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Transmembrane helix folding and self-association play important roles in biological signaling and transportation pathways across biomembranes. With molecular simulations, studies to explore the structural biochemistry of this process have been limited to focusing on individual fragments of this process - either helix formation or dimerization. While at an atomistic resolution, it can be prohibitive to access long spatio-temporal scales, at the coarse grained (CG) level, current methods either employ additional constraints to prevent spontaneous unfolding or have a low resolution on sidechain beads that restricts the study of dimer disruption caused by mutations. To address these research gaps, in this work, we apply our recent, in-house developed CG model ( ProMPT ) to study the folding and dimerization of Glycophorin A (GpA) and its mutants in the presence of Dodecyl-phosphocholine (DPC) micelles. Our results first validate the two-stage model that folding and dimerization are independent events for transmembrane helices and found a positive correlation between helix folding and DPC-peptide contacts. The wild type (WT) GpA is observed to be a right-handed dimer with specific GxxxG contacts, which agrees with experimental findings. Specific point mutations reveal several features responsible for the structural stability of GpA. While the T87L mutant forms anti-parallel dimers due to an absence of T87 interhelical hydrogen bonds, a slight loss in helicity and a hinge-like feature at the GxxxG region develops for the G79L mutant. We note that the local changes in the hydrophobic environment, affected by the point mutation, contribute to the development of this helical bend. This work presents a holistic overview of the structural stability of GpA in a micellar environment, while taking secondary structural fluctuations into account. Moreover, it presents opportunities for applications of computationally efficient CG models to study conformational alterations of transmembrane proteins that have physiological relevance.

Anion exchanger-1 (AE1) is the main erythroid Cl - /HCO 3 - transporter that supports CO 2 transport. Glycophorin A (GPA), a component of the AE1 complexes, facilitates AE1 expression and anion transport, but Glycophorin B (GPB) does not. Here, we dissected the structural components of GPA/GPB involved in glycophorin-AE1 trafficking by comparing them with three GPB variants-GPBhead (lacking the transmembrane domain [TMD]), GPBtail (mainly the TMD), and GP.Mur (glycophorin B-A-B hybrid). GPB-derived GP.Mur bears an O-glycopeptide that encompasses the R18 epitope, which is present in GPA but not GPB. By flow cytometry, AE1 expression in the control erythrocytes increased with the GPA-R18 expression; GYP.Mur + / + erythrocytes bearing both GP.Mur and GPA expressed more R18 epitopes and more AE1 proteins. In contrast, heterologously expressed GPBtail and GPB were predominantly localized in the Golgi apparatus of HEK-293 cells, whereas GBhead was diffuse throughout the cytosol, suggesting that glycophorin transmembrane encoded an ER/Golgi retention signal. AE1 coexpression could reduce the ER/Golgi retention of GPB, but not of GPBtail or GPBhead. Thus, there are forward-trafficking and transmembrane-driven ER/Golgi retention signals encoded in the glycophorin sequences. How the balance between these opposite trafficking signals could affect glycophorin sorting into AE1 complexes and influence erythroid anion transport remains to be explored.

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Low plasma levels of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) are associated with decreased low-density lipoprotein (LDL) cholesterol and a reduced risk of cardiovascular disease. PCSK9 binds to the epidermal growth factor-like repeat A (EGFA) domain of LDL receptors (LDLR), very low-density lipoprotein receptors (VLDLR), apolipoprotein E receptor 2 (ApoER2), and lipoprotein receptor-related protein 1 (LRP1) and accelerates their degradation, thus acting as a key regulator of lipid metabolism. Antibody and RNAi-based PCSK9 inhibitor treatments lower cholesterol and prevent cardiovascular incidents in patients, but their high-cost hampers market penetration. We sought to develop a safe, long-term and one-time solution to treat hyperlipidemia. We created a cDNA encoding a chimeric protein in which the extracellular N- terminus of red blood cells (RBCs) specific glycophorin A was fused to the LDLR EGFA domain and introduced this gene into mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). Following transplantation into irradiated mice, the animals produced RBCs with the EGFA domain (EGFA-GPA RBCs) displayed on their surface. These animals showed significantly reduced plasma PCSK9 (66.5% decrease) and reduced LDL levels (40% decrease) for as long as 12 months post-transplantation. Furthermore, the EGFA- GPA mice remained lean for life and maintained normal body weight under a high-fat diet. Hematopoietic stem cell gene therapy can generate red blood cells expressing an EGFA-glycophorin A chimeric protein as a practical and long-term strategy for treating chronic hyperlipidemia and obesity., Competing Interests: H.F.L. and H.L.P. served as scientific advisors and have/had equity in Rubius, a biotechnology company that seeks to exploit engineered red blood cells as therapeutics but does not provide financial support for the technology described in this paper. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The MARTINI model is a widely used coarse-grained force field popular for its capacity to represent a diverse array of complex biomolecules. However, efforts to simulate increasingly realistic models of membranes, involving complex lipid mixtures and multiple proteins, suggest that membrane protein aggregates are overstabilized by the MARTINI v2.2 force field. In this study, we address this shortcoming of the MARTINI model. We determined the free energy of dimerization of four transmembrane protein systems using the nonpolarizable MARTINI model. Comparison with experimental FRET-based estimates of the dimerization free energy was used to quantify the significant overstabilization of each protein homodimer studied. To improve the agreement between simulation and experiment, a single uniform scaling factor, α, was used to enhance the protein-lipid Lennard-Jones interaction. A value of α = 1.04-1.045 was found to provide the best fit to the dimerization free energies for the proteins studied while maintaining the specificity of contacts at the dimer interface. To further validate the modified force field, we performed a multiprotein simulation using both MARTINI v2.2 and the reparameterized MARTINI model. While the original MARTINI model predicts oligomerization of protein into a single aggregate, the reparameterized MARTINI model maintains a dynamic equilibrium between monomers and dimers as predicted by experimental studies. The proposed reparameterization is an alternative to the standard MARTINI model for use in simulations of realistic models of a biological membrane containing diverse lipids and proteins.

