Avidin Gene Research Articles

Egg white composition, antioxidant capacity, serum and yolk lipids and oxidative damage of the oviduct magnum in laying hens fed diets contaminated with different concentrations of cadmium

Total of 384 healthy 38-week-old hens were randomly divided into four treatments. Layers in four groups were fed a basal diet and a basal diet with Cd at the level of 15, 30 and 60 mg/kg for nine weeks. Results showed that Cd could dose-dependently deposited in magnum and a positive correlation occurred between the accumulation of Cd in magnum and Cd content in egg whites. When compared with control group, 60 mg/kg Cd decreased magnum ovomucin, ovalbumin, lysozyme and ovomucoid gene expressions, but increased avidin gene expression obviously. Hens receiving 60 mg/kg Cd exhibited a significant decrease in egg white ovalbumin content and its proportion in total protein and increase in total cholesterol and LDLC levels in serum and yolk. The level of GSH and activities of T-SOD and CAT in magnum and GSH-Px activity in egg white were decreased significantly, while MDA level in both magnum and egg white was increased obviously in 60 mg/kg Cd group. Besides, Cd induced oxidative stress, inflammation, endoplasmic reticulum stress (ERS) and mitophagy accompanied by a significant up-regulation of the expressions of Keap1, IL1β, TNFα, NF-κB, p65, p-p65, Grp78, CHOP, ATF4, ATF6, IRE1α, Grp94, PINK1, Parkin, Fundc1, GRP75, VDAC1, MCU, MFF, Bnip3 and LC3I/II and a significant down-regulation of the expressions of Nrf2, HO-1, SOD1, SOD2, SOD3, MFN1, MFN2 and PGC1α. These results indicated that Cd can increase serum and yolk cholesterol level and induce magnum oxidative stress, inflammation, ERS and mitophagy through Nrf2/NF-κB and IRE1α/IP3R/GRP75/VDAC1/MCU pathways, thus disrupting egg white composition. Highlights Cd exposure disrupted the synthesis of egg white protein in oviduct magnum and destroyed antioxidant status and protein composition in egg white. Cd exposure significant enhanced serum and yolk total cholesterol and LDL cholesterol levels. Cd exposure induced magnum oxidative stress, inflammation, ERS and mitophagy through Nrf2/NF-κB and IRE1α/IP3R/GRP75/VDAC1/MCU pathways.

Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects.

BackgroundWheat is considered the most important cereal crop all over the world. The wheat weevil Sitophilus granarius is a serious insect pests in much of the wheat growing area worldwide and is responsible for significant loss of yield. Avidin proteins has been proposed to function as plant defense agents against insect pests.ResultsA synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. The presence and expression of the transgene in six selected T0 transgenic wheat lines were confirmed at the molecular level. Accumulation of avidin protein was detected in transgenic plants compared to non-transgenic plants. Avidin transgene was stably integrated, transcribed and translated as indicated by Southern blot, ELISA, and dot blot analyses, with a high level of expression in transgenic wheat seeds. However, no expression was detected in untransformed wheat seeds. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days.ConclusionTransgenic wheat plants had improved resistance to Sitophilus granarius.

Characterization and Application of Oviductal Epithelial Cells In Vitro in Gallus domesticus1

Chicken oviductal epithelium produces large quantities of egg white protein in daily cycles. In this study, we cultured and characterized oviductal epithelial cells (OECs) from juvenile (10-wk-old) chickens and from actively laying (30-wk-old) hens. The juvenile OECs were maintained over passage 25 and were positive for toluidine blue, lectin-ConA, HPA, UEA-1, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR, whereas the adult OECs were cultured over passage 6 and were positive for toluidine blue, periodic acid-Schiff, lectin-ConA, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR. To investigate the optimal concentration of steroid hormones for inducing egg white protein genes in vitro, we examined the effects of estrogen, diethylstilbestrol, progesterone, and corticosterone on OECs. Results showed that oviduct-specific levels of avidin, ovalbumin, ovomucin, lysozyme, ESR1, and PGR gene expression were significantly elevated in steroid hormone-treated OECs compared with those of untreated cells (P < 0.05). Ovalbumin protein was also secreted into culture medium from hormone-treated OECs. In addition, to examine the application of OECs for avian transgenesis, we introduced human thrombopoietin (THPO)-expressing lentiviral vector controlled by a 3.5-kb ovalbumin promoter into cultured OECs, and THPO expression was significantly induced with diethylstilbestrol or progesterone in juvenile OECs (P < 0.05) and in adult OECs (P < 0.05). In conclusion, these data demonstrate the potential of cultured OECs as a model system for providing a better understanding of the regulation of gene expression and for the production of an avian transgenic bioreactor.

