Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342.

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.     

Construction and expression of alpha 1-proteinase inhibitor mutants and the effects of these mutations on secretion of the variant inhibitors

Human alpha 1-proteinase inhibitor (A1Pi) deficiency, associated with the Z variant A1Pi gene, results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of replacement of Glu342 with Lys. To investigate the effect of the amino acid occupying position 342 on secretion of A1Pi, we have used oligonucleotide-directed mutagenesis of A1Pi cDNA to randomly change the codon specifying this amino acid. Since replacement of Glu342 by Lys leads to a change in the predicted secondary structure for this protein, we also tested the possibility that defective secretion of A1PiZ is the result of this type of alteration. For this purpose, site-directed mutagenesis was used to produce sequences encoding A1Pi retaining Glu342 but predicted to have A1PiZ type secondary structure. The effects of 10 different amino acids occupying position 342 on the secretion of A1Pi were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of medium from transiently transfected COS cells. Results of these studies show that secretion of A1Pi is most efficient when position 342 is occupied by a negatively charged amino acid, efficient but somewhat less so when occupied by a neutral amino acid, and least efficient when a positively charged residue is present. The mutation designed to alter secondary structure had no effect on the secretion of A1Pi. As indicated by immunofluorescence microscopy and mobility of intracellular A1Pi on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lowered secretion is accompanied by accumulation of A1Pi in the endoplasmic reticulum of the transfected cells. These results are compatible with the ideas that secretion of A1Pi is directly influenced by the amino acid occupying position 342, that a positively charged amino acid in this position is especially detrimental to secretion of this protein, and that the rate-limiting step in the secretion of the altered forms is transport from endoplasmic reticulum to Golgi.     

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.     

Alpha 1-antitrypsin deficiency—A defect in secretion

A naturally occurring point mutation in the human alpha 1-antitrypsin gene leads to the synthesis of a variant of the protein which is poorly secreted from hepatocytes. This Z; mutation codes for a glutamic acid to lysine substitution at residue 342 in the polypeptide chain. The mutant protein is correctly translocated into the lumen of the endoplasmic reticulum and core glycosylated but inefficiently transported beyond the ER compartment. Experiments using Xenopus oocytes as a surrogate secretory cell show that abberant secretion of the variant is not confined to hepatocytes and glycosylation of the polypeptide is not obligatory for the block in secretion. Site-directed mutagenesis can be used to examine the effect of natural mutations on protein structure and the relationship between structure and intraceltular transport.     

Alpha(1)-antitrypsin deficiency,liver disease and emphysema

alpha(1)-Antitrypsin is a member of the serine proteinase inhibitor (serpin) superfamily and a potent inhibitor of neutrophil elastase. The most important deficiency variant of alpha(1)-antitrypsin arises from the Z mutation (Glu342Lys). This mutation perturbs the protein's tertiary structure to promote a precise, sequential intermolecular linkage that results in polymer formation. These polymers accumulate within the endoplasmic reticulum of the hepatocyte forming inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and adult hepatocellular carcinoma. The resultant secretory defect leads to plasma deficiency of alpha(1)-antitrypsin. This exposes lung tissue to uncontrolled proteolytic attack from neutrophil elastase, culminating in alveolar destruction. Thus, the Z alpha(1)-antitrypsin homozygote is predisposed to developing early onset basal, panacinar emphysema. In this review, we summarise the current understanding of the pathobiology of alpha(1)-antitrypsin deficiency and the associated liver cirrhosis and emphysema. We show how this knowledge has led to the development of novel therapeutic approaches to treat this condition.     

A frameshift mutation results in a truncated alpha 1-antitrypsin that is retained within the rough endoplasmic reticulum