The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Objectives: MNS is a highly polymorphic blood group comprising 49 antigens recognized by International Society of Blood Transfusion, some of which may have been generated by genomic recombination among the closely linked genes GYPA, GYPB and GYPE. The GYPE gene has an almost identical sequence to GYPA*01 allele in exon 2 (99% homology), which accounts for M antigen. We investigated an unusual glycophorin molecule with protease-resistant M antigen., Methods: Blood samples were screened by an automated blood typing system (PK7300) using bromelain-treated red blood cells (RBCs) and murine monoclonal anti-M. The M-positive RBC samples were analysed by immunoblotting using anti-M as the primary antibody. GYPA, GYPB and GYPE genes were analysed by polymerase chain reaction (PCR), cloning and sequencing using reticulocyte mRNA and genomic DNA., Results: Serological tests and immunoblotting revealed that 103 of the 193 009 individuals (0·0534%) expressed protease-resistant M-active glycophorin having a molecular weight of 20 kDa. All the 103 individuals were S+ s- or S- s+. When reticulocyte mRNA from the individuals with M-active glycophorin (20 kDa) was examined by PCR and cloning followed by sequencing, a novel GYPE-B hybrid transcript was identified. Long-range PCR and sequencing using genomic DNA revealed that the individuals had a GYPB-E(2-4)-B hybrid gene. This hybrid gene was predicted to encode a 59-amino-acid mature glycoprotein that expresses no S or s antigens CONCLUSIONS: The prevalence of the M-active glycophorin (20 kDa) in the Japanese population is 0·0534%. This glycophorin is predicted to be a 59 amino acids polypeptide encoded by the novel GYPB-E(2-4)-B hybrid gene., (© 2020 International Society of Blood Transfusion.)

Transmembrane helix association is a fundamental step in the folding of helical membrane proteins. The prototypical example of this association is formation of the glycophorin dimer. While its structure and stability have been well-characterized experimentally, the detailed assembly mechanism is harder to obtain. Here, we use all-atom simulations within phospholipid membrane to study glycophorin association. We find that initial association results in the formation of a non-native intermediate, separated by a significant free energy barrier from the dimer with a native binding interface. We have used transition-path sampling to determine the association mechanism. We find that the mechanism of the initial bimolecular association to form the intermediate state can be mediated by many possible contacts, but seems to be particularly favoured by formation of non-native contacts between the C-termini of the two helices. On the other hand, the contacts which are key to determining progression from the intermediate to the native state are those which define the native binding interface, reminiscent of the role played by native contacts in determining folding of globular proteins. As a check on the simulations, we have computed association and dissociation rates from the transition-path sampling. We obtain results in reasonable accord with available experimental data, after correcting for differences in native state stability. Our results yield an atomistic description of the mechanism for a simple prototype of helical membrane protein folding., Competing Interests: The authors have declared that no competing interests exist.

The biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Due to the absence of a transcellular cytoskeleton, RBC shape is determined by the membrane skeleton, a network of actin filaments cross-linked by spectrin and attached to membrane proteins. While the physical properties of a uniformly distributed actin network interacting with the lipid bilayer membrane have been assumed to control RBC shape, recent experiments reveal that RBC biconcave shape also depends on the contractile activity of nonmuscle myosin IIA (NMIIA) motor proteins. Here, we use the classical Helfrich-Canham model for the RBC membrane to test the role of heterogeneous force distributions along the membrane and mimic the contractile activity of sparsely distributed NMIIA filaments. By incorporating this additional contribution to the Helfrich-Canham energy, we find that the RBC biconcave shape depends on the ratio of forces per unit volume in the dimple and rim regions of the RBC. Experimental measurements of NMIIA densities at the dimple and rim validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple than the rim to produce the observed membrane curvatures. Furthermore, we predict that RBC membrane tension and the orientation of the applied forces play important roles in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance may also have implications for shape maintenance in different cell types., Competing Interests: The authors have declared that no competing interests exist.

