as compared with the normal alpha1AT gene, no QOclayton protein is secreted. Protein trafficking and double-label immunofluorescence demonstrate that the QOclayton protein is retained in the rough endoplasmic reticulum or pre-Golgi compartment and is degraded (t1/2 = 6.5 h). Since QOmattawa, QObolton, and QOsaarbruecken have similar termination sites in the alpha1AT mRNA, they may share a similar intracellular fate.

Abnormal synthesis of dolichol-linked oligosaccharides in carbohydrate-deficient glycoprotein syndrome.

Carbohydrate-deficient glycoprotein syndrome (CDGS) is a rare metabolic disorder presenting in infancy with severe neurologic involvement and variable multisystemic abnormalities. Diagnosis relies upon the detection of abnormal serum glycoprotein isoforms on isoelectric focusing (IEF) gels. Carbohydrate structural analyses were performed on the N-linked oligosaccharides of serum alpha 1-antitrypsin (alpha-1AT) from two Danish children with classical type I CDGS. Following preparative gel electrophoresis of alpha-1AT isoforms, oligosaccharide charge and monosaccharide composition analyses revealed increased glycosylation heterogeneity in CDGS compared with normal alpha-1AT. CDGS alpha-1AT isoforms bore N-glycans co-migrating with monosialylated standards, while normal alpha-1AT oligosaccharides co-migrated with both mono- and disialylated standards. While the monosaccharide contents of normal alpha-1AT isoforms were relatively uniform, those of CDGS alpha-1AT isoforms varied widely, and many were relatively mannose enriched. The mannose-rich oligosaccharides of CDGS alpha-1AT were not typical oligomannose structures since they were not released by endo-beta-N-acetylglucosaminidase H (endo H) digestion. Metabolic labelling of CDGS fibroblasts with [3H]mannose showed lower than normal intracellular total mannose, free mannose and phosphorylated mannose species, as well as diminished [3H]mannose incorporation into dolichol-linked and protein-linked oligosaccharides. In addition, the glycans liberated from CDGS dolichol-linked oligosaccharides were significantly truncated compared with those from normal fibroblasts. These data suggest that our type I CDGS patients produce abnormal N-linked oligosaccharides due to impaired biosynthesis of dolichol-oligosaccharide precursors.

Deletion/frameshift mutation in the alpha 1-antitrypsin null allele, PI*QObolton.

The most common deficiency allele of the protease inhibitor (PI) alpha 1-antitrypsin (alpha 1AT) is PI*Z. Other rare deficiency alleles of alpha 1AT are of two types: those producing low but detectable amounts of alpha 1AT (less than 20% of normal serum concentrations), and null alleles producing less than 1% of normal alpha 1AT and therefore not detectable by routine quantitative methods. We have previously used DNA polymorphisms and family data to determine heterozygosity in an individual producing low levels of serum alpha 1AT (12% of normal) of PI type Mmalton. By DNA analysis we observed the typical haplotype associated with PI*Mmalton and a unique null haplotype associated with the allele PI*QObolton. The QObolton allele produces no detectable serum alpha 1AT. We have cloned and sequenced the QObolton allele from a phage genomic library. Deletion of a single cytosine residue near the active site of alpha 1 AT in exon V results in a frameshift causing an in-frame stop codon downstream of the deletion. This stop codon leads to premature termination of protein translation at amino acid 373, resulting in a truncated protein. The truncated protein is predicted to have an altered carboxy terminus (amino acids 363-372) and will lack structurally important amino acids.

Molecular Basis of the Liver and Lung Disease Associated with the α1-Antitrypsin Deficiency Allele Mmalton

Abstract Alpha 1-Antitrypsin (alpha 1AT) deficiency is characterized by reduced serum levels of alpha 1AT and a risk for the development of emphysema and liver disease. However, whereas there is an increased risk for emphysema associated with at least 10 alpha 1AT deficiency and null alleles, the hepatic disease is observed only in a subset of these alleles, suggesting that it is not the reduced serum levels of alpha 1AT per se which cause the liver disease. The present study characterizes the alpha 1AT deficiency allele Mmalton, an allele that like the common Z deficiency mutation (Glu342----Lys) is associated with both alpha 1AT deficiency and hepatic disease. Capitalizing on the identification of the homozygous inheritance of the rare Mmalton alpha 1AT deficiency allele, it was demonstrated that although caused by a very different mutation, the Mmalton allele shares with the Z allele the association of liver disease with the same type of abnormalities of alpha 1AT biosynthesis. Cloning of the Mmalton gene and sequence analysis demonstrated that it differs from the normal alpha 1AT M2 allele by deletion of the entire codon (TTC) for residue Phe52. Liver biopsy of the Mmalton homozygote revealed inflammation, mild fibrosis, and intrahepatocyte accumulation of alpha 1AT. Evaluation of de novo alpha 1AT biosynthesis in alpha 1AT-synthesizing cells of this individual demonstrated normal levels of alpha 1AT mRNA transcripts but abnormal intracellular accumulation of newly synthesized alpha 1AT at the level of the rough endoplasmic reticulum with consequent reduced alpha 1AT secretion. Finally, retroviral gene transfer of a normal alpha 1AT cDNA and an alpha 1AT cDNA with the Mmalton Phe52 deletion into murine cells demonstrated that the Mmalton cells reproduced the abnormal accumulation of newly synthesized alpha 1AT, thus directly demonstrating that the deletion mutation is responsible for the intracellular accumulation of the newly synthesized alpha 1AT. Thus, not only is the liver disease associated with alpha 1AT deficiency restricted to a subset of alpha 1AT deficiency alleles, it appears to be restricted to those alleles associated with intracellular accumulation of newly synthesized alpha 1AT, suggesting that it is the abnormal intrahepatocyte alpha 1AT accumulation which incites the liver injury.

