Major Membrane Protein Research Articles

Immunomodulatory Nanoparticles from Elastin-Like Recombinamers: Single-Molecules for Tuberculosis Vaccine Development

This study investigates both the physicochemical properties and immunogenicity of a genetically engineered elastin-like block corecombinamer (ELbcR) containing a major membrane protein sequence from Mycobacterium tuberculosis. The recombinant production of this ELbcR allows the production of large quantities of safe, antigenic particle-based constructs that directly and reversibly self-assemble into highly biocompatible, multivalent, monodisperse, and stable nanovesicles with a diameter of 55 nm from the same gene product using a highly efficient and cost-effective inverse transition cycling (ITC) procedure. The compositional complexity of these vesicles is retained after secondary processes such as endotoxin removal, sterilization, and lyophilization. An initial pro-chemotactic cytokine response (IL-1β) followed by a pro-Th2/IL-5 response was observed in mice plasma following subcutaneous administration of the antigen-loaded nanovesicles in mice. This biphasic model of cytokine production was coupled with humoral isotype switching from IgM- to IgG-specific antibodies against the antigen, which was only observed in the presence of both the antigen and the polymer in the same construct and in the absence of additional adjuvants.

結核ワクチン作製の試み

A novel recombinant BCG (BCG-DHTM), that was deficient in urease, expressed with gene encoding the fusion of BCG-derived HSP70 and M. tuberculosis-derived major membrane protein (MMP)-II, was constructed for use as a vaccine against tuberculosis. BCG-DHTM efficiently activated dendritic cells (DC) to induce cytokine production, including IL-12, TNFalpha and IL-1beta and phenotypic changes. The DC infected BCG-DHTM was more potent in activation of native T cells of CD4 and CD8 subsets than those infected vector control BCG. The activation of naïve T cells by BCG-DHTM was closely associated with phagomal maturation, and that of naïve CD8+ T cells by BCG-DHTM was induced by the activation of cytosolic cross-presentation pathway. Further, BCG-DHTM seemed to activate native CD4+ T cells and native CD8+ T cells by antigen-specific fashion. The primary infection of BCG-DHTM in C57BL/6 mice for 12 weeks efficiently produced T cells responsive to in vitro secondary stimulation with MMP-II, HSP70 and H37Rv-derived cytosolic protein and inhibited with multiplication of subsequently challenged M. tuberculosis in lungs at least partially. The effect of BCG-DHTM as a vaccine for tuberculosis is not fully convincing and need the improvement, however, our strategy in the development of new recombinant BCG for tuberculosis seems to provide useful tool.

Parallel regulation of renin and lysosomal integral membrane protein 2 in renin-producing cells: further evidence for a lysosomal nature of renin secretory vesicles

The protease renin is the key enzyme in the renin-angiotensin system (RAS) that regulates extracellular volume and blood pressure. Renin is synthesized in renal juxtaglomerular cells (JG cells) as the inactive precursor prorenin. Activation of prorenin by cleavage of the prosegment occurs in renin storage vesicles that have lysosomal properties. To characterize the renin storage vesicles more precisely, the expression and functional relevance of the major lysosomal membrane proteins lysosomal-associated membrane protein 1 (LAMP-1), LAMP-2, and lysosomal integral membrane protein 2 (LIMP-2) were determined in JG cells. Immunostaining experiments revealed strong coexpression of renin with the LIMP-2 (SCARB2), while faint staining of LAMP-1 and LAMP-2 was detected in some JG cells only. Stimulation of the renin system (ACE inhibitor, renal hypoperfusion) resulted in the recruitment of renin-producing cells in the afferent arterioles and parallel upregulation of LIMP-2, but not LAMP-1 or LAMP-2. Despite the coregulation of renin and LIMP-2, LIMP-2-deficient mice had normal renal renin mRNA levels, renal renin and prorenin contents, and plasma renin and prorenin concentrations under control conditions and in response to stimulation with a low salt diet (with or without angiotensin-converting enzyme (ACE) inhibition). No differences in the size or number of renin vesicles were detected using electron microscopy. Acute stimulation of renin release by isoproterenol exerted similar responses in both genotypes in vivo and in isolated perfused kidneys. Renin and the major lysosomal protein LIMP-2 are colocalized and coregulated in renal JG cells, further corroborating the lysosomal nature of renin storage vesicles. LIMP-2 does not appear to play an obvious role in the regulation of renin synthesis or release.