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ∼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane-protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.

The Band 3 (AE1, SLC4A1) membrane protein is found in red blood cells and in kidney where it functions as an electro-neutral chloride/bicarbonate exchanger. In this study, we have used molecular dynamics simulations to provide the first realistic model of the dimeric membrane domain of human Band 3 in an asymmetric lipid bilayer containing a full complement of phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2) and cholesterol, and its partner membrane protein Glycophorin A (GPA). The simulations show that the annular layer in the inner leaflet surrounding Band 3 was enriched in phosphatidylserine and PIP2 molecules. Cholesterol was also enriched around Band 3 but also at the dimer interface. The interaction of these lipids with specific sites on Band 3 may play a role in the folding and function of this anion transport membrane protein. GPA associates with Band 3 to form the Wright (Wr) blood group antigen, an interaction that involves an ionic bond between Glu658 in Band 3 and Arg61 in GPA. We were able to recreate this complex by performing simulations to allow the dimeric transmembrane portion of GPA to interact with Band 3 in a model membrane. Large-scale simulations showed that the GPA dimer can bridge Band 3 dimers resulting in the dynamic formation of long strands of alternating Band 3 and GPA dimers., Competing Interests: The authors have declared that no competing interests exist.

Atomistic simulations have recently been shown to be sufficiently accurate to reversibly fold globular proteins and have provided insights into folding mechanisms. Gaining similar understanding from simulations of membrane protein folding and association would be of great medical interest. All-atom simulations of the folding and assembly of transmembrane protein domains are much more challenging, not least due to very slow diffusion within the lipid bilayer membrane. Here, we focus on a simple and well-characterized prototype of membrane protein folding and assembly, namely the dimerization of glycophorin A, a homodimer of single transmembrane helices. We have determined the free energy landscape for association of the dimer using the CHARMM36 force field. We find that the native structure is a metastable state, but not stable as expected from experimental estimates of the dissociation constant and numerous experimental structures obtained under a variety of conditions. We explore two straightforward approaches to address this problem and demonstrate that they result in stable dimers with dissociation constants consistent with experimental data.

Erythrocyte binding antigens 175 (EBA-175) and 140 (EBA-140) play key roles in erythrocyte invasion by binding to glycophorin A (GPA) and C (GPC) respectively in human malaria. Since antigenic variation in malaria endemic region is a major barrier to development of effective vaccine, we explore the nature and pattern of sequence diversity of these two vaccine candidates in Kolkata, India. Population genetic parameters based on parasite sequences representing region II of Pfeba-175 and Pfeba-140 genes were estimated using DnaSP V.5.10 and MEGA version 6.0. A novel molecular docking approach was implemented to assess the binding affinities of Kolkata Pfeba-175 variants with GPA. P. falciparum Kolkata isolates experienced a recent population expansion as documented by negative Tajima's D, Fu & Li's statistics, unimodal mismatch distribution and star-like median-joining network for both loci. Positive selection seemed to play a major role in shaping the diversity of Pfeba-175 (d N /d S =2.45, and McDonald-Kreitman P-value=0.04) with successive accumulation of Q584K/E, E592A and R664S deriving high frequency haplotypes designated here as F2KH3 and F2KH1. In silico molecular docking demonstrated that polypeptides encoded by F2KH1 and F2KH3 were capable of engaging the parasite ligand into energetically favorable interaction with GPA. Our data demonstrated emergence of Pfeba-175 sequences harboring selectively advantageous nonsynonymous substitutions on Pf3D7 sequence background in the Kolkata parasite isolates. A contrasting pattern of Pf3D7-centric expansion of parasite sequences was noted for Pfeba-140. Together, this study provides a firm genetic and biological support favoring a dominant role of EBA-175 in erythrocyte invasion., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The GxxxG motif is frequently found at the dimerization interface of a transmembrane structural motif called GAS right , which is characterized by a short interhelical distance and a right-handed crossing angle between the helices. In GAS right dimers, such as glycophorin A (GpA), BNIP3, and members of the ErbB family, the backbones of the helices are in contact, and they invariably display networks of 4 to 8 weak hydrogen bonds between Cα-H carbon donors and carbonyl acceptors on opposing helices (Cα-H···O═C hydrogen bonds). These networks of weak hydrogen bonds at the helix-helix interface are presumably stabilizing, but their energetic contribution to dimerization has yet to be determined experimentally. Here, we present a computational and experimental structure-based analysis of GAS right dimers of different predicted stabilities, which show that a combination of van der Waals packing and Cα-H hydrogen bonding predicts the experimental trend of dimerization propensities. This finding provides experimental support for the hypothesis that the networks of Cα-H hydrogen bonds are major contributors to the free energy of association of GxxxG-mediated dimers. The structural comparison between groups of GAS right dimers of different stabilities reveals distinct sequence as well as conformational preferences. Stability correlates with shorter interhelical distances, narrower crossing angles, better packing, and the formation of larger networks of Cα-H hydrogen bonds. The identification of these structural rules provides insight on how nature could modulate stability in GAS right and finely tune dimerization to support biological function.