Construction of Chimeric Dual-Chain Avidin by Tandem Fusion of the Related Avidins

BackgroundAvidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications.Principal FindingsTo overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation.SignificanceThe improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

Avidin related protein 2 shows unique structural and functional features among the avidin protein family

BackgroundThe chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.ResultsIn the present study, we have employed multiple biochemical methods to better understand the structure-function relationship of AVR proteins focusing on AVR2. Firstly, we have solved the high-resolution crystal structure of AVR2 in complex with a bound ligand, D-biotin. The AVR2 structure reveals an overall fold similar to the previously determined structures of avidin and AVR4. Major differences are seen, especially at the 1–3 subunit interface, which is stabilized mainly by polar interactions in the case of AVR2 but by hydrophobic interactions in the case of AVR4 and avidin, and in the vicinity of the biotin binding pocket. Secondly, mutagenesis, competitive dissociation analysis and differential scanning calorimetry were used to compare and study the biotin-binding properties as well as the thermal stability of AVRs and avidin. These analyses pinpointed the importance of residue 109 for biotin binding and stability of AVRs. The I109K mutation increased the biotin-binding affinity of AVR2, whereas the K109I mutation decreased the biotin-binding affinity of AVR4. Furthermore, the thermal stability of AVR2(I109K) increased in comparison to the wild-type protein and the K109I mutation led to a decrease in the thermal stability of AVR4.ConclusionAltogether, this study broadens our understanding of the structural features determining the ligand-binding affinities and stability as well as the molecular evolution within the protein family. This novel information can be applied to further develop and improve the tools already widely used in avidin-biotin technology.

High-resolution crystal structure of an avidin-related protein: insight into high-affinity biotin binding and protein stability

The chicken avidin gene belongs to an extended gene family encoding seven avidin-related genes (AVRs), of which only avidin is expressed in the chicken. The sequences of AVR4 and AVR5 are identical and the common protein (AVR4) has been expressed both in insect and bacterial systems. The recombinant proteins are similarly hyperthermostable and bind biotin with similarly high affinities. AVR4 was crystallized in the apo and biotin-complexed forms and their structures were determined at high resolution. Its tertiary and quaternary structures are very similar to those of avidin and streptavidin. Its biotin-binding site shows only a few alterations compared with those of avidin and streptavidin, which account for the observed differences in binding affinities. The increased hyperthermostability can be attributed to the conformation of the critical L3,4 loop and the extensive network of 1-3 inter-monomeric interactions. The loop contains a tandem Pro-Gly sequence and an Asp-Arg ion pair that collectively induce rigidity, thus maintaining its closed and ordered conformation in both the apo and biotin-complexed forms. In addition, Tyr115 is present on the AVR4 1-3 monomer-monomer interface, which is absent in avidin and streptavidin. The interface tyrosine generates inter-monomeric interactions, i.e. a tyrosine-tyrosine pi-pi interaction and a hydrogen bond with Lys92. The resultant network of interactions confers a larger 1-3 dimer-dimer contact surface on AVR4, which correlates nicely with its higher thermostability compared with avidin and streptavidin. Several of the proposed thermostability-determining factors were found to play a role in strengthening the tertiary and quaternary integrity of AVR4.

Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family.

BackgroundA chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings.ResultsTwo separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin.ConclusionWe describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

Design and Construction of Highly Stable, Protease-resistant Chimeric Avidins

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.

Avidin Expressed in Transgenic Rice Confers Resistance to the Stored-Product Insect PestsTribolium confusumandSitotroga cerealella

Rice (Oryza sativa var. Nipponbare) was transformed with an artificial avidin gene. The features of this construct are as follows: (1) a signal peptide sequence derived from barley alpha amylase was added at the N-terminal region, (2) codon usage of the gene was optimized for rice, and (3) the gene was driven by rice glutelin GluB-1, an endosperm-specific promoter. Avidin was produced in the grain of the transgenic rice but not in the leaves. The concentration of avidin in the kernels was about 1,800 ppm. All larvae of the confused flour beetle (Tribolium confusum) and Angoumois grain moth (Sitotroga cerealella) died when fed transgenic avidin rice powder or kernels, respectively, whereas most of the test insects developed into adults when they were fed a nontransgenic rice control diet. Avidin extracted from the transgenic rice kernel lost most biotin-binding activity after 5 min heating at 95 degrees C.

Efficient Expression and Mutation of Avidin and Streptavidin as Host Proteins for Enantioselective Catalysis

Avidin and structurally related streptavidin are attractive host proteins for the creation of artificial metalloenzymes displaying features reminiscent both of homogeneous catalysts and enzymes. The main advantages are that both proteins have been cloned and expressed in several organisms and possess a deep hydrophobic binding pocket capable of hosting biotinylated catalyst precursors. An optimized artificial avidin gene in Pichia pastoris is expressed. The high level of active protein produced in the extracellular medium is suitable for the performance of high-throughput screening in 96-well plate format. Biologically active recombinant streptavidin is expressed in E. coli. Mutations have been introduced both in avidin and streptavidin genes and both wild type and mutated proteins have been utilized to explore the role of the second coordination sphere in enantioselective catalysis.

Copy-Number Fluctuation by Unequal Crossing-Over in the Chicken Avidin Gene Family

The chicken avidin gene (AVD) forms a closely clustered gene family together with several avidin-related genes (AVRs). In this study, we used fluorescence in situ hybridization on extended DNA fibers (fiber-FISH) to show that the number of the AVD and AVR genes differs between individuals. Furthermore, the gene copy-number showed wide somatic variation in white blood cells of the individuals. The molecular mechanism underlying the fluctuation is most probably unequal crossing-over and/or unequal sister chromatid exchange, as judged by the Gaussian distribution of the gene counts. By definition, an increase in gene number on one locus should be accompanied by a decrease on the other locus in unequal sequence exchange. The results suggest that copy-number lability may be more common among gene families than previously thought. The chicken avidin gene family also provides an excellent model for studying the mechanisms of recombination and gene conversion.

Sequence Features and Evolutionary Mechanisms in the Chicken Avidin Gene Family

The chicken avidin gene family comprises the avidin gene (avd) and several homologous avidin-related genes (avrs). The sequences of the avr genes are nearly identical to each other but exhibit nonrandomly distributed, frequently nonsynonymous nucleotide substitutions compared to avd. In this study, we determined the genetic distances and the phylogeny of the avd and avr genes and found differences between different exons and introns. Our results suggest the involvement of biased gene conversion in the evolution of the genes. Furthermore, one of the genes was identified as a putative fusion gene. The occurrence of both gene conversion and recombination supports the models suggesting a common initiation mechanism for conversion and crossing-over. The existence of avidin-related proteins (AVRs) is currently unknown, but the putative AVRs are expected to bind biotin similarly to avidin. However, the observed sequence differences may affect the stability and glycosylation patterns of the putative AVR proteins.

Characterization and chromosomal localization of the chicken avidin gene family.

Chicken avidin is a biotin-binding protein expressed under inflammation in several chicken tissues and in the oviduct after progesterone induction. The gene encoding avidin belongs to a family that has been shown to include multiple genes homologous to each other. The screening and chromosomal localization studies performed to reveal the structure and organization of the complete avidin gene family is described. The avidin gene family is arranged in a single cluster within a 27-kb genomic region. The cluster is located on the sex chromosome Z on band q21. The organization of the genes was determined and two novel avidin-related genes, AVR6 and AVR7, were cloned and sequenced.

Development of drug targeting based on recombinant expression of the chicken avidin gene.

The chemistry required for covalent biotinylation of drugs, radiopharmaceuticals and other ligands is highly developed, and a large number of biotinylated reagents can be readily synthesized. In order to investigate whether expression of avidin cDNA in mammalian cells might be useful as part of a drug targeting strategy, we transiently expressed the avidin gene in two human tumor cell lines (the cervical carcinoma cell line, HeLa, and the liver derived line, Hep G2). Avidin protein as detected by either immunohistochemistry or binding of streptavidin-biotin complexes was present and functional following transient expression. This result indicated that the mechanisms underlying avidin oligomerization which are necessary for proper protein folding are present within mammalian carcinoma cell lines. Next, we generated a producer cell line (derived from psi2) capable of releasing a recombinant retrovirus encoding chicken avidin, and a tumorigenic murine breast cancer cell line (16/C) with stable avidin expression. We show that these cell lines are suitable for conferring functional expression of avidin in vitro. These experiments establish a means by which avidin gene expression can be explored as a mechanism for targeted gene delivery of biotin-derivitized drugs in vitro, and have important implications for utilization of this strategy in vivo.

