Japanese Quail Ovomucoid Research Articles

Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of ascidian trypsin inhibitor (ATI), a 55 amino acid residue protein with four disulfide bridges, was determined by means of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The resulting structure of ATI was characterized by an alpha-helical conformation in residues 35-42 and a three-stranded antiparallel beta-sheet in residues 22-26, 29-32, and 48-50. The presence of an alpha-helical conformation was predicted from the consensus sequences of the cystine-stabilized alpha-helical (CSH) motif, which is characterized by an alpha-helix structure in the Cys-X(1)-X(2)-X(3)-Cys portion (corresponding to residues 37-41), linking to the Cys-X-Cys portion (corresponding to residues 12-14) folded in an extended structure. The secondary structure and the overall folding of the main chain of ATI were very similar to those of the Kazal-type inhibitors, such as Japanese quail ovomucoid third domain (OMJPQ3) and leech-derived tryptase inhibitor form C (LDTI-C), although ATI does not show extensive sequence homology to these inhibitors except for a few amino acid residues and six of eight half-cystines. On the basis of these findings, we realign the amino acid sequences of representative Kazal-type inhibitors including ATI and discuss the unique structure of ATI with four disulfide bridges.

Interaction of second and third domains of Japanese quail ovomucoid with ten mammalian trypsins

Second and third domains were prepared from Japanese quail ovomucoid and association equilibrium constants, K as, were measured at 25°C and pH 8 for these domains with trypsins from ten mammalian species: cat, cow, dog, guinea pig, hog, horse, man, rabbit, rat, and sheep. The values ranged from 10 8 M −1 to 10 10 M −1 for the second domain–trypsin associations and from 10 6 M −1 to 10 8 M −1 for the third domain–trypsin associations. Changes in K a values for the interactions between the trypsins and each domain are attributed to slight changes in surface conformation caused by the residue changes in the inhibitor-binding region other than the S 1 pocket of the trypsin species. The representative of such residue changes is assumed to be the one observed at residue 217 of trypsin molecule. Concerning each trypsin, the K a value with the second domain was always higher than that with the third domain. However, the ratios between the two equilibrium constants varied from 3 to 60 depending upon trypsin species. This means that amino acid changes in enzyme-contact residues other than the P 1 site of the Kazal-type inhibitor can make it possible to recognize even a slight difference in inhibitor-binding surface among the enzymes with the same S 1 pocket and highly similar overall three-dimensional structure.

Glycation of Proteinous Inhibitors: Loss in Trypsin Inhibitory Activity by the Blocking of Arginine and Lysine Residues at Their Reactive Sites

Lysine-type trypsin inhibitors [Japanese quail ovomucoid (QOM) and Bowman−Birk inhibitor (BBI)], and arginine-type trypsin inhibitors [chicken ovomucoid (COM) and Kunitz soybean trypsin inhibitor (KSTI)] were modified with glucose by amino−carbonyl reaction at 50 °C and 65% relative humidity for 15 days. Free amino groups of inhibitors were blocked rapidly and decreased to <20% within 5 days. On the other hand, their free guanidino groups began to decrease after a time lag for a few days, and 6−40% of the free guanidino groups remained after the 15-day incubation. Both lysine- and arginine-type trypsin inhibitors were inactivated by the reaction. A linear relationship between the loss in inhibitory activities for the lysine-type inhibitors but not for the arginine-type inhibitors and the decrease of their free amino groups was observed. The soybean trypsin inhibitors were almost completely inactivated by the 15-day incubation with glucose without severe structural or chemical damage. Denaturation of COM and KSTI incubated with glucose was scarcely observed with immunoassay, though protein polymerization of COM−, QOM−, and BBI−glucose complexes was detected by SDS−PAGE. Keywords: Chicken ovomucoid; Japanese quail ovomucoid; Kunitz soybean trypsin inhibitor; Bowman−Birk inhibitor; amino−carbonyl reaction; Maillard reaction; protein modification; inactivation; trypsin inhibitory activity

High-resolution three-dimensional structure of a single zinc finger from a human enhancer binding protein in solution

