Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus.

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

Prevalence of Rh, Duffy, Kell, Kidd & MNSs blood group antigens in the Indian blood donor population.

BACKGROUND & OBJECTIVES: Little data are available regarding the frequencies of the blood group antigens other than ABO and RhD in the Indian population. Knowledge of the antigen frequencies is important to assess risk of antibody formation and to guide the probability of finding antigen-negative donor blood, which is especially useful when blood is required for a patient who has multiple red cell alloantibodies. This study was carried out to determine the frequencies of the D, C, c, E, e, K, k, Fy(a), Fy(b), Jk(a), Jk(b), M, N, S and s antigens in over 3,000 blood donors. METHODS: Samples from randomly selected blood donors from Delhi and nearby areas (both voluntary and replacement) were collected for extended antigen typing during the period January 2009 to January 2010. Antigens were typed via automated testing on the Galileo instrument using commercial antisera. RESULTS: A total of 3073 blood samples from donors were phenotyped. The prevalence of these antigens was found to be as follows in %: D: 93.6, C: 87, c: 58, E: 20, e: 98, K: 3.5, k: 99.97, F(a) : 87.4, Fy(b) : 57.6, Jk(a) : 81.5, Jk(b) : 67.4, M: 88.7, N: 65.4, S: 54.8 and s: 88.7. INTERPRETATION & CONCLUSIONS: This study found the prevalence of the typed antigens among Indian blood donors to be statistically different to those in the Caucasian, Black and Chinese populations, but more similar to Caucasians than to the other racial groups.

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes.

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-ß-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.

Identification of an erythrocyte component carrying the Duffy blood group Fya antigen.

The erythrocyte component carrying the Duffy blood group antigen Fya has been identified as a 35- to 43-kilodalton protein. The protein is degraded by proteases, chymotrypsin, and Pronase, which destroy its antigenicity on intact erythrocytes. Its unusual property of aggregating on being boiled in 5 percent sodium dodecyl sulfate with 5 percent 2-mercaptoethanol distinguishes it from other erythrocyte membrane proteins described to date.

Postcapillary venule endothelial cells in kidney express a multispecific chemokine receptor that is structurally and functionally identical to the erythroid isoform, which is the Duffy blood group antigen.

The human erythrocyte chemokine receptor has recently been shown to be identical to the Duffy blood group antigen and is expressed in multiple organs, including kidney. Here we have examined the molecular properties of the renal isoform. Immunoblot analysis of erythrocyte and kidney detergent lysates, with a monoclonal antibody (Fy6) to the Duffy antigen, revealed that the renal isoform had a molecular mass of 43-45 kD, which could be distinguished from that observed in erythroid cells (38-47 kD). Chemical cross-linking of kidney membranes to 125I-melanoma growth stimulatory activity (MGSA) indicated that the renal chemokine receptor had a molecular mass of 38-45 kD. Binding of 125I-labeled MGSA to kidney membranes was competitively inhibited by the addition of unlabeled MGSA, IL-8, regulated on activation, normal T expressed and secrted, and monocyte chemotactic protein-1. Scatchard analysis of MGSA binding showed that the chemokine receptor from renal tissues had a binding affinity of 3.5 nM similar to that observed for the erythroid isoform (5-10 nM). The primary structure of the renal chemokine receptor predicted from the nucleotide sequence of cDNA from renal tissues is identical to that reported for the erythroid isoform. Immunocytochemical staining of kidney with Fy6 localized expression to endothelial cells present in postcapillary venules. These studies implicate the Duffy antigen/chemokine receptor in the complex interactions between postcapillary endothelial cells and granulocytes, which are modulated by pro-inflammatory chemokines.

High-resolution melting analysis for genotyping Duffy, Kidd and Diego blood group antigens.

High-resolution melting (HRM) analysis is a simpler genotyping method than allele-specific PCR, PCR-restriction fragment length polymorphism and multiplex PCR. Duffy, Kidd and Diego are clinically important blood group antigens. We used a novel method to genotype these three blood group antigens. Purified genomic DNA extracts of blood samples (354 Duffy, 347 Kidd and 457 Diego) were amplified using specific amplification primers. HRM curves were obtained by HRM analysis. Results were in complete concordance with those obtained for previous phenotypes and genotypes. Nucleotide substitutions for these blood group antigens were differentiated by the HRM curves. HRM analysis is a simple genotyping method and is an alternative to serological typing. Our results suggest that HRM analysis can also be used for genotyping blood group antigens whose allotype specificity is determined by single nucleotide substitutions.