The major physiological role of the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) is to protect elastic fibers in the lung from excessive hydrolysis by neutrophil elastase. Genetic deficiency of alpha 1-AT predisposes individuals toward the development of emphysema. We have cloned and characterized a mutant alpha 1-AT gene from an individual exhibiting a total absence of immunoreactive alpha 1-AT in serum. Nucleotide sequence analysis of this "null" allele has demonstrated a TC dinucleotide deletion within the codon for Leu318 in exon IV. This frame-shift mutation results in the generation of a premature termination codon at residue 334, which is upstream of the active inhibitory site. To determine the biochemical basis of the null phenotype, the mutant and normal genes were transferred into mouse hepatoma cells for expression analysis. Pulse-chase experiments demonstrated that the mutant gene is expressed into a truncated protein of 45 kDa, which is retained within the rough endoplasmic reticulum. The complete lack of secretion of the truncated protein is consistent with the absence of immunoreactive alpha 1-AT in the patient's serum. In addition, a G to A transition was identified in exon II of the mutant gene, changing the codon for Arg101 to His101. Finally, an A to C transversion was identified in exon V changing the codon for Glu376 to Asp376. Since the latter conservative amino acid substitution has previously been identified in the common PiM2 variant, the frame-shift mutation might have occurred on a PiM2 background chromosome. Using the birthplace of this index case, this mutant alpha 1-AT allele has been designated "nullHong Kong."     

An alpha 1-antitrypsin variant, Pi B Alhambra (Lys to Asp, Glu to Asp), with rapid anodal electrophoretic mobility.

A variant of human alpha 1-antitrypsin (alpha 1 AT) was found by acid starch gel electrophoresis and by thin-layer electrofocusing. The variant has an anodal migration velocity almost identical to PiB. It is designated as Pi B Alhambra. Pi B Alhambra was purified to homogeneity from a heterozygous PiM1/PiB Alhambra subject. Specific trypsin inhibitory activity and composition of amino acids and carbohydrates were similar to those of normal PiM1. The structural difference between the normal and the variant inhibitors was elucidated by peptide mapping of their tryptic digests. Two amino acid substitutions, Lys to Asp and Glu to Asp, were found. The amino acid substitution, Gly to Asp, has been found in a common PiM2 variant [1]. The Pi B Alhambra variant presumably originated by two steps of mutation: generation of PiM2 from wild type PiM1 by the substitution Gly to Asp, and subsequent generation of Pi B Alhambra from PiM2 by another substitution, Lys to Asp.     

Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins: solution structures of mouse CRYPTDIN-4 and (E15D)-CRYPTDIN-4

alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu(15) residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.     

Intracellular degradation of the transport-impaired human PiZ alpha 1-antitrypsin variant. Biochemical mapping of the degradative event among compartments of the secretory pathway

The naturally occurring PiZ and Pi NullHong Kong variants of the human secretory protein alpha 1-antitrypsin (AAT) are retained within an early compartment of the secretory pathway. Intracellular degradation of these transport-impaired secretory proteins is initiated 30-45 min following their synthesis and translocation into the endoplasmic reticulum (ER). Interestingly, the overall rate of degradation of the retained mutant protein is significantly accelerated when all subcellular compartments are buffered at pH 6. In contrast, degradation is virtually abolished when intravesicular compartments are buffered at pH 8. However, despite this pH sensitivity neither lysosomotrophic amines, leupeptin, or leucine methyl ester have an apparent effect on the intracellular removal of the PiZ variant. The inability of a variety of inhibitors of ER-to-Golgi protein trafficking to hinder the degradative process suggests that degradation of the PiZ variant occurs prior to its delivery to the Golgi complex. To biochemically map the subcellular site of the degradation of the retained mutant protein, a recombinant truncated PiZ variant containing the tetrapeptide KDEL at its carboxyl terminus (a signal for sorting luminal proteins from a post-ER compartment back to the ER) was expressed in cells. Attachment of this ER-recycling signal to the recombinant protein prevented its intracellular degradation. These findings indicate that degradation of the PiZ variant occurs following its export from the ER.     

Identification of a second mutation in the protein-coding sequence of the Z type alpha 1-antitrypsin gene

