Gonzalez et al. Respiratory Research (2022) 23:339 https://doi.org/10.1186/s12931-022-02271-8 RESEARCH Open Access New variants of alpha‑1‑antitrypsin: structural simulations and clinical expression Angel Gonzalez1†, Irene Belmonte2†, Alexa Nuñez3, Georgina Farago3, Miriam Barrecheguren3, Mònica Pons4, Gerard Orriols2, Pablo Gabriel‑Medina2, Francisco Rodríguez‑Frías2,5,6,7, Marc Miravitlles3,8* and Cristina Esquinas3,9 Abstract Background: Alpha-1 antitrypsin deficiency (AATD) is characterized by reduced serum levels of the AAT protein and predisposes to liver and lung disease. The characterization at structural level of novel pathogenic SERPINA1 mutants coding for circulating AAT could provide novel insights into the mechanisms of AAT misfolding. The present study aimed to provide a practical framework for the identification and analysis of new AAT mutations, combining structural simulations and clinical data. Methods: We analysed a total of five mutations (four not previously described) in a total of six subjects presenting moderate to severe AATD: Gly95Alafs*18, Val210Glu, Asn247Ser, Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile. Clinical data, genotyping and phenotyping assays, structural mapping, and conformational characterization through molecular dynamic (MD) simulations were developed and combined. Results: Newly discovered AAT missense variants were localized both on the interaction surface and the hydropho‑ bic core of the protein. Distribution of mutations across the structure revealed Val210Glu at the solvent exposed s4C strand and close to the “Gate” region. Asn247Ser was located on the accessible surface, which is important for glycan attachment. On the other hand, Asp341His, Leu383Phe were mapped close to the “breach” and “shutter” regions. MD analysis revealed the reshaping of local interactions around the investigated substitutions that have varying effects on AAT conformational flexibility, hydrophobic packing, and electronic surface properties. The most severe struc‑ tural changes were observed in the double- and triple-mutant (Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile) molecular models. The two carriers presented impaired lung function. Conclusions: The results characterize five variants, four of them previously unknown, of the SERPINA1 gene, which define new alleles contributing to the deficiency of AAT. Rare variants might be more frequent than expected, and therefore, in discordant cases, standardized screening of the S and Z alleles needs complementation with gene sequencing and structural approaches. The utility of computational modelling for providing supporting evidence of the pathogenicity of rare single nucleotide variations is discussed. † Angel Gonzalez and Irene Belmonte contributed equally and should be considered first authors *Correspondence: marcm@separ.es 3 Pneumology Department, Hospital Universitari Vall d’Hebron, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, P. Vall d’Hebron 119‑129, 08035 Barcelona, Spain Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑ mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gonzalez et al. Respiratory Research (2022) 23:339 Page 2 of 13 Keywords: Alpha-1 antitrypsin deficiency, SERPINA1 novel variants, Structural mapping, Molecular dynamic simulations Introduction polymers [17–19]. While the discovery of rare variants A known genetic risk factor for chronic obstructive pul- is continually increasing, especially in individuals with monary disease (COPD) is severe alpha-1antitrypsin lower AAT concentrations, most remain of unknown deficiency (AATD), a hereditary co-dominant autosomal clinical significance [20, 21]. disorder [1]. AATD is characterized by reduced serum In vitro characterization to define the pathogenic- levels of the alpha-1 antitrypsin (AAT) protein and pre- ity of all AAT mutations identified is unlikely to be per- disposes to liver and lung disease [2]. AAT is synthesized formed in some diagnostic laboratories [22]. However, at high levels by hepatocytes, and at lower concentrations computational modelling has shown to be a useful tool by intestinal epithelial cells, neutrophils, lung epithelial to characterize the potential deleterious effects of these cells and alveolar macrophages [3]. It is an important not yet characterized variants and to provide a more pre- acute phase protein and the major serine protease inhibi- cise diagnosis. In this regard, the combination of experi- tor in human serum, in which it shows strong affinity to mental and computational techniques has proven useful neutrophil elastase. The main site of AAT activity is in the to study the influence of amino acid substitutions on the lung, protecting the fragile connective tissue of the lower structure and function of mutated proteins through sev- respiratory tract from the uncontrolled proteolysis trig- eral examples [23]. Thus, the characterization at struc- gered by neutrophils during inflammation. Importantly, tural level of novel pathogenic SERPINA1 mutations AAT has also emerged as a multifunctional protein com- coding for circulating AAT could provide novel insight bining its inhibitory properties with immunomodulatory into the mechanisms of AAT misfolding in liver and lung and anti-inflammatory activities [4]. disease, with important implications for molecular diag- AAT is encoded by the highly polymorphic SERPINA1 nosis and therapeutic development [24, 25]. gene, which is located on chromosome 14q32.1 and com- The present study aimed to provide a practical frame- prises four coding exons (II–V), three untranslated exons work for the identification and analysis of new AAT (Ia–Ic) and six introns [5]. The native AAT comprises 418 mutations in a clinical context, combining clinical data, amino acids of which the initial 24 amino acids are a sig- genotyping and phenotyping assays, structural mapping nal peptide and are subsequently removed. This results in and molecular dynamic (MD) simulations. Using this a 394 amino acid mature protein of 52 kDa which is nor- approach, we describe six novel uncharacterized AAT mally glycosylated at Asn46, Asn83 and Asn247 residues variants and delineate their potential role in the confor- [6, 7]. The tertiary structure of AAT consists of 3 β-sheets mational dynamics of this protease inhibitor. (A–C), 9 α-helices (A–I) and a reactive central loop (RCL), which is an important region for initial interaction Methods with the target protease [8]. This protein is characterized Subjects by a metastable native fold that is particularly sensitive to All the patients were recruited from the outpatient clinic mutations [9, 10]. Single amino acid substitutions at spe- of the respiratory department of the Vall d’Hebron Uni- cific positions of SERPINA1 could favor the transition to versity Hospital (Barcelona, Spain) [26]. They were iden- inactive or polymeric dysfunctional folds linked to dis- tified following the diagnostic algorithm for AATD used ease [11, 12]. in our laboratory, which comprises three strategies: Over 150 SERPINA1 mutations have been identified to serum AAT quantification, protein phenotyping and gen- date, and of these, 60 are implicated in the pathogenesis otyping [27]. of disease [13, 14]. The normal and non-disease-causing Analysis by sequencing the entire encoding region of allele of AAT is called Pi*M and is present in more than the SERPINA1 gene is undertaken in patients with dis- 90% of the population. The most frequent deficient alleles crepancies between AAT concentration and phenotype are Pi*S (Glu264Val) and Pi*Z (Glu342Lys) [15, 16]. In pattern or allele-specific genotype. We identified six particular, carriers of two Z alleles (Pi*ZZ) have strongly carriers of five variants (four not previously described): reduced circulating levels of AAT (up to 10–20% of nor- Gly95Alafs*18 in patient 1, Val210Glu in patients 2 and mal, which is 1.3–2 g/L). Other rare alleles of AAT are 3, Asn247Ser in patient 4, Pi*S + Asp341His in patient 5, also associated with reduced serum concentrations of and Pi*S + Leu383Phe + Lys394Ile in patient 6. the AAT protein, such as the Pi*Mmalton (Phe52del) The study was approved by the Ethics Committee of and Pi*Siiyama (Ser53Phe), which also form intracellular the Vall d’Hebron Hospital (Barcelona, Spain) with the Gonzalez et al. Respiratory Research (2022) 23:339 Page 3 of 13 number PR(AG) 157/2016, and signed informed consent monoclonal antibody has been used to capture CP was obtained from all subjects for participation in the (HM2289 Alpha-1 Antitrypsin mAb 2C1), coating plates study. at 2 ug/ml overnight. The next day, the plates were incu- bated 2 h with 300ul/well with blocking solution. Stand- Clinical and molecular analysis ards and samples were diluted in blocking buffer, added Clinical data and laboratory testing to the plate, and incubated 2 h at room temperature. Clinical characteristics and lung function data (forced 3C11 monoclonal antibody labelled with horserad- expiratory volume in the 1st second (FEV1), forced ish peroxidase has been used to detect bound polymers vital capacity (FVC) and the FEV1/FVC ratio) were col- (HM2358 Alpha-1 antitrypsin Human mAb3C11-HRP). lected. Liver function was determined by analysis of liver After 80 min of incubation, 50ul/well of 3,3’,5,5’-tetra- enzymes: aspartate-aminotransferase (AST), alanine- methylbenzidine (TMB) substrate solution were added aminotransferase (ALT), and gamma-glutamyl trans- (T0440, MERCK). AAT polymers (μg/mL) were deter- ferase (GGT). In addition, an enhanced liver fibrosis mined by interpolation of absorbance values on the (ELF) test was performed. The ELF test (Siemens Health- standard curve. Then, AAT polymers (%) were deter- care Diagnostics, Vienna, Austria) is a fibrosis biomarker mined in all samples. which consists of three components: type III procollagen peptide, hyaluronic acid, and tissue inhibitor of metallo- Database search and computational modelling proteinase-1 [28]. Allele frequency We investigated the presence and population frequency AAT serum concentrations and phenotyping of the newly identified AAT variants in the Short Genetic Quantitative measurement of AAT levels was determined Variations database (Single Nucleotide Polymorphism by immune nephelometry, and phenotyping was per- Database—dbSNP) (https://​www.​ncbi.​nlm.​nih.​gov/​ formed with the isoelectric focusing (IEF) assay in serum snp/) and the Genome Aggregation Database (https://​ samples in a pH gradient of 4.2–4.9 using the Hydragel gnomad.​broad​insti​tute.​org/). 