Unique Group Of Proteins Research Articles

Interactions between EB1 and Microtubules

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low-copy-number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre-fractionation were digested and then analyzed by LC-MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un-fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un-fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre-fractionation with MS-based proteomic analysis is simple, low-cost, and effective, providing the prospect of broad application in proteomics.

A novel fractionation method prior to MS-based proteomics analysis using cascade biomimetic affinity chromatography

This is the first report that combines cascade biomimetic affinity fractionation with MS-based proteomics analysis. Our lab has constructed an affinity ligand library composed of thousands of ligands with different protein-binding properties. Structural differences between these ligands result in different non-bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first screened out three affinity ligands with large difference in protein-binding properties. Next, cascade combination of these ligands was applied to fractionate tissue sample into simple subgroups prior to trypsin digestion and LC–MS/MS analysis. In this study, 391 non-redundant protein groups were identified in unfractionated rat liver cytosol, 499 protein groups were identified in 2 fractions of the first affinity fractionation, 616 in 4 fractions of the second fractionation, and 738 in 8 fractions of the third fractionation (an 88.74% increase). Ultimately, a total of 859 unique protein groups were identified in all cascade fractions (a 119.6% increase compared with unfractionated sample). The proteins detected in each fraction were bioinformatically categorized according to their physicochemical characteristics (relative molecular mass, p I, GRAVY value and TM helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes.

Off-Line Two-Dimensional Liquid Chromatography with Maximized Sample Loading to Reversed-Phase Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry for Shotgun Proteome Analysis

We demonstrate a strategy of maximizing the performance of reversed-phase (RP) liquid chromatography (LC) tandem mass spectrometry (MS/MS) for efficient shotgun proteome analysis by optimizing the sample loading to the instrument in an off-line two-dimensional (2D) LC tandem MS platform. To determine the quantity of peptides present in a proteome digest or fractionated peptides from strong-cation exchange (SCX) separation, an automated system based on RPLC with a rapid step solvent gradient for peptide elution and ultraviolet (UV) detection was developed. This system also allowed the purification of the peptides by removing salts and other impurities present in a sample. It was found that controlling the amount of peptides injected into a RPLC MS/MS system was critical to achieve the maximum efficiency in peptide and protein identification. With the use of off-line 2D-LC-MS/MS, peptide fractions from the first dimension of separation were desalted and quantified, followed by injecting the optimal amount of the sample into RPLC-MS/MS for peptide sequencing. The application of this strategy was demonstrated in the proteome profiling of breast cancer MCF-7 cells. From the analysis of 28 SCX fractions with each injecting 1 microg of sample into a 75 mum x 100 mm C18 column interfaced to a quadrupole/time-of-flight mass spectrometer, a total of 2362 unique proteins or protein groups were identified with a false positive peptide identification rate of 0.19%, as determined by target-decoy proteome sequence searches. Replicate 2 h runs of individual fractions with the exclusion of precursor ions of peptides already identified in the first runs resulted in the identification of an additional 549 unique proteins or protein groups with a false positive identification rate of 0.60%. This example illustrated that off-line 2D-LC-MS/MS, with maximal sample injection to the RPLC-MS, is an effective method for shotgun proteome analysis. Finally, the advantages and limitations of this method, compared to other methods, are discussed.

Potential for phosphonoacetate utilization by marine bacteria in temperate coastal waters

Phosphonates are organic compounds that contain a C-P bond and are a poorly characterized component of the marine phosphorus cycle. They may represent a potential source of bioavailable phosphorus, particularly in oligotrophic conditions. This study has investigated the distribution of the phnA gene which encodes phosphonoacetate hydrolase, the enzyme that mineralizes phosphonoacetate. Using newly designed degenerate primers targeting the phnA gene we analysed the potential for phosphonoacetate utilization in DNA and cDNA libraries constructed from a phytoplankton bloom in the Western English Channel during July 2006. Total RNA was isolated and reverse transcribed and phosphonoacetate hydrolase (phnA) transcripts were PCR amplified from the cDNA with the degenerate primers, cloned and sequenced. Phylogenetic analysis demonstrated considerable diversity with 14 sequence types yielding five unique phnA protein groups. We also identified 28 phnA homologues in a 454-pyrosequencing metagenomic and metatranscriptomic study from a coastal marine mesocosm, indicating that > 3% of marine bacteria in this study contained phnA. phnA homologues were also present in a metagenomic fosmid library from this experiment. Finally, cultures of four isolates of potential coral pathogens belonging to the Vibrionaceae contained the phnA gene. In the laboratory, these isolates were able to grow with phosphonoacetate as sole P and C source. The fact that the capacity to utilize phosphonoacetate was evident in each of the three coastal environments suggests the potential for widespread utilization of this bioavailable P source.