Production of a new murine monoclonal antibody with Fy6 specificity and characterization of the immunopurified N-glycosylated Duffy-active molecule.

A murine monoclonal antibody (mAb i3A; IgG1, kappa light chain) was obtained using human red blood cells as immunogen. The antibody showed Fy6 specificity since it agglutinated all but Fy(a-b-)-untreated red cells and failed to agglutinate chymotrypsin-treated cells. An erythrocyte membrane protein of 42-46 kD was revealed as the major component recognized by the antibody on immunoblots. The antibody also bound to 92- to 95- and 200-kD proteins, tentatively identified as oligomers of the 42- to 46-kD monomeric form. The affinity-purified Fy6-active protein was converted to a sharp band of 35 kD after N-glycanase treatment. The molecule appeared as a slightly broadly band after neuraminidase treatment but was not further altered by O-glycanase. The i3A mAb bound to 6,000 +/- 1,000 receptor sites on either Fy(a-b+), Fy (a+b+) and Fy(a+b-) red cells with an affinity constant in the range of 3-6 x 10(8) M-1. No binding was observed to other blood cells nor to several cells (B, T, myelomonocytic and erythro-leukemia cell lines). Also, the bulk of i3A-Fy6 immune complexes could be dissociated from the red cell membrane with as low as 0.2% Triton X-100, showing that the Fy6-active glycoprotein is not tightly associated with the membrane skeleton. Our data obtained with a new monoclonal antibody directed to the Fy6 antigen demonstrate that the blood group Duffy-active component is a red cell-specific glycoprotein carrying one or more N-linked oligosaccharides.

A family of chimeric erythrocyte binding proteins of malaria parasites.

Proteins sequestered within organelles of the apical complex of malaria merozoites are involved in erythrocyte invasion, but few of these proteins and their interaction with the host erythrocyte have been characterized. In this report we describe MAEBL, a family of erythrocyte binding proteins identified in the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei. MAEBL has a chimeric character, uniting domains from two distinct apical organelle protein families within one protein. MAEBL has a molecular structure homologous to the Duffy binding-like family of erythrocyte binding proteins located in the micronemes of merozoites. However, the amino cysteine-rich domain of MAEBL has no similarity to the consensus Duffy binding-like amino cysteine-rich ligand domain, but instead is similar to the 44-kDa ectodomain fragment of the apical membrane antigen 1 (AMA-1) rhoptry protein family. MAEBL has a tandem duplication of this AMA-1-like domain, and both of these cysteine-rich domains bound erythrocytes when expressed in vitro. Differential transcription and splicing of the maebl locus occurred in the YM clone of P. yoelii yoelii. The apical distribution of MAEBL suggested localization within the rhoptry organelles of the apical complex. We propose that MAEBL is a member of a highly conserved family of erythrocyte binding proteins of Plasmodium involved in host cell invasion.

Poblaciones costeras de Chile: marcadores genéticos en cuatro localidades / Coastal Chilean populations: genetic markers in 4 locations

Background: Historical and anthropological data suggest the presence of descendents of Changos, Cuncos, Chonos and Yamanas, South American indian populations, in certain Chilean coastal villages. Aim: To assess the degree of South American indian admixture in Chilean coastal villages using protein markers, to complete the assessment of human biological diversity in Chile. Subjects and methods: AB0, Rh, MNS, Duffy and Kidd blood group systems were assessed in 47, 48, 55 and 24 individuals from Paposo, Carelmapu, Laitec and Ukika respectively. Phenotypic and gene frequencies were calculated. The degree of South American indian admixture was estimated from the AB0*0 allele and Rh*dce haplotypes. Results: High frequencies of AB0*0, Fy*a, Jk*b alleles, Dce and Ms haplotypes were found in all villages, consistent with the pattern expected for South AmericanAboriginal populations. The highest presence of South American indian admixture was present in Laitec with 80 per cent and in Ukika with 74 per cent. The figures for Paposo and Carelmapu were 60 and 65 per cent respectively. Conclusions: Accordin g to South American indian admixture estimates, the genetic isolation of coastal populations is lower than that of inland subjects, suggesting thatsea proximity facilitates gene flow

Composición genética de la población chilena: los atacameños de la comuna de San Pedro de Atacama / Genetic composition of chilean population: the atacameños from San Pedro de Atacama

This work describes the genetic composition of atacameños from San Pedro de Atacama. The results show that a) the contribution of non-indigenous genes is relatively low, in relation to the spanish inmigration period. b) the Hardy-Weinberg genetics disequilibrium for MNSs system should have biological implications. c) the variant for esterasa D enzyme may be the same found in other chilean populations