18 A1AT Isofocusing kit (Sebia). Comparison with sera of known phenotypes allowed determining whether the Structural modelling of AAT variants amino acid change affected AAT protein migration. Molecular models corresponding to missense AAT variants Val210Glu, Asn247Ser, Pi*S + Asp341His and Genotyping of new alpha1‑antitrypsin variants Pi*S + Leu383Fhe + Lys394Ile were constructed with the For molecular studies, DNA was isolated from whole Mutagenesis Wizard module of PyMOL 2.0.5 using the peripheral blood using a commercial extraction kit X-Ray crystal structures of the intact native wild-type (Qiamp DNA blood, Qiagen). Sanger automated human AAT at 1.8 Å and 2.0 Å resolution (PDBids: 1QLP, sequencing of all coding exons (II-V) of the AAT​ gene 3NE4) [29, 30]. Residues N-terminal to position 23 of the was performed as described previously [15]. New vari- mature chain were disordered and lacked electron den- ants were identified comparing obtained sequences sity in the template structures and were not modelled. with the reference SERPINA1 sequence (NG_008290.1, Assignment of ionization states and hydrogens at physi- NM_000295.4). ological pH was conducted with the Protonate 3D appli- cation [31]. Molecular models were refined by energy Cloning of the new alleles minimization using the Amber12EHT forcefield to a gra- The genetic background of the Val210Glu, dient of 0.1 kcal/mol/Å within the Molecular Operating Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile Environment (MOE) software suite (version 2020.09). was determined by cloning experiments. Polymer- ase chain reaction (PCR) products of exon II (Val- Molecular dynamic (MD) simulations 210Glu) and exons III to V (Pi*S + Asp341His and Molecular dynamics simulations in explicit sol- Pi*S + Leu383Phe + Lys394Ile) from the respective vent of wild type AAT and Val210Glu, Asn247Ser, patients’ DNA were cloned into a Zero Blunt TOPO vec- Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile tor, transformed into One Shot Top10 chemically compe- mutant structures were conducted with GROMACS [32]. tent Escherichia coli (Invitrogen) and sequenced by the Molecular systems were solvated with a simple-point usual protocol. charge water model in a rhombic dodecahedron box with monoatomic Na + and Cl− ions (0.2 M) and equilibrated Circulating polymers of AAT​ with positional restraints on the protein backbone atoms To quantify circulating polymers (CP), a sandwich using the Amber ff99SB-ILDN force field [33]. After ELISA has been performed using serum samples. 2C1 relaxation, 1 microsecond of unbiased MD trajectories Gonzalez et al. Respiratory Research (2022) 23:339 Page 4 of 13 were produced at a constant temperature of 300 K and diagnosis and normal lung function after referral to the time step of 2 femtosecond. Analysis of MD trajectories respiratory unit. was performed using GROMACS tools. Patient 3 was a 23-year-old non-smoker female with a serum AAT concentration of 66.8 mg/dl. In this case, Electrostatic surface calculation the digestive unit requested AAT quantification due to Electrostatic potentials of wild-type and mutant struc- a likely liver disease and a mild hyper-transaminasemia tures were obtained from the Adaptive Poisson-Boltz- (AST: 20 U/L; ALT: 40 U/L; GGT: 55 U/L). The patient mann Solver (APBS) implemented in PyMol v2.5.0. showed normal lung function. (Schrödinger, LLC). Briefly, the APBS method uses the Patient 4 carried the Asn247Ser mutation and was a finite element method to numerically solve the nonlinear 58-year-old male former smoker of 10 pack-years with Poisson-Boltzmann equation, representing the electro- a serum AAT concentration of 98.4 mg/dL. He was static interactions between molecules in aqueous envi- referred from the digestive unit because of mild hepatic ronments [34]. The solvent dielectric constant was 80, steatosis and hepatomegaly with normal liver enzymes and the protein dielectric was approximated as 2.0. The and no evidence of lung disease. isoelectric potential of − 5kT and 5kT was mapped onto Patient 5 carried the double mutation Pi*S + Asp341His the solvent accessible surface (1.4 Å sphere radii) of all and was a 45-year-old male former smoker with serum the molecular models. AAT levels of 19.8 mg/dL. He was initially diagnosed as a PI*ZZ phenotype at the age of 21. The patient suffered Results from bronchiectasis and recurrent pneumonias with mild Identification of new alpha1‑antitrypsin variants airflow obstruction (FEV1/FVC: 63% and FEV1(%): 81% The entire coding sequence of the SERPINA1 gene predicted). No signs of liver disease were detected. Due showed five novel variants (four not previously described) to the very low levels of AAT and the lack of genotyping carried by six patients with AATD: four missense and at the time of diagnosis 24 years previously, the samples one non-sense (Table 1). The clinical characteristics and of the patient were tested again. the final genotype of the patients carrying the new vari- Patient 6 carrying the S + Leu383Phe + Lys394Ile vari- ants are summarized in Table 2. ant was a 64-year-old male never smoker with AAT lev- Patient 1 with the Gly95Alafs*18 variant in heterozy- els of 69.8 mg/dL. The individual showed alcoholic liver gosity with the normal M allele was a 49-year-old female disease and the liver unit requested AATD analysis. The former smoker, with an accumulated exposure of 13 patient presented severe airflow obstruction (FEV1/FVC: pack-years, and reduced AAT serum concentrations of 52% and FEV1 (%): 45.6% predicted). 