This study reports the entire nucleotide sequence of the protein coding region sequence of the alpha 1-antitrypsin (alpha 1AT) Z gene, a common form of the alpha 1AT gene associated with serum alpha 1AT deficiency. In addition to Glu342 to Lys342 mutation in exon V which has been previously identified by peptide analysis, another point mutation (GTG to GCG in exon III) in the gene sequence predicts a second amino acid substitution (Val213 to Ala213) in the Z protein. This Val213 to Ala213 mutation was confirmed to be a general finding in Z type alpha 1AT gene by evaluating genomic DNA from 40 Z haplotypes using synthetic oligonucleotide gene probes directed toward the mutated exon III sequences in the Z gene. Furthermore, the exon III Val213 to Ala213 mutation eliminates a BstEII restriction endonuclease site in the alpha 1AT Z gene, allowing rapid identification of this Val213 to Ala213 substitution at the genomic DNA level. Surprisingly, when genomic DNA samples from individuals thought to be homozygous for the M1 gene (the most common alpha 1AT normal haplotype) were evaluated with BstEII, 23% of the M1 haplotypes were BstEII site negative, thus identifying a new form of M1 (i.e. M1(Ala213], likely identical to M1 but with an isoelectric focusing "silent" amino acid substitution (Val213 to Ala213). Although the relative importance of the newly identified exon III Val213 to Ala213 mutation to the pathogenesis of the abnormalities associated with the Z gene is not known, it is likely that M1(Ala213) gene represents a common "normal" polymorphism of the alpha 1AT gene that served as an evolutionary intermediate between the M1(Val213) and Z genes.     

Aggregation of plasma Z type alpha 1-antitrypsin suggests basic defect for the deficiency

The abnormal type of alpha 1-antitrypsin, PI (protease inhibitor) type Z, is associated with inclusion bodies in the liver, which contain non-secreted alpha 1-antitrypsin. Our studies show that Z protein has an inherent tendency to aggregate, even in plasma. Depending upon conditions, from 15 to 70% of the Z protein in plasma was in a high-Mr form, compared with 1.5% of M type alpha 1-antitrypsin. The high-Mr complex in plasma cannot be disaggregated using Triton X detergent or reducing conditions. This increased tendency to aggregate can be explained by the mutation affecting, tertiary structure and salt bridge formation in Z protein. We have observed this same tendency to aggregate for Mmalton alpha 1-antitrypsin, a rarer variant also associated with a plasma deficiency.     

Hypersensitive mousetraps,alpha1-antitrypsin deficiency and dementia

Alpha(1)-antitrypsin functions as a "mousetrap" to inhibit its target proteinase, neutrophil elastase. The common severe Z deficiency variant (Glu(342)-->Lys) destabilizes the mousetrap to allow a sequential protein-protein interaction between the reactive-centre loop of one molecule and beta-sheet A of another. These loop-sheet polymers accumulate within hepatocytes to form inclusion bodies that are associated with juvenile cirrhosis and hepatocellular carcinoma. The lack of circulating protein predisposes the Z alpha(1)-antitrypsin homozygote to emphysema. Loop-sheet polymerization is now recognized to underlie deficiency variants of other members of the serine proteinase inhibitor (serpin) superfamily, i.e. antithrombin, C1 esterase inhibitor and alpha(1)-antichymotrypsin, which are associated with thrombosis, angio-oedema and emphysema respectively. Moreover, we have shown recently that the same process in a neuron-specific protein, neuroserpin, underlies a novel inclusion-body dementia, known as familial encephalopathy with neuroserpin inclusion bodies. Our understanding of the structural basis of polymerization has allowed the development of strategies to prevent the aberrant protein-protein interaction in vitro. This must now be achieved in vivo if we are to treat the associated clinical syndromes.     

Expression of PiM-and PiZ-mutated forms of the human alpha 1-antitrypsin gene in transfected monkey COS1 cells

The PiZ mutation of the gene coding for alpha 1-antitrypsin results in a serum deficiency of this protein leading to early onset emphysema and liver disease. The PiZ gene has a Z-specific point mutation in exon V together with a point mutation in exon III which is also present in some normal (PiM) individuals. There has thus far been no system to study the effects of PiZ point mutations in tissue culture. We constructed plasmids containing alpha 1-antitrypsin cDNA synthetically altered at either exon III or exon V mutation sites and linked to simian virus 40 promoter sequences. Such constructs with the exon V mutation were transfected into monkey COS1 cells followed by analysis of expression of alpha 1-antitrypsin gene products. COS1 cells normally synthesize virtually no alpha 1-antitrypsin mRNA or protein. alpha 1-Antitrypsin mRNA is transcribed at high levels in cells transfected with either M or Z plasmids. Immunologic staining of COS1 cells within 48 h of transfection localizes alpha 1-antitrypsin protein to specific regions of the cytoplasm. This extranuclear localization is also observed with human HepG2 hepatoma cells, which synthesize alpha 1-antitrypsin at high levels, and with human SK-Hep1 hepatoma cells transfected with an M plasmid. The cloned synthetically altered alpha 1-antitrypsin genes provide a system for dissecting contributions of distinct point mutations to the pathological effects of the PiZ protein.     

