Vitelline Membrane Outer Layer Research Articles

Urine screening by Seldi-Tof, followed by biomarker identification, in a Brazilian cohort of patients with Renal Cell Carcinoma (RCC)

To screen proteins/peptides in urine of Renal Cell Carcinoma (RCC) patients by SELDI-TOF (Surface Enhanced Laser Desorption Ionization - Time of Flight) in search of possible biomarkers. Sixty-one urines samples from Clear Cell RCC and Papillary RCC were compared to 29 samples of control urine on CM10 chip. Mass analysis was performed in a ProteinChip Reader PCS 4,000 (Ciphergen Biosystems, Fremont, CA) with the software Ciphergen Express 3.0. All chips were read at low and at high laser energy. For statistical analysis the urine samples were clustered according to the histological classification (Clear Cell and Papillary Carcinoma). For identification urine was loaded on a SDS PAGE gel and bands of most interest were excised, trypsinized and identified by MS/MS. Databank searches were performed in Swiss-Prot database using the MASCOT search algorithm and in Profound. Proteins that were identified from urine of controls included immunoglobulin light chains, albumin, secreted and transmembrane 1 precursor (protein K12), mannan-binding lectin-associated serine protease-2 (MASP-2) and vitelline membrane outer layer 1 isoform 1. Identification of immunoglobulins and isoforms of albumin are quite common by proteomics and therefore cannot be considered as possible molecular markers. K12 and MASP-2 play important physiological roles, while vitellite membrane outer layer 1 role is unknown since it was never purified in humans. The down expression of Protein K-12 and MASP-2 make them good candidates for RCC urine marker and should be validated in a bigger cohort including the other less common histological RCC subtypes.

A transcriptomic analysis of American lobster (Homarus americanus) immune response during infection with the bumper car parasite Anophryoides haemophila

Anophryoides haemophila is an important protistan parasite of American lobster, Homarus americanus, as it has been found to infect lobsters in the wild as well as causing major losses of lobsters maintained in commercial holding facilities. Expression of over 14,500 H. americanus hepatopancreatic genes were monitored during an A. haemophila infection challenge in order to elucidate molecular mechanisms involved in the lobster immune response. One hundred and forty-five genes were found to be differentially expressed during infection. For many genes, this study is the first to link their expression to an immune response to a known lobster pathogen. Several of the genes have previously been linked to crustacean or invertebrate immune response including: several anti-lipopolysaccharide factor isoforms (ALFHa), acute phase serum amyloid protein A (SAA), a serine protease inhibitor, a toll-like receptor, several haemocyanin subunits, phagocyte signaling-impaired protein, vitelline membrane outer layer protein-1, trypsin, and a C-type lectin receptor. Microarray results were verified using RT-qPCR and agreement was good between the two methods. The expression of six ALFHa isoforms was monitored via microarray where ALFHa-1, ALFHa-2, ALFHa-4 and ALFHa-6 were differentially expressed while ALFHa-3 and ALFHa7 were not. RT-qPCR analysis confirmed that ALFHa-1, ALFHA-2 and ALFHa-4 expression increased during infection with a peak at 5–7weeks for ALFHa-1 and 10weeks for ALFHa-2 and ALFHa-4. This suggests that different ALFHa isoforms are temporally expressed during A. haemophila infection. Importantly, these results provide evidence that different ALFHa isoforms have more significant roles in responding to A. haemophila infection. Significant increases in SAA gene expression were also found, corroborating previous findings of increased SAA expression during Aerococcus viridans infections; highlighting the importance of SAA as a marker of H. americanus immune activation and potential indicator of H. americanus health.

Comparisons of liver proteomes in the European flounder Platichthys flesus from three contrasted estuaries

Estuaries are important areas highly susceptible to anthropogenic degradations like pollution. Estuarine species have thus to cope with many types of constraints depending on the estuaries' characteristics. The European flounder Platichthys flesus is considered as a sentinel species for the monitoring of estuarine water quality. In this study, juvenile flounders (0+ group) were sampled from three contrasted Channel estuaries, i.e. the Seine, the Canche and the Tamar, and we characterized their liver proteomes by using a two-dimensional electrophoresis based proteomic approach.We showed that 27 protein spots differentially accumulated between the 3 populations. Six of these proteins were identified by MALDI TOF–TOF mass spectrometry. Flounders from the Seine and from the Tamar, two highly polluted estuaries, displayed common differences, i.e. an increase of the energetic- and the glutathione-metabolism. The most accumulated protein in the Seine's samples (6.7-fold) was a Vitelline Membrane Outer layer protein 1 homolog, suggesting oogenesis deregulation in these juvenile (sexually immature) flounders. Future works applying this kind of proteomic approach on flounders experimentally exposed to conditions that mimic environmental constraints will help to better understand the significance of these environmental proteomic signatures.

