The serum sialyltransferase activity in alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin phenotypes Pi M and Z, purified by the thiol-disulfide exchange procedure, were desialylated by treatment with neuraminidase covalently coupled to Sepharose and used as acceptors of sialic acid in an assay system for serum sialic acid transferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity. Both asialoantitrypsins were equally effective as acceptors in contrast to native Pi Z antitrypsin which did not accept any sialic acid. Serum sialyltransferase activity was determined in 38 adult alpha 1-antitrypsin deficient individuals (Pi Z, MZ, FZ, SZ) with normal liver function and was found to be of the same magnitude as the activity in normal individuals (Pi M). Equal activities were also found in 5 Pi Z patients with cirrhosis of the liver. The results strongly argue against the concept that sialyltransferase deficiency provides the molecular basis for alpha 1-antitrypsin deficiency.     

Demonstration of alpha 1-antitrypsin by immunofluorescence on paraffin-embedded hepatic and pancreatic tissue.

Studies were performed in an attempt to improve current immunohistological techniques for the demonstration of alpha 1-antitrypsin (A1AT) in formalin-fixed paraffin-embedded tissues. The unwanted fluorescence (UF) commonly occurring in such procedures was found to be effectively eliminated by immunoadsorption of A1AT antisera with human serum lacking A1AT (Pi-null phenotype) coupled in solid phase to glutaraldehyde-activated aminohexyl-Sepharose 4B. Specificity of the antisera for A1AT was established by subsequent solid phase immunoadsorption against normal human serum bound to AH-Sepharose 4B. Using these techniques, immunoreactive A1AT was demonstrated in the cytoplasm of hepatocytes in liver biopsies obtained from patients with Z and MZ serum phenotypes, and in the cytoplasm of normal pancreatic islet cells.     

The molecular structure of different polymorphic forms of canine C3 and C4

The subunit composition of different electrophoretic forms of canine C3 and C4 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of reduced immune precipitates. Canine C4 comprises alpha, beta, and gamma chains of approximate molecular weight 90 000–95 000, 72 000, and 33 000, respectively. The molecular weight of the alpha chain of the C4 1 allelic product was approximately 95 000, but 90 000 for the C4 2 and C4 4 allotypes. No differences were observed in the molecular weights of the beta or gamma chains of any canine C4 phenotype tested. Canine C3 appears to be encoded by a single locus. The subunit composition comprises an alpha and beta chain with molecular weights of approximately 106 000 and 71000, respectively. Unlike C4, no differences in the molecular weights of the subunits were observed in different electrophoretic forms of canine C3.     

Distribution of alpha-1-antitrypsin and haptoglobin phenotypes in bladder cancer patients

Frequencies of the alpha 1-antitrypsin (Pi) alleles and haptoglobin phenotypes have been determined in a series of 264 North-German patients with bladder cancer. Compared to a healthy control population, we found a statistically significant decrease of Hp 2-2 phenotype in the patient series. A significant increase of the serum Pi Z allele, as previously shown for patient groups with certain other tumours, could also be confirmed for bladder cancer. Furthermore, a distinct association between a lowered M 3 allele and bladder carcinoma was observed.     

Segregation distortion of the alpha 1-antitrypsin Pi Z allele.

alpha 1-antitrypsin (alpha 1AT) of the Pi type Z is associated with two diseases: pulmonary emphysema and cirrhosis of the liver. We report 23 families with both parents heterozygous for the PiZ allele, characterized from our own analysis and from world literature sources. All families were identified through members expressing disease. From the extended pedigrees, 18 backcross families (parents with Pi types MM and MZ) were identified. Analysis of the backcross families reveals a significant increase in Pi MZ offspring (.73) among families where the male is heterozygous. The distortion is not detected among families where the female is heterozygous. Among the matings where both parents are heterozygous, we found 0.43 Pi ZZ from families where one or more members expressed hepatic cirrhosis, and 0.40 Pi ZZ for total families studied. This contrasts to the 0.25 Pi ZZ expected, but is consistent with the distortion observed in backcross matings. The implications of various statistical approaches are discussed, and we point out why our findings differ from previous reports. We suggest a possible biological explanation residing in the fertilization process.     

