Analysis of the Alpha-1-Antitrypsin Deficient Alleles M3S, MZ, and ZZ by Biochemical and Molecular Methods: A Family Study

Deficiency of alpha-1-antitrypsin (α₁-AT, a major protease inhibitor controlling tissue degradation) is a genetic disorder transmitted in a codominant autosomal form. It has more than 100 genetically determined variants. This study attempted to determine the degree of association between serum α₁-AT levels and phenotypes and to provide a strategy for reliable laboratory evaluation of deficiencies. The study group consisted of a 38-year-old male proband with clinical features of emphysema, his first-degree relatives, and healthy controls. Family history revealed a four-generation pedigree. Genomic DNA was isolated from peripheral blood leukocytes. Alpha-1-AT levels were determined from human serum by immunonephelometry. Phenotypes were determined by isoelectric focusing of blood samples. DNA sequences of coding exons were analyzed by the amplification DNA technique and direct sequencing. Inheritance and plasma levels of the ZZ, MM, M3S, and MZ phenotypes were confirmed by the family study. In the family members with deficiencies, plasma concentrations were 22.55% ± 5.15 (ZZ), 84.18% ± 5.18 (M3S), and 61.06% ± 7.15 (MZ) of the normal MM level. We found a close association between α₁-AT level and genotype. A combination of genotyping, quantification, and phenotyping is the optimal strategy for the laboratory evaluation of α₁-AT deficiency.     

Genetic Heterogeneity of Rabbit Alpha-1-Antitrypsin

Sixteen inbred or partially inbred strains of rabbits were investigated for electrophoretic and quantitative variations of alpha-1-antitrypsin (A-1-AT). We found interindividual differences in the electrophoretic A-1-AT patterns as well as quantitative differences in the concentrations of A-1-AT and the serum trypsin-inhibiting activity.

Three electrophoretic phenotypes were distinguished: M, P and MP. M was characterized by a predominant anodal A-1-AT band, and P had a major cathodal component. The MP pattern can be explained by the occurrence of the M and P components in the same serum due to heterozygosity.

The P pattern was associated with an A-1-AT concentration of approximately 56% of that in sera with the M phenotype. The levels of A-1-AT in sera with the MP phenotype were intermediate between those in M and P types.

In addition to the type-specific quantitative variation, we found a quantitative sexual dimorphism of a moderate degree: Female rabbits had A-1-AT concentrations 16% less than males.

     

Blood groups of bonnet macaques (Macaca radiata), with a brief introduction to seroprimatology

The human-type A-B-O blood groups of 52 bonnet macaques (Macaca radiata) were determined. Application of method of population genetics indicated the gene frequences to be O = 0.173, A = 0.480 and B = 0.347. Cross testing of sera and red cells of the bonnet macaques revealed two blood-type-specific isoagglutinins, one of them strong enough for use as a blood typing reagent. No blood group polymorphism was revealed by testing bonnet macaque red cells with isoantisera produced in rhesus monkeys (M. mulatta) and in crab-eating macaques (M. fascicularis). The rhesus and crab-eating macaque isoantisera reacted either with all or with none of the bonnet macaque red cells tested.     

Blood groups of crab-eating macaques (Macaca fascicularis) demonstrated by isoimmune rhesus monkey (Macaca mulatta) sera.

Twenty-one isoimmune sera produced in rhesus monkey (Macaca mulatta) containing type-specific antibodies for simian-type red cell antigens were tested for their cross-reactivity with red cells from crab-eating macaques (M. fascicularis). The majority of the antisera gave cross-reactions determining polymorphisms in the red cells of crab-eating macaques, homologous to those of rhesus monkeys. These results attest to the close taxonomic realationship between the two species of macaques, and have the practical implication that isoimmune sera produced for blood typing can also be used for typing red cells from related species, as has been also observed in studies on apes.     