Lectin-glycan interactions play important roles in many biological systems, but the nature of glycoprotein counter-receptors expressed on cell membranes is often poorly understood. To help overcome this problem, we developed a method based on proximity labeling technology. Using a peroxidase-coupled lectin, addition of H2O2 and tyramide-biotin substrates leads to generation of short-range biotin radicals that biotinylate proteins in the immediate vicinity of the bound lectin, which can subsequently be identified. As a proof-of-principle, sialoadhesin-horseradish peroxidase-human IgG1 Fc recombinant protein constructs were precomplexed with anti-Fc antibodies, bound to human erythrocytes and reacted with H2O2 and tyramide-SS-biotin. The erythrocyte membrane protein with strongest biotinylation was identified as glycophorin A, in agreement with early studies using lectin overlay and reglycosylation approaches. As a further test of the method, the plant lectin MAL II was conjugated with horseradish peroxidase and used in proximity labeling of human erythrocytes. Glycophorin A was again selectively labeled, which is consistent with previous reports that MAL II has high affinity for glycophorin. This method could be applied to other lectins to identify their membrane counter-receptors., (© The Author 2017. Published by Oxford University Press.)

The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria., (Copyright © 2017, American Association for the Advancement of Science.)

For many membrane proteins, self-association serves both structural and functional roles. Studies of such association can be simplified by switching to micelles as the membrane-mimicking environment, but native interaction is not preserved in all detergents. The selection of suitable conditions for biochemical experiments would be greatly facilitated by a quantitative high-throughput assay. Here we showed that the fluorescence polarization reduction, which resulted from homo-Förster resonance energy transfer and was measured in a high-throughput compatible format, can be used to determine both association states and constants for membrane proteins in micelles.

The assembly of integral membrane protein complexes is frequently supported by transmembrane domain (TMD) interactions. Here, we present the BLaTM assay that measures homotypic as well as heterotypic TMD-TMD interactions in a bacterial membrane. The system is based on complementation of β-lactamase fragments genetically fused to interacting TMDs, which confers ampicillin resistance to expressing cells. We validated BLaTM by showing that the assay faithfully reports known sequence-specific interactions of both types. In a practical application, we used BLaTM to screen a focussed combinatorial library for heterotypic interactions driven by electrostatic forces. The results reveal novel patterns of ionizable amino acids within the isolated TMD pairs. Those patterns indicate that formation of heterotypic TMD pairs is most efficiently supported by closely spaced ionizable residues of opposite charge. In addition, TMD heteromerization can apparently be driven by hydrogen bonding between basic or between acidic residues.

Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides.

Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016., (© 2015 Wiley Periodicals, Inc.)

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

The mechanisms of assembly and function for many important type I/II (single-pass) transmembrane (TM) receptors are proposed to involve the formation and/or alteration of specific interfaces among their membrane-embedded α-helical TM domains. The application of lipidic cubic phase (LCP) bilayer media for crystallization of single-α-helical TM complexes has the potential to provide valuable structural and mechanistic insights into many such systems. However, the fidelity of the interfaces observed in crowded crystalline arrays has been difficult to establish from the very limited number of such structures determined using X-ray diffraction data. Here we examine this issue using the glycophorin A (GpA) model system, whose homodimeric TM helix interface has been characterized by solution and solid-state NMR and biochemical techniques but never crystallographically. We report that a GpA-TM peptide readily crystallized in a monoolein cubic phase bilayer, yielding a dimeric α-helical structure that is in excellent agreement with previously reported NMR measurements made in several different types of host media. These results provide compelling support for the wider application of LCP techniques to enable X-ray crystallographic analysis of single-pass TM interactions.

We present an efficient all-atom/coarse-grained hybrid model and apply it to membrane processes. This model is an extension of the all-atom/ELBA model applied previously to processes in water. Here, we improve the efficiency of the model by implementing a multiple-time step integrator that allows the atoms and the coarse-grained beads to be propagated at different timesteps. Furthermore, we fine-tune the interaction between the atoms and the coarse-grained beads by computing the potential of mean force of amino acid side chain analogs along the membrane normal and comparing to atomistic simulations. The model was independently validated on the calculation of small-molecule partition coefficients. Finally, we apply the model to membrane peptides. We studied the tilt angle of the Walp23 and Kalp23 helices in two different model membranes and the stability of the glycophorin A dimer. The model is efficient, accurate, and straightforward to use, as it does not require any extra interaction particles, layers of atomistic solvent molecules or tabulated potentials, thus offering a novel, simple approach to study membrane processes.