Alpha 1-antitrypsin Wbethesda: molecular basis of an unusual alpha 1-antitrypsin deficiency variant

Molecular analysis of alpha 1-antitrypsin (alpha 1AT) Wbethesda revealed that it differs from the normal M1 (Ala213) allele by a single base mutation causing an amino acid substitution Ala336 GCT----Thr ACT. Evaluation of alpha 1AT biosynthesis directed by the Wbethesda allele showed that although Wbethesda alpha 1AT mRNA was translated normally in vitro, transfection of the Wbethesda cDNA into COS-I cells was associated with human alpha 1AT secretion of 50% that of cells transfected with a normal alpha 1AT cDNA. The pattern of alpha 1AT biosynthesis was not intracellular accumulation as observed with the common Z alpha 1AT deficiency allele, but reduced intracellular alpha 1AT, suggesting intracellular degradation of the newly synthesized Wbethesda molecule. Together these observations suggest that in heterozygous combination with a Z or Null alpha 1AT allele, the Wbethesda variant causes "alpha 1AT deficiency", thus classifying it as an alpha 1AT "at risk" allele for emphysema.     

Alpha-1-antitrypsin (PI) polymorphism in France, with special regard to the PI*Z allele.

Alpha-1-antitrypsin (PI) phenotypes were studied in a sample of more than 5,000 individuals from cities throughout France. Special interest was paid to the PI*Z allele whose average frequency, based on the present work plus results from the literature, was 0.0130. This figure was used to estimate the number of PI Z homozygotes in France. In accordance with previous studies, the frequency of the PI*S allele was found to increase towards the southern parts of France.     

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.     

6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.

Conformational diseases such as amyloidosis, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. These diseases are typified by the Z variant of alpha(1)-antitrypsin (E342K), which causes the retention of protein within hepatocytes as inclusion bodies that are associated with neonatal hepatitis and cirrhosis. The inclusion bodies result from the Z mutation perturbing the conformation of the protein, which facilitates a sequential interaction between the reactive center loop of one molecule and beta-sheet A of a second. Therapies to prevent liver disease must block this reactive loop-beta-sheet polymerization without interfering with other proteins of similar tertiary structure. We have used reactive loop peptides to explore the differences between the pathogenic Z and normal M alpha(1)-antitrypsin. The results show that the reactive loop is likely to be partially inserted into beta-sheet A in Z alpha(1)-antitrypsin. This conformational difference from M alpha(1)-antitrypsin was exploited with a 6-mer reactive loop peptide (FLEAIG) that selectively and stably bound Z alpha(1)-antitrypsin. The importance of this finding is that the peptide prevented the polymerization of Z alpha(1)-antitrypsin and did not significantly anneal to other proteins (such as antithrombin, alpha(1)-antichymotrypsin, and plasminogen activator inhibitor-1) with a similar tertiary structure. These findings provide a lead compound for the development of small molecule inhibitors that can be used to treat patients with Z alpha(1)-antitrypsin deficiency. Furthermore they demonstrate how a conformational disease process can be selectively inhibited with a small peptide.     

Properties of isolated human alpha1-antitrypsins of Pi types M, S and Z.

1. alpha1-Antitrypsin contains a single thiol group partly blocked in native plasma and reactive after mild reduction. 2. Human alpha1-antitrypsins of Pi types F, M, S and Z have been isolated with native microheterogeneity using thiol-disulfide (SH-SS) interchange reactions utilizing the reactive thiol group. 3. The pI of the various microheterogeneous fractions are given for protein M. Stepwise desialylation of alpha1-antitrypsin indicates that the charge difference between the major fractions is one sialic acid residue between each. This is further supported by the pI changes obtained on substitution of the single thiol with positively or negatively charged compounds. 4. Desialyation of purified proteins from each Pi type converts the individual microheterogeneous fractions to one major fraction. The pI shift for the variants studied indicate a difference of plus or minus one or two charge units between protein M and the variants. 5. A difference of one sialic acid residue was obtained for proteins M and Z by the thiobarbituric assay, but stepwise removal of sialic acid with neuraminidase revealed almost identical stepwise change of pattern of both proteins indicating the same number of sialic acid residues. 6. Electrofocusing has been used to identify CNBr fragments from proteins M, S and Z. 7. An amino acid substitution has been found to be located in one of the eight CNBr fragments, glutamic acid in protein M is substituted by lysine in protein Z. 8. The average concentration of alpha1-antityprsin in plasma from healthy males was found to be 1.32 g/1.     

The effect of the Glu342Lys mutation in alpha1-antitrypsin on its structure, studied by molecular modelling methods.