Loss of XRN4 Function Can Trigger Cosuppression in a Sequence-Dependent Manner

OLE1 encodes an oleosin isoprotein, a major membrane protein of the lipid-reserve organelle in seeds known as the oil body. Transgenic Arabidopsis were generated to contain an artificial chimeric transgene composed of OLE1 and green fluorescent protein (GFP). Overexpression of the fusion protein allowed visualization of the oil body size and structure in living cells using fluorescence microscopy. Two mutants, xrn4-8(OleG) and xrn4-9(OleG), accumulating enlarged oil bodies with reduced GFP fluorescence were isolated from the mutagenized progeny of a transgenic plant. Both mutants contained a defect in EXORIBONUCLEASE4 (XRN4), a gene known to encode a ribonuclease that specifically degrades uncapped mRNAs. Transgene expression was silenced in these mutants, as demonstrated by the reduced levels of the transgene mRNA and its product, OLE1-GFP. XRN4 loss of function also triggered cosuppression, i.e. simultaneous reduction in expression of the transgene and an endogenous OLE1 gene that shared a region of identical sequence. The enlarged oil bodies exhibiting reduced GFP fluorescence were formed in the xrn4-8(OleG) and xrn4-9(OleG) mutants due to the reduction of the endogenous OLE1 and the transgene product, OLE1-GFP, respectively. Cosuppression triggered by the xrn4 mutation also occurs for other genes such as PYK10, which encodes an endoplasmic reticulum (ER) body-resident β-glucosidase. The overall results indicate that a loss of XRN4 function can potentially trigger the cosuppression in a sequence-dependent manner.

Implication of perturbed axoglial apparatus in early pediatric multiple sclerosis

Cerebrospinal fluid samples collected from children during initial presentation of central nervous system inflammation, who may or may not subsequently be diagnosed as having multiple sclerosis (MS), were subjected to large-scale proteomics screening. Unexpectedly, major compact myelin membrane proteins typically implicated in MS were not detected. However, multiple molecules that localize to the node of Ranvier and the surrounding axoglial apparatus membrane were implicated, indicating perturbed axon-glial interactions in those children destined for diagnosis of MS.

Does Prohibitin Expression Regulate Sperm Mitochondrial Membrane Potential, Sperm Motility, and Male Fertility?

Prohibitin (PHB) is a highly conserved major sperm mitochondrial membrane protein whose absence in somatic cells is associated with mitochondrial membrane depolarization and increased generation of reactive oxygen species (ROS). Our recent findings suggest that high levels of oxidants in human semen may contribute to male infertility and that sperm motility could be the earliest and most sensitive indicator of oxidative damage. Based on PHB's roles in mitochondrial sub-compartmentalization and respiratory chain assembly, we examine sperm PHB expression and mitochondrial membrane potential (MITO) in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). Here, we demonstrate that MITO is significantly lower in sperm from A and OA subjects than in normospermic (N) subjects; the decrease is more severe for OA than for A subjects. PHB expression is also significantly lower in sperm from A and OA subjects. Significantly positive correlations are found among PHB expression, MITO, and sperm motility in normospermic, asthenospermic, and oligoasthenospermic subjects. Collectively, our observations lead to the hypothesis that PHB expression is an indicator of sperm quality in infertile men, and that it regulates sperm motility via an alteration in MITO and increased ROS levels.

Vaccinia Virus A6 Is Essential for Virion Membrane Biogenesis and Localization of Virion Membrane Proteins to Sites of Virion Assembly

Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.

