Objective:To analyze the sequencing results, protein structure model, and impact of mutations on the dynamic stability of glycosyltransferase (GTA) in a case with Aw26 blood group subtype.Methods:ABO phenotype was determined by serological testing (anti-A, anti-B, anti-H, and reverse typing). Potential variant of the ABO gene was identified by Sanger sequencing, and the haploid sequence of the variant site was analyzed by TOPOT-A cloning. Molecular models of the GTA was generated by PyMol, and 100-ns molecular dynamics (MD) was simulated with GROMACS software to assess the conformational stability using root mean square deviation (RMSD), radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen bonding, and binding free energy.Results:Serological assays confirmed the proband as an Aw subtype, whose genotype was identified as ABO*Aw.26/ABO*O.01.02 with variants including p. Pro156Leu, p. Arg176His and p. Pro354ArgfsTer23. Haploid sequencing validated the results of direct sequencing. Molecular modeling showed that the p. Arg176His variant could reduce water-mediated hydrogen bonds from six (wild-type) to one (variant). MD simulation revealed the wild type system could achieve equilibrium within 10 ns (mean RMSD ≈ 0.30 nm), whilst the mutant system required 50 ns to equilibrate and exhibited greater fluctuation (mean RMSD ≈ 0.40 nm). Root mean square fluctuation (RMSF) analysis confirmed significantly increased flexibility in the mutant′s N-terminal loop (residues 63-76). The mutant Rg displayed an expansion-contraction transition within 0 ~ 40 ns, and its SASA value has increased. The number of hydrogen bonds and binding energy of the mutant had decreased (wild-type: 5 to 8, binding energy: -11.53 kcal/mol; mutant: 2 to 5, binding energy: -8.52 kcal/mol). Conclusion:An Aw26 subtype was identified. The p. Arg176His and p. Pro354Argfs*23p variants could synergistically compromise the structural stability of GTA and its substrate binding capacity by disrupting the hydrogen-bond network, increasing local flexibility, and reducing the overall conformational stability.

OBJECTIVE: To analyze the sequencing results, protein structure model, and impact of mutations on the dynamic stability of glycosyltransferase (GTA) in a case with Aw26 blood group subtype. METHODS: ABO phenotype was determined by serological testing (anti-A, anti-B, anti-H, and reverse typing). Potential variant of the ABO gene was identified by Sanger sequencing, and the haploid sequence of the variant site was analyzed by TOPOT-A cloning. Molecular models of the GTA was generated by PyMol, and 100-ns molecular dynamics (MD) was simulated with GROMACS software to assess the conformational stability using root mean square deviation (RMSD), radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen bonding, and binding free energy. RESULTS: Serological assays confirmed the proband as an Aw subtype, whose genotype was identified as ABO*Aw.26/ABO*O.01.02 with variants including p.Pro156Leu, p.Arg176His and p.Pro354ArgfsTer23. Haploid sequencing validated the results of direct sequencing. Molecular modeling showed that the p.Arg176His variant could reduce water-mediated hydrogen bonds from six (wild-type) to one (variant). MD simulation revealed the wild type system could achieve equilibrium within 10 ns (mean RMSD ≈ 0.30 nm), whilst the mutant system required 50 ns to equilibrate and exhibited greater fluctuation (mean RMSD ≈ 0.40 nm). Root mean square fluctuation (RMSF) analysis confirmed significantly increased flexibility in the mutant's N-terminal loop (residues 63-76). The mutant Rg displayed an expansion-contraction transition within 0 ~ 40 ns, and its SASA value has increased. The number of hydrogen bonds and binding energy of the mutant had decreased (wild-type: 5 to 8, binding energy: -11.53 kcal/mol; mutant: 2 to 5, binding energy:-8.52 kcal/mol). CONCLUSION An Aw26 subtype was identified. The p.Arg176His and p.Pro354Argfs*23p variants could synergistically compromise the structural stability of GTA and its substrate binding capacity by disrupting the hydrogen-bond network, increasing local flexibility, and reducing the overall conformational stability.
ABO Blood-Group System/chemistry* ; Humans ; Molecular Dynamics Simulation ; Models, Molecular ; Mutation ; Genotype ; Protein Conformation ; Glycosyltransferases/chemistry* ; Male