Induction of chicken avidin and related mRNAs after bacterial infection

Inflammation-mediated avidin gene expression in the chicken was studied using hybridization analysis, polymerase chain reaction (RT-PCR) and sequencing. The results indicate the presence of avidin mRNA in the oviduct and intestine after Escherichia coli infection. The mRNA for the inflammation-induced avidin was mainly encoded by the avidin gene, but the avidin-related genes, avr2 and avr3, were also transcribed at a low level in the oviduct and intestine, respectively. Because avidin is also induced in the chicken oviduct by progesterone, our results indicate a multifactorial regulation of avidin gene expression.

Inducibility of the avidin gene by progesterone is suppressed during estrogen-induced cytodifferentiation

We have studied epithelial differentiation of the chick oviduct as induced by diethylstilbestrol (DES) and 17β-estradiol (E 2). The proportion of goblet cells in the oviduct was slightly higher after E 2 than after DES treatment. Also avidin induction by progesterone was stronger following DES than E 2 priming. In the estrogen pretreated oviduct epithelium, avidin expression was induced by progesterone in the surface epithelial cells, protodifferentiated gland cells and tubular gland cells, but not in goblet cells. During prolonged estrogen treatment, however, the inducibility of avidin by progesterone ceased in tubular gland cells but not in surface epithelial cells. The estrogen action on the expression of avidin could be explained by estrogen-induced terminal differentiation of the epithelial gland cells or by a direct effect of estrogen on the progesterone action, for instance interaction of estrogen receptor and progesterone receptor in the regulation of transcription.

Induction of avidin messenger ribonucleic acid in the chick oviduct by progesterone and other steroids.

Avidin gene expression was analyzed using an avidin immunoassay and RNA hybridization analysis. To ascertain whether the induction of the avidin gene by progesterone remains specific also during secondary restimulation with diethylstilbestrol, chicks were given different steroid hormones or hormone combinations. Progesterone-specific induction of avidin protein and messenger RNA (mRNA) was 15- to 30-fold over the control even after secondary restimulation with diethylstilbestrol. A functional difference between the progesterone response element and glucocorticoid response element was suggested, since dexamethasone alone did not induce avidin in vivo. In spite of progesterone specificity, a combination of progesterone with other steroids nevertheless generated a synergistic increase in the amount of avidin mRNA. This may indicate that binding of progesterone receptor to the progesterone response element may be important to alter the functional activity of other hormone response elements present on the avidin gene. The time response curve of the avidin mRNA induction by progesterone was also determined. Avidin mRNA was detectable 8 h after progesterone induction, and its amount was maximal after 16-24 h. This would indicate that the avidin gene belongs in the so-called late responder genes, which also include chicken ovalbumin, ovomucoid, and lysozyme genes.

Induction of avidin messenger ribonucleic acid in the chick oviduct by progesterone and other steroids

Avidin gene expression was analyzed using an avidin immunoassay and RNA hybridization analysis. To ascertain whether the induction of the avidin gene by progesterone remains specific also during secondary restimulation with diethylstilbestrol, chicks were given different steroid hormones or hormone combinations. Progesterone-specific induction of avidin protein and messenger RNA (mRNA) was 15- to 30-fold over the control even after secondary restimulation with diethylstilbestrol. A functional difference between the progesterone response element and glucocorticoid response element was suggested, since dexamethasone alone did not induce avidin in vivo. In spite of progesterone specificity, a combination of progesterone with other steroids nevertheless generated a synergistic increase in the amount of avidin mRNA. This may indicate that binding of progesterone receptor to the progesterone response element may be important to alter the functional activity of other hormone response elements present on the avidin gene. The time response curve of the avidin mRNA induction by progesterone was also determined. Avidin mRNA was detectable 8 h after progesterone induction, and its amount was maximal after 16-24 h. This would indicate that the avidin gene belongs in the so-called late responder genes, which also include chicken ovalbumin, ovomucoid, and lysozyme genes.

Avidin induction by tissue injury and inflammation in male and female chickens

1. 1. The occurrence and inducibility of the biotin-binding egg white protein (avidin) in the chicken is not restricted to the oviduct. 2. 2. Inflammatory treatments (intestinal injury, actinomycin D) induced avidin in a number of tissues of young and adult hens and roosters, but not of female rats and mice. Highest avidin concentrations were found in the organs containing epithelial cells and serous membrane. 3. 3. The expression of the avidin gene by tissue injury and inflammation suggests that avidin has a significant function in the injured and inflamed chicken tissues.