Chicken Ovoinhibitor Research Articles

Polymorphisms in the ovoinhibitor gene (OIH) and their association with egg quality of Xinhua E-strain chickens

ABSTRACT1. Chicken ovoinhibitor has been reported to prevent contamination and affect storage for eggs. The objective of the present study was to conduct an association analysis between ovoinhibitor gene (OIH) polymorphisms and egg quality traits in the population of Xinhua E-strain chickens and explore its expression characteristics in different tissues.2. Three single nucleotide polymorphisms (SNPs) were identified and genotyped, in which one synonymous mutation was located in exon16 (G9810A), one in intron7 (A4363G) and one in intron14 (C8937G). The A4363G and C8937G polymorphisms significantly influenced Haugh unit (HU) and albumen height (AH), while HU and AH in AG and CT heterozygous birds were significantly higher than in birds with homozygous genotypes. Diplotype association analysis showed individuals with an H1H4 diplotype had the highest AH and HU values in the investigated population.3. RT-qPCR results showed that the expression level of the OIH gene was higher in the oviduct and liver than other tissues. During the development of liver at different phases, a drastic decrease occurred during the period of first ovulation event, which suggests the regulation of some unknown factors effected it.4. Our results indicated that OIH plays a vital role in egg quality. The combinations of A4363G and C8937G might be potential advantageous molecular markers for improving HU of chicken eggs.

Ovoinhibitor in the chicken bursa of Fabricius: identification, isolation, and localization.

A monoclonal antibody (Mab) developed against a partially purified bursal protein extract was found to bind specifically to a single cell type in the cortico-medullary border region of the chicken bursa of Fabricius. These cells were microscopically similar to the bursal secretory dendritic-like cells. A product with an apparent molecular weight of approximately 56 kDa on SDS-polyacrylamide gel electrophoresis was immunopurified from bursal extracts by utilizing this Mab. This product was subjected to peptide digestion and protein sequencing. The two resulting sequences perfectly matched the known sequence of chicken ovoinhibitor. Gene-specific polymerase chain reaction (PCR) primers were designed for the ovoinhibitor, RNA was purified from chicken bursae, and reverse transcription/PCR was performed. Two amplicons with the expected size for ovoinhibitor mRNA were obtained. These data suggest that the gene for ovoinhibitor is expressed in the bursa of Fabricius, and that the bursal secretory dendritic-like cells may be a previously unreported source of ovoinhibitor.

The chicken pituitary expresses an ovoinhibitor-like protein in subpopulations of some, but not all, hormone-producing cell types

Ovoinhibitor is a serine protease-inhibiting protein that was originally purified from egg whites. It is secreted by the oviduct under the control of estrogen and progesterone and it specifically inhibits serine proteinases such as trypsin and chymotrypsin. During recent attempts to raise monoclonal antibodies (Mabs) against chicken bursa of Fabricius proteins, one Mab was produced that specifically recognized chicken ovoinhibitor. This was the first demonstration of ovoinhibitor in an avian immune organ. We presently report on the expression of an ovoinhibitor-like molecule by the pituitary of the chicken as revealed by immunocytochemistry and RT-PCR. Immunofluorescent dual staining experiments using the mouse anti-ovoinhibitor Mab in conjunction with polyclonal antibodies against various hypophysial hormones revealed partial co-localization of an ovoinhibitor-like molecule with growth hormone (GH), luteinizing hormone (LH), and pro-opiomelanocortin (POMC), in a subset of the respective hormone producing cells. By contrast, no co-localization with prolactin (PRL) could be reliably demonstrated. RT-PCR of hypophysial mRNA using ovoinhibitor gene-specific primers yielded an amplicon that was 20% shorter than predicted on the basis of the published ovoinhibitor sequence. Sequencing revealed that of the represented exons only the central portion was expressed in the pituitary and that both 5′ and 3′ ends of each exon had been truncated. While expression of ovalbumin-like serine protease inhibitors (serpins) has been previously reported in the rat pituitary, to our knowledge, this is the first report of a Kazal-type serine protease inhibitor in the vertebrate neuroendocrine system.