78.1 mg/dL (Table 1). She was referred from the respira- tory unit showing normal lung and liver function. Protein migration phenotype Two unrelated patients carried the Val210Glu variant Figure 1 shows the phenotype determined by IEF assay in in heterozygosity. Patient 2 was 40-year-old non-smoker serum samples. Patient 1 carrying the null variant (Gly- male. The liver unit carried out the AAT request due to 95Alafs*18) showed a PI*MM phenotype, corresponding mild hepatic steatosis showing elevated hepatic enzymes to the PI*M variant carried by the patient. This variant (AST: 72 U/L; ALT: 108 U/L; GGT: 98 U/L). The subject seems to produce complete degradation of the truncated showed AAT serum levels of 76.3 mg/dL at the time of protein, a mechanism usually observed in null alleles. Table 1 Molecular description and final genotype of the new non well-described variants Patient ID Aa change in mature protein * Aa change in precursor Sequence position and Exon Genotype (NP_001121175.1) change (NM_00025.5) Patient 1 Gly95fs Gly119fs 355/356, GGC > GCT​ II G95Afs18* – M Patient 2 Val210Glu Val234Glu 701, GTG > GAG​ III V210E + H16H – M Patient 3 Val210Glu Val234Glu 701, GTG > GAG​ III V210E + H16H – M Patient 4 Asn247Ser Asn271Ser 812, AAT > AGT​ III N247S – M Patient 5 S + Asp341His S + Asp365His 1093, GAC > CAC​ V S + D341H – Z Patient 6 S + Leu383Phe + Lys394Ile S + Leu407Phe + Lys418Ile 1219, CTC > T TC V S + L407F + K418I – M 1253, AAA > ATA​ Aa amino acid * All variants are reported without the 24 amino acid corresponding to the signal peptide Table 2 Clinical characteristics of patients carrying the new non well-described variants Gonzalez et al. Respiratory Research Patient ID Sex Age [AAT] (mg/dL) AAT AAT polymers% Genotype Smoking status Pulmonary/hepatic status Lung function (%) Liver enzymes ELF polymers (IU/L) (ug/ml) FVC FEV1 FEV1/FVC AST ALT GGT​ Patient 1 Female 49 78.1 3.65 0.47 G95Afs18* – M Former smoker – No evidence of liver 93 93 81 27 16 – 8.25 disease (2022) 23:339 Patient 2 Male 40 76.3 2.52 0.30 V210E + H16H – M Never smoker – Mild hepatic steatosis 93 101 87 72 108 98 8.67 – Elevated hepatic enzymes – No evidence of lung disease Patient 3 Female 23 66.8 3.44 0.52 V210E + H16H – M Never smoker – Elevated hepatic enzymes 99 102 107 20 40 55 8.81 – No evidence of lung disease Patient 4 Male 58 98.4 5.20 0.53 N247S – M Former smoker – Mild hepatic steatosis and 121 124 81 32 43 35 8.89 hepatomegaly – Normal hepatic enzymes – No evidence of lung disease Patient 5 Male 45 19.8 4.08 2.06 S + D341H – Z Former smoker – Mild COPD, Bronchiectasis 98.6 81 62.9 16 18 17 7.62 and recurrent pneumonia – No evidence of liver disease Patient 6 Male 64 69.8 2.44 0,35 S + L407F + K418I – M Never smoker – Severe COPD 65.8 45.6 52 28 47 106 NA Alcoholic liver disease AAT​alpha-1 antitrypsin, AST aspartate-aminotransferase, ALT alanine-aminotransferase, GGT​gamma-glutamyl transferase, ELF enhanced liver fibrosis, NA data not available, FVC forced vital capacity, FEV1 forced expiratory volume in the first second, COPD chronic obstructive pulmonary disease Normal ranges in serum: AAT: 116–2.00 mg/dL; AST: female 10 to 35 IU/L and male 10 to 50 IU/L; ALT: female 7 to 35 IU/L and male 8 to 50 IU/L; GGT: female 6–38 IU/L and male 9–55 IU/L; ELF score < 7.7: none to mild liver fibrosis, >  = 7.7 to < 9.8: moderate and >  = 9.8 sever Page 5 of 13 Gonzalez et al. Respiratory Research (2022) 23:339 Page 6 of 13 Fig. 1 Isoelectric focusing electrophoresis of serum samples from subjects carrying the newly described variants and individuals carrying the most frequent normal and deficient genotypes. Serum from the V210E/M genotype showed an additional band (marked with asterisk) not present in the MM control. As expected, G95fs/M displayed a MM phenotype. Variants carrying a new mutation with S allele in cis configuration (S + L407F + K418I/M, S + D341H /Z) had null phenotype behavior, showing a MM and ZZ phenotype, respectively. The N247S/ genotype presented a multiband phenotype, different from the pattern in MM, MS, MZ, SZ or ZZ. Numbers at the right and left represent the migration of major (4 and 6) and minor (7 and 8) bands from the MM and ZZ genotypes Patient 2 with the Val210Glu variant displayed an IEF initial diagnosis of this patient. The combination of the pattern similar to that of PI*MM. However, an extra band S allele with Asp341His seems to produce a null pheno- was observed between the M7 and M8 bands. The two type, in which only the PI*Z allele, in trans-configuration bands after M8 that migrate more cathodally are often with the new variant, is observed. observed in the PI*M allele. Regrettably, a plasma sample Finally, Patient 6 (Pi*S + Leu383Phe + Lys394Ile) was from patient 3 carrying the Val210Glu variant was not phenotyped as PI*MM. Similar to patient 5, the combi- available for IEF. nation of the S variant in cis-configuration with the new Patient 4 (Asn247Ser) showed a multiband pattern variants had features of a null phenotype behavior, in with bands associated with PI*M and PI*Z alleles, among