Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency

Objective:Glucose-6-phosphate isomerase(GPI)deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants.This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics,often posing challenges for precise diagnoses using conventional methods.To this end,this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family.Methods:The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis.Novel compound heterozygous variants of the GPI gene,c.174C>A(p.Asn58Lys)and c.1538G>T(p.Trp513Leu),were identified using whole-exome and Sanger sequencing.The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure.Results:By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study,we found that most variants were located in exons 3,4,12,and 18,with a few localized in exons 8,9,and 14.This study identified novel compound heterozygous variants associated with GPI deficiency.These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids.Conclusion:Early family-based sequencing analyses,especially for patients with congenital anemia,can help increase diagnostic accuracy for GPI deficiency,improve child healthcare,and enable genetic counseling.

Protein Disulfide Isomerase and Its Potential Function on Endoplasmic Reticulum Quality Control in Diatom Phaeodactylum tricornutum

PDI is a molecular chaperone and plays an important role in Endoplasmic Reticulum quality control (ERQC).PDI participates in the refolding of the misfolded/unfolded proteins to maintain cellular homeostasis under differentstresses. However, bioinformatic characteristics and potential functions of PDIs in diatom Phaeodactylumtricornutum (Pt) are still unknown so far. Hence, the genome-wide characteristics of PtPDI proteins in P. tricornutumwere first studied via bioinformatic and transcriptomic methods. 42 PtPDI genes were identified from thegenome of P. tricornutum. The motif, protein structure, classification, number of introns, phylogenetic relationship,and the expression level of 42 PtPDI genes under the tunicamycin stress were analyzed. A pair of tandemduplicated genes (PtPDI15 and PtPDI18) was observed in P. tricornutum. The 42 PtPDIs with different genecharacteristics were divided into three independent clades, indicating different evolutional relationships and functionsof these PtPDIs. The 14 up-regulated PtPDI genes under the tunicamycin treatment might have a positiveeffect on the ER quality control of the unfolded/misfolded proteins, while the 7 down-regulated PtPDIs mightnegatively affect the ERQC. The characteristics of all 42 PtPDIs and their proposed working model here providea comprehensive understanding of the PtPDIs gene family. The differential expression of 21 PtPDIs will be usefulfor further functional study in the ERQC.

Protein Disulfide Isomerase A2 Is Correlated with Immune Infiltrates and Is a Novel Prognostic Biomarker in Glioma Patients

Objective Protein disulfide isomerase A2(PDIA2),a member of the protein disulfide isomerase family,plays a key role in the folding of nascent proteins in the endoplasmic reticulum by forming disulfide bonds,together with enzymes such as thiol isomerase,oxidase,and reductase.This study investigated the clinical significance and potential functions of PDIA2 in glioma.Methods The expression of PDIA2 in gliomas was explored using The Cancer Genome Atlas and Gene Expression Omnibus databases.We analyzed the clinical characteristics of glioma patients and the prognostic and diagnostic value of PDIA2 expression.Kaplan-Meier and Cox regression analyses were used to examine the effect of PDIA2 expression on overall survival,progression-free interval,and disease-specific survival.Furthermore,we performed Gene Set Enrichment Analysis and immune infiltration analysis to investigate the functions of PDIA2.PDIA2 mRNA and protein expression was evaluated in cell lines and glioma tissues.Results PDIA2 was expressed at low levels in glioma patients.Kaplan-Meier survival analysis showed that glioma patients with low PDIA2 levels had a worse prognosis than those with high PDIA2 levels.Receiver operating characteristic curve analysis indicated the diagnostic and prognostic ability of PDIA2(area under the curve=0.918).Pathways associated with PD1,PI3K/AKT,cancer immunotherapy via PD1 blockade,Fceri-mediated NF-kB activation,FOXM1,and DNA repair were enriched in glioma patients with low levels of PDIA2.PDIA2 expression levels were negatively correlated with immune cell infiltrate levels.Conclusion PDIA2 levels are significantly downregulated in glioma.PDIA2 expression may be a potential biomarker for the diagnosis and prognosis of glioma patients.