Expression and characterization of chicken ovoinhibitor in Pichia pastoris.

Chicken ovoinhibitor cDNA was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) using chicken oviduct mRNA. The ovoinhibitor cDNA was successfully cloned downstream from the AOXI promoter of pPICZalphaA plasmid vector to facilitate its expression in the methylotrophic yeast Pichia pastoris. The pPICZalphaA carrying the ovoinhibitor cDNA was integrated into the Pichia genome. The secreted recombinant ovoinhibitor was purified by ion-exchange chromatography on a DEAE sepharose column. The recombinant ovoinhibitor had a molecular mass of 49 kDa, as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and time of flight-mass spectrometry (TOF-MS) analyses. The recombinant ovoinhibitor, just as the native ovoinhibitor, showed inhibitory activity against trypsin, chymotrypsin and elastase.

Purification of two low-molecular-mass serine proteinase inhibitors from chicken liver

Two serine proteinase inhibitors, designated clTI-1 and clTI-2 were purified from livers of chickens to apparent homogeneity by a combination of ethanol-acetone fractionation, gel filtration and ion-exchange chromatography on CM-cellulose and Mono S columns. The inhibitor clTI-1 is a single polypeptide chain, low-molecular-mass protein (Mr about 6200), very stable to heat and ethanol. It inhibits chicken, porcine and bovine trypsins as well as human plasmin. The second protein, lcTI-2 of Mr 17 000 was shown to be a very effective inhibitor of both trypsins and human cathepsin G. Since both inhibitors are sensitive to arginine modification with phenylglyoxal it is assumed that this amino acid residue is present at the P1 position of the reactive site peptide bond. The N-terminal amino acid sequence of 28 residues of clTI-2 (SVDVSKYPSTVSKDGRTLVACPRILSPV) revealed a high homology of this protein to the third domain of the chicken ovoinhibitor, whereas, the clTI-1 (APPAAEKYYSLPPGAPRYYSPVV) has some sequence identity to a fragment of the human inter-α-trypsin inhibitor.

Purification and partial characterization of elastase activity from rat alveolar and peritoneal macrophages

Macrophage elastase was purified from conditioned media from alveolar and thioglycollate-elicited peritoneal macrophages. The enzyme was purified to apparent electrophoretic homogeneity by preparative isoelectric focusing after a purification step consisting of low ionic strength dialysis and sequential batch fractionation on DEAE-Sephadex A-50. The proteinase activities isolated from alveolar and peritoneal macrophages showed the same physical and biochemical properties. This fact suggests that the same enzyme activity is present in rat macrophages of two different anatomical sites. The molecular weight and isoelectric point of the enzyme were estimated to be 22,500 and 8.3, respectively. The enzyme, characterized as a metallo proteinase, had elastolytic activity, as well as activity toward Suc-(Ala) 3-NA. It is inhibited by o-phenanthroline, chicken ovoinhibitor, and EDTA, but not by phenylmethylsulfonyl fluoride or soybean trypsin inhibitor. The macrophage enzyme possesses biochemical and biophysical properties different from the rat pancreatic and granulocyte elastases (which are serine proteinases), and from the metallo proteinase with elastolytic activity isolated from rat platelets.

Ovoinhibitor introns specify functional domains as in the related and linked ovomucoid gene.