Contribution of surface salt bridges to protein stability

The role of surface salt bridges in protein stabilization has been a source of controversy. Here we present the NMR structure of a hyperthermophilic rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges by double mutant cycles. This analysis shows that the surface side chain to side chain salt bridge between Lys 6 and Glu 49 does not stabilize PFRD-XC4. The main chain to side chain salt bridge between the N-terminus and Glu 14 was, however, found to stabilize PFRD-XC4 by 1. 5 kcal mol(-)(1). The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.     

The Conserved Residue Arg46 in the N-Terminal Heptad Repeat Domain of HIV-1 gp41 Is Critical for Viral Fusion and Entry

During the process of HIV-1 fusion with the target cell, the N-terminal heptad repeat (NHR) of gp41 interacts with the C-terminal heptad repeat (CHR) to form fusogenic six-helix bundle (6-HB) core. We previously identified a crucial residue for 6-HB formation and virus entry - Lys63 (K63) in the C-terminal region of NHR (aa 54-70), which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121) in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46), in the N-terminal region of NHR (aa 35-53), which forms a hydrogen bond with a polar residue, Asn43 (N43), in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137), in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A) or the negatively charged residue Glu (R46E) resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB's stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A) or Arg (E137R) also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.     

The effect of amino acid substitutions at position 342 on the secretion of human alpha 1-antitrypsin from Xenopus oocytes

A glutamic acid to lysine change in the Z variant of human alpha 1-antitrypsin is associated with a failure to secrete the protein from synthesising cells. The block in export of the protein may be caused either by the loss of an acidic residue or the introduction of a basic one at this point in the polypeptide chain. Site-directed mutagenesis has been used to construct novel alpha 1-antitrypsin mutants which show that the side chain interactions from Glu-342 are not obligatory for protein export and it is rather the introduction of a basic residue at this point which produces the intracellular accumulation of the protein.     

Alpha-1 antitrypsin gene polymorphism in Chronic Obstructive Pulmonary Disease (COPD)

Alpha-1-antitrypsin (AAT) plays an important role in the pathogenesis of emphysema, the pathological lesion underlying the majority of the manifestations of Chronic Obstructive Pulmonary Disease (COPD). In this study we tested the hypothesis that common AAT polymorphisms influence the risk of developing COPDs. We investigated PiM1 (Ala213Val), PiM2 (Arg101His), PiM3 (Glu376Asp), PiS (Glu264Val) and PiZ (Glu342Lys) SERPINA1 alleles in 100 COPD patients and 200 healthy controls. No significant differences were observed in allele frequencies between COPD patients and controls, neither did haplotype analysis show significant differences between the two groups. A cross-sectional study revealed no significant relationship between common SERPINA1 polymorphisms (PiM1, PiM2, PiM3) and the emphysematous type of COPD. In addition, FEV(1) annual decline, determined during a two-year follow up period, revealed no difference among carriers of the tested polymorphisms.     

Structural difference between the normal PiM1 and the common PiM2 variant of human alpha 1-antitrypsin.

The common PiM2 variant of human alpha 1-antitrypsin (alpha 1-AT) which can be distinguished from the wild type PiM1 by isoelectric focusing (IEF) in a narrow pH gradient, was purified to homogeneity from plasma of a homozygous PiM2/PiM2 subject. The specific trypsin inhibitory activity and the amino acid and carbohydrate composition of the normal PiM1 and the variant PiM2 are very similar. The structural difference between the normal and the variant inhibitors was elucidated by peptide mapping of their tryptic digests. An amino acid substitution of glutamic acid in the normal inhibitor by aspartic acid in the variant inhibitor was found. The same amino acid substitution was found in PiMN, which was presumed to be identical to PiM2 based on their IEF patterns.     

Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.