Egg white lysozyme is the major protein of the hen's egg vitelline membrane

Lysozyme accounts for 37% of the proteins of the hen's egg vitelline membrane. It can be extracted by salt solutions and purified by gel filtration on Sephadex G-50. There are no differences between the chemical and enzymic properties of egg white and vitelline membrane lysozymes. Vitelline membranes of ovarian eggs do not contain lysozyme. It is thus concluded that lysozyme is localized in the outer layer. Vitelline membranes from fertilized and unfertilized eggs contain the same amount of lysozyme; its percentage decreases after two days of incubation.     

Rapid evolution of outer egg membrane proteins in the Drosophila melanogaster subgroup: a case of ecologically driven evolution of female reproductive traits

Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids, therefore leaving the possibility of rapid evolution through gametic interaction wide open. At this point, however, our results support previous suggestions that divergences in ecology, particularly, ovipositional substrate divergences may be a strong force driving the evolution of eggshell proteins.     

Penetration of Lysozyme and Cytochrome C in Lipid Bilayer: Fluorescent Study

Lysozyme and cytochrome c (CytC) are well-investigated proteins. Their specific interactions with lipid membranes, however, keep surprising secrets. Lysozyme destroys bacterial membrane; CytC binds hydrophobically to alkyl chains of the membrane lipid tails, indicating that both proteins are able to interact directly with the inner membrane components, especially with the fatty acyl chains of membrane lipids. The degrees of integration, depth of localization in the hydrophobic interior of different types of model membranes, and the type of interaction of lysozyme and CytC with surrounding lipids were investigated by fluorescent spectroscopy. Three different fluorescent markers, located at approximately 6.5, 9, and 18 Å into the lipid bilayer, were used. In addition, liposomes were designed as electrically neutral or positively or negatively charged to unravel the importance of the net electrical charge for lipid/protein interaction. CytC penetrates deeper into the lipid bilayer in comparison with lysozyme, and data are discussed in the terms of Stern–Volmer quenching of fluorescence.     

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila

Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.     

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.     

Germline and somatic vitelline proteins colocalize in aggregates in the follicular epithelium of Drosophila ovaries

Nasrat and Polehole, two Drosophila proteins related functionally and by sequence, are secreted from the oocyte and incorporated into the vitelline membrane, where they play a role in the integrity of the same and in the activation of embryonic Torso RTK. In addition, they also accumulate in a punctate pattern in the follicular epithelium. Here we show that their accumulation at the follicle cells depends on their gene expression in the germline, indicating that these proteins move from the oocyte to the follicle cells in a process that does not require endocytosis. Finally we used cell markers to examine the distribution of these proteins at the follicle cells and show they accumulated in aggregates with vitelline membrane proteins in close association with the plasmatic membrane. We propose that these aggregates represent spatially restricted sinks for vitelline membrane proteins that fail to be incorporated into vitelline bodies and later on into the vitelline membrane.     

Proteomic analysis of the chicken egg vitelline membrane

The avian vitelline membrane (VM) is a multilayered proteinaceous structure separating egg white from yolk. The innermost layer of the VM, deposited onto the oocyte plasma membrane in the ovary, corresponds to the mammalian zona pellucida (ZP). The outer layer is produced in the infundibulum, the first section of the oviduct. Using high-throughput, high-end LC-MS(n) 137 proteins were identified, only 13 of which were known previously to be components of the VM. Depending on the washing protocol, two largely overlapping, but not identical, sets of identified proteins were produced from water-washed and salt-washed VMs. Most of the components of the VM were known previously from other egg compartments, such as, for instance, the egg white proteins lysozyme C, ovalbumin, ovotransferrin, and ovomucin. Specific components of the VM not identified previously in other egg compartments included eight ZP proteins, oviductin protease, and two ATPases. The vitelline outer membrane protein (VMO) VMO II was identified as beta-defensin-11. The list of VM proteins presented in this report is by far the most comprehensive dataset available at present and complements proteomic analyses of chicken egg compartments published previously.     

A method for observing protein-protein interaction.