Novel transcripts in the maxillary venom glands of advanced snakes

Venom proteins are added to reptile venoms through duplication of a body protein gene, with the duplicate tissue-specifically expressed in the venom gland. Molecular scaffolds are recruited from a wide range of tissues and with a similar level of diversity of ancestral activity. Transcriptome studies have proven an effective and efficient tool for the discovery of novel toxin scaffolds. In this study, we applied venom gland transcriptomics to a wide taxonomical diversity of advanced snakes and recovered transcripts encoding three novel protein scaffold types lacking sequence homology to any previously characterised snake toxin type: lipocalin, phospholipase A2 (type IIE) and vitelline membrane outer layer protein. In addition, the first snake maxillary venom gland isoforms were sequenced of ribonuclease, which was only recently sequenced from lizard mandibular venom glands. Further, novel isoforms were also recovered for the only recently characterised veficolin toxin class also shared between lizard and snake venoms. The additional complexity of snake venoms has important implications not only for understanding their molecular evolution, but also reinforces the tremendous importance of venoms as a diverse bio-resource.

Analysis and Comparison of Proteomic Profiles of Tear Fluid from Human, Cow, Sheep, and Camel Eyes

To investigate the tear proteome profiles of human, cow, sheep, and camel comparatively and to explore the difference of tear protein profiles among different species. Tears were collected from both eyes of 25 clinically healthy volunteers, 50 cows, 25 sheep, and 50 camels. Pooled tear protein samples were separated by SDS-PAGE and two-dimensional electrophoresis. Protein spots of differential expression were excised and subjected to in-gel digestion and identification by matrix assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrum analysis. Because of the incomplete genomic data of cow, sheep, and camel, a combined strategy of de novo sequencing and BLAST (Best Local Alignment Search Tool) homology searching was also used for protein identification. The differentially expressed proteins were validated by Western blot analysis. On comparison with human tears (182 ± 6 spots), 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in triphenylmethane dye-stained gels of cow, sheep, and camel tears, respectively. Similar high-abundant proteins (lactoferrin, lysozyme, etc.) were found in all tear fluids. Tear lipocalins have been identified in cow and sheep tears. BLAST searching revealed a 21-kDa protein, identical with human vitelline membrane outer layer protein 1 (VMO1) homolog, in camel tears. The Western blot confirmed that VMO1 homolog was present in both camel and sheep tears but not in human and cow tears. The comparative proteomic analyses of tears from healthy humans, cows, sheep, and camels were first reported. Differential protein expression existed in the tear among species, offering useful information for further study on tear proteins and the related ocular diseases.

VMO-II Mediates the Binding of the Chalaziferous Layer with the Vitelline Membrane in Quail Eggs

Electron microscopic observations have revealed an electron density gradient in the chalaziferous layer of avian egg. The density is highest in the innermost sub-layer, which is bound with the vitelline membrane, and is thus traditionally referred to as the vitelline membrane outer layer (VMO). After high-concentration salt treatment, two proteins, VMO-I and VMO-II, are liberated from the egg envelopes of quail (Coturnix japonica), which results in the separation of the chalaziferous layer from the vitelline membrane. VMO-I and VMO-II were purified through gel-filtration and subjected to antisera produced in rabbits. The molecular sizes of these two proteins were estimated to be 9 to 15kDa for VMO-II and 18kDa for VMO-I. Their amino acid sequences were very similar to those of chickens. Immunofluorescent and immunoelectron micrographs showed that VMO-II was produced by the luminal epithelium and VMO-I by both the luminal and glandular epithelia of the infundibulum. Immunogold particles for the labeling of both proteins were distributed throughout the chalaziferous layer, as well as in the chalazae, with a density gradient similar to that mentioned above. In salt-treated envelopes, immunogold labeling was completely absent when stained with anti-VMO-II antiserum and weak with anti-VMO-I antiserum. Immunohistochemical studies revealed that purified VMO-II bound with vitelline membranes of salt-treated envelopes, and ligand blotting tests revealed that ZP1 and ZP3 were the molecules bound by VMO-II; the binding was inhibited by various kinds of sugars. VMO-II might play a role in the binding of the chalaziferous layer with the vitelline membrane, a process that leads to the anchoring of the chalaza cord.