Western blot detection of PMI protein in transgenic rice

Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain （about 0.08 mg）. PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.

Stress protein expression in early phase spinal cord ischemia/reperfusion injury

Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins （n 〉 2） in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initially improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradually decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6-12 hours, showing a characterization of induction-inhibition-induc- tion. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons.

Phosphoglucose isomerase gene expression as a prognostic biomarker of gastric cancer

Objective: Tumor heterogeneity renders identification of suitable biomarkers of gastric cancer(GC)challenging. Here, we aimed to identify prognostic genes of GC using computational analysis.Methods: We first used microarray technology to profile gene expression of GC and paired nontumor tissues from 198 patients. Based on these profiles and patients’ clinical information, we next identified prognostic genes using novel computational approaches. Phosphoglucose isomerase, also known as glucose-6-phosphate isomerase(GPI), which ranked first among 27 candidate genes, was further investigated by a new analytical tool namely enviro-geno-pheno-state(E-GPS) analysis. Suitability of GPI as a prognostic marker, and its relationship with physiological processes such as metabolism, epithelial-mesenchymal transition(EMT), as well as drug sensitivity were evaluated using both our own and independent public datasets.Results: We found that higher expression of GPI in GC correlated with prolonged survival of patients.Particularly, a combination of CDH2 and GPI expression effectively stratified the outcomes of patients with TNM stage Ⅱ/Ⅲ. Down-regulation of GPI in tumor tissues correlated well with depressed glucose metabolism and fatty acid synthesis, as well as enhanced fatty acid oxidation and creatine metabolism, indicating that GPI represents a suitable marker for increased probability of EMT in GC cells.Conclusions: Our findings strongly suggest that GPI acts as a novel biomarker candidate for GC prognosis,allowing greatly enhanced clinical management of GC patients. The potential metabolic rewiring correlated with GPI also provides new insights into studying the relationship between cancer metabolism and patient survival.

The Study of Food-Grade Induced Expression and Enzymatic Properties of L-Arabinose Isomerase from Lactobacillus plantarum WU14 with High D-Tagatose Yield

L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60&degC, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60&degC. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50&degC, pH 7.17 and D-galactose concentration was 0.6 mol/L.

Phosphoglucose Isomerase Deficiency in <i>Escherichia coli</i>K-12 Is Associated with Increased Spontaneous Mutation Rate

Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strain with a deleted pgi gene (Δpgi) and shown that this strain in comparison with the parental strain 1) accumulates higher amount of G6Ph, 2) grows slowly, and 3) exhibits higher spontaneous mutation frequency to rifampicin resistance (Rifr), when grown on high glucose minimal medium. Intriguingly, the spontaneous mutation rate to Rifr was inversely related to the degree of E. coli chromosomal DNA modification with sugar derivatives. We measured higher concentrations of Amadori products, fluorophores (360 nm excitation/440 nm emission) and carboxymethyl residues in the chromosomal DNA of the E. coli parental strain than in DNA of the isogenic Δpgi strain. To explain this apparent paradox we hypothesized that PGI might be implicated in repair of G6Ph-derived lesions in DNA. In favor of our hypothesis, we further demonstrate that protein extract from the E. coli PGI proficient strain but not from the PGI deficient strain catalyzes the release of G6Ph from G6Ph-modified single stranded DNA oligonucleotide and from its hybrid duplex with a complementary peptide nucleic acid.

Molecular Docking of Selective Binding Affinity of Sulfonamide Derivatives as Potential Antimalarial Agents Targeting the Glycolytic Enzymes: GAPDH, Aldolase and TPI

The parasite Plasmodium falciparum is responsible for the major world scourge malaria, a disease that affects 3.3 billion people worldwide. The development of new drugs is critical because of the diminished effectiveness of current antimalarial agents mainly due to parasitic resistance, side effects and cost. Molecular docking was used to explore structural motifs responsible for the interactions between triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase (ALD) from human and Plasmodium cells with 8 novel sufonylamide derivatives. All the ligands modeled, interact with all three enzymes in the micromolar range. The top ligand (sulfaE) shows a 70-fold increase in selective binding to pfTPI compared to hTPI (dissociation constant-KI of 7.83 μM and 0.177 μM for hTPI and pfTPI respectively), on par with antimalarial drug chloroquine.ALD and GAPDH form complexes with similar binding sites, comprising amino acids of similar chemical properties and polarities. Human TPI and pfTPI bind sulfonamide derivatives using two distinct binding sites and residues. Key residues at the dimer interface of pfTPI (VAL44, SER45, TYR48, GLN64, ASN65, VAL78) form a tight pocket with favorable polar contacts. The affinity with TPI is the most specific, stable, and selective suggesting pfTPI is a candidate for development of antimalarial drugs.

Characterization of an algal phosphomannose isomerase gene and its application as a selectable marker for genetic manipulation of tomato

Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells.The existence of such selectable marker genes in genetically modified foods has long been criticized.Plant cells generally exhibit too low an activity of phosphomannose isomerase(PMI)to grow with mannose as a sole carbon source.In this study,we characterized PMI from the green microalga Chlorococcum sp.and assessed its feasibility as a selectable marker for plant biotechnology.Chlorococcum sp.PMI(ChlPMI)was shown to be closely related to higher plants but more distant to bacterial counterparts.Overexpression of ChlPMI in tomato induced callus and shoot formation in media containing mannose(6 g/L)and had an average transformation rate of 3.9%.Based on this transformation system,a polycistronic gene cluster containing crtB,HpBHY,CrBKT and SlLCYB(BBBB)was co-expressed in a different tomato cultivar.Six putative transformants were achieved with a transformation rate of 1.4%,which produced significant amounts of astaxanthin due to the expression of the BBBB genes.Taken together,these findings indicate that we have established an additional tool for plant biotechnology that may be suitable for genetically modifying foods safely.

Using the Phosphomannose Isomerase (PMI) Gene from Saccharomyces cerevisiae for Selection in Rice Transformation

The phosphomannose isomerase （PMI） gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice （Oryza sativa L.） via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. 13- Glucuronidase （GUS） activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.

Evolutionary Relationship of Wheat Protein Disulphide Isomerase (PDI) Gene Promoter Sequence Based on Phylogenetic Analysis

Protein disulphide isomerase (PDI) is an oxidoreductase enzyme abundant in the endoplasmic reticulum (ER). In plants, PDIs have been shown to assist the folding and deposition of seed storage proteins during the biogenesis of protein bodies in the endosperm. Cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv. Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species were reported in our previous publications. Promoter sequences of three homoeologous genes encoding typical PDI, located on chromosome group 4 of bread wheat, and PDI promoter sequence analysis of Triticum urartu, Aegilops speltoides and Aegilops tauschii had also been reported previously. In this study, we report the isolation and sequencing of a ~700 bp region, comprising ~600 bp of the putative promoter region and 88 bp of the first exon of the typical PDI gene, in five accessions each from Triticum urartu (AA), Aegilops speltoides (BB) and Aegilops tauschii (DD). Sequence analysis indicated large variation among sequences belonging to the different genomes, while close similarity was found within each species and with the corresponding homoeologous PDI sequences of Triticum aestivum cv. CS (AABBDD) resulting in an overall high conservation of the sequence conferring endosperm-specific expression.

The diagnostic significance of glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibody in rheumatoid arthritis patients

Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA activity states. Methods: The levels of G6PI antigens and anti-G6PI Abs in sera from 176 RA patients in different states, 35 non-RA patients and 100 healthy donors and in synovia fluids from 33 patients and 11 non-RA patients were measured by ELISA. Results: The sensitivity and specificity of G6PI antigens in the RA patients were 75.0% and 93.3%, respectively. The levels of serum G6PI antigens in 176 RA patients were significantly higher than non-RA patients and the health controls. Especially, there was a significant difference between the active phase and the inactive phase in G6PI antigens levels. The levels of G6PI antigens in synovia fluid were also significantly higher in RA groups than in non-RA patients. With the values of the anti-G6PI Abs in sera, there were no marked differences among RA, non-RA patients and health controls. Also, there was no significant difference between the active phase and the inactive phase in RA patients. However, there were no significant differences of G6PI and anti-G6PI between RA patients and health controls in synovial fluid. Conclusions: G6PI is highly correlated with the activity states of RA, and could be applied for a clinical biomarker with high sensitivity and specificity for the diagnosis of RA.