A method is proposed for observation of the interaction between charged macromolecules such as proteins. The method is based on the fact that the pK of an ionizable reporter group attached to a macromolecule can be altered by the electrostatic effect of another charged macromolecule which associates with the former. The effectiveness of the method was shown in the study of the association of bovine serum albumin with hen egg lysozyme [EC 3.2.1.17]. The errors inherent in this method in obtaining the equilibrium constant of the association reaction and procedures for their correction are discussed.     

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell.     

Interaction Between Equally Charged Membrane Surfaces Mediated by Positively and Negatively Charged Macro-Ions

In biological systems, charged membrane surfaces are surrounded by charged molecules such as electrolyte ions and proteins. Our recent experiments in the systems of giant phospholipid vesicles indicated that some of the blood plasma proteins (macro-ions) may promote adhesion between equally charged membrane surfaces. In this work, theory was put forward to describe an IgG antibody-mediated attractive interaction between negatively charged membrane surfaces which was observed in experiments on giant phospholipid vesicles with cardiolipin-containing membranes. The attractive interactions between negatively charged membrane surfaces in the presence of negatively and positively charged spherical macro-ions are explained using functional density theory and Monte Carlo simulations. Both, the rigorous solution of the variational problem within the functional density theory and the Monte Carlo simulations show that spatial and orientational ordering of macro-ions may give rise to an attractive interaction between negatively charged membrane surfaces. It is also shown that the distinctive spatial distribution of the charge within the macro-ions (proteins) is essential in this process.     

Electrostatic repulsion between molecules of like charge can be misinterpreted as binding

Spectroscopic methods have shown that Ca2+ chelators interact with Ca2(+)-binding proteins. These spectral alterations have been interpreted as evidence for the binding of chelator by the proteins. We show by direct examination of EDTA interaction with calmodulin and alpha-lactalbumin that these proteins repel EDTA rather than bind it. The repulsion is reduced by increased salt concentration but is unaffected by Ca2+ binding to the proteins. The acidic protein, alpha-lactalbumin, repells the negatively charged EDTA and inorganic phosphate whereas the basic protein, lysozyme, repells the positively charged spermine. Thus, spectroscopic changes induced by negatively charged Ca2+ chelators on negatively charged Ca2(+)-binding proteins are due to electrostatic repulsion, and not to binding. These observations underscore the possible pitfalls of using spectroscopic methods alone to analyze protein-ligand interactions.     

Purification of the basic protein avidin using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, pI 10, from chicken egg white. Using a charge based separation at pH 9.0, the high pI of avidin and lysozyme (pI 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60-65% from one egg white with minimal lysozyme activity detected.     

Interaction of the nuclear proteins protamine and histone with charged bilayer membranes]

The interaction of nuclear proteins of protamine and histone with neutral and charged BLM was studied. Anion and cation detergents were used to create the surface charge. The surface density of charges in BLM was comparable with that in biomembranes. Protamine and histone increased the electroconductivity of negatively charged BLM for anions and cations correspondingly. It is suggested that the surface charge of the membrane may influence the ion transport directly and indirectly due to the interaction of the membrane structures with charged proteins present in the surrounding medium.     

closca, a new gene required for both Torso RTK activation and vitelline membrane integrity. Germline proteins contribute to Drosophila eggshell composition

The Drosophila eggshell is a specialised extracellular matrix (ECM) that surrounds and protects the oocyte and the embryo until its eclosion. In addition, the vitelline membrane, the innermost layer of the eggshell, holds the local determinant required to activate the Torso RTK pathway, which establishes the embryonic terminal regions. Here we report the identification and characterisation of closca, a gene encoding a new member of a group of proteins that act non-redundantly in vitelline membrane biogenesis and in Torso signalling. We also show that the Nasrat protein, another member of this group, is incorporated into the vitelline membrane, thereby indicating that the eggshell is a shared ECM that receives contributions from both follicle cells and the germline. This observation also provides a new scenario that accounts for the long known contribution of germline products to vitelline membrane biogenesis and to the follicle cell-dependent activation of the Torso receptor.     