which only the M allele was observed. others. This migration pattern may be explained by the alteration of protein glycosylation due to the omission Genomic location and allele frequency of the N-glycosylation site at position 247 of the mature Patient 1; Gly95Alafs*18 protein (see below). Molecular study of the SERPINA1 gene revealed a G Patient 5 carrying the Pi*S + Asp341His variant showed base deletion in exon II at nucleotide position 355 or 356 a PI*ZZ phenotype pattern, which could explain the (NM_000295.5, c.355/356delG), resulting in a frameshift Gonzalez et al. Respiratory Research (2022) 23:339 Page 7 of 13 that gives rise to a new stop codon downstream the dele- 1219 (NM_000295.5, c.1219 C > T) and an T > A trans- tion site at amino acid position 112, leading to a pre- version at position 1253 (NM_000295.5, c.1253 A > T) mature termination of protein translation. This variant leading to leucine to phenylalanine (Leu383Phe) and occurred in heterozygosity with the presence of one nor- lysine to isoleucine (Lys394Ile) changes in the mature mal M allele and has not been previously reported. The protein, respectively. Background analysis confirmed dbSNP entry rs749295615 indicates three different amino that both mutations were in cis-configuration with the acid substitutions at this location. S variant and in trans-configuration with the M allele. Variant Leu383Phe was not found in genomic databases, Patients 2–3; Val210Glu but entries rs767700105 and rs1011476837 documented DNA sequencing revealed an A > T transversion in exon one synonymous and one missense (Leu383Arg) substi- III (NM_000295.5, c.701 T > A) in heterozygous state tution at this position. On the other, hand, the mutation with the M variant. This change produces a substitution Lys394Ile is described with a low frequency (< 1/10,000) of valine to glutamic acid (p.Val210Glu) in the mature in dbSNP (rs1566747286). protein. Moreover, both patients carried a synonymous mutation in exon II (c.120 T > C, p.His16His), and cloning Circulating polymer concentrations in the different AATD experiments using a sample from patient 2 demonstrated genotypes that this change was in cis-configuration with the Val- Serum samples from all patients were analyzed. Patients 210Glu variant. This mutation was previously detected 4 and 5 presented higher CP concentrations. However, in heterozygous state with the Z variant in a 75-year-old the concentration had to be normalized taking into male who had atelectasis and COPD with serum AAT account the AAT levels of each patient. Considering the levels of 42 mg/dL and was named M1pierre-bénite [35]. percentage of total AAT, patient 5 (variant S + D341H – The Val210Glu change has been reported with a low fre- Z) presented the highest CP concentration (2.06%). The quency (< 1/10,000) in dbSNP (rs746197812). other variants presented between 0.3% and 0.5% of CP concentrations (Table 2). Patient 4; Asn247Ser This amino acid mutation is characterized by an A to G Structural mapping and molecular dynamic (MD) transition in exon III (NM_000295.5, c.812 A > G), result- simulations ing in an asparagine to serine substitution (p.Asn247Ser). AAT is a conformationally flexible protein with several This single nucleotide variation (SNV) was found in regions involved in a large structural rearrangement nec- heterozygous form with M and has not been previously essary for its protease inhibitory function. Therefore, this reported. However, the accession rs755851961 docu- protein is susceptible to mutations that cause misfolding, mented a change of asparagine to aspartic at the same inactivation or intracellular accumulation of pathogenic position. Asn247 is one of the three N-glycosylation sites polymers [36–38]. identified in AAT [6]. Newly discovered AAT missense variants were local- ized both on the interaction surface and the hydrophobic Patient 5; Pi*S + Asp341His core of the protein (Figs. 2 and 3). Distribution of muta- Gene sequencing demonstrated that this patient was a tions across the structure revealed Val210Glu at the sol- carrier of three alleles: PI*S, PI*Z, and the new variant. vent exposed s4C strand and close to the “gate” region. This new allele consisted of a G > C transversion in exon Asn247Ser was located on the accessible surface, which V (NM_000295.5, c.1093 G > C), changing aspartic acid is important for glycan attachment. On the other hand, for histidine at codon 341 (p.Asp341His) in the mature Asp341His, Leu383Phe and Lys394Ile were mapped close protein. Cloning experiments showed that this new vari- to the “breach” and “shutter” regions (Fig. 2). ant was in cis-configuration with the S allele and in trans- The MD simulation method was applied for analyz- configuration with the Z variant. The change Asp341His ing the structural effect of the mutational changes. The is found with a low frequency (< 1/10,000) in dbSNP small deviation of the protein backbone atoms during 1 (rs143370956). This position seems to be highly polymor- microsecond of simulation indicates that all amino acid phic as rs864622046 and rs201774333 indicate additional replacements were tolerated, with an overall root mean substitutions at this site. square deviation < 2 Å in all molecular structures (see Table 3 and Additional file 1: Fig. S1). Most fluctuating Patient 6; Pi*S + Leu383Phe + Lys394Ile regions correspond to the reactive center loop (RCL, Exon sequencing of the SERPINA1 gene showed two sin- residues 344–362 of the mature protein) and other non- gle base substitutions at the end of exon V. This new allele structured regions in accordance with the weak electron consisted of a C > T transition at nucleotide position density observed in the X-Ray structures [39] (see Fig. 3). Gonzalez et al. Respiratory Research (2022) 23:339 Page 8 of 13 Fig. 2 Structural mapping and analysis of the AAT variants identified. The central image is a cartoon representation of the molecular coordinates of human AAT1, with the serpin RCL shown in black, alpha-helices in red and beta-sheet regions A, B and C in yellow, cyan, and green, respectively. Mutated residues are highlighted in vdW surface (light violet = new variants; deep purple = Pi*Z and Pi*S). Structural comparisons by superposition of the AAT wild type and mutant molecular coordinates appear highlighted in the square insets (a to f). In d, reference (GlcNAc)2 glycan from (PDBid: 7API) is displayed in sticks. Residues (in sticks) are numbered according to the processed mature form of the protein MD analysis revealed the reshaping of local interac- The most severe structural changes were observed in tions around the investigated substitutions that have the double- and triple-mutant (Pi*S + Asp341His and varying effects on AAT conformational flexibility, Pi*S + Leu383Phe + Lys394Ile) molecular models. In hydrophobic packing, and electronic surface properties both cases, the change of the negatively charged Glu (see Figs. 3 and 4). Substitution of the hydrophobic Val by the hydrophobic Val at position 264 disrupted a salt by the negatively charged Glu at position 210 promotes bridge interaction with Lys387 and modified the relative new electrostatic interactions of the s3C-s4C β-hairpin orientation of helix hG (Figs. 2c and 3). In addition, the with the C-terminal (Fig. 2a). This change increases substitution of the negatively charged Asp341 by a posi- the negative charge on the “gate” region regarding the tive His modified the molecular interactions and electro- wild-type protein (Fig. 4a, b). On the other hand, the static landscape proximal to the “breach” region, which asparagine by serine mutation at the surface accessible is essential for RCL insertion upon proteolytic cleavage N-glycosylation motif (Asn247-x-Thr) showed a mod- (Figs. 2b, 4a, d). erate impact on the structural dynamics of the protein Finally, the changes at position 383 reshaped the hydro- (Fig. 3). However, this position is crucial in the glyco- phobic core and affect the electrostatic interactions sylation profile of the protein and the loss of the gly- towards the C-terminal region of the mutated protein. can attachment site could affect the plasma half-life and The Leu383 form part of a highly conserved sequence of stability of the protein (Fig. 2d) [40]. 9 amino acids (residues 383 through 391) in the central core of the protein, chain s5B (Fig. 2f ). As observed in Gonzalez et al. Respiratory Research (2022) 23:339 Page 9 of 13 Fig. 3 Molecular dynamics (MD) trajectory analysis of human AAT1 and identified mutants. A Root mean square fluctuation (RMSF) of Cα atom coordinates throughout 1 μs of MD for the wild-type (WT) human AAT and mutants V210E, N247S, Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile molecular models (color legend). B Cartoon representation of the WT and mutant’s average structures colored according to the fluctuation of atomic coordinates during MD production. Mutated residues at each monomer are displayed as yellow vdW spheres the MD simulation experiments, the bulky aromatic Phe residue in this position induced interactions with other hydrophobic/aromatic residues (e.g., Leu103, Leu112, Phe190 and Phe384) modifying the hydrophobic pocket Table 3 Root mean square deviation (RMSD, in Å) of topology and the flexibility in the nearby hD-s2A region protein backbone atomic coordinate displacement during (Figs. 3, 4a, e). On the other hand, the functional role of 1 μs MD simulations for the Wild-Type (WT) human AAT and mutants V210E, N247S, Pi*S + Asp341His and Lys394 is uncertain due to the great flexibility observed in Pi*S + Leu383Phe + Lys394Ile water-solvated molecular systems. the MD experiments at the C-terminus residues (Figs. 2e, Average (Avg.), Standard deviation (Sd.) and minimum and 3). This residue is not conserved among different spe- maximum values (min. max.) cies and its location and interactions are not preserved in time, suggesting a minor functional role [41]. MD simulation structural Avg. Sd. (Å) min. (Å) max. (Å) models ­RMSDbkbn (Å) Discussion The repertoire of AATD causing mutations has expanded WT-AAT​ 1.838 0.175 0.562 2.275 over the last years, and to date, it is acknowledged that V210E 1.850 0.217 0.536 2.382 along with the major disease risk genotypes, ZZ and SZ, N247S 1.754 0.178 0.396 2.260 there are many others resulting from different combina- Pi*S + Asp341His 1.996 0.258 0.477 2.733 tions with rare deficient alleles. Indeed, previous studies Pi*S + Leu383Phe + Lys394Ile 1.936 0.297 1.141 2.909 have shown that rare mutations can be detected in up to Gonzalez et al. Respiratory