We have isolated cDNA clones and determined the gene structure of chicken ovoinhibitor, a seven domain Kazal serine proteinase inhibitor. Using RNA blot hybridization analysis, the gene was identified initially as a region 9-23 kilobases upstream of the gene for the related inhibitor ovomucoid. Ovoinhibitor RNA appears in oviduct and liver. cDNA clones were identified by screening an oviduct cDNA library with a nick-translated DNA restriction fragment which contained an exon of the gene. The mature protein sequence derived from a cDNa clone is in excellent agreement with that which we obtained from direct sequencing of purified ovoinhibitor. The protein-sequencing strategy is reported. The P1 amino acids of the Kazal domains are consistent with the known broad inhibitory specificity of ovoinhibitor. The gene is about 10.3 kilobases in length and consists of 16 exons. Each Kazal domain is encoded by two exons. Like ovomucoid, introns fall between the coding sequences of the ovoinhibitor domains, an arrangement which may have facilitated domain duplication. The intradomain intron occurs in an identical position in all of the ovoinhibitor and ovomucoid Kazal domains, suggesting that this intron was present in the primordial inhibitor gene. We discuss the location of the intradomain intron in relation to the known structure of four Kazal inhibitors and suggest a scheme for the evolution of the ovoinhibitor gene.

Chymotrypsin-specific protease inhibitors decrease H2O2 formation by activated human polymorphonuclear leukocytes.

Stimulated phagocytic cells generate active oxygen species which are known to contribute to inflammatory diseases, necrosis of surrounding tissues, mutagenicity and carcinogenicity. Until now, it was not certain whether protease inhibitors are capable of decreasing the production of those oxygen species, and if they are, what type of protease inhibitor is the most active. In this work we monitored formation of H2O2 by 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated polymorphonuclear leukocytes (PMNs) because H2O2 is the immediate precursor of the actual damaging species. These determinations were carried out in the absence or presence of protease inhibitors and/or superoxide dismutase (SOD). The protease inhibitors tested were: potato inhibitors 1 (PtI-1) and 2 (PtI-2), a chymotrypsin-inhibitory fragment of PtI-2 (PCI-2), chicken ovoinhibitor (COI), turkey ovomucoid ovoinhibitor (TOOI), Bowman-Birk inhibitor (BBI), lima bean inhibitor (LBI) and soybean (Kunitz) trypsin inhibitor (SBTI). The order of activity, as measured by inhibition of H2O2 formation by TPA-activated PMNs during incubation at 37 degrees C for 30 min, was (in descending order): PtI-1 greater than or equal to PCI-2 greater than PtI-2 greater than COI greater than BBI greater than or equal to TOOI greater than LBI greater than SBTI. Thus, the most effective were the chymotrypsin-specific inhibitors PtI-1 and PCI-2, followed by the bifunctional inhibitors recognizing both chymotrypsin and trypsin, and the least active was SBTI, a predominantly trypsin inhibitor. At the higher concentrations of protease inhibitors tested, the inhibitory activity was similar in both the absence and presence of SOD. These results show that protease inhibitors specific for chymotrypsin but not those that are trypsin-specific are capable of inhibiting formation of active oxygen species during the oxidative burst of stimulated human PMNs.

Sialic acid glycoproteins inhibit in vitro and in vivo replication of rotaviruses.

We investigated the interactions of rotaviruses with glycoproteins and cells that support rotaviral replication. We found that a wide range of naturally occurring glycoproteins, including ovalbumins and ovomucoids from chicken and turkey eggs, and mucin derived from bovine submaxillary glands, inhibit the replication of rotaviruses in MA-104 cells. Our studies further indicated that the glycoproteins bind directly to rotaviruses and that virus-glycoprotein binding is dependent largely upon interactions with sialic acid oligosaccharides. We found that accessible sialic acid oligosaccharides are required for efficient rotavirus infection of MA-104 cells, thus demonstrating that sialic acid oligosaccharides play an important role in the interactions of rotaviruses with both glycoproteins and cells that support rotaviral replication. Bovine submaxillary mucin and chicken ovoinhibitor can also prevent the shedding of rotavirus antigen and the development of rotavirus gastroenteritis in a mouse model of rotavirus infection. Our findings document that a range of glycoproteins inhibit the in vivo and in vitro replication of rotaviruses and suggest that the alteration in the quantity or chemical composition of intestinal glycoproteins is a potential means for the modulation of enteric infections.

Purification and characterization of a collagenolytic serine proteinase from the catfish pancreas.