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.

Characterization of the M1(Ala213) type of alpha 1-antitrypsin, a newly recognized, common "normal" alpha 1-antitrypsin haplotype.

alpha 1-Antitrypsin (alpha 1AT) is a highly pleomorphic 52-kDa serum glycoprotein that functions as the major inhibitor of neutrophil elastase. Of these, the most common normal alpha 1AT haplotypes identified by isoelectric focusing (IEF) of serum are those of the M family, including M1, M2, and M3. In the course of studying the alpha 1AT type Z gene, we identified a restriction endonuclease BstEII polymorphism in the M1 gene that predicted the existence of a previously unidentified, but relatively common, haplotype of M, referred to as M1(Ala213) [Nukiwa, T., Satoh, K., Brantly, M. L., Ogushi, F., Fells, G. A., Courtney, M., & Crystal, R. G. (1986) J. Biol. Chem. 261, 15989-15994]. In this study we have cloned both alpha 1AT genes from an individual heterozygous for the M1(Ala213) and M1(Val213) haplotypes. Sequencing of the coding exons of both demonstrated that they are identical except for the Ala-Val difference at residue 213. The codominant transmission of the M1(Ala213) gene was demonstrated in a family study. Evaluation of 39 genomic samples of Caucasians with the IEF haplotype M1 demonstrated haplotype frequencies of 68% for M1(Val213) and 32% for M1(Ala213). alpha 1AT serum levels of individuals inheriting the M1(Ala213) gene in a homozygous fashion were in the same range as those for homozygous M1(Val213) as was the rate of association of the M1(Ala213) protein with neutrophil elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT.

A new allele of alpha 1-antitrypsin: PI NADELAIDE.

The allele PI NADELAIDE (PI NADE) was named in accord with nomenclature guidelines and specifies a new co-dominant variant of alpha 1AT. Discovery was achieved by IEF and the isoelectric point of NADE is between N and NHAM. Familial inheritance of PI NADE was demonstrated and both PI M2NADE and PI M3NADE phenotypes were observed. The mobility of PI NADE is identical to PI M by both starch and agarose electrophoresis. PI NADE apparently confers normal alpha 1AT serum concentrations and is probably unrelated to disease.

Alpha1-Antitrypsin: 1. Evidence for two genes causing low concentrations in serum; 2. Association of heterozygosity and Chronic obstructive lung disease

Alpha1-antitrypsin (alpha1-at) is one of several proteinase inhibitors in human serum. It is a glycoprotein with molecular weight of approximately 50,000. The normal concentration in serum is 213±32 mg/100 ml. The corresponding trypsin inhibiting capacity is between 1.0 and 1.3 mg of trypsin inhibited by one ml of serum. During recent years, different phenotypes of alpha1-at have been identified and a genetic model with several codominant alleles at one locus has been developed. Two phenotypes can be distinguished quantitatively and electrophoretically while others can be recognized by electrophoresis only. I consider here only the phenotypes with lower than normal alpha1-at concentrations in serum. The method most commonly used to determine the total trypsin inhibiting capacity of serum is an inhibition assay with H,α-benzoyl-arginine-p-nitroanilide as substrate for trypsin. A convenient method for a specific determination of alpha1-at is the radial immunodiffusion employing an antiserum against alpha1-at. For the electrophoretic distinction of the different phenotypes starch gel electrophoresis at an acidic pH (4.95) in combination with a second electrophoresis at an alkaline pH (8.65) in agarose gel that contains an antiserum at alpha1-at is a sensitive method. With this technique, the alpha1-at phenotypes of heterozygotes and homozygotes for the two genes, Pin and Pz (nomenclature of Fagerhol), that cause lower concentrations of serum alpha1-at, can be recognized as a characteristic pattern of bands. The protein that is determined by the gene Piz moves on electrophoresis more slowly towards the anode than that determined by Pi”, but both are moving slower than the normal protein controlled by the most common gene PiM. In heterozygotes, both patterns of alpha1-at can be recognized. The resulting phenotypes are called: M/M, S/S, Z/Z, M/S, M/Z, etc. The alpha1-at concentrations for these phenotypes are in mg/100 ml of serum: M/M; 213±32, S/S; 122±10, Z/Z; 25±6, M/S; 159±39, M/Z; 125±46. These concentrations are only found under normal physiologic conditions. In the presence of an inflammation, during pregnancy, and due to certain steroid hormones, the alpha1-at concentration of normal individuals can rise to levels 100 percent above the starting value, and heterozygotes can show values in the normal range of 200 mg percent. The electrophoretic characteristics are, however, independent of concentration and for that reason the electrophoretic determination of the alpha1-at phenotype is more reliable than the quantitative one, particularly in patients with inflammation.