Enzymes responsible for the bactericidal effect in extracts of vitelline and fertilisation envelopes of rainbow trout eggs

Extracts from both the vitelline envelope (VE) and fertilisation envelopes (FE) of rainbow trout eggs have the ability to exert a bactericidal effect on Gram-positive and -negative bacteria. The effect may be due to the presence of phospholipase D (PLD), lysozyme, proteinase and DNases, as the extracts contain these enzyme activities. The intensity of chorionic PLD and lysozyme activities in the VE extract was maintained in the FE without any alteration in activity even after transformation in the course of the cortical reaction, as components of a fundamental architecture of the envelope. Both extracts also contain different types of proteinase activities. Treatment with VE or FE extract seriously damaged the outer membrane of Gram-negative bacteria and the plasma membrane of Gram-positive and -negative bacteria at the ultrastructural level. Chorionic DNases probably degrade DNA of bacterial cells killed by virtue of the action of PLD and/or lysozyme and contribute to the transmigration of nucleosides and/or nucleotides produced by degrading bacterial DNA after degradation of bacterial components by the actions of the chorionic PLD, lysozyme and proteinase. These results suggest that the bactericidal process manifested by the VE or FE extract may start with the action of PLD and/or lysozyme against bacteria and be completed by subsequent degradation of constitutive proteins and DNA by the action of proteinases and DNases, respectively. Thus the VE and FE are able to protect the egg itself and the embryo, respectively, from bacterial infection in the internal or external environments.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

An ovarian follicular epithelium protein of the silkworm (Bombyx mori) that associates with the vitelline membrane and contributes to the structural integrity of the follicle

We have cloned and functionally characterized a novel protein, BmVMP30, which is synthesized by the cells of the follicular epithelium of the ovarian follicles of the domesticated silkworm Bombyx mori, secreted from them and associated with the vitelline membrane. BmVMP30 is a 30 kDa protein that bears limited structural features reminiscent of other insect vitelline membrane proteins. Although BmVMP30 does not share pronounced similarities or signature motifs with other reported proteins, its temporal and spatial expression and its behavior throughout oogenesis suggest that it is a novel member of the insect vitelline membrane protein family. The protein is expressed exclusively in the cells of the follicular epithelium during stages -15 to -1 of vitellogenesis, secreted from them and, ultimately, localized at the junction between the oocyte and the eggshell, where the vitelline membrane is located. Treatment of follicles with an antisense oligonucleotide that encompasses the translation initiation codon results in the production of an N-terminally truncated protein and disruption of the integrity of the follicular epithelium. Antisense oligonucleotide treatment, however, has no effect on the implementation of the developmental program that directs the autonomous progression of ovarian follicles through the last stages of vitellogenesis and choriogenesis.     

Evidence for divalent cation (Ca2+)-stabilized oligomeric proteins and covalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum.

Two unusual characteristics of some outer membrane proteins of Rhizobium leguminosarum are described. First, most of the major outer membrane proteins could only be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after lysozyme treatment of the isolated cell envelopes, suggesting a very strong, possibly covalent, interaction of these proteins with the peptidoglycan. These peptidoglycan-associated outer membrane proteins belonged to two distinct groups of immunologically related proteins, groups II and III, as defined by typing with monoclonal antibodies. As members of both groups of proteins could be radioactively labeled by growing cells in the presence of N-[3H]acetylglucosamine, we propose that variation in the apparent molecular weight of the antigens within each group is caused by varying numbers of peptidoglycan subunit residues on only two or three different outer membrane proteins. Second, group III outer membrane proteins, with masses of 35 to 46 kilodaltons, formed oligomers stabilized by divalent cations which resisted complete denaturation in 2% sodium dodecyl sulfate at 100 degrees C. Reconstitution experiments showed that of the divalent cations tested, Ca2+ and, to a lesser extent, Mn2+ and Sr2+ were the best stabilizers.     

An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.     

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture,model of the Escherichia coli cytoplasmic membrane

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.