The three-dimensional structure of a 30-residue synthetic peptide containing the carboxy-terminal "zinc finger" motif of a human enhancer binding protein has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on 487 approximate interproton distance and 63 torsion angle (phi, psi, and chi 1) restraints. A total of 40 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 29 and 30 which are ill-defined) is 0.4 A for the backbone atoms, 0.8 A for all atoms, and 0.41 A for all atoms excluding the lysine and arginine side chains, which are disordered. The solution structure of the zinc finger consists of two irregular antiparallel beta-strands connected by an atypical turn (residues 3-12) and a classical alpha-helix (residues 14-24). The zinc is tetrahedrally coordinated to the sulfur atoms of two cysteines (Cys-5 and Cys-8) and to the N epsilon 2 atoms of two histidines (His-21 and His-27). The two cysteine residues are located in the turn connecting the two beta-strands (residues 5-8); one of the histidine ligands (His-21) is in the alpha-helix, while the second histidine (His-27) is at the end of a looplike structure (formed by the end of the alpha-helix and a turn). The general architecture is qualitatively similar to two previously determined low-resolution Cys2-His2 zinc finger structures, although distinct differences can be observed in the beta-strands and turn and in the region around the two histidines coordinated to zinc. Comparison of the overall polypeptide fold of the enhancer binding protein zinc finger with known structures in the crystallographic data base reveals a striking similarity to one region (residues 23-44) of the X-ray structure of proteinase inhibitor domain III of Japanese quail ovomucoid [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I., & Laskowski, M. (1982) J. Mol. Biol. 158, 515-537], which could be superimposed with a backbone atomic rms difference of 0.95 A on residues 3-25 (excluding residue 6) of the zinc finger from the enhancer binding protein. The presence of structural homology between two proteins of very different function may indicate that the so-called zinc finger motif is not unique for a class of DNA binding proteins but may represent a general folding motif found in a variety of proteins irrespective of their function.

Method of modeling protein structure by the two-dimensional nuclear magnetic resonance spectroscopy data; application to the proteinase inhibitor BUSI IIA from bull seminal plasma.

A new approach is suggested to model the spatial structure of protein molecules in solution based on combined use of the methods of theoretical conformational analysis and NMR spectroscopy data. At the first stage, special means are used to convert d connectivity information into the most probable values of dihedral angles. This allows search for possible spatial structures in the limited regions of the conformational space at further stages using the methods of the theoretical conformational analysis. The suggested approach was verified in reconstructing the spatial backbone structure of the fragment 17-57 of the proteinase inhibitor BUSI IIA from the bull seminal plasma. The structural model parameters are compared with the corresponding characteristics obtained from the X-ray analysis data for the homologic proteinase inhibitor from the Japanese quail ovomucoid. The suggested approach is shown to correctly reproduce both the general molecule topology and the conformations of individual amino acid residues.

The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts.

While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.

Interaction between Trypsin-Like Enzyme from Streptomyces erythraeus and Japanese Quail Ovomucoid

We examined the interaction between a trypsin-like enzyme from Streptomyces erythraeus (TLE-Se) and Japanese quail ovomucoid (QO) as a model of the interaction between a serine protease and a Kazal-type inhibitor. Both the second domain (Domain II) and the third domain (Domain III) of QO inhibited TLE-Se. But there was a great difference in the apparent association rate constant (kappa a) between Domain II and Domain III at pH 8.0 (the optimum pH of the enzymatic activity), and Domain III associated with TLE-Se about 10(5) times faster than Domain II. The pH dependency of kappa a for Domain III and TLE-Se was maximum around pH 8, but in the case of Domain II and TLE-Se the maximum was around pH 4. This suggested that some acidic amino acid residues had some influence upon the association of Domain II with TLE-Se. When we examined the interaction between TLE-Se and amidated Domain II in which most of the aspartic and glutamic acids were amidated for conversion into asparagine and glutamine with NH4Cl and carbodiimide, the pH dependence of kappa a greatly differed from that obtained for TLE-Se and native Domain II, and an almost constