Polymorphic Residues Research Articles

HIV-1 group O integrase displays lower enzymatic efficiency and higher susceptibility to raltegravir than HIV-1 group M subtype B integrase.

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.

Use of immunodampening to overcome diversity in the malarial vaccine candidate apical membrane antigen 1.

Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli, refolded in vitro, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.

Three-dimensional modeling and diversity analysis reveals distinct AVR recognition sites and evolutionary pathways in wild and domesticated wheat Pm3 R genes.

The Pm3 gene confers resistance against wheat powdery mildew. Studies of Pm3 diversity have shown that Pm3 alleles isolated from southern populations of wild emmer wheat located in Lebanon, Jordan, Israel, and Syria are more diverse and more distant from bread wheat alleles than alleles from the northern wild wheat populations located in Turkey, Iran, and Iraq. Therefore, southern populations from Israel were studied extensively to reveal novel Pm3 alleles that are absent from the cultivated gene pool. Candidate Pm3 genes were isolated via a polymerase chain reaction cloning approach. Known and newly identified Pm3 genes were subjected to variation analysis and polymorphic amino acid residues were superimposed on a three-dimensional (3D) model of PM3. The region of highest interspecies diversity between Triticum aestivum and T. dicoccoides lies in leucine-rich repeats (LRR) 19 to 24, whereas most intraspecies diversity in T. aestivum is located in LRR 25 to 28. Interestingly, these two regions are separated by one large LRR whose propensity for flexibility facilitates the conformation of the PM3 LRR domain into two differently structured models. The combination of evolutionary and protein 3D structure analysis revealed that Pm3 genes in wild and domesticated wheat show different evolutionary histories which might have been triggered through different interactions with the powdery mildew pathogen.

Distinct assembly profiles of HLA-B molecules.

MHC class I polymorphisms are known to influence outcomes in a number of infectious diseases, cancers, and inflammatory diseases. Human MHC class I H chains are encoded by the HLA-A, HLA-B, and HLA-C genes. These genes are highly polymorphic, with the HLA-B locus being the most variable. Each HLA class I protein binds to a distinct set of peptide Ags, which are presented to CD8(+) T cells. HLA-disease associations have been shown in some cases to link to the peptide-binding characteristics of individual HLA class I molecules. In this study, we show that polymorphisms at the HLA-B locus profoundly influence the assembly characteristics of HLA-B molecules and the stabilities of their peptide-deficient forms. In particular, dependence on the assembly factor tapasin is highly variable, with frequent occurrence of strongly tapasin-dependent or independent allotypes. Several polymorphic HLA-B residues located near the C-terminal end of the peptide are key determinants of tapasin-independent assembly. In vitro refolded forms of tapasin-independent allotypes assemble more readily with peptides compared to tapasin-dependent allotypes that belong to the same supertype, and, during refolding, reduced aggregation of tapasin-independent allotypes is observed. Paradoxically, in HIV-infected individuals, greater tapasin-independent HLA-B assembly confers more rapid progression to death, consistent with previous findings that some HLA-B allotypes shown to be tapasin independent are associated with rapid progression to multiple AIDS outcomes. Together, these findings demonstrate significant variations in the assembly of HLA-B molecules and indicate influences of HLA-B-folding patterns upon infectious disease outcomes.

1028-LBP: Association of mismatched polymorphic amino acid residues in HLA with chronic rejection of kidney transplants

The success of kidney transplantation relies heavily upon the compatibility of the HLA genes between donor and recipient, but the high degree of polymorphism among these genes in the population makes finding an ideal match difficult. However, it is unclear whether specific amino acids are particularly critical in determining histocompatibility. To investigate this possibility, we introduce a novel method of comparing donor and recipient genotypes and explore its efficacy in predicting transplant rejection. Using typing data of the HLA-A, -B, -C, and -DRB1 genes for 580 paired donors and recipients of a kidney transplant, we developed a method of allele estimation that used population frequency information to estimate amino acid sequence information. Using this sequence data we tested a number of metrics for measuring differences between donor and recipient genotypes, and looked for associations between mismatched residues and chronic rejection. Preliminary analysis of a cohort of 348 transplants with Caucasian recipients indicate a high association between residue mismatches in HLA-B and chronic rejection, primarily in residues 69 and 71 with p < 0.005 and α < 0.16, and in residue 66 with p < 0.001 and α < 0.2. Residues 69 and 71 are not directly involved in peptide binding, but residue 66 is active in binding pockets A and B. All three residues are polymorphic, with variants that differ significantly in their properties. Residue mismatches at any of these sites generally co-occur with broad motif mismatches across multiple binding pockets. Association between HLA residue mismatches and chronic rejection was observed in cases where the residues’ properties differed significantly, and when the value of the residue is linked to broader motif changes. Future investigations will consider the impact of functional changes in residues in addition to statistical characterization of these predictive sites, and how these factors relate to transplant compatibility assessment.

Update of the HLA class I eplet database in the website based registry of antibody-defined HLA epitopes.

Eplets are small configurations of polymorphic amino acid residues on human leukocyte antigen (HLA) molecules and are considered as essential components of HLA epitopes recognized by antibodies. This report describes a new design of the eplet repertoire in HLA-ABC alleles used in Luminex kits for antibody testing. There were three steps (1): identify all combinations of polymorphic residues with HLA molecular modeling within a 3-Å radius, (2) determine polymorphic residue compositions of 3 Å patches from amino acid sequences of HLA alleles in Luminex panels and (3) annotate eplets from one or more patches present on one allele or shared by the same group of alleles. There are now 270 HLA-ABC eplets in the Registry, of which 219 are in antibody-accessible positions on the molecular surface and 51 are defined solely by residue polymorphisms located below the molecular surface. Each eplet has a list of Luminex and non-Luminex alleles for which mismatch acceptability can be determined.

First report on the antibody verification of HLA-ABC epitopes recorded in the website-based HLA Epitope Registry.

The International Registry of Antibody-Defined HLA Epitopes ( http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to human leukocyte antigen (HLA) mismatches. These epitopes are defined structurally by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. So-called eplets represent essential components of HLA epitopes and they are defined by polymorphic residues. A major goal is to identify HLA epitopes that have been verified experimentally with informative antibodies. Our analysis has also included data in many publications. As of 1 November 2013, 95 HLA-ABC antibody-verified epitopes have been recorded, 62 correspond to eplets and 33 are defined by eplets paired with other residue configurations. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

Shared Molecular Eplet Stimulates Acute Antibody-Mediated Rejection in a Kidney Transplant Recipient With Low-Level Donor-Specific Antibodies: A Case Report

HLA antibodies usually recognize epitopes rather than antigens. This case report reveals that acute antibody-mediated rejection (AMR) that occurred in a kidney transplant recipient with low-level donor-specific antibodies (DSAs) could be explained by shared epitope. A 39-year-old woman received a first kidney transplant from a deceased donor (HLA-DRB1*11:06, *12:02, DRB3*02:02, *03:01). She developed acute AMR confirmed by kidney biopsy on day 4 after transplantation. Antibody testing with pretransplant serum showed anti-DR11 DSA below cutoff level (mean fluorescence intensity [MFI], 702; cutoff >1,000). However, high-level DSAs were detected on day 5 after transplantation (anti-DR11 MFI, 8,531; anti-DR12 MFI, 3,146). We hypothesized that the sharp rise in DSA levels was a result of anamnestic response with donor-antigen sensitization that occurred during pregnancy. High-resolution HLA-DR typing of her husband showed HLA-DRB1*03:01, *15:02:01, DRB3*02:02, DRB5*01:02. No sharing between donor HLAs eliciting reactive antibodies and her husband's HLAs was detected. Nevertheless, we speculated that shared epitope, not antigen, was the cause of allosensitization. To identify the shared epitope recognized by patient's antibodies, we used HLAmatchmaker, a computer algorithm that considers small configurations of polymorphic residues referred to as eplets as essential components of HLA epitopes for analysis. The results showed that 149H, which was the eplet shared by HLA-DRB1*03:01 (from her husband) and DRB1*11:06, DRB1*12:02, DRB3*03:01 (from donor), was the most prevalent eplet on DRB1 reactive alleles in Luminex assay. In conclusion, pretransplant low-level DSAs can induce AMR early after transplantation as a result of shared epitopes with a previous immunizer.

Two Polymorphisms Facilitate Differences in Plasticity between Two Chicken Major Histocompatibility Complex Class I Proteins

Major histocompatibility complex class I molecules (MHC I) present peptides to cytotoxic T-cells at the surface of almost all nucleated cells. The function of MHC I molecules is to select high affinity peptides from a large intracellular pool and they are assisted in this process by co-factor molecules, notably tapasin. In contrast to mammals, MHC homozygous chickens express a single MHC I gene locus, termed BF2, which is hypothesised to have co-evolved with the highly polymorphic tapasin within stable haplotypes. The BF2 molecules of the B15 and B19 haplotypes have recently been shown to differ in their interactions with tapasin and in their peptide selection properties. This study investigated whether these observations might be explained by differences in the protein plasticity that is encoded into the MHC I structure by primary sequence polymorphisms. Furthermore, we aimed to demonstrate the utility of a complimentary modelling approach to the understanding of complex experimental data. Combining mechanistic molecular dynamics simulations and the primary sequence based technique of statistical coupling analysis, we show how two of the eight polymorphisms between BF2*15∶01 and BF2*19∶01 facilitate differences in plasticity. We show that BF2*15∶01 is intrinsically more plastic than BF2*19∶01, exploring more conformations in the absence of peptide. We identify a protein sector of contiguous residues connecting the membrane bound α3 domain and the heavy chain peptide binding site. This sector contains two of the eight polymorphic residues. One is residue 22 in the peptide binding domain and the other 220 is in the α3 domain, a putative tapasin binding site. These observations are in correspondence with the experimentally observed functional differences of these molecules and suggest a mechanism for how modulation of MHC I plasticity by tapasin catalyses peptide selection allosterically.

Inhibition of Hepatitis C Virus in Chimeric Mice by Short Synthetic Hairpin RNAs: Sequence Analysis of Surviving Virus Shows Added Selective Pressure of Combination Therapy

We have recently shown that a cocktail of two short synthetic hairpin RNAs (sshRNAs), targeting the internal ribosome entry site of hepatitis C virus (HCV) formulated with lipid nanoparticles, was able to suppress viral replication in chimeric mice infected with HCV GT1a by up to 2.5 log10 (H. Ma et al., Gastroenterology 146:63-66.e5, http://dx.doi.org/10.1053/j.gastro.2013.09.049) Viral load remained about 1 log10 below pretreatment levels 21 days after the end of dosing. We have now sequenced the HCV viral RNA amplified from serum of treated mice after the 21-day follow-up period. Viral RNA from the HCV sshRNA-treated groups was altered in sequences complementary to the sshRNAs and nowhere else in the 500-nucleotide sequenced region, while the viruses from the control group that received an irrelevant sshRNA had no mutations in that region. The ability of the most commonly selected mutations to confer resistance to the sshRNAs was confirmed in vitro by introducing those mutations into HCV-luciferase reporters. The mutations most frequently selected by sshRNA treatment within the sshRNA target sequence occurred at the most polymorphic residues, as identified from an analysis of available clinical isolates. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNA interference (RNAi) mechanism of action. This study presents a detailed analysis of the impact of treating a hepatitis C virus (HCV)-infected animal with synthetic hairpin-shaped RNAs that can degrade the virus's RNA genome. These RNAs can reduce the viral load in these animals by over 99% after 1 to 2 injections. The study results confirm that the viral rebound that often occurred a few weeks after treatment is due to emergence of a virus whose genome is mutated in the sequences targeted by the RNAs. The use of two RNA inhibitors, which is more effective than use of either one by itself, requires that any resistant virus have mutations in the targets sites of both agents, a higher hurdle, if the virus is to retain the ability to replicate efficiently. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNAi mechanism of action.

Polymorphism p.Val231Ile alters substrate selectivity of drug-metabolizing arylamine N-acetyltransferase 2 (NAT2) isoenzyme of rhesus macaque and human

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes mediating the biotransformation of arylamine/arylhydrazine xenobiotics, including pharmaceuticals and environmental carcinogens. The NAT1 and NAT2 genes, and their many polymorphic variants, have been thoroughly studied in humans by pharmacogeneticists and cancer epidemiologists. However, little is known about the function of NAT homologues in other primate species, including disease models. Here, we perform a comparative functional investigation of the NAT2 homologues of the rhesus macaque and human. We further dissect the functional impact of a previously described rhesus NAT2 gene polymorphism, causing substitution of valine by isoleucine at amino acid position 231. Gene constructs of rhesus and human NAT2, bearing or lacking non-synonymous polymorphism c.691G>A (p.Val231Ile), were expressed in Escherichia coli for comparative enzymatic analysis against various NAT1- and NAT2-selective substrates. The results suggest that the p.Val231Ile polymorphism does not compromise the stability or overall enzymatic activity of NAT2. However, substitution of Val231 by the bulkier isoleucine appears to alter enzyme substrate selectivity by decreasing the affinity towards NAT2 substrates and increasing the affinity towards NAT1 substrates. The experimental observations are supported by in silico modelling localizing polymorphic residue 231 close to amino acid loop 125–129, which forms part of the substrate binding pocket wall and determines the substrate binding preferences of the NAT isoenzymes. The p.Val231Ile polymorphism is the first natural polymorphism demonstrated to affect NAT substrate selectivity via this particular mechanism. The study is also the first to thoroughly characterize the properties of a polymorphic NAT isoenzyme in a non-human primate model.

A Mechanistic Basis for the Co-evolution of Chicken Tapasin and Major Histocompatibility Complex Class I (MHC I) Proteins

MHC class I molecules display peptides at the cell surface to cytotoxic T cells. The co-factor tapasin functions to ensure that MHC I becomes loaded with high affinity peptides. In most mammals, the tapasin gene appears to have little sequence diversity and few alleles and is located distal to several classical MHC I loci, so tapasin appears to function in a universal way to assist MHC I peptide loading. In contrast, the chicken tapasin gene is tightly linked to the single dominantly expressed MHC I locus and is highly polymorphic and moderately diverse in sequence. Therefore, tapasin-assisted loading of MHC I in chickens may occur in a haplotype-specific way, via the co-evolution of chicken tapasin and MHC I. Here we demonstrate a mechanistic basis for this co-evolution, revealing differences in the ability of two chicken MHC I alleles to bind and release peptides in the presence or absence of tapasin, where, as in mammals, efficient self-loading is negatively correlated with tapasin-assisted loading. We found that a polymorphic residue in the MHC I α3 domain thought to bind tapasin influenced both tapasin function and intrinsic peptide binding properties. Differences were also evident between the MHC alleles in their interactions with tapasin. Last, we show that a mismatched combination of tapasin and MHC alleles exhibit significantly impaired MHC I maturation in vivo and that polymorphic MHC residues thought to contact tapasin influence maturation efficiency. Collectively, this supports the possibility that tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading in vivo.

Atypische Tumorregression

A 67-year-old man presented with a poorly differentiated squamous cell carcinoma of the esophagus diagnosed by biopsy. After neoadjuvant radiochemotherapy the gastroesophagectomy specimen showed diffuse polymorphic and anuclear cell residues ranging from 35 µm to 46 µm in size. Immunohistochemically, PanCK and AE1-3 revealed a positive staining while CD68 and MIB1 showed a negative staining. The retrospective anamnesis revealed that the patient had chronic polyarthritis as underlying illness, for which reason he had been taking humira and methotrexate, a cytostatic drug, for many years. Therefore, the development of the tumor might have been enhanced by these drugs. Electron microscopic analysis confirmed that the avital akaryote cell residues represented a special type of tumor regression. Complete tumor regression level IV without vital rest tumor tissue based on Baldus et al. was diagnosed.

Analysis of Polymorphic Residues Reveals Distinct Enzymatic and Cytotoxic Activities of the Streptococcus pyogenes NAD+ Glycohydrolase

The Streptococcus pyogenes NAD(+) glycohydrolase (SPN) is secreted from the bacterial cell and translocated into the host cell cytosol where it contributes to cell death. Recent studies suggest that SPN is evolving and has diverged into NAD(+) glycohydrolase-inactive variants that correlate with tissue tropism. However, the role of SPN in both cytotoxicity and niche selection are unknown. To gain insight into the forces driving the adaptation of SPN, a detailed comparison of representative glycohydrolase activity-proficient and -deficient variants was conducted. Of a total 454 amino acids, the activity-deficient variants differed at only nine highly conserved positions. Exchanging residues between variants revealed that no one single residue could account for the inability of the deficient variants to cleave the glycosidic bond of β-NAD(+) into nicotinamide and ADP-ribose; rather, reciprocal changes at 3 specific residues were required to both abolish activity of the proficient version and restore full activity to the deficient variant. Changing any combination of 1 or 2 residues resulted in intermediate activity. However, a change to any 1 residue resulted in a significant decrease in enzyme efficiency. A similar pattern involving multiple residues was observed for comparison with a second highly conserved activity-deficient variant class. Remarkably, despite differences in glycohydrolase activity, all versions of SPN were equally cytotoxic to cultured epithelial cells. These data indicate that the glycohydrolase activity of SPN may not be the only contribution the toxin has to the pathogenesis of S. pyogenes and that both versions of SPN play an important role during infection.

Humoral Responses to Islet Antigen-2 and Zinc Transporter 8 Are Attenuated in Patients CarryingHLA-A*24Alleles at the Onset of Type 1 Diabetes

The HLA-A*24 allele has shown negative associations with autoantibodies to islet antigen-2 (IA-2) and zinc transporter 8 (ZnT8) in patients with established type 1 diabetes. Understanding how this HLA class I allele affects humoral islet autoimmunity gives new insights into disease pathogenesis. We therefore investigated the epitope specificity of associations between HLA-A*24 and islet autoantibodies at disease onset. HLA-A*24 genotype and autoantibody responses to insulin (IAA), glutamate decarboxylase (GADA), IA-2, IA-2β, and ZnT8 were analyzed in samples collected from patients with recent-onset type 1 diabetes. After correction for age, sex, and HLA class II genotype, HLA-A*24 was shown to be a negative determinant of IA-2A and ZnT8A. These effects were epitope specific. Antibodies targeting the protein tyrosine phosphatase domains of IA-2 and IA-2β, but not the IA-2 juxtamembrane region, were less common in patients carrying HLA-A*24 alleles. The prevalence of ZnT8A specific or cross-reactive with the ZnT8 tryptophan-325 polymorphic residue, but not those specific to arginine-325, was reduced in HLA-A*24-positive patients. No associations were found between HLA-A*24 and IAA or GADA. Association of an HLA class I susceptibility allele with altered islet autoantibody phenotype at diagnosis suggests CD8 T-cell and/or natural killer cell–mediated killing modulates humoral autoimmune responses.

Diversity of T Cell Epitopes in Plasmodium falciparum Circumsporozoite Protein Likely Due to Protein-Protein Interactions

Circumsporozoite protein (CS) is a leading vaccine antigen for falciparum malaria, but is highly polymorphic in natural parasite populations. The factors driving this diversity are unclear, but non-random assortment of the T cell epitopes TH2 and TH3 has been observed in a Kenyan parasite population. The recent publication of the crystal structure of the variable C terminal region of the protein allows the assessment of the impact of diversity on protein structure and T cell epitope assortment. Using data from the Gambia (55 isolates) and Malawi (235 isolates), we evaluated the patterns of diversity within and between epitopes in these two distantly-separated populations. Only non-synonymous mutations were observed with the vast majority in both populations at similar frequencies suggesting strong selection on this region. A non-random pattern of T cell epitope assortment was seen in Malawi and in the Gambia, but structural analysis indicates no intramolecular spatial interactions. Using the information from these parasite populations, structural analysis reveals that polymorphic amino acids within TH2 and TH3 colocalize to one side of the protein, surround, but do not involve, the hydrophobic pocket in CS, and predominately involve charge switches. In addition, free energy analysis suggests residues forming and behind the novel pocket within CS are tightly constrained and well conserved in all alleles. In addition, free energy analysis shows polymorphic residues tend to be populated by energetically unfavorable amino acids. In combination, these findings suggest the diversity of T cell epitopes in CS may be primarily an evolutionary response to intermolecular interactions at the surface of the protein potentially counteracting antibody-mediated immune recognition or evolving host receptor diversity.

Evolution of HLA class II through diversification in protein stability levels (P5009)

Abstract Allelic diversity in human leukocyte antigens (HLA) have been explained by balancing selection acting on polymorphic residues in the peptide binding groove of HLA that diversify binding spectrum for pathogenic epitopes. Here we show that HLA class II (HLA-DQ) allele products are extremely diverse in protein stability levels. We have profiled the relative stability of DQ proteins by a cell-based assay that measures cell surface MHC protein density (surface density index; SDI). The SDI varies over 100-fold among DQ allele products, indicating large stability differences. Through mutagenesis, we identified polymorphic residues that regulate the intrinsic stability of DQ protein, which includes variations on the exterior of the peptide-binding groove that mediate interdomain hydrogen bonds. One of the major stability regulator in DQA1 achieves high ratio of synonymous to non-sysnonymous substitutions per site (ds/dn), suggesting that variations at this site have been maintained through balancing selection. DQ genes of non-human primates and mammals also maintain these variatns. These data suggest that HLA and MHC have been evolving through diversifications in protein stability levels. Our study provides new functional insights into the mechanism of HLA evolution.

Population genetics, sequence diversity and selection in the gene encoding the Plasmodium falciparum apical membrane antigen 1 in clinical isolates from the south-east of Iran

The Plasmodium falciparum apical membrane antigen1 (AMA1) is a leading malaria vaccine candidate antigen. In the present investigation, for the first time, the almost full length of the ama1 gene covering domain I (DI), DII and DIII was PCR amplified and sequenced in 21 P. falciparum isolates collected from the southeastern parts of Iran. The result showed the low genetic diversity of Iranian PfAMA1 with 11 PfAMA1 haplotypes in which nine out of 11 haplotypes are novel and have been reported for the first time. The Iranian P. falciparum population indicated a moderate level of genetic differentiation. The difference among the rates of non-synonymous and synonymous mutations, Tajima’s D and McDonald–Kreitman tests suggested that the diversity at DI is due to positive natural selection. In addition, recombination contributes to the diversity of Iranian PfAMA1 and this is supported by the decline of the linkage disequilibrium index R2 with increasing the nucleotide distance. The highly polymorphic residues (positions: 187, 197, 200, 230 and 243) were polymorphic; however, most of the SNPs in non-polymorphic residues were conserved except the residue at position 395. Nevertheless, no mutation was found in the DII loop of the Iranian PfAMA1, indicating that it is subjected to purifying selection. In conclusion, the low genetic diversity in PfAMA1 among Iranian isolates supports and provides valuable information for the development of a PfAMA1-based malaria vaccine.

Burial of the Polymorphic Residue 129 in Amyloid Fibrils of Prion Stop Mutants

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

Intramolecular Interaction Influences Binding of the Flax L5 and L6 Resistance Proteins to their AvrL567 Ligands

L locus resistance (R) proteins are nucleotide binding (NB-ARC) leucine-rich repeat (LRR) proteins from flax (Linum usitatissimum) that provide race-specific resistance to the causal agent of flax rust disease, Melampsora lini. L5 and L6 are two alleles of the L locus that directly recognize variants of the fungal effector AvrL567. In this study, we have investigated the molecular details of this recognition by site-directed mutagenesis of AvrL567 and construction of chimeric L proteins. Single, double and triple mutations of polymorphic residues in a variety of AvrL567 variants showed additive effects on recognition strength, suggesting that multiple contact points are involved in recognition. Domain-swap experiments between L5 and L6 show that specificity differences are determined by their corresponding LRR regions. Most positively selected amino acid sites occur in the N- and C-terminal LRR units, and polymorphisms in the first seven and last four LRR units contribute to recognition specificity of L5 and L6 respectively. This further confirms that multiple, additive contact points occur between AvrL567 variants and either L5 or L6. However, we also observed that recognition of AvrL567 is affected by co-operative polymorphisms between both adjacent and distant domains of the R protein, including the TIR, ARC and LRR domains, implying that these residues are involved in intramolecular interactions to optimize detection of the pathogen and defense signal activation. We suggest a model where Avr ligand interaction directly competes with intramolecular interactions to cause activation of the R protein.