Exocytosis and proteomic analysis of the vesicle content of granular hemocytes from a crayfish

The circulating blood cells (hemocytes) of invertebrates are important in cellular immune reactions and to deliver immune factors synthesized in these cells to the external milieu. Previously, we have shown that release of vesicle contents is involved in a regulated exocytosis and here we show which proteins in the vesicles are most abundant and which are released by triggering of exocytosis using a calcium ionophore, lipopolysaccharides–peptidoglycan and peroxinectin, a cell adhesion and degranulation factor from the hemocytes. The ionophore caused release of nine proteins and six of them were characterized and found to be a masquerade-like protein, a masquerade-like serine proteinase, a mannose receptor protein, a vitelline membrane outer layer protein I, and two anti-microbial peptides. The released protein band with a mass of 76 kDa is more likely pro-phenoloxidase and/or peroxinectin. When peroxinectin was used as a trigger of exocytosis, seven proteins could be identified and for the lipopolysaccharides–peptidoglycan six proteins could be identified and all of them were also released by the ionophore treatment. Interestingly, several anti-microbial peptides were the most abundant proteins and were efficiently released by all treatments as were two masquerade-like proteins one of which is functioning as an opsonic protein.

Identification of the major proteins of the organic matrix of emu (Dromaius novaehollandiae) and rhea (Rhea americana) eggshell calcified layer

1. The eggshell is a composite consisting of 95% calcite and an organic matrix. 2. While many proteins of the chicken eggshell matrix have already been identified, little is known about the matrix of other birds. 3. Isolation of the emu and rhea eggshell matrix and analysis of its major constituents showed that the predominant components were C-type lectin-like proteins related to those of ostrich, chicken and goose. 4. Serum albumin, vitelline membrane outer layer protein I (VMO-I) and the turpentine-induced acute phase serum protein 18-B were identified as minor components of the emu shell matrix. Both eggshell matrices also contained a novel proline- and alanine-rich protein. 5. Like ostrich, and unlike chicken and goose, both emu and rhea eggshell matrix contained two different C-type lectin-like proteins as major components, indicating that the occurrence of two proteins of this family may be widespread among ratites.

タンパク質結晶学と構造生物学 新しいタンパク質立体構造 ニワトリ卵黄膜由来の蛋白質ＶＭＯ‐Ｉの結晶構造解析

The crystal structure of vitelline membrane outer layer protein I (VMO-I), which was isolated from the outer layer of the vitelline membrane of hen's eggs, has been determined by X-ray analysis. VMO-I is composed of three homologous structures, each containing a β-sheet forming Greek key motif, which are in accordance with the three repeats in the sequence. These three homologous structures are related by a pseudo three-fold symmetry. This new folding motif, recently designated as the β-prism, has also been observed in the second domain of δ-endotoxin. Thus, VMO-I and δ-endotoxin constitute a new family with a novel folding motif. The VMO-I molecule has a groove-like cavity. This region contains invariant acidic residues in the three repeats. VMO-I is assumed to be an oligosaccharide binding protein like lysozyme, although details of its catalytic function remain to be solved. A docking model of an oligosaccharide to VMO-I has been constructed. This model shows that the cavity has a sufficient space to bind roughly pentamer saccharides. The structural features strongly suggest that the negatively-charged cavity in the top region of the protein may be involved in an unknown catalytic reaction.

The β-prism: a new folding motif

A new protein fold with internal symmetry has been observed in two proteins: vitelline membrane outer layer protein I (VMO-I) and delta-endotoxin. Despite lacking any discernible sequence similarity, both proteins have similar three-dimensional structures as well as a carbohydrate-binding site in the top region of the common fold.

Characterization of vitelline membrane outer layer protein I, VMO-I: amino acid sequence and structural stability.

Vitelline membrane outer layer protein I (VMO-I) tightly bound to ovomucin fibrils of hen's egg yolk membrane was characterized in terms of its amino acid sequence and structural stability. The deduced sequence of VMO-I using the conventional sequencing method is: RTREYTSVITVPNGGHWGKWGIRQFCHSGYANGFALKVEPSQFGRDDTALNGIRLRCLD- GSVIESLVGKWGTWTSFLVCPTGYLVSFSLRSEKSQGGGDDTAANNIQFRCSDEAVLVGD- DLSWGRFGPWSKRCKICGLQTKVESPQGLRDDTALNNVRFFCCK. Thus, VMO-I is composed of 163 amino acid residues with a calculated molecular weight of 17,979. The sequence confirms the cDNA sequence of VMO-I we recently determined and does not show any significant similarity to proteins compiled in the NBRF database. Two of the four disulfide bonds found in VMO-I were estimated to lie between Cys26 and Cys57 and between Cys79 and Cys110. The sequence analyses show that VMO-I contains three 53-residue internal repeats that contain distinctive regions of turns flanked by beta-sheets consistent with the recent finding that the molecule contains a new beta-fold motif, the beta-prism. The molecular characteristics of VMO-I in solution were examined by CD spectroscopy in the far and near ultraviolet regions, NMR spectroscopy, and high sensitive differential scanning calorimetry (DSC). CD spectra in the far UV region at room temperature were similar to that assigned to a random coil, while in the near UV region, small positive peaks were observed. The ellipticity in both regions decreased on raising the temperature. Proton NMR experiments showed the native structure unfolds to unordered conformations at 70 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and sequencing of hen magnum cDNAs encoding vitelline membrane outer layer protein I (VMO-I

Two cDNAs encoding hen vitelline membrane outer layer protein I (VMO-I), which is classified as a new type of multi-β-sheet assembly, were cloned and sequenced. Northern blot analysis using vmo-I cDNA as a probe showed the presence of three mRNA species. Strikingly, expression of these mRNAs was restricted to a specific region of the hen oviduct, the area joining the infundibulum to the magnum.

Crystal structure of vitelline membrane outer layer protein I (VMO-I): a folding motif with homologous Greek key structures related by an internal three-fold symmetry.

The crystal structure of vitelline membrane outer layer protein I (VMO-I), which is isolated from the vitelline membrane outer layer of hen's eggs, has been determined by the multiple isomorphous replacement method and refined to an R-factor of 18.8% at 2.2 A resolution. The main chain folds into an unusual structure that consists of three beta-sheets forming Greek key motifs, which are related by an internal pseudo three-fold symmetry. The internal portion surrounded by these three beta-sheets is filled with hydrophobic side chains. This conformational feature coincides with three internal repeats in the sequence. Although a similar fold exists in the second domain of delta-endotoxin, there are significant structural differences between the two proteins, with the three-fold symmetry being most regular in VMO-I.

Crystallization and Preliminary Crystallographic Data of Vitelline Membrane Outer Layer Protein, I VMO-I

The vitelline membrane outer layer protein I (VMO-I), which is isolated from the vitelline membrane outer layer of hen's eggs, has been crystallized from an acetate buffer solution by the hanging-drop method. The crystals belong to the orthorhombic space group P2 12 12 1, with unit cell dimensions a=62·42 Å, b=110·52 Å, c=44·15 Å. There are two molecules ( M r = 18,000) per asymmetric unit. The crystals diffract to at least 2·2 Å Bragg spacings.

Isolation of a novel protein from the outer layer of the vitelline membrane

The outer layer of the vitelline membrane from hen egg yolk consists of ovomucin, vitelline membrane outer layer protein I (VMOI) and lysozyme. Here we report the occurrence of a further basic protein (pI 11.5) in the outer layer, which was designated as vitelline membrane outer layer protein II (VMOII). It was dissociated from the outer layer in a 10% (w/v) NaCl solution and purified to homogeneity by ion-exchange chromatography. VMOII is a simple protein with a molecular mass of 6000 Da, as determined by sedimentation equilibrium analysis. The amino acid composition of VMOII was characterized by the absence of Met and high contents of cystine (half) (14%) and basic amino acids (6% Arg, 6% Lys and 3% His). Analysis of carboxymethylated VMOII indicated that all cysteine residues were involved in disulphide bonding, which appears to facilitate the binding of SDS to the protein. Sequence comparison of the N-terminal 20 residues revealed no identity with other known proteins. VMOII contained a small amount of alpha-helix and was quite resistant to heat denaturation.