The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association.

Membranes are central for cells as borders to the environment or intracellular organelle definition. They are composed of and harbor different molecules like various lipid species and sterols, and they are generally crowded with proteins. The membrane system is very dynamic and components show lateral, rotational and translational diffusion. The consequence of the latter is that phase separation can occur in membranes in vivo and in vitro. It was documented that molecular dynamics simulations of an idealized plasma membrane model result in formation of membrane areas where either saturated lipids and cholesterol (liquid-ordered character, Lo) or unsaturated lipids (liquid-disordered character, Ld) were enriched. Furthermore, current discussions favor the idea that proteins are sorted into the liquid-disordered phase of model membranes, but experimental support for the behavior of isolated proteins in native membranes is sparse. To gain insight into the protein behavior we built a model of the red blood cell membrane with integrated glycophorin A dimer. The sorting and the dynamics of the dimer were subsequently explored by coarse-grained molecular dynamics simulations. In addition, we inspected the impact of lipid head groups and the presence of cholesterol within the membrane on the dynamics of the dimer within the membrane. We observed that cholesterol is important for the formation of membrane areas with Lo and Ld character. Moreover, it is an important factor for the reproduction of the dynamic behavior of the protein found in its native environment. The protein dimer was exclusively sorted into the domain of Ld character in the model red blood cell plasma membrane. Therefore, we present structural information on the glycophorin A dimer distribution in the plasma membrane in the absence of other factors like e.g. lipid anchors in a coarse grain resolution.

Interactions between membrane proteins are of great biological significance and are consequently an important target for pharmacological intervention. Unfortunately, it is still difficult to obtain detailed views on such interactions, both experimentally, where the environment hampers atomic resolution investigation, and computationally, where the time and length scales are problematic. Coarse grain simulations have alleviated the later issue, but the slow movement through the bilayer, coupled to the long life times of nonoptimal dimers, still stands in the way of characterizing binding distributions. In this work, we present DAFT, a Docking Assay For Transmembrane components, developed to identify preferred binding orientations. The method builds on a program developed recently for generating custom membranes, called insane (INSert membrANE). The key feature of DAFT is the setup of starting structures, for which optimal periodic boundary conditions are devised. The purpose of DAFT is to perform a large number of simulations with different components, starting from unbiased noninteracting initial states, such that the simulations evolve collectively, in a manner reflecting the underlying energy landscape of interaction. The implementation and characteristic features of DAFT are explained, and the efficacy and relaxation properties of the method are explored for oligomerization of glycophorin A dimers, polyleucine dimers and trimers, MS1 trimers, and rhodopsin dimers. The results suggest that, for simple helices, such as GpA and polyleucine, in POPC/DOPC membranes series of 500 simulations of 500 ns each allow characterization of the helix dimer orientations and allow comparing associating and nonassociating components. However, the results also demonstrate that short simulations may suffer significantly from nonconvergence of the ensemble and that using too few simulations may obscure or distort features of the interaction distribution. For trimers, simulation times exceeding several microseconds appear needed, due to the increased complexity. Similarly, characterization of larger proteins, such as rhodopsin, takes longer time scales due to the slower diffusion and the increased complexity of binding interfaces. DAFT and its auxiliary programs have been made available from http://cgmartini.nl/ , together with a working example.

Glycophorins C and D are highly glycosylated integral sialoglycoproteins of human red blood cell membranes carrying the Gerbich blood group antigens. The O- and N-glycosidic chains of the major erythrocyte glycoprotein (Lisowska E. 2001, Antigenic properties of human glycophorins - an update. Adv Exp Med Biol, 491:155-169; Tomita M and Marchesi VT. 1975, Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci USA, 72:2964-2968.) are well characterized but the structure of GPC N-glycans has remained unknown. This problem became important since it was reported that GPC N-glycans play an essential role in the interaction with Plasmodium falciparum EBA-140 merozoite ligand. The elucidation of these structures seems essential for full characterization of the GPC binding site for the EBA-140 ligand. We have employed detailed structural analysis using sequential mass spectrometry to show that many GPC N-glycans contain H2 antigen structures and several contain polylactosamine structures capped with fucose. The results obtained indicate structural heterogeneity of the GPC N-glycans and show the existence of structural elements not found in glycophorin A N-glycans. Our results also open a possibility of new interpretation of the data concerning the binding of P. falciparum EBA-140 ligand to GPC. We hypothesize that preferable terminal fucosylation of N-glycosidic chains containing repeating lactosamine units of the GPC Gerbich variant could be an explanation for why the EBA-140 ligand does not react with GPC Gerbich and an indication that the EBA-140 interaction with GPC is distinctly dependent on the GPC N-glycan structure., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA- band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs., (© 2015 by The American Society of Hematology.)