A collagenolytic enzyme was purified to homogeneity from the activated pancreatic extract of the catfish Parasilurus asotus by chromatography on DEAE-cellulose, hydroxylapatite, and Cellulofine columns. The molecular weight of the enzyme was estimated to be 29,500 by SDS-polyacrylamide gel electrophoresis. The enzyme is capable of degrading native, reconstituted calf skin collagen fibrils at pH 7.5 and 37 degrees C, and also of reducing the viscosity of native calf skin collagen at pH 7.5 and 20 degrees C. The SDS-polyacrylamide gel electrophoresis of thermally denatured enzyme-collagen reaction mixtures showed that the enzyme can cleave peptide bonds in the non-helical and triple-helical regions of the collagen. The enzyme was inhibited by DFP, PMSF, soybean trypsin inhibitor, and chicken ovoinhibitor, but not by metal-chelating reagents EDTA, EGTA, o-phenanthroline, or L-cysteine. These results indicate that the enzyme is a unique collagenolytic proteinase belonging to the group of serine proteinases and is a new member of the class of pancreatic enzymes.

Purification and properties of protease F, a bacterial enzyme with chymotrypsin and elastase specificities

It has been previously demonstrated that commercial bacterial fibrinolysin (EC 3.4.21.7) selectively cleaves the bond between Met-53 and Ala-54 in ovine prolactin (199 amino acids). A one-step purification procedure on DEAE-cellulose for Protease F, which is the active component of bacterial fibrinolysin, and properties of the purified enzyme are reported. The enzyme is homogeneous as judged by acrylamide gel electrophoresis. Its molecular weight, calculated from gel filtration experiments on Sephadex G-100, is around 13,800. Amino acid analyses do not reveal the presence of any half-cystines. The presence of one tryptophan residue per enzyme molecule was resolved from the fluorescence spectrum. Amino terminal analysis showed that leucine was at the amino terminal position. Protease F hydrolyzes casein and synthetic specific substrates for chymotrypsin and elastase esterases but not for trypsin esterases. It is fully inhibited by phenylmethylsulfonyl fluoride, by chicken ovoinhibitor, and by Chymotrypsin Inhibitor I from potatoes but not by the trypsin-chymotrypsin inhibitors from soybeans and chick peas or by tosyl- l-phenylalanine chloromethyl ketone. The enzyme is stable at room temperature and in the cold, it is not affected by dialysis or by freezing and thawing, but it is inactivated during freeze-drying. The circular dichroism spectra of Protease F indicate an ~20% α-helix content of the enzyme with a considerable similarity to those of subtilisin, elastase, and β-trypsin. The relatively low molecular weight of Protease F, the absence of intrachain disulfide bridges, and the fact that it is inhibited by several, but not all, chymotrypsin inhibitors suggest that it may differ phylogenetically from the known serine proteases.

Studies on pancreatic enzymes in fish - V. Characterization of catfish pancreatic elastase.

An elastolytic enzyme, designated as elastase B, from the pancreas of the catfish Parasilurus asotus was characterized and compared with the well-established elastase from the porcine pancreas. The approximate molecular weight of catfish elastase B was estimated to be 26, 000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of catfish elastase B was similar to that of porcine elastase. Elastase B had high activities toward elastin, casein, hemoglobin, and Suc-(Ala)5 -NA, but very low activities toward Tos-Arg-OMe and Bz-Tyr-OEt. Catfish elastase B was strongly inhibited by DFP, PMSF, elastatinal, soybean trypsin inhibitor (Kunitz), and chicken ovoinhibitor. These findings indicate that catfish elastase B belongs to the group of serine proteinases and is closely related to porcine elastase.

Ozone inactivation of anti-elastase activity of chicken ovoinhibitor and human α-l-proteinase inhibitor

Summary Treatment of chicken ovoinhibitor with ozone inhibits the anti-elastase activity more than anti-trypsin and anti-chymotrypsin. Treatment of diluted human serum with ozone also shows that anti-elastase is inactivated more than anti-trypsin and anti-chymotrypsin. Treatment of purified human α -1-proteinase inhibitor with a 9 fold molar excess of ozone, inhibited anti-elastase 55%, anti-chymotrypsin 29% and anti-trypsin 18%.

Inhibition of porcine elastase II by chicken ovoinhibitor.

Interaction of porcine elastase II with native and modified chicken egg-white ovoinhibitor was studied by determining the residual activity of the partially inhibited enzyme and by direct measurement of the stoichiometry of interaction using affinity chromatography, electrophoresis and gel filtration. It was found that the chymotrypsin binding site that is not modified by mild oxidation with N-chlorosuccinimide (Shechter et al., Biochemistry, (1977) 16, 992-997) is capable of binding elastase II as well. The binding of chymotrypsin and elastase II is mutually exclusive and the affinity of chymotrypsin is stronger. Binding of 2 mol trypsin as 1 mol elastase I by ovoinhibitor does not interfere with the binding of elastase II. There is also an indication that the second binding site for chymotrypsin is capable of forming a complex with an additional molecule of elastase II, but the binding is so weak that it could be detected only by electrophoresis.

Stoichiometry of interaction of chicken ovoinhibitor with pancreatic trypsin, chymotrypsin and elastase I.

1. The interaction of chicken ovoinhibitor with pancreatic trypsin, chymotrypsin and elastase I was studied in the absence of substrate by affinity chromatography, electrophoresis and gel filtration. 2. It was found that ovoinhibitor has, at least, five separate and non-overlapping binding sites: two for trypsin, two for chymotrypsin and one for elastase. Most likely, all five sites may be occupied simultaneously. 3. Electrophoretic studies indicated that two binding sites for trypsin and probably also for chymotrypsin differ in their affinity toward the respective enzyme.

Independent heat stabilization of proteases associated with multiheaded inhibitors. Complexes of chymotrypsin, subtilisin and trypsin with chicken ovoinhibitor and with lima bean protease inhibitor

The heat stabilization resulting from specific association of serine proteases with either of two multiheaded protease inhibitors, chicken ovoinhibitor or lima bean protease inhibitor, was determined at pH 6.7 in a differential scanning calorimeter. The 2 : 1 complex of either bovine α-chymotrypsin or subtilisin BPN with ovoinhibitor showed two major denaturation endotherms; each 1 : 1 complex showed one major endotherm. Association with ovoinhibitor increased the kinetic thermal stabilities over those of the free chymotrypsin or subtilisin. Association with lima bean protease inhibitor stabilized bovine β-trypsin > porcine β-trypsin > bovine α-chymotrypsin. Complexes having different proteases bound to the same inhibitor, such as chymotrypsin · ovoinhibitor · subtilisin (1 : 1 : 1) or trypsin · inhibitor · chymotrypsin (1 : 1 : 1), denatured like mixtures of the 1 : 1 complexes. These results show more clearly that 2 : 1 association with multiheaded inhibitors stabilizes the two bound protease molecules independently. Each bound protease and the domain(s) of the inhibitor influenced by specific binding of this protease are denatured as a unit. Thus, 2 : 1 complexes comprise at least two new denaturing units. The extent of heat stabilization appears roughly proportional to the K assoc determined by other methods. The results are consistent with other evidence that binding sites for proteases on multi-headed inhibitors are relatively independent in structure and function.

Independent heat stabilization of proteases associated with multiheaded inhibitors. Trypsins bound at nonequivalent sites on chicken ovoinhibitor.

Independent heat stabilization of proteases associated with multiheaded inhibitors. Trypsins bound at nonequivalent sites on chicken ovoinhibitor.

Effect of oxidation of methionine residues in chicken ovoinhibitor on its inhibitory activities against trypsin, chymotrypsin, and elastase.