Research (2022) 23:339 Page 10 of 13 Fig. 4 Surface representation of the wild-type (WT) human AAT and mutants V210E, N247S, Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile molecular structures (a and e). Molecular solvent accessible surfaces are colored by the electrostatic potential calculated using the APBS program with nonlinear Poisson-Boltzmann equation and contoured at ± 5 kT/e (negatively and positively charged surface areas in red and blue, respectively). Black arrows indicate the location of the substitutions in each mutant 17% of clinically cases and, as for Z and S alleles, several changes, except for the premature stop codon at posi- low-frequency pathogenic variants have been segregating tion 95 of the mature protein, were studied at a struc- in human populations for hundreds or thousands of years tural level. The AAT missense mutations investigated [42, 43]. were localized at interaction surfaces as well as at the The emergence of new genomic technologies has hydrophobic core of the protein. Three variants, such as boosted the identification of rare genetic variants with the Val210Glu, modify the electrostatic surface of the high accuracy and at a very reasonable cost, opening protein. Compared with Val, the Glu residue displays a the way to the precision medicine healthcare approach. negative charge, which reduces the extent of hydrophobic However, the major bottleneck for the effective use of this interactions by the s3C-s4C connecting turn close to the information in clinical practice lies in accurate interpre- “gate” region. This observation agrees with other com- tation of the functional consequences of the newly iden- putational modelling studies that indicated an elevated tified variants. Consequently, in this study we developed pathogenic potential for this change [21]. a method that combines biochemical, molecular biology A newly identified variant was also located on the and computational structural analysis for the interpreta- surface of the protein. Human plasma AAT is normally tion of individuals carrying mutations on the SERPINA1 fully glycosylated at three asparagine residues (46, 83, gene. and 247) [6]. Thus, the Asn247Ser substitution inter- We analysed a total of five mutations (four not previ- feres with the glycosylation signal, which is readily seen ously described) in six subjects who presented moderate by IEF experiment, showing the observed band pat- to severe AATD: Gly95Alafs*18, Val210Glu, Asn247Ser, tern to be altered. This change is distanced form the Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile. All RCL and loss of the glycan groups has not been shown Gonzalez et al. Respiratory Research (2022) 23:339 Page 11 of 13 to alter the metastable state of AAT. However, it has Owing to the considerable diversity of AAT-deficient been shown that some mutant variants which are gly- variants, the present study aimed to provide a practical cosylated differently compared to normal AAT may be framework for the identification and analysis of new AAT proteolytically degraded preferentially in the endoplas- mutations. This approach successfully combines the use mic reticulum [44, 45]. Moreover, changes in glycan of clinical data derived from standard laboratory tests, pattern could affect the half-life of the protein, aggrega- phenotyping, genotyping assays, and supporting compu- tion kinetics and the immune modulatory properties of tational modelling tools to delineate the impact of new AAT [7]. The detection of CP was very low and similar SNV in the population. In this way, the determination of to the concentrations observed in normal individuals the functional impact of these rare deficient phenotypes PiMM. will help to establish the requirements for the clinical Two other variants appeared in cis-configuration with management of serpinopathies. Additionally, the identi- the S variant and had a null-phenotype behaviour. The fication of these novel mutations should be incorporated patient carrying the Pi*S + Asp341His presented severe in the diagnostic tools for the screening of AATD [47]. AATD and he also presented the highest concentration of CP comparing with the other variants being simi- Conclusions lar to the concentration observed in patients PiSZ [19]. Our results characterized five variants of the SERPINA1 His genotype was a combination of three variants: PI*S gene, four of them previously unkonwn, which define in cis-configuration with the new mutation Asp341His new alleles contributing to the deficiency of AAT. Rare (allele 1) and PI*Z (allele 2). This patient was a former variants might be more frequent than expected, and smoker and had been diagnosed with bronchiectasis with therefore, in discordant cases, standardized screening mildly impaired lung function and recurrent pneumo- of the S and Z alleles’ should be complemented by gene nia. According to AAT serum levels and the phenotype sequencing and structural analysis. The use of conven- obtained, the combination of the S allele with Asp341His tional MD simulations, albeit useful to describe the led to a new variant with features of null-phenotype changes on local interactions produced by mutations, is behaviour. These results are in accordance with those limited by the time-scale and impossibility to overcome