Identification of major milk fat globule membrane proteins from pony mare milk highlights the molecular diversity of lactadherin across species

Although several studies have been devoted to the colloidal and soluble protein fractions of mare milk (caseins and whey proteins), to date little is known about the milk fat globule membrane (MFGM) protein fraction from mare milk. The objective of this study was thus to describe MFGM proteins from Equidae milk and to compare those proteins to already described MFGM proteins from cow and goat milk. Major MFGM proteins (namely, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already described in cow or goat milk were identified in mare milk using mass spectrometry. However, species-specific peculiarities were observed for 2 MFGM proteins: butyrophilin and lactadherin. A highly glycosylated 70-kDa protein was characterized for equine butyrophilin, whereas proteins of 64 and 67 kDa were characterized for cow and goat butyrophilin, respectively. Prominent differences across species were highlighted for lactadherin. Indeed, whereas 1 or 2 polypeptide chains were identified, respectively, by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis for caprine and bovine lactadherin, 4 isoforms (60, 57, 48, and 45 kDa) for lactadherin from mare milk were identified by 10% sodium dodecyl sulfate-PAGE. Polymerase chain reaction experiments on lactadherin transcripts isolated from milk fat globules revealed the existence of 2 distinct lactadherin transcripts in the horse mammary gland. Cloning and sequencing of both transcripts encoding lactadherin showed an alternative use of a cryptic splice site located at the end of intron 5 of the equine lactadherin-encoding gene. This event results in the occurrence of an additional alanine (A) residue in the protein that disrupts a putative atypical N-glycosylation site (VNGC/VNAGC) described in human lactadherin. Liquid chromatography coupled with tandem mass spectrometry analyses confirmed the existence of both lactadherin variants in mare MFGM. We show here that lactadherin from Equidae milk is much more complex than that from Bovidae milk (i.e., cow and goat milk), therefore raising questions regarding the precise function of these different isoforms, if any, in the equine mammary gland.

Arterivirus Minor Envelope Proteins Are a Major Determinant of Viral Tropism in Cell Culture

Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly.

未感作T細胞を強く活性化するウレアーゼ欠損HSP70-MMP-II融合蛋白産生性リコンビナントBCGの作出

To activate naïve T cells convincingly using Mycobacterium bovis BCG (BCG), rBCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4+ and CD8+ subsets of naïve T cells than rBCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DC) to induce cytokine production and phenotypic changes, and activated CD4+ T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pre-treatment of DC with chloroquine inhibited both surface expression of MMP-II on DC and the activation of T cells by BCG-D70M-infected APCs. The naïve CD8+ T cell activation was inhibited by treatment of DC with brefeldin A and lactacystin so that the T cells was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naïve CD8+ T cells, effector T cells producing perforin and memory T cells having migration markers, were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70, and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II and urease depletion may provide useful tool for inducing better activation of naïve T cells.

The sodium pump controls the frequency of action-potential-induced calcium oscillations

Calcium plays a significant role in a number of cellular processes, like muscle contraction, gene expression, synaptic plasticity, signal transduction, but the significance of calcium oscillations (CaOs) is not yet completely understood in most of the cell types. It is a widely accepted fact that CaOs are a frequency encoded signal that allows a cell to use calcium as a second messenger while avoiding its toxic effects. These intracellular CaOs are primarily driven by some agonist-dependent pathways or fluctuations in membrane potential. The present mathematical model is of the latter type. The model incorporates expression for all major intracellular ionic species and membrane proteins. Especially, it integrates the coupling effect of sodium pump and Na+ / Ca2+ exchanger over CaOs. By varying sodium pump current, it is found that, sodium pump is a key player in modulating intracellular CaOs. The model predicts that the sodium pump can play a decisive role in regulating intercellular cell signaling process. The present study forms the basis for sodium pump controlled intercellular signaling process and requires further experimental verification. Mathematical subject classification: 34M10, 92C20.