Oxidation of methionine residues of chicken ovoinhibitor with N-chlorosuccinimide resulted in a selective loss of its inhibitory activities. While trypsin inhibiting activity was not affected at all, half of the chymotrypsin-inhibiting activity and all of the elastase inhibiting activity were lost. Electrophoretic and affinity chromatography studies indicated that the 50% loss of the chymotrypsin-inhibiting activity resulted from the inactivation of one of its two chymotrypsin-inhibiting sites rather than from a decrease in the binding constants of both sites. Oxidation of ovoinhibitor-chymotrypsin and ovoinhibitor-elastase complexes with excess of N-chlorosuccinimide indicated that the complex formation in each case protected the site that binds the enzyme which participated in the complex, but did not protect the site that binds the other enzyme. Quantitative estimation of the number of oxidized methionine residues in the ovoinhibitor isolated from the complexes has shown that in each complex about one methionine residue was protected from oxidation. Nitrophenyl-sulfenylation of the single tryptophan residue of ovoinhibitor did not affect its inhibitory activities at all.

Preferred binding of bovine and porcine trypsins at two different sites on chicken ovoinhibitor. Reduced dissociation of mixed trypsin complexes.

Dissociation of mixed trypsin (bovine plus porcine trypsin) complexes with chicken ovoinhibitor was used to investigate the nonequivalence of the two binding sites for trypsin on the inhibitor. Previous work has shown that 1 mol of trypsin dissociates much more rapidly than the 2nd from unmixed trypsin complexes, those containing 2 mol of one kind of trypsin, bovine or porcine, per mol of inhibitor. However, only approximately 0.5 to 0.6 mol of trypsin dissociated in the rapid step from the mixed trypsin complexes, those containing 1 mol each of bovine and porcine trypsin. Rates of the slow dissociation steps for the two types of complexes did not differ appreciably from each other. A general dissociation scheme is proposed, in which each of the 2:1 complexes can lose a trypsin molecule from either in two parrallel first order reactions, producing two different 1:1 complexes, which subsequently dissociate to yield free ovoinhibitor and a second trypsin molecule. In this scheme, both the earlier results with unmixed trypsin complexes and the preponderance (approximately 3:1) of slow dissociation from the mixed trypsin complexes can be rationalized if bovine trypsin is retained preferentially at one of the two trypsin binding sites on chicken ovoinhibitor, and porcine trypsin at the other. That is, one site allows rapid dissociation of porcine trypsin and slow dissociation of bovine trypsin, whereas the other allows rapid dissociation of bovine trypsin and slow dissociation of porcine trypsin.

Evidence That the Two Binding Sites for Trypsin on Chicken Ovoinhibitor Are Not Equivalent: DISSOCIATION OF THE COMPLEXES WITH PORCINE TRYPSIN

Abstract Complexes between chicken ovoinhibitor (OI) and porcine trypsin were allowed to dissociate in the presence of the active site titrant, p-nitrophenyl p'-guanidinobenzoate, which reacts extremely rapidly with free active trypsin (T). The rate of the slower, postburst reaction with titrant, which is a measure of the rate of dissociation of the complexes, showed a distinct break not observed previously with bovine trypsin (Zahnley, J. C., and Davis, J. G. (1970) Biochemistry 9, 1428–1433). The OI-trypsin complexes dissociate according to the scheme OI-T2 ⇌ OI-T ⇌ OI + T + T With porcine trypsin, rate constants for dissociation of the OI-T2 and OI-T complexes were 7.5 x 10-2 and 4 to 8 x 10-4 s-1, respectively. The OI-T2 complex obtained with porcine trypsin dissociated 6 to 8 times faster than that obtained with bovine trypsin, perhaps reflecting structural differences between the two trypsins. The greater ratio of dissociation rate constants, kd, with porcine trypsin (g 100, against 9 for bovine trypsin) shows clearly that the two binding sites for trypsin on the single polypeptide chain of chicken ovoinhibitor are not equivalent. This appears to be due to intrinsic differences between the two sites, rather than to changes induced by the binding of the first trypsin molecule. An association rate constant, ka, of 8 x 104 m-1 s-1 for formation of the OI-bovine trypsin (1:1) complex was calculated from the inhibition constant (Ki) and the kd. This value of ka is lower than those previously reported for other naturally occurring trypsin inhibitors.