obtained by Matamala et al. who described PI*S-plus large energy barriers. Thus, the application of enhancing alleles which modify the properties of the Pi*S allele in sampling methods (e.g. metadynamics) should be con- terms of higher intracellular retention [18]. In contrast to sidered for the study of the conformational dynamics of null alleles, which cause a complete abrogation of AAT AAT in future developments. Nevertheless, we believe production, these ’PI*S-plus’ described by the authors, the computational analysis presented here, even with produced the AAT protein, but this protein is strongly its limitations, provide valuable information that can be retained intracellularly and for that reason is not detected used in hypothesis generation of the pathogenicity of rare in the serum of carriers. According to the MD analy- SNVs. All of the SNVs were previously recorded in the sis, Asp341His modify the structural packing and sur- database of the National Center for Biotechnology Infor- face electrostatic properties of the protein around the mation of single nucleotide polymorphisms (dbSNP) “breach” region. Similar to what occurs in the Pi*S and and/or in the literature [19, 20, 22–26]. Pi*Z variants, these changes would lead to aberrant con- formations, disrupting the folding pathway of the pro- tein [12]. The last variant causing a null-phenotype was Abbreviations AAT​: Alpha-1 antitrypsin; AATD: Alpha-1 antitrypsin deficiency; ALAT: Alanine- the Pi*S + Leu383Phe + Lys394Ile. The carrier of these aminotransferase alanine-aminotransferase; AST: Aspartate-aminotransferase; changes presented low AAT serum levels and low CP APBS: Adaptive Poisson-Boltzmann Solver; COPD: Chronic obstructive pulmo‑ concentrations, similar to the concentrations observed nary disease; CP: Circulating polymers; CRP: C-reactive protein; dbSNP: Single Nucleotide Polymorphism Database; ELF: Enhanced liver fibrosis; FEV1: Forced in PIMM individuals. This patient also presented severe expiratory volume in one second; FVC: Forced vital capacity; IEF: Isoelectric COPD, alcoholic liver disease and a clear MM IEF pat- focusing; PI*: Protease inhibitor; GGT​: Gamma-glutamyl transferase; MD: tern, indicating that the combination of the S variant Molecular dynamics; MOE: Molecular Operating Environment; RCL: Reactive central loop; SPC: Simple-point charge. with the two novel mutations modified the secretion of the protein. Both changes were mapped on the C-ter- Supplementary Information minal region, Lys394 being the last amino acid of the The online version contains supplementary material available at https://​doi.​ protein. In our computational experiments, this mutant org/​10.​1186/​s12931-​022-​02271-8. modified the environment of the “hydrophobic pocket” and increased the dynamics of the RCL. Thus, changes in Additional file 1: Figure S1. Stability of the AAT molecular dynam‑ this preserved region may have significant effects on the ics trajectories. a. The root mean square deviation (RMSD) of backbone protein atoms during the 1 μs of MD simulations for the wild type (WT) secretion of the protein [46]. Gonzalez et al. Respiratory Research (2022) 23:339 Page 12 of 13 Received: 29 March 2022 Accepted: 1 December 2022 and mutant structures (color legend). b. Radius of gyration (RG) of protein coordinates as a function of time for each simulated structure. Acknowledgements Mònica Pons is the recipient of a Juan Rodes contract in the 2022 Strate‑ gic Action Health Call from the Instituto de Salud Carlos III for the years References 2019–2020. Alexa Núñez is the recipient of a Rio Hortega contract in the 2019 1. Menga G, Fernandez Acquier M, Echazarreta AL, Sorroche PB, Lorenzon Strategic Action Health Call from the Instituto de Salud Carlos III for the years MV, Fernandez ME, Saez MS, Grupo de estudio DA. Prevalence of alpha-1 2020–2022. antitrypsin deficiency in COPD patients in Argentina. The DAAT.AR Study. Arch Bronconeumol (Engl Ed) 2020, 56:571–577. Author contributions 2. Stolk J, Seersholm N, Kalsheker N. Alpha1-antitrypsin deficiency: cur‑ The study concept and design: AG, AN, IB, MB, FR-F, MM, CE. The acquisition of rent perspective on research, diagnosis, and management. Int J Chron the data: AG, IB, AN, EM, MB, GF, EL, GO, MP, ER. Analysis or interpretation of the Obstruct Pulmon Dis. 2006;1:151–60. data: CE, AG, IB, AN, FR-F, JG, GO, PG-M, MM. The drafting of the manuscript: 3. Greene CM, Marciniak SJ, Teckman J, Ferrarotti I, Brantly ML, Lomas DA, AN, MM. Critical revision and approval for submission: IB, EM, MB, GF, EL, MP, Stoller JK, McElvaney NG. Alpha1-Antitrypsin deficiency. Nat Rev Dis Prim‑ FR-F, GO, PG-M, ER, JG, CE. All authors read and approved the final manuscript. ers. 2016;2:16051. 4. Jonigk D, Al-Omari M, Maegel L, Muller M, Izykowski N, Hong J, Hong K, Funding Kim SH, Dorsch M, Mahadeva R, et al. Anti-inflammatory and immu‑ This study was supported by funding from Grifols to the Catalan Center for nomodulatory properties of alpha1-antitrypsin without inhibition of Research in Alpha-1 antitrypsin deficiency of the Vall d’Hebron Research Insti‑ elastase. Proc Natl Acad Sci U S A. 2013;110:15007–12. tute (VHIR) in the Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain 5. 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