As traditional detergents might destabilize or even denature membrane proteins, amphiphilic polymers have moved into the focus of membrane-protein research in recent years. Thus far, Amphipols are the best studied amphiphilic copolymers, having a hydrophilic backbone with short hydrophobic chains. However, since stabilizing as well as destabilizing effects of the Amphipol belt on the structure of membrane proteins have been described, we systematically analyze the impact of the most commonly used Amphipol A8-35 on the structure and stability of a well-defined transmembrane protein model, the glycophorin A transmembrane helix dimer. Amphipols are not able to directly extract proteins from their native membranes, and detergents are typically replaced by Amphipols only after protein extraction from membranes. As Amphipols form mixed micelles with detergents, a better understanding of Amphipol-detergent interactions is required. Therefore, we analyze the interaction of A8-35 with the anionic detergent sodium dodecyl sulfate and describe the impact of the mixed-micelle-like system on the stability of a transmembrane helix dimer. As A8-35 may highly stabilize and thereby rigidify a transmembrane protein structure, modest destabilization by controlled addition of detergents and formation of mixed micellar systems might be helpful to preserve the function of a membrane protein in Amphipol environments.

The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA.

Structural information of a transmembrane (TM) helix dimer is useful in understanding molecular mechanisms of important biological phenomena such as signal transduction across the cell membrane. Here, we describe an umbrella sampling (US) scheme for predicting the structure of a TM helix dimer in implicit membrane using the interhelical crossing angle and the TM-TM relative rotation angles as the reaction coordinates. This scheme conducts an efficient conformational search on TM-TM contact interfaces, and its robustness is tested by predicting the structures of glycophorin A (GpA) and receptor tyrosine kinase EphA1 (EphA1) TM dimers. The nuclear magnetic resonance (NMR) structures of both proteins correspond to the global free-energy minimum states in their free-energy landscapes. In addition, using the landscape of GpA as a reference, we also examine the protocols of temperature replica-exchange molecular dynamics (REMD) simulations for structure prediction of TM helix dimers in implicit membrane. A wide temperature range in REMD simulations, for example, 250-1000 K, is required to efficiently obtain a free-energy landscape consistent with the US simulations. The interhelical crossing angle and the TM-TM relative rotation angles can be used as reaction coordinates in multidimensional US and be good measures for conformational sampling of REMD simulations., (Copyright © 2013 Wiley Periodicals, Inc.)

Erythropoietin is a signaling glycoprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domains. Here we describe the total synthesis of homogeneous erythropoietin with consensus carbohydrate domains incorporated at all of the native glycosylation sites. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild-type primary sequence, themselves obtained by solid-phase peptide synthesis. The glycopeptidyl constructs were joined by chemical ligation, followed by metal-free dethiylation, and subsequently folded. This homogeneous erythropoietin glycosylated at the three wild-type aspartates with N-linked high-mannose sialic acid-containing oligosaccharides and O-linked glycophorin exhibits Procrit-level in vivo activity in mice.

Membrane protein interactions, which underlie biological function, take place in the complex cellular membrane environment. Plasma membrane derived vesicles are a model system which allows the interactions between membrane proteins to be studied without the need for their extraction, purification, and reconstitution into lipid bilayers. Plasma membrane vesicles can be produced from different cell lines and by different methods, providing a rich variety of native-like model systems. With these choices, however, questions arise as to how the different types of vesicle preparations affect the interactions between membrane proteins. Here we address this question using the glycophorin A transmembrane domain (GpA) as a model system. We compare the dimerization of GpA in six different vesicle preparations derived from Chinese hamster ovary (CHO), Human Embryonic Kidney 293T (HEK 293T) and A431 cells. We accomplish this with the use of a FRET-based method which yields the FRET efficiency, the donor concentration, and the acceptor concentration in each vesicle. We show that the vesicle preparation protocol has no statistically significant effect on GpA dimerization. Based on these results, we propose that any of the six plasma membrane preparations investigated here can be used as a model system for studies of membrane protein interactions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Giant unilamellar vesicles (GUVs) are an attractive experimental model for studying various membrane-related phenomena. The procedure for GUV electroformation from erythrocyte ghosts under physiological conditions was introduced recently; however, it allows preparation of a limited number of GUVs. Here we describe an efficient, reliable, and simple method for electroformation of GUVs from native erythrocyte membranes at low salt concentration, which enables the formation of higher amounts of large, spherical GUVs. GUVs prepared according to the new procedure may not retain original lipid asymmetry; however, they preserved native proteins, lipids, and oligosaccharide heterogeneity and could be a suitable system for functional studies for which larger amounts of GUVs of complex composition are needed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183-190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a "click chemistry" reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175- glycophorin A complex.

Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.