The structure of native alpha1-antitrypsin, the most abundant protease inhibitor in human plasma, is characterised primarily by a reactive loop containing the centre of proteinase inhibition, and a beta-sheet composed of five strands. Mobility of the reactive loop is confined as a result of electrostatic interactions between side chains of Glu342 and Lys290, both located at the junction of the reactive loop and the beta structure. The most common mutation in the protein, resulting in its inactivation, is Glu342-->Lys, named the Z mutation. The main goal of this work was to investigate the influence of the Z mutation on the structure of alpha1-antitrypsin. Commonly used molecular modelling methods have been applied in a comparative study of two protein models: the wild type and the Z mutant. The results indicate that the Z mutation introduces local instabilities in the region of the reactive loop. Moreover, even parts of the protein located far apart from the mutation region are affected. The Z mutation causes a relative change in the total energy of about 3%. Relatively small root mean square differences between the optimised structures of the wild type and the Z mutant, together with detailed analysis of 'conformational searching' process, lead to the hypothesis that the Z mutation principally induces a change in the dynamics of alpha1-antitrypsin.     

The effect of neuraminidase on genetic variants of alpha anitrypsin.

Sera of Pi types M, F, S, Z, IM, FM, MS, and MZ were incubated with neuraminidase and the reaction products followed by electrophoresis. The alpha1 antitrypsin components showed a series of changes in mobility as sialic residues were removed. Removal of sialic acid was confirmed by chemical assay. Results of studies with two different electrophoretic systems suggested that the Z type alpha1 antitrypsin has less sialic acid than the M, F, and S types. There was no evidence that other genetic variants have a reduced sialic acid content. The two major bands of alpha1 antitrypsin seen in certain electrophoretic systems may reflect a difference of one sialic acid residue. It is proposed that the Z protein lacks a carbohydrate chain with two terminal sialic acid residues. This carbohydrate deficiency results in lack of secretion of type Z alpha1 antitrypsin from the endoplasmic reticulum, perhaps because of binding to sites specific for the incomplete glycoprotein or because of aggregation of the Z asialo protein. A carbohydrate chain could be prevented from attaching to the Z type either because of a conformational change or because of the replacement of a carbohydrate-binding asparagine residue in the Z protein.     

The N-terminal amino acid sequence from alpha 1-antitrypsin isolated from liver inclusion bodies

The alpha 1-antitrypsin from the liver of a subject with alpha i-antitrypsin deficiency was purified and subjected to automated Edman degradation. The N-terminal amino acid sequence from position 1 to 12 was identical to that in plasma alpha 1-antitrypsin, type Z. This result precludes that the intrahepatic accumulation of Z alpha 1-antitrypsin is due to a defective removal of a signal peptide.     

Molecular multiplicity of nuclease TT1 from Thermus thermophilus HB8

Purified nuclease TT1 from Thermus thermophilus HB8 has multimolecular weight forms, each of which is composed of three different subunits, alpha (10.8 x 10(4)), beta (7.8 x 10(4)), and gamma (4.1 x 10(4)). The molecular weights of this enzyme were estimated by gel filtration, polyacrylamide gel electrophoresis and equilibrium sedimentation. It was found that most of the enzyme has a molecular weight of about 22 x 10(4) being a monomer having the subunit composition of alpha beta gamma. The remaining part of the enzyme has larger molecular weights and is considered to be size-isomers of alpha beta gamma. The alpha-helical content, 5.5--6.5%, and the beta-structure, about 28%, were estimated from the CD spectrum at 4 degrees C.     

Differential effects of flavonols on inactivation of alpha1-antitrypsin induced by hypohalous acids and the myeloperoxidase-hydrogen peroxide-halide system

Alpha1-antitrypsin is well known for its ability to inhibit human neutrophil elastase. Pretreatment of alpha1-antitrypsin with hypohalous acids HOCl and HOBr as well as with the myeloperoxidase-hydrogen peroxide-chloride (or bromide) system inactivated this proteinase. The flavonols rutin, quercetin, myricetin, and kaempferol inhibited the inactivation of alpha1-antitrypsin by HOCl and HOBr with rutin having the most pronounced effect. In contrast, these flavonols did not remove the proteinase inactivation by the myeloperoxidase-hydrogen peroxide-halide system. Taurine did not protect against the inactivation of alpha1-antitrypsin by HOCl, HOBr, or the myeloperoxidase-hydrogen peroxide-halide system, while methionine was efficient in all systems. A close association between myeloperoxidase and alpha1-antitrypsin was revealed by native gel electrophoresis and in-gel peroxidase staining. In addition, alpha1-antitrypsin binds to the myeloperoxidase components transferred after SDS-PAGE on a blotting membrane. With this complex formation, myeloperoxidase overcomes the natural antioxidative protective system of plasma and prevents the inactivation of alpha1-antitrypsin.     

Rare deficiency types of alpha 1-antitrypsin: electrophoretic variation and DNA haplotypes.

A deficiency of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT), is usually associated with the deficiency allele PI*Z. However, other alleles can also produce a deficiency. Some of these rare deficiency alleles produce a low concentration (3%-15% of normal) of alpha 1AT and include Mmalton, Mduarte, Mheerlen, and Mprocida. Null, or nonproducing, alleles are associated with trace amounts (less than 1%) of plasma alpha 1AT. We have identified, using isoelectric focusing, the deficiency alleles in 222 patients (68 children and 154 adults) with alpha 1AT deficiency. In addition to PI*Z, we found low-producing alleles PI*Mmalton and PI*Mcobalt and four null (PI*QO) alleles. On the basis of a population frequency of .0122 for PI*Z, frequencies for other deficiency alleles are 1.1 x 10(-4) for PI*Mmalton, 2.5 x 10(-5) for PI*Mcobalt (which may be the same as that for PI*Mduarte, and 1.4 x 10(-4) for all null alleles combined. Using 12 polymorphic restriction sites with seven different restriction enzymes, we have obtained DNA haplotypes for each of the rare deficiency types. All of the rare deficiency alleles can be distinguished from PI*Z by their DNA haplotype, and most can be distinguished from each other. DNA haplotypes are useful to indicate the presence of new types of null alleles, to identify genetic compounds for rare deficiency alleles, and to identify the original normal allele from which each deficiency allele is derived.     

Sequence data of the rare deficient alpha 1-antitrypsin variant PI Zaugsburg.

Weidinger et al. recognized a rare deficient PI-variant, named PI Zaugsburg (PI Zaug), by using isoelectric focusing with a narrow pH gradient. The serum alpha 1-antitrypsin (alpha 1AT) level determined quantitatively in an individual carrying the phenotype PI M1Zaug revealed a value of 50%-60% of the normal range. The frequency of the deficient PI*Zaug allele is still unknown. Haplotyping the Zaug-affected chromosome, we found a pattern different from the common PI*Z allele described by Cox et al. Therefore, we directly sequenced the coding exons of both genes (M1 and Zaug) after PCR amplification. Zaug sequence data analysis showed the presence of the common PI*Z allele-specific mutation (M1 Glu342 GAG to Z Lys342 AAG) surprisingly occurring in an M2 ancestral gene. This is not consistent with the heretofore common finding, by Nukiwa et al. and others, that this mutation is derived from an M1 (Ala213) background gene. No further mutations were found in the PI Zaug gene.     

Interaction between human pancreatic elastase and plasma protease inhibitors

1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.     

Population genetics of alpha-1-antitrypsin polymorphism in US whites, US blacks and African blacks.

An isoelectric focusing (IEF) procedure in an ultra-narrow pH range, 4.2-4.9, has been utilized to detect alpha 1-antitrypsin or alpha 1-protease inhibitor (PI) allele products in 2 US white and 3 US black populations as well as 1 native African black population. In addition to the 3 common alleles PI*M1, PI*M2 and PI*M3, products of the 4th allele PI*M4 have been identified in US whites at low-level frequency. The presence of the PI*S, PI*Z and PI*I alleles has also been verified in our population samples. While the PI*S allele is present at a polymorphic level in US whites, it is only present sporadically in US blacks and is completely absent in African blacks. The PI*Z allele was not detected in the black populations tested. The PI allele frequency data have been used to calculate white admixture in US blacks.     

Comparative Studies on S-Glycoproteins Purified from Different S-Genotypes in Self-Incompatible BRASSICA Species I. Purification and Chemical Properties

S-glycoproteins, i.e. stigma glycoproteins that are heritable in correlation with S allele in self-incompatible Brassica species, were apparently purified for three S alleles in B. oleracea. From SDS gel electrophoresis, the estimated molecular weight for two of the S-glycoproteins was 57,000. The other S-glycoprotein was considered to be heterogeneous with molecular weights of 60,000 and 65,000. Distinct differences in amino acid content were found; in general, cysteine, methionine and histidine were low, and serine, glutamate, glycine, leucine, arginine and aspartate were high and variable between the S-glycoproteins. Differences in the isoelectric point were mainly attributed to the amino acid composition of each S-glycoprotein.