Objective:To analyze the sequencing results, protein structure model, and impact of mutations on the dynamic stability of glycosyltransferase (GTA) in a case with Aw26 blood group subtype.Methods:ABO phenotype was determined by serological testing (anti-A, anti-B, anti-H, and reverse typing). Potential variant of the ABO gene was identified by Sanger sequencing, and the haploid sequence of the variant site was analyzed by TOPOT-A cloning. Molecular models of the GTA was generated by PyMol, and 100-ns molecular dynamics (MD) was simulated with GROMACS software to assess the conformational stability using root mean square deviation (RMSD), radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen bonding, and binding free energy.Results:Serological assays confirmed the proband as an Aw subtype, whose genotype was identified as ABO*Aw.26/ABO*O.01.02 with variants including p. Pro156Leu, p. Arg176His and p. Pro354ArgfsTer23. Haploid sequencing validated the results of direct sequencing. Molecular modeling showed that the p. Arg176His variant could reduce water-mediated hydrogen bonds from six (wild-type) to one (variant). MD simulation revealed the wild type system could achieve equilibrium within 10 ns (mean RMSD ≈ 0.30 nm), whilst the mutant system required 50 ns to equilibrate and exhibited greater fluctuation (mean RMSD ≈ 0.40 nm). Root mean square fluctuation (RMSF) analysis confirmed significantly increased flexibility in the mutant′s N-terminal loop (residues 63-76). The mutant Rg displayed an expansion-contraction transition within 0 ~ 40 ns, and its SASA value has increased. The number of hydrogen bonds and binding energy of the mutant had decreased (wild-type: 5 to 8, binding energy: -11.53 kcal/mol; mutant: 2 to 5, binding energy: -8.52 kcal/mol). Conclusion:An Aw26 subtype was identified. The p. Arg176His and p. Pro354Argfs*23p variants could synergistically compromise the structural stability of GTA and its substrate binding capacity by disrupting the hydrogen-bond network, increasing local flexibility, and reducing the overall conformational stability.

Objective:To analyze the genetic sequences of two patients with a rare Ael blood subgroup.Methods:Two female patients undergoing treatment respectively for adenomyoma of the uterus and gastritis at the Second Affiliated Hospital, Yuying Children′s Hospital of Wenzhou Medical University in June 2019 and September 2020 were selected as the study subjects. Their Ael subtypes were identified with a saline tube agglutination assay and absorption-emission assay. Sequence of the ABO gene Ael subtypes was determined by the Sanger method. The impact of genetic variants on the structural stability of N-acetylgalactosaminyl transferase (GTA) was analyzed with PyMOL software by constructing a structure predicted model. Results:Both patients were determined as Ael blood subgroup. Sequencing result of patient 1 was ABO* O.01.02/ ABO* O.01.02, which has resulted in a p. Thr88Profs*31 amino acid substitution. The sequencing result of patient 2 was ABO* Ael.06/ ABO* O.01.02, in which c. 425C>T and c. 467C>T variants in exon 7 have led to p. Met142Thr and p. Pro156Leu substitutions. Prediction of the protein model speculated that the p. Met142Thr not only can change the binding of GTA protein with water molecules, but also the local hydrogen bond network of GTA, which may lead to decreased enzymatic activity. By contrast, the p. Pro156Leu variant has trivial effect on the structural stability of GTA. Conclusion:The molecular structure of Ael subtypes can be diverse. The genotypes of the two patients have been respectively determined as ABO* O.01.02/ ABO* O.01.02 with a G261 deletion and ABO* Ael.06/ ABO* O.01.02.