Lab Source Anomalous Scattering Using Cr Kα Radiation

High-throughput crystallography requires a method by which the structures of proteins can be determined quickly and easily. Experimental phasing is an essential technique in determining the three-dimensional protein structures using single-crystal X-ray diffraction. In macromolecular crystallography, the phases are derived either by Molecular Replacement (MR) method using the atomic coordinates of a structurally similar protein or by locating the positions of heavy atoms that are intrinsic to the protein or that have been added (MIR, MIRAS, SIR, SIRAS, MAD and SAD). Availability of in-house lab data collection sources (Cu Kα and Cr Kα radiation), cryo-crystallography and improved software for heavy atom location and density modification have increased the ability to solve protein structures using SAD. SAD phasing using intrinsic anomalous scatterers like sulfur, chlorine, calcium, manganese and zinc, which are already present in the protein becomes increasingly attractive owing to the advanced phasing methods. An analysis of successful SAD phasing on three proteins, lysozyme, glucose isomerase and thermolysin based on the signal of weak anomalous scatterers such as sulfur atom and chloride ion have been carried out. This analysis also proves that even the anomalous signal provided or present naturally in a macromolecule is good enough to solve crystal structures successfully using lab source chromium-generated X-ray radiation.

Ce-SAD Phasing of Glucose Isomerase and Thermolysin Using Cu <i>Kα</i>Radiation

Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 A) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.

Enhancing Lab Source Anomalous Scattering Using Cr Kα Radiation for Its Potential Application in Determining Macromolecular Structures

Obtaining phase information for the solution of macromolecular structures is a bottleneck in X-ray crystallography. Anomalous dispersion was recognized as a powerful tool for phasing macromolecular structures. It was used mainly to supplement the isomorphous replacement or to locate the anomalous scatterer itself. The first step in solving macromolecular structures by SAD (single-wavelength anomalous diffraction) is the location of the anomalous scatterers. The SAD method for experimental phasing has evolved substantially in the recent years. A phasing tool, 5-amino-2,4,6- triiodoisophthalic acid (I3C—magic triangle), was incorporated into three proteins, lysozyme, glucose isomerase and thermolysin using quick-soaking and co-crystallization method in order to understand the binding of metal ion with proteins. The high quality of the diffraction data, the use of chromium anode X-ray radiation and the required amount of anomalous signal enabled way for successful structure determination and automated model building. An analysis and/or comparison of the sulfur and iodine anomalous signals at the Cr Kα wavelength are discussed.

Evidence That Protein Disulfide Isomerase in Yeast Saccharomyces cerevisiae Is Transported from the ER to the Golgi Apparatus

Newly synthesized membrane and secretory proteins in cells undergo folding in the endoplasmic reticulum with the introduction of disulfide bonds and acquire the correct three-dimensional structure. Disulfide bonds are especially important for protein folding. It has been thought that formation of protein disulfide bonds in eukaryotes is mainly carried out by an enzyme called protein disulfide isomerase. Proteins, bearing the C-terminus of amino acids sequences with His-Asp-Glu-Leu (HDEL) sequence in yeast, in the endoplasmic reticulum (ER), which is a eukaryotic cellular organelle involved in protein synthesis, processing, and transport, have been considered to recycle between ER and Golgi apparatus. The proposal for this recycling model derives from the study of an HDEL-tagged fusion protein. Here, the localization and oligosaccharide modification of protein disulfide isomerase were investigated in yeast, and showed the first direct evidence that this intrinsic ER protein transports from ER to Golgi. Results suggest that this native protein is also accessible to post-ER enzymes, and yet accumulates in the ER.