The organization of the plasma membrane is both highly complex and highly dynamic. One manifestation of this dynamic complexity is the lateral mobility of proteins within the plane of the membrane, which is often an important determinant of intermolecular protein-binding interactions, downstream signal transduction, and local membrane mechanics. The mode of membrane protein mobility can range from random Brownian motion to immobility and from confined or restricted motion to actively directed motion. Several methods can be used to distinguish among the various modes of protein mobility, including fluorescence recovery after photobleaching, single-particle tracking, fluorescence correlation spectroscopy, and variations of these techniques. Here, we present both a brief overview of these methods and examples of their use to elucidate the dynamics of membrane proteins in mammalian cells-first in erythrocytes, then in erythroblasts and other cells in the hematopoietic lineage, and finally in non-hematopoietic cells. This multisystem analysis shows that the cytoskeleton frequently governs modes of membrane protein motion by stably anchoring the proteins through direct-binding interactions, by restricting protein diffusion through steric interactions, or by facilitating directed protein motion. Together, these studies have begun to delineate mechanisms by which membrane protein dynamics influence signaling sequelae and membrane mechanical properties, which, in turn, govern cell function., (© 2013 Elsevier Inc. All rights reserved.)

Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cytochrome b(559)' is a transmembrane protein formed by homodimerization of the 44-residue PsbF polypeptide and noncovalent binding of a heme cofactor. The PsbF polypeptide can dimerize in the absence and presence of heme. To monitor structural alterations associated with binding of heme to the apo-cytochrome, we analyzed the apo- and holo-cytochrome structure by electron paramagnetic resonance spectroscopy. Spin labeling of amino acids located close to the heme binding domain of the cytochrome revealed that the structure of the heme binding domain is unconstrained in the absence of heme. Heme binding restricts the conformational dynamics of the heme binding domain, resulting in the structurally more constricted holo-cytochrome structure.

Interaction of protein 4.1 (4.1R) with the transmembrane protein glycophorin C (GPC) regulates the functions of erythrocyte membrane. Fluorescence correlation spectroscopy (FCS) was used to define the interaction of EGFP-4.1R with DsRed-GPC on transport vesicles (TVs) by measuring their fluctuation in living cells. DsRed-GPC expressed in HeLa cells was delivered to the plasma membrane through slow vesicle transport. EGFP-4.1R, which freely diffused in the cytosol when expressed alone, diffused slowly when co-expressed with DsRed-GPC, indicating association of EGFP-4.1R with TVs. Fluorescence cross-correlation spectroscopy (FCCS) showed direct interaction of EGFP-4.1R with DsRed-GPC on TVs. The present study demonstrates that 4.1R binds to GPC on TVs in living cells., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background and Objectives: Appropriate screening for irregular red-cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells., Materials and Methods: We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of 143 501 patients tested from 2004 to 2010 were retrieved and divided into two groups: period-1 (2004-2008) and period-2 (2009-2010). During period-1, standard screening cells were used while in period-2, MUT+Mur+ KODE(™) transformed red cells (kodecytes) were used., Results: Four per cent of samples tested during period-2 were positive on antibody screening compared to 3·2% in period-1. Specific antibodies, excluding anti-D, were identified in 1·66% and 1·52% of patients in period-2 and -1, respectively. When confined to antibodies of clinical significance only, period-2 showed higher alloantibody prevalence of 1·16% as compared to 0·66% in period-1. Antibodies to glycophorin variants of MNS (vMNS) were more commonly detected while antibodies to Lewis antigens declined during period-2., Conclusion:   Antibodies to vMNS antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red-cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein-detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.

Membrane proteins have evolved to fold and function in a lipid bilayer, so it is generally assumed that their stability should be optimized in a natural membrane environment. Yet optimal stability is not always in accord with optimization of function, so evolutionary pressure, occurring in a complex membrane environment, may favor marginal stability. Here, we find that the transmembrane helix dimer, glycophorin A (GpATM), is actually much less stable in the heterogeneous environment of a natural membrane than it is in model membranes and even common detergents. The primary destabilizing factors are electrostatic interactions between charged lipids and charged GpATM side chains, and nonspecific competition from other membrane proteins. These effects overwhelm stabilizing contributions from lateral packing pressure and excluded volume. Our work illustrates how evolution can employ membrane composition to modulate protein stability.