O-glycosylation modulates integrin and FGF signalling by influencing the secretion of basement membrane components

Extracellular microenvironments have crucial roles in modulating cell interactions during development. Here we discover that a conserved protein modification (O-glycosylation) influences extracellular matrix composition during mammalian organogenesis, affecting integrin signalling and fibroblast growth factor-mediated cell proliferation. Specifically, mice deficient for an enzyme (Galnt1) that adds sugars to proteins during early stages of organogenesis resulted in intracellular accumulation of major basement membrane proteins and endoplasmic reticulum stress, with resultant effects on fibroblast growth factor signalling, epithelial cell proliferation and organ growth. Exogenous addition of basement membrane components rescued fibroblast growth factor signalling and the growth defects in a β1-integrin-dependent manner. Our work demonstrates for the first time that O-glycosylation influences the composition of the extracellular matrix during mammalian organ development, influencing specific aspects of the endoplasmic reticulum stress response, cell signalling, cell proliferation and organ growth. Our work provides insight into the role of this conserved protein modification in both development and disease.

Characterization of the antigenic and functional domains of a Mycoplasma synoviae variant vlhA gene

The Mycoplasma synoviae haemagglutinin gene, vlhA, encodes two major immunodominant and surface-exposed membrane proteins, MSPB and MSPA. Both products are antigenically variable but only MSPA mediates binding to erythrocytes. Previously we have shown that M. synoviae type strain WVU 1853 could express a variant vlhA gene, referred to as MS2/28.1, with a considerably shorter and divergent MSPA region. A finding that prompted detailed characterization of its antigenic and functional properties. Here we mutagenized each of the six opal codons of the variant MS2/28.1 vlhA member into tryptophan, thus allowing its expression in Escherichia coli as well as its cleavage products, MSPB and MSPA. In addition, we expressed 5 contiguous regions of MS2/28.1 extending from the last part of MSPB to the C-terminus of MSPA. Colony immunostaining with region-specific antisera mapped antigenic variation to the N-terminal half of MS2/28.1 MSPA. No haemagglutinating activity was observed for MSPB, but consistent haemadsorption inhibition was mapped to the region extending from amino acid 325 to 344. Inhibition of both haemagglutination and haemadsorption activities were obtained with sera directed against the C-terminal region of MSPA, with the highest titers (1/320 and 1/160, respectively) being recorded for its last 60 residues. Importantly, antibodies to this region also yielded the highest metabolic inhibition titer of 1/1280. Overall, aside from mapping the functional domains of a M. synoviae highly divergent haemagglutinin gene, this study shows that the C-terminal half of its MSPA region induced the highest titers of antibodies inhibiting haemagglutination, haemadsorption, and metabolism.

Bovine milk exosome proteome

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.

Spatial analysis of human lens aquaporin-0 post-translational modifications by MALDI mass spectrometry tissue profiling

Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 μm. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.

Cloning, expression analysis, and sequence diversity of genes encoding two different immunodominant membrane proteins in poinsettia branch-inducing phytoplasma (PoiBI)

Poinsettia branch-inducing phytoplasma (PoiBI) is a phytopathogenic bacterium that infects poinsettia, and is associated with the free-branching morphotype (characterized by many axillary shoots and flowers) of many commercially grown poinsettias. The major membrane proteins of phytoplasmas are classified into three general types, that is, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). These membrane proteins are often used as targets for the production of antibodies used in phytoplasma detection. Herein, we cloned and sequenced the imp and idpA genes of PoiBI strains from 26 commercial poinsettia cultivars. Although the amino acid sequences of the encoded IdpA proteins were invariant, those of the encoded Imp varied among the PoiBI isolates, with no synonymous nucleotide substitution. Western blotting and immunohistochemical analyses revealed that the amount of Imp expressed exceeded that of IdpA, in contrast to the case of a related phytoplasma-disease, western X-disease, for which the major membrane protein appears to be IdpA, not Imp. These results suggest that even phylogenetically close phytoplasmas express different types of major membrane proteins.

Antifungal action of the oxathiolone‐fused chalcone derivative

AMG-148, an oxathiolone-fused chalcone derivative, exhibited in vitro antifungal activity against several strains of human pathogenic yeast, with minimum inhibitory concentration values within the range of 1-16 μg ml(-1) and a fungicidal effect was observed at higher concentrations. Presence of major drug-effluxing membrane proteins Cdr1p, Cdr2p or Mdr1p, did not affect substantially the fungistatic activity of this compound against clinical Candida albicans strains. Studies on the mode of action revealed that AMG-148 inhibited chitin and β(1→3)glucan biosynthesis and was in vitro an inhibitor of β(1→3)glucan synthase. Inhibition of chitin biosynthesis was responsible for fungistatic activity, while the fungicidal effect was a consequence of disturbance of β(1→3)glucan synthase function. The chalcone derivative may be a useful lead compound for the development of novel antifungal agents.

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.

Elastase release during the hemodialysis procedure seems to induce changes in red blood cell membrane proteins

To the Editor: Elastase is a serine proteinase, expressed mainly by neutrophils, and also by monocytes and mast cells. It is a component of the primary azurophil granules of the neutrophils. Within the cell, it is involved in the degradation of microorganisms that are phagocytosed, and, outside the cell, it may degrade local extracellular matrix proteins, favoring neutrophil migration into or through tissues. 1,2 Recently, we showed that the red blood cell (RBC) membrane protein composition is altered in hemodialysis (HD) patients, particularly in nonresponders to recombinant human erythropoietin (rhEPO) therapy. 3,4 We also showed that the HD procedure induces neutrophil activation. 5,6 Therefore, we hypothesized that the RBC changes could be due to the release of elastase by neutrophils during HD. Our aim was to study the effect of elastase in RBC membrane protein composition from healthy controls and from HD patients under rhEPO therapy (responders and non-responders) to strengthen this hypothesis. We performed in vitro assays using RBC from 18 HD patients (10 responders and 8 nonresponders) and of 8 healthy controls. Classification of the patients, as responders or non-responders, was performed in accordance with the European Best Practice Guidelines, which defines resistance to rhEPO as a failure to achieve target Hb levels (between 11 and 12g/dL) with maintained doses of rhEPO higher than 300IU/kg/wk of epoietin or 1.5mg/kg/wk of darbopoietin-a. The two groups of patients were matched for age, gender, weight, body mass index, mean time under HD, urea reduction ratio, urea Ktv, and parathyroid hormone serum levels. Hemodialysis patients (10 males, 8 females; mean age 62.1 15.7 years) were under therapeutic HD 3 times per week, for 3‐5 hours, for a median period of time of 35 months. All patients used the highflux polysulfone FX-class dialysers of Fresenius (Fresenius Medical Care, Bad Homburg, Germany). The causes of renal failure in patient’s population were as follows: diabetic nephropathy (n=5), chronic glomerulonephritis (n=3), polycystic kidney disease (n=2), hypertensive nephrosclerosis (n=2), obstructive nephropathy (n=1), and chronic renal failure of uncertain etiology (n=5). Patients with autoimmune disease, malignancy, hematological disorders, and acute or chronic infection were excluded. All patients gave their informed consent to participate in this study. In HD patients, this assay used RBCs collected before and immediately after the HD procedure. Red blood cell suspensions (5 10 8 cell/mL; 10mL PBS, pH 7.4) were incubated for 3 hours at 371C, under the following assay conditions: without elastase and with 0.03, 0.1, and 0.5mg/mL of elastase. After incubation, the relative amount of each major RBC membrane protein was evaluated by densitometry. 3 We found no significant differences in the membrane protein composition of the RBCs from healthy controls and from responders and nonresponders after the HD procedure, when incubated without and with different elastase concentrations. However, the RBCs from responders and nonresponders HD patients, collected before the HD procedure, showed some susceptibility to elastase; the RBCs from responders incubated with 0.5mg/mL of elastase showed a significant decrease in ankyrin [7.0 (6.5‐7.5%) vs. 6.0 (5.9‐6.5%), P=0.024] and trends toward a decrease in spectrin [25.6 (25.1‐ 26.9%) vs. 24.7 (24.4‐25.6%), P=0.073) and an increase in band 3 [36.6 (34.8‐37.6%) vs. 39.1 (36.9‐39.4%), P=0.077) as compared with RBCs incubated without elastase. Similar changes were found for the RBCs incubated with 0.1mg/mL of elastase. In nonresponder patients, the RBCs incubated with 0.1 and 0.5mg/mL of elastase showed a significant decrease in spectrin [25.5 (24.9‐25.9%) and 25.3 (24.8‐26.2%), respectively, vs.