From structure to function for the characterization of ERAP1 active site in Behçet syndrome. A novel polymorphism associated with known gene variations. (January 2020)

• Record Type: Journal Article
• Authors: Padula, Maria Carmela; Leccese, Pietro; Lascaro, Nancy; Carbone, Teresa; Limongi, Antonina Rita; Radice, Rosa Paola; Padula, Angela Anna; D'Angelo, Salvatore; Martelli, Giuseppe
• Is Part Of: Molecular immunology. Volume 117(2020:Jan.)
• Page Start: 155
• Page End: 159
• Publication Date: 2020-01
• Subjects: BS Behçet syndrome -- CI confidence interval -- Ct cycle threshold -- ERAP1 Endoplasmic reticulum aminopeptidase protein 1 -- GWAS Genome-wide association study -- HC healthy controls -- HLA Human Leukocyte Antigen -- ISG International Study Group -- MHC Major Histocompatibility Complex -- MIQE Quantitative Real-Time PCR Experiments -- OR odds ratio -- PCR Polymerase Chain Reaction -- SNPs single nucleotide polymorphisms -- qPCR Quantitative Real-Time PCR; Behçet syndrome -- ERAP1 -- Genotyping -- Gene expression -- SNP; Immunochemistry -- Periodicals; Molecular biology -- Periodicals; Immunochemistry -- Periodicals; Allergy and Immunology -- Periodicals; Molecular Biology -- Periodicals; Immunochimie -- Périodiques; Biologie moléculaire -- Périodiques; Immunochemistry; Molecular biology; Periodicals; Electronic journals; 571.96
• DOI: 10.1016/j.molimm.2019.11.005
• Languages: English
• ISSNs: 0161-5890

Abstract

Highlights: Genotyping of Italian patients with Behçet syndrome showed a novel ERAP1 polymorphism. The novel p.Phe360Cys SNP is within the Zn-binding region of ERAP1 active site. The novel SNP is predicted to be damaging (maximun score of deleteriousness). The SNP could affect the structurally conserved region for the enzymatic function. The down-regulation of mRNA ERAP1 was recognized in presence of p.Phe360Cys SNP. Abstract: Introduction: ERAP1 has been recently proposed as risk marker of Behçet syndrome (BS). Gene single nucleotide polymorphisms (SNPs) could affect the enzymatic activity and the conserved active site is pivotal for the aminopeptidase function. This study aims to characterize the ERAP1 active site in a cohort of BS patients vs healthy controls (HC) integrating genomics, transcriptomics and bioinformatics approach. Materials and methods: We recruited 109 consecutive Italian BS patients (63M:46 F; mean age: 45.07 ± 12.28 years) and 106 matched HC (55M:51 F; mean age: 42.57 ± 12.29 years). DNA was isolated and amplified using PCR with home made-primer pairs. PCR products were directly sequenced and computational analyses were performed to search active site SNPs (NCBI-BlastN tool), to predict SNPs functional effect (PolyPhen-2 software) and to obtain protein 3D modelling (Protean3D software). In a second phase of analysis, RNA was extracted and reverse transcribed. Quantitative Real-Time PCR (qPCR) was performed to assess ERAP1 mRNA level in presence (target) and in absence (control) of gene polymorphisms. The Fold change was calculated for the relative quantification of gene expression. Results: A novel coding variation (NG_027839.1:g.25637 T > G; NP_057526.3:p.Phe360Cys, HGSV nomenclature) was found in heterozygosity state in 5/109 BS patients (4.59 % of cases) and none of HC. It was recognized in association with rs2287987, rs30187, rs17482078, and rs27044 BS-related polymorphisms for 4 out of 5 patients. All patients carrying the novel SNP were HLA-B*51-positive. The novel SNP was released in GenBank database with MK140632.1 ID. The SNP was predicted to be damaging and resides within the Zn-binding HEXXH(X)18 E region of the active site, changing the structurally conserved region for the amminopeptidase function. In fact, the change in energy (ΔE) score between wild-type and SNP-containing protein showed a less stable protein in presence of p.Cys360 (ΔE:3.584) (Protean3D prediction). Preliminary qPCR results underlined a significant difference in fold change value when target and control values were compared (p < 0.05), suggesting a reduced expression of ERAP1 mRNA in presence of the novel SNP. Conclusions: Our study strengthens the association between ERAP1 and BS. The most significant point was the localization of the novel p.Phe360Cys SNP within the Zn-binding region of protein active site that was predicted to affect its function, causing protein destabilization. Our findings need to be tested in larger genetic studies.