Malaria has been a very strong selection pressure in recent human evolution, particularly in Africa. Of the one million deaths per year due to malaria, more than 90% are in sub-Saharan Africa, a region with high levels of genetic variation and population substructure. However, there have been few studies of nucleotide variation at genetic loci that are relevant to malaria susceptibility across geographically and genetically diverse ethnic groups in Africa. Invasion of erythrocytes by Plasmodium falciparum parasites is central to the pathology of malaria. Glycophorin A (GYPA) and B (GYPB), which determine MN and Ss blood types, are two major receptors that are expressed on erythrocyte surfaces and interact with parasite ligands. We analyzed nucleotide diversity of the glycophorin gene family in 15 African populations with different levels of malaria exposure. High levels of nucleotide diversity and gene conversion were found at these genes. We observed divergent patterns of genetic variation between these duplicated genes and between different extracellular domains of GYPA. Specifically, we identified fixed adaptive changes at exons 3-4 of GYPA. By contrast, we observed an allele frequency spectrum skewed toward a significant excess of intermediate-frequency alleles at GYPA exon 2 in many populations; the degree of spectrum distortion is correlated with malaria exposure, possibly because of the joint effects of gene conversion and balancing selection. We also identified a haplotype causing three amino acid changes in the extracellular domain of glycophorin B. This haplotype might have evolved adaptively in five populations with high exposure to malaria., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

In criminal investigations, there are three stages involved when studying bloodstains: search and orientation, confirmation, and individualization. Confirmatory tests have two aims: to show that the stain contains a human biological fluid and to confirm the type of biological fluid. The need to determine the nature of the evidence is reflected in the latest bibliography, where the possibility of employing mRNA and miRNA markers for this purpose is proposed. While these new proposals are being investigated, the kits for determining human hemoglobin currently provide a simple solution for resolving this issue. With these kits, the possibility of obtaining false positives and false negatives is well known. However, recently, a new problem has been detected. This involves the interference caused by new cleaning products that contain sodium percarbonate (or active oxygen) when determining human hemoglobin. With the aim to resolve this problem, this work studied the ability of the human glycophorin A test to determine human blood in samples that have been treated with active oxygen. Our results show that the human glycophorin A test has a greater resistance to the destructive effect of the new detergents containing active oxygen; consequently, it provides an alternative to be taken into consideration in the confirmatory diagnoses of bloodstains.

The MNS system was the second blood group system discovered and at least 16 of the 46 antigens in the MNS system result from genetic recombination, producing a hybrid glycophorin. The incidence of these hybrid glycophorins is highest in East Asian populations. MNS system antigens defined by hybrid glycophorins are immunogenic with alloimmune IgG responses developing after transfusion or pregnancy; with reports originating from Asia, Europe, the Americas, and Australia. This demonstrates the global nature of problems associated with these antibodies. Since the initial report that production of anti-Mi(a) was a cause of hemolytic disease of the fetus and newborn (HDFN), antibodies to antigens defined by hybrid glycophorins have been reported in 27 cases of HDFN (1 fatal) and 8 cases of hemolytic transfusion reaction (HTR) (1 fatal). In at least 40% of these clinical cases, the disease was reported as severe. Hyporegenerative fetal anemia is a common feature of the reported HDFN cases. In all published cases, the causative antibodies were identified by reference laboratory investigative tests following clinical presentation. The failure to detect these antibodies by routine testing highlights the need for consideration of the medical importance of these antibodies when defining antibody screening practices and reagents. The aim of this review is to raise awareness of severe disease caused by antibodies to MNS antigens defined by hybrid glycophorins and, thus, to improve diagnosis and patient management., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Object: Preoperative clinical risk classification of carotid artery (CA) stenosis anticipates the outcome of CA intervention. A higher incidence of neurological morbidity was noted after CA stenting (CAS) in patients with medical risks than in those without risks. However, little is known about the correlation between clinical risks and plaque composition. The purpose of this study was to characterize the CA plaque histology in 3 groups of patients who were classified based on clinical risks for carotid endarterectomy (CEA). Furthermore, the authors examined whether the plaque with high embolic potential after CA intervention, particularly CAS, could be predicted based on clinical risks for CEA., Methods: Patients were divided into 4 groups, according to the CEA risk classification system, and 3 groups with more than 10 cases were enrolled in this study as follows: absence of all angiographic, medical, and neurological risks (Grade I, 27 cases); presence of medical risk, but no neurological risk (Grade III, 31 cases); and presence of neurological risk (Grade IV, 17 cases). Histopathological characteristics of CA plaques, including fibrous cap thickness, plaque disruption, thrombus formation, intraplaque hemorrhage (IPH), and adipophilin expression were examined without information regarding clinical status., Results: Plaques in patients in Grades III and IV demonstrated a thin fibrous cap and enhanced IPH, compared with those in Grade I. Plaques in patients in Grade IV showed more adipophilin-expressing macrophages in the fibrous cap than in those of the other groups., Conclusions: Plaques in Grades III and IV patients were characterized by thin fibrous cap atheroma with IPH. Adipophilin-positive macrophage infiltration in the fibrous cap might be correlated with instability in neurological status. The plaque morphology in patients with medical and neurological risks needs to be examined carefully with the aid of imaging modalities. In plaques demonstrating a thin fibrous cap and IPH, the CAS procedure should be avoided and CEA should be performed instead.