Augmentation of lung antineutrophil elastase capacity with recombinant human alpha-1-antitrypsin

To evaluate the potential use of recombinant DNA-produced alpha-1-antitrypsin (alpha-1-AT) to augment the lung antineutrophil elastase defenses in alpha-1-AT deficiency, we compared the kinetics of intravenously administered recombinant produced alpha-1-AT (r alpha-1-AT) and purified normal human plasma alpha-1-AT (p alpha-1-AT) in the blood and lung of rhesus monkeys. The r alpha-1-AT was produced in yeast transformed with an expressing plasmid containing a full-length human alpha-1-AT complementary deoxyribonucleic acid and purified to greater than 99% homogeneity. The r alpha-1-AT has a molecular weight of 45,000, no carbohydrates, and is identical in sequence to normal plasma alpha-1-AT except for an additional N-terminal acetylmethionine. Despite its lack of carbohydrates, the r alpha-1-AT inhibited human neutrophil elastase with an association rate constant similar to that of p alpha-1-AT. Rhesus monkeys were infused intravenously with 120 mg/kg of r alpha-1-AT (n = 13) or p alpha-1-AT (n = 12) and the serum, urine, and lung epithelial lining fluid (ELF) concentrations of these molecules quantified at various intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood groups of macaques:a comparative study.

Distribution of the human-type and of the simian-type blood groups in rhesus, crab-eating, bonnet, pig-tailed and stump-tailed macaques revealed significant similarities and differences among these species. Human-type A--B-O blood groups cut across taxonomic lines and seem less value for taxonomic purposes than the simian-type blood groups detected by cross-reacting isoimmune rhesus monkey sera.     

Blood groups of pig-tailed macaques (Macaca nemestrina).

The human-type A-B-O blood groups of 57 pig-tailed macaques (Macaca nemestrina) were determined and the calculated gene frequencies, O = 0.8908, A = 0.0825 and B = 0.0267, gave excellent fit with the hypothesis of inheritance by triple allelic genes. In tests for simian-type blood groups with rhesus, baboon and crab-eating macaque immune antisera, it was shown that the red cells of pig-tailed macaques are polymorphic for several simian-type specificities defined by those cross-reacting sera. Pig-tailed macaques share with other macaque species the complex Drh-graded blood group system, which seems to occupy a special role among red cell antigens of macaques. Normal sera of three female macaques contained spontaneous isoagglutinins which selectively agglutinated the red cells of some pig-tailed as well as stump-tailed macaques.     

Probability of paternity exclusion and the number of loci needed to determine the fathers in a troop of macaques

We calculated the probability of paternity exclusion (P) in 6 troops of rhesus and Japanese macaques housed in open enclosures and 68 wild troops of Japanese, crab-eating, and toque macaques using 33 genetic loci which encoded the blood protein variations detected by electrophoretic techniques. In the open enclosures, especially of rhesus troops, we obtained a fairly high probability of paternity exclusion and succeeded in determining the fathers of offspring. However, we found significant differences between the observed and calculated probabilities in most of the troops. These differences were ascribed to a situation whereby the Hardy-Weinberg equilibrium had not been attained in the troops and/or the numbers of variable loci were too small. In the wild troops of Japanese, crab-eating, and toque macaques, the means ofP were 0.2274 (0.0192–0.5017), 0.4635 (0.1676–0.7151), and 0.7382 (0.6266–0.7954), respectively. We also estimated the number of loci needed to determine the fathers of offspring with a probability of 0.8 assuming that ten males were present in the troop. The estimated number was about 13.5 times, 5 times and 1.8 times the number of loci examined on average in the troops of Japanese, crab-eating and toque macaques, respectively. This means that determination of most of the fathers of offspring in wild troops of these macaques, even of toque macaques which have a rather high probability of paternity exclusion, is difficult so long as we employ only electrophoretic techniques.     