ITRAQ analysis of Singapore grouper iridovirus infection in a grouper embryonic cell line

We report, here, the first proteomics study of a grouper embryonic cell line (GEC) infected by Singapore grouper iridovirus (SGIV). The differential proteomes of GEC with and without viral infection were studied and quantified with iTRAQ labelling followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Forty-nine viral proteins were recognized, of which 11 were identified for the first time. Moreover, 743 host proteins were revealed and classified into 218 unique protein groups. Fourteen host proteins were upregulated and five host proteins were downregulated upon viral infection. The iTRAQ analysis of SGIV infection in GEC provides an insight to viral and host gene products at the protein level. This should facilitate further study and the understanding of virus-host interactions, molecular mechanisms of viral infection and pathogenesis.

A unique group of inactive serine protease homologues from snake venom

A number of inactive serine protease homologues (SPHs), which have poorly understood functions, have been identified in invertebrates and vertebrates. Recently, several SPH transcripts have been reported from snake venom glands, which provide potential new tools for the study of the functions of SPHs. Herein we report for the first time a snake venom serine protease homologue (svSPH) protein, designated as TjsvSPH, isolated from the venom of Trimeresurus jerdonii. Despite its high sequence similarity to snake venom serine proteases (SVSPs), TjsvSPH is devoid of arginine esterase and proteolytic activity. This is probably due to the replacement of Arg-43 by His-43 in the catalytic triad. TjsvSPH did not influence the coagulation time of human plasma, induce human platelet aggregation, inhibit adenosine diphosphate/thrombin-induced human platelet aggregation or increase capillary permeability. Phylogenetic analysis showed that svSPHs were separated from SVSPs and formed an independent group. Structural analysis revealed that the structures of svSPHs are quite different from those of SPHs previously reported. These results indicate that snake venoms contain a unique group of svSPH proteins.

Biomineralization: A structural perspective

Biomineralization is an inherently structural subject; the structure of the mineral phase, the structure of the matrix composed of macromolecules and especially the structure of the interphase zone between them. Studies of the dynamics of mineral formation have revealed that a widespread strategy used by many organisms is to first form a disordered mineral phase. Only when it is in place and has adopted its appropriate shape, is it induced to crystallize. Matrix studies have highlighted the importance of a unique group of proteins that are rich in aspartic acid. These are involved in controlling mineral formation. Relating structure to function in mineralized tissues, often involves an understanding of mechanical properties in terms of not only the hierarchical structure of the tissue, but also the graded structure that varies from one location to another. Structure is thus in many respects the foundation upon which the field of biomineralization rests.

Association of PAT Proteins with Lipid Storage Droplets in Term Fetal Membranes

As depots for neutral lipids, lipid storage droplets (LDs) accumulate with advancing gestation within the fetal membranes. Little is currently known about the proteins associated with the LDs of these cells. The PAT family [ perilipin, adipose differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47)] represents a unique group of proteins thought to contribute to LD formation and function. We examined the association of each of the PAT proteins with LDs of term fetal membranes. We found that large LDs of amnion epithelial cells were reactive for neutral lipid stains and simultaneously encoated with ADRP and TIP47, but not perilipin. Within the remaining cell types, LDs were frequently co-labeled with antibodies recognizing ADRP and TIP47; however, in cells harboring only small LDs, the majority of TIP47 labeling was cytoplasmic. Structures labeled with perilipin antibodies were present only in chorion laeve trophoblasts. Gene and protein expression analyses suggested this to be a small molecular weight perilipin isoform, such as that seen in steroidogenic cells. We conclude that LDs are heterogeneous among differing cell types of the fetal membranes. Subclassification of LDs based on associated proteins suggests that these organelles may serve specialized functions within individual cells.

Apolipoprotein B is conformationally flexible but anchored at a triolein/water interface: A possible model for lipoprotein surfaces

Apolipoprotein B (apoB) is one of a unique group of proteins that form and bind to fat droplets, stabilize the emulsified fat, and direct their metabolism. ApoB, secreted on lipoproteins (emulsions), remains bound during lipid metabolism yet exhibits conformational flexibility. It has amphipathic beta-strand (AbetaS)-rich domains and amphipathic alpha-helix (AalphaH)-rich domains. We showed that two consensus AbetaS peptides of apoB bound strongly to hydrophobic interfaces [triolein/water (TO/W) and dodecane/water], were elastic, and were not pushed off the interface when the surface was compressed. In contrast, an AalphaH peptide modeling helical parts of apoB was forced off the TO/W interface by compression and readsorbed when the interface was expanded. In this report, the surface behavior of apoB-100 was studied at the TO/W interface. Solubilized apoB lowered the interfacial tension of TO/W in a concentration-dependent fashion. At equilibrium tension, if the surface was compressed, part of apoB was pushed off but quickly readsorbed when the surface was expanded. Even when the surface area was compressed by approximately 55%, part of the apoB molecule remained bound. The maximum surface pressure that apoB could withstand without being partially ejected was 13 mN/m. ApoB showed high elasticity at the TO/W interface. Based on studies of the consensus AbetaS and AalphaH peptides, we suggest that AbetaSs anchor apoB and are its nonexchangeable motif, whereas its conformational flexibility arises from both the elastic nature of the AbetaS and the ability of AalphaH domains of the molecule to desorb and readsorb rapidly in response to surface pressure changes.

Identification and functional characterization of mouse TPO1 as a myelin membrane protein

TPO1 is a member of the AIGP family, a unique group of proteins that contains 11 putative transmembrane domains. Expression of the rat TPO1 gene is upregulated in cultured oligodendrocytes (OLs) during development from pro-oligodendroblasts to postmitotic OLs. However, the distribution of native TPO1 protein in cultured OLs and in the brain has not been elucidated. We investigated the distribution and cellular function of TPO1 in myelinating cells of the nervous system. In mice, TPO1 gene expression was detected in the central (CNS) and peripheral (PNS) nervous systems and was markedly upregulated at postnatal days 10–20, an early phase of myelination in the mouse brain. To investigate TPO1 localization, we generated affinity-purified antibodies to synthetic peptides derived from mouse TPO1. Immunohistochemical analysis showed that TPO1 was expressed in OLs and Schwann cells but not in neurons and astrocytes. Schwann cells from trembler mice, which lack PNS myelin, had significantly decreased TPO1 expression and an altered localization pattern, suggesting that TPO1 is a functional myelin membrane protein. In OL lineage cell cultures, TPO1 was detected in A2B5(+) bipolar early progenitors, A2B5(+) multipolar Pro-OLs, GalC(+) immature OLs and MBP(+) mature OLs. The subcellular localization of TPO1 in OL lineage cells was mapped to the GM130(+) Golgi in cell bodies and Fyn(+) cell processes and myelin-like sheets. Furthermore, TPO1 selectively colocalized with non-phosphorylated Fyn and promoted Fyn autophosphorylation in COS7 cells, suggesting that TPO1 may play a role in myelin formation via Fyn kinase activation in the PNS and CNS.

Lectins as Bioactive Plant Proteins: A Potential in Cancer Treatment

Plant lectins, a unique group of proteins and glycoproteins with potent biological activity, occur in foods like wheat, corn, tomato, peanut, kidney bean, banana, pea, lentil, soybean, mushroom, rice, and potato. Thus, dietary intakes by humans can be significant. Many lectins resist digestion, survive gut passage, and bind to gastrointestinal cells and/or enter the circulation intact, maintaining full biological activity. Several lectins have been found to possess anticancer properties in vitro, in vivo, and in human case studies; they are used as therapeutic agents, preferentially binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis, and inhibition of tumor growth. These compounds can become internalized into cells, causing cancer cell agglutination and/or aggregation. Ingestion of lectins also sequesters the available body pool of polyamines, thereby thwarting cancer cell growth. They also affect the immune system by altering the production of various interleukins, or by activating certain protein kinases. Lectins can bind to ribosomes and inhibit protein synthesis. They also modify the cell cycle by inducing non-apoptotic G1-phase accumulation mechanisms, G2/M phase cell cycle arrest and apoptosis, and can activate the caspase cascade. Lectins can also downregulate telomerase activity and inhibit angiogenesis. Although lectins seem to have great potential as anticancer agents, further research is still needed and should include a genomic and proteomic approach.

Biochemical Adaptation to the Freezing Environment - the Biology of Fish Antifreeze Proteins

Many organisms are known to survive in icy environments. These include both over wintering terrestrial insects and plants as well the marine fish inhabiting high latitudes. The adaptation of these organisms is both a fascinating and important topic in biology. Marine teleosts in particular, can encounter ice-laden seawater that is approximately <TEX>$1^{\circ}C$</TEX> colder than the colligative freezing point of their body fluids. These animals produce a unique group of proteins, the antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) that absorb the ice nuclei and prevent ice crystal growth. Presently, there are at least four different AFP types and one AFGP type that are isolated from a wide variety of fish. Despite their functional similarity, there is no apparent common protein homology or ice-binding motifs among these proteins, except that the surface-surface complementarity between the protein and ice are important for binding. The remarkable diversity of these proteins and their odd phylogenetic distribution would suggest that these proteins might have evolved recently in response to sea level glaciations just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Winter flounder, Pleuronectes americanus, has been used as a popular model to study the regulation of AFP gene expression. It has a built-in annual cycle of AFP expression controlled negatively by the growth hormone. The signal transduction pathways, transcription factors and promoter elements involved in this process have been studied in our laboratory and these studies will be presented.

The Kinesin-Related Protein MCAK Is a Microtubule Depolymerase that Forms an ATP-Hydrolyzing Complex at Microtubule Ends

MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 μM−1·s−1), perhaps by diffusion along the microtubule lattice, and, once there, removes ∼20 tubulin dimers at a rate of 1 s−1. We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.

Structure-Function Studies of the Lustrin A Polyelectrolyte Domains, RKSY and D4

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins not only exhibit elastomeric behavior within the mineralized matrix, but also adhesion to the aragonite-containing composite layer. One member of the lustrin superfamily, Lustrin A, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesized to perform separate functions (i.e., force unfolding, mineral adhesion, intermolecular binding) within the Lustrin A protein. Using nuclear magnetic resonance (NMR) and in vitro mineralization assays, we investigated structure-function relationships for two Lustrin A putative mineral binding domains, the 30 AA Arg, Lys, Tyr, Ser-rich (RKSY) and the 24 AA Asp-rich (D4) sequence regions domain of the Lustrin A protein. The results indicate that both sequences adopt open, unfolded structures that represent either extended or random coil states. Using geologic calcite overgrowth assays and scanning electron microscopic analyses, we observe that the RKSY polypeptide does not significantly perturb calcium carbonate growth. However, the D4 domain does influence crystal growth in a concentration-dependent manner. Collectively, our data indicate that D4, and not the RKSY domain, exhibits structure-function activity consistent with a mineral binding region.

Structure-Function Studies of the Lustrin A Polyelectrolyte Domains, RKSY and D4

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins not only exhibit elastomeric behavior within the mineralized matrix, but also adhesion to the aragonite-containing composite layer. One member of the lustrin superfamily, Lustrin A, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesized to perform separate functions (i.e., force unfolding, mineral adhesion, intermolecular binding) within the Lustrin A protein. Using nuclear magnetic resonance (NMR) and in vitro mineralization assays, we investigated structure-function relationships for two Lustrin A putative mineral binding domains, the 30 AA Arg, Lys, Tyr, Ser-rich (RKSY) and the 24 AA Asp-rich (D4) sequence regions domain of the Lustrin A protein. The results indicate that both sequences adopt open, unfolded structures that represent either extended or random coil states. Using geologic calcite overgrowth assays and scanning electron microscopic analyses, we observe that the RKSY polypeptide does not significantly perturb calcium carbonate growth. However, the D4 domain does influence crystal growth in a concentration-dependent manner. Collectively, our data indicate that D4, and not the RKSY domain, exhibits structure-function activity consistent with a mineral binding region.

Bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician?

Bone Morphogenetic Proteins (BMPs) form a unique group of proteins within the Transforming Growth Factor beta (TGF-beta) superfamily of genes and have pivotal roles in the regulation of bone induction, maintenance and repair. They act through an autocrine or paracrine mechanism by binding to cell surface receptors and initiating a sequence of downstream events that have effects on various cell types. Differentiation of osteoprogenitor mesenchymal cells and up-regulation of osteoblastic features occur under the influence of cytokines and growth factors that are expressed with the direct or indirect guidance of BMPs acting at the transcriptional level or higher. The Smads family of proteins has been identified as the downstream propagator of BMP signals, whereas hedgehog genes are possible modulators of BMP expression. The inflammatory response observed during wound repair and fracture healing, results in by-products that interact with BMPs and affect their biologic potential. Additive, negative or synergistic effects are observed when homodimeric or heterodimeric forms of BMPs interact with BMP receptors. Storage within the bone matrix allows for their involvement in the modeling/remodeling process by mediating coupling of osteoblasts and osteoclasts. Micro-environmental conditions, dose, possible carrier materials and geometrical parameters of delivery matrix are critical determinants of the pharmacokinetics of BMP action and the biologic outcome during wound repair. Because of their osteogenic potential, BMPs are of tremendous interest as therapeutic agents for healing fractures of bones, preventing osteoporosis, treating periodontal defects and enhancing bone formation around alloplastic materials implanted in bone.

Structural Analyses of Polyelectrolyte Sequence Domains within the Adhesive Elastomeric Biomineralization Protein Lustrin A

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins exhibit elastomeric and adhesive behavior within the mineralized matrix. One member of the lustrin superfamily, Lustrin A, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesized to perform separate functions (e.g., force unfolding, mineral interaction, intermolecular binding) within the Lustrin A protein. Using NMR, CD spectrometry, and model peptides, we investigated the solution structure of two Lustrin A polyelectrolyte modular domains that represent putative sites for Lustrin A−nacre component interaction: the 30-AA Arg, Lys, Tyr, Ser-rich (RKSY), and 24-AA Asp-rich (D4) domains. The results indicate that both sequences adopt open unfolded structures, with RKSY exhibiting structural features of an extended conformer, and D4, a more labile, random-coil conformation. These results suggest that the Lustrin A protein possesses open, unfolded regions that could act as putative sites for Lustrin A−mineral or Lustrin A−macromolecular interactions that lead to the observed adhesive properties of the nacre organic matrix.

Model peptide studies of sequence regions in the elastomeric biomineralization protein, Lustrin A. I. The C‐domain consensus—PG—, —NVNCT—motif

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layer) in mollusk shell. Recent atomic force microscopy (AFM) pulling studies have demonstrated that the lustrin-containing organic nacre layer in the abalone, Haliotis rufescens, exhibits a typical sawtooth force-extension curve with hysteretic recovery. This force extension behavior is reminiscent of reversible unfolding and refolding in elastomeric proteins such as titin and tenascin. Since secondary structure plays an important role in force-induced protein unfolding and refolding, the question is, What secondary structure(s) exist within the major domains of Lustrin A? Using a model peptide (FPGKNVNCTSGE) representing the 12-residue consensus sequence found near the N-termini of the first eight cysteine-rich domains (C-domains) within the Lustrin A protein, we employed CD, NMR spectroscopy, and simulated annealing/minimization to determine the secondary structure preferences for this sequence. At pH 7.4, we find that the 12-mer sequence adopts a loop conformation, consisting of a "bend" or "turn" involving residues G3-K4 and N7-C8-T9, with extended conformations arising at F1-G3; K4-V6; T9-S10-G11 in the sequence. Minor pH-dependent conformational effects were noted for this peptide; however, there is no evidence for a salt-bridge interaction between the K4 and E12 side chains. The presence of a loop conformation within the highly conserved -PG-, -NVNCT- sequence of C1-C8 domains may have important structural and mechanistic implications for the Lustrin A protein with regard to elastic behavior.

Cloning of rainbow trout egg envelope proteins: members of a unique group of structural proteins.

All vertebrate eggs are surrounded by an extracellular envelope that protects the egg and is vital for a successful fertilization. The terminology and functions of the egg envelope vary in different vertebrate groups, but the envelope itself is consistently composed of a few major proteins that are deposited around the oocyte during oocyte growth. Here, we describe the deduced amino acid sequences and tissue expression patterns of the three major egg envelope proteins for rainbow trout (Oncorhynchus mykiss). All three vitelline envelope proteins (VEPs) are expressed in the livers of both male and female fish, with higher expression in females. In addition, VEPgamma mRNA is also detected in the female gonads. To our knowledge, this is the first time that expression of a VEP protein gene has been demonstrated to occur in more than one organ. Sequence comparison reveals that all three VEP proteins share distinct homology with their amphibian, avian, and mammalian counterparts. Whereas mammalian zona pellucida protein 3 isoforms contain two conserved serines needed for sperm binding, these are not conserved in teleost species, in which sperm entry is restricted to the micropyle. Besides the difference in VEPgamma sperm-binding function, the high sequence homology suggests that the egg envelope proteins from these distinct vertebrate groups share a common ancestry and form a unique group of structural proteins.