Structural mechanism of a dual-functional enzyme Dgp A/B/C as both a C-glycoside cleaving enzyme and an O-to C-glycoside isomerase

The C-glycosidic bond that connects the sugar moiety with aglycone is difficult to be broken or made due to its inert nature.The knowledge of C-glycoside breakdown and synthesis is very limited.Recently,the enzyme Dgp A/B/C cascade from a human intestinal bacterium PUE was identified to specifically cleave the C-glycosidic bond of puerarin(daidzein-8-C-glucoside).Here we investigated how puerarin is recognized and oxidized by Dgp A based on crystal structures of Dgp A with or without substrate and biochemical characterization.More strikingly,we found that apart from being a C-glycoside cleaving enzyme,Dgp A/B/C is capable of efficiently converting O-to C-glycoside showing the activity as a structure isomerase.A possible mechanistic model was proposed dependently of the simulated complex structure of Dgp B/C with 3’’-oxo-daidzin and structure-based mutagenesis.Our findings not only shed light on understanding the enzyme-mediated C-glycosidic bond breakage and formation,but also may help to facilitate stereospecific C-glycoside synthesis in pharmaceutical industry.

Engineering the thermostability of D-lyxose isomerase from Caldanaerobius polysaccharolyticus via multiple computer-aided rational design for efficient synthesis of D-mannose

D-Mannose is an attractive functional sugar that exhibits many physiological benefits on human health.The demand for low-calorie sugars and sweeteners in foods are increasingly available on the market.Some sugar isomerases,such as D-lyxose isomerase(D-LIase),can achieve an isomerization reaction between D-mannose and D-fructose.However,the weak thermostability of D-LIase limits its efficient conversion from D-fructose to D-mannose.Nonetheless,few studies are available that have investigated the molecular modification of D-LIase to improve its thermal stability.In this study,computer-aided tools including FireProt,PROSS,and Consensus Finder were employed to jointly design D-LIase mutants with improved thermostability for the first time.Finally,the obtained five-point mutant M5(N21G/E78P/V58Y/C119Y/K170P)showed high thermal stability and cat-alytic activity.The half-life of M5 at 65◦C was 10.22 fold,and the catalytic efficiency towards 600 g/L of D-fructose was 2.6 times to that of the wild type enzyme,respectively.Molecular dynamics simulation and intramolecular forces analysis revealed a thermostability mechanism of highly rigidity conformation,newly formed hydrogen bonds andπ-cation interaction between and within protein domains,and redistributed surface electrostatic charges for the mutant M5.This research provided a promising D-LIase mutant for the industrial production of D-mannose from D-fructose.

Three amino acid substitutions contributing to thermostability of phosphoglucose isomerase in the Glanville fritillary butterfly

Temperature is one of the most important environmental factors that affect organisms,especially ectotherms,due to its effects on protein stability.Understanding the general rules that govern thermostability changes in proteins to adapt high-temperature environments is crucial.Here,we report the amino acid substitutions of phosphoglucose isomerase(PGI)related to thermostability in the Glanville fritillary butterfly(Melitaea cinxia,Lepidoptera:Nymphalidae).The PGI encoded by the most common allele in M.cinxia in the Chinese population(G3-PGI),which is more thermal tolerant,is more stable under heat stress than that in the Finnish population(D1-PGI).There are 5 amino acid substitutions between G3-PGI and D1-PGI.Site-directed mutagenesis revealed that the combination of amino acid substitutions of H35Q,M49T,and I64V may increase PGI thermostability.These substitutions alter the 3D structure to increase the interaction between 2 monomers of PGI.Through molecular dynamics simulations,it was found that the amino acid at site 421 is more stable in G3-PGI,confining the motion of theα-helix 420-441 and stabilizing the interaction between 2 PGI monomers.The strategy for hightemperature adaptation through these 3 amino acid substitutions is also adopted by other butterfly species(Boloria eunomia,Aglais urticae,Colias erate,and Polycaena lua)concurrent with M.cinxia in the Tianshan Mountains of China,i.e.,convergent evolution in butterflies.