Alpha-1-antitrypsin types and pulmonary disease among employees at a sulphite pulp factory in northern Sweden

Alpha-1-antitrypsin (alpha 1-AT) phenotypes and serum levels were measured in 518 employees at a sulphite pulp factory. There were 439 men and 79 women with the mean age of 42 years (range 18-65 years). Mean time of employment at the factory was 17.5 years and 216 (42%) individuals had been employed for more than 20 years. Chronic bronchitis was present in 47 (9.1%) individuals. alpha 1-AT rare types (MZ, MS, MF) were present in 12.8% of the individuals with chronic bronchitis compared to 8.4% in employees with no respiratory symptoms, the difference being not statistically significant. Individuals with chronic bronchitis and rare types were evenly distributed with regard to work place at the factory. Serum levels of alpha 1-AT were somewhat higher in smokers compared to non-smokers, but the difference was not statistically significant. Exposure to SO2 and chlorine did not seem to affect the serum levels of alpha 1-AT in M type individuals. In the present study, individuals heterozygous for alpha 1-AT deficiency phenotypes (MZ, MS, MF) did not seem to have an increased rate of chronic bronchitis. However, the rate of chronic bronchitis in factory employees was significantly increased compared to that among non-employees in the surrounding community. This increase appears to be due to a higher rate of smoking and to occupational exposure (SO2 and chlorine) among the sulphite pulp factory workers.     

Comparison of the chemical, physical, and survival properties of normal and Z-variant alpha-1-antitrypsins.

A procedure has been developed for the purification of Z-type alpha-1-antitrypsin (alpha-1-AT) which is rapid, gentle, and results in good yields. From 4 units (750 ml) of fresh human plasma, obtained from two individuals possessing the Pizz phenotype, 53 mg of pure Z-type alpha-1-AT was obtained. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis, both in the presence and absence of sodium dodecyl sulfate, and by analytical ultracentrifugation. When compared to pure alpha-1-AT from plasma of individuals possessing the normal PiMM phenotype, the two proteins were indistinguishable with respect to amino acid composition, sedimentation coefficient (s20w of 3.33 for both M and Z), molecular weight (51,000 by sodium dodecyl sulfate gel electrophoresis and 47,000 by sedimentation equilibrium for both M and Z), and trypsin-combining ratio (0.91 for Z and 0.99 for M). The only difference which was observed between the variant forms of alpha-1-AT was in the carbohydrate composition. The Z-type alpha1-AT contains between 20 and 25% less carbohydrate than the M-type alpha-1-AT. Specifically, the Z-type alpha-1-AT is deficient in 1 glucosamine residue, 3 neutral sugar residues (1 mannose and 2 galactose), and 2 sialic acid residues. Although the Z-variant is deficient in sialic acid, its survival time in the serum of a rabbit was not significantly different from that of M-type alpha-1-AT.     

Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.     

Genetic polymorphism of the vitamin D-binding protein (DBP) in crab-eating macaques (Macaca fascicularis)

Vitamin D-binding protein (DBP) of crab-eating macaques (Macaca fascicularis) was examined by means of three electrophoretic methods. DBP phenotypes were observed to be one or two bands in each method. All of DBP molecular variants could be detected by the simultaneous typing with these three methods. Family analysis suggested that DBP variants followed the mode of autosomal codominant inheritance. A total of 17 phenotypes governed by at least 11 alleles were observed in the populations of Malaysia, Indonesia, and the Philippines. The genetic variability was high in Malaysian and Indonesian populations but low in the Philippine population.     

Inactivation of human alpha-1-antitrypsin by a tissue-destructive protease of Legionella pneumophila

Three extracellular proteases produced by Legionella pneumophila during growth in liquid medium were examined for their effects on human alpha-1-antitrypsin (alpha-1-AT). One of these proteases, tissue-destructive protease (TDP) destroyed completely the trypsin-inhibitory capacity of alpha-1-AT at protease: inhibitor molar ratios down to 0.002:1. After inactivation by TDP, the Mr of alpha-1-AT was reduced by 5000 in SDS-PAGE. This suggested that inactivation entailed only limited cleavage.     

Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity

Alpha-1 giardin is an immunodominant protein in the intestinal protozoan parasite Giardia lamblia. The Triage((R)) parasite panel, used to detect copro-antigens in stool from giardiasis patients, reacts with an epitope between amino acids 160 and 200 in alpha-1 giardin. This region of the protein is also highly immunogenic during human infections. Alpha-1 giardin is related to annexins and like many other annexins it was shown to be plasma membrane associated. Immunoelectron and immunofluorescence microscopy revealed that some alpha-1 giardin are displayed on the surface of recently excysted cells. Recombinant alpha-1 giardin displayed a Ca(2+)-dependent binding to glycosaminoglycans (GAGs), in particular heparan sulphate, a common GAG in the intestinal tract. Recombinant alpha-1 giardin bound to thin sections of human small intestine, a binding which could be inhibited by adding increasing concentrations of sulphated sugars. A surface associated trypsin activated Giardia lectin (taglin) has been suggested to be important for G. lamblia attachment. In this study we show that a monoclonal antibody that inhibits taglin recognises alpha-1 and alpha-2 giardin. Thus, alpha-1 giardin is a highly immunoreactive GAG-binding protein, which may play a key role in the parasite-host interaction. Our results further show a conserved function of annexins from lower to higher eukaryotes.     

Isolation, chemical, and physical properties of alpha-1-antitrypsin.

A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.     

Analysis of immune response: comparison of immunoblots after isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytoplasmic protein extract from Brucella

Analysis of the immune response towards the facultative intracellular bacterium, Brucella melitensis, was studied by immunoblotting after either isoelectric focusing (IEF) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cytoplasmic extract (CPE) of Brucella melitensis was used as antigen to analyse the response in 17 sera from naturally infected goats. CPE analysed by IEF exhibited 25 proteins within the pH range of 4.35 to 6. Immunoblotting revealed most of the stained bands around pH 4.5-5.4. CPE analysed by SDS-PAGE showed more than 20 silver stained proteins in the molecular range of 16-18 kDa to 70 kDa but immunoblotting revealed only 1 to 6 bands according to the sera tested. Because proteins are preserved in their native state with IEF, in contrast to SDS-PAGE treatment, this technique may be best suited for analysis of the overall response to natural infection.     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Summary. Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed.
Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

Genetic polymorphism of alpha 1-antitrypsin in green monkeys studied by isoelectric focusing and family analysis

24 variants of alpha 1-antitrypsin (alpha 1-AT) were recognized in sera of 120 wild and capture-born African green monkeys by isoelectrofocusing in Ampholine PAG-plates (pH 4-6.5) and western blotting with antihuman alpha 1-AT serum. All variants had much more cathodal position than human alpha 1-AT and revealed very high microheterogeneity which was slightly different from the observed in human alpha 1-AT. The alpha 1-AT banding pattern allowed to postulate existence of 10 codominant alleles in the Pi locus of African green monkeys. The reality of 8 alleles was proved by family analysis which included 45 monkey birth cases. Two other alleles were absent in the parents available. Thus, alpha 1-AT is the most polymorphic among the known serum proteins of African green monkeys. The latter can be useful for molecular systematics of these primates.     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed. Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

The use of proteomics in the discovery of serum biomarkers from patients with severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) is a new infectious disease with a global impact. Understanding its pathogenesis and developing specific diagnostic methods for its early diagnosis are crucial for the effective management and control of this disease. By using proteomic technology, truncated forms of alpha(1)-antitrypsin (TF-alpha(1)-AT) were found to increase significantly and consistently in sera of SARS patients compared to control subjects. The result showed a sensitivity of 100% for SARS patients and a specificity of 92.8% for controls. Furthermore, the levels of these proteins significantly correlated with certain clinico-pathological parameters. The dramatic increase in TF-alpha(1)-AT may be the result of degradation of alpha(1)-AT. As alpha(1)-AT plays an important role in the protection of lung function, its degradation may be an important factor in the pathogenesis of SARS. These findings indicate that increased TF-alpha(1)-AT may be therapeutically relevant, and may also be a useful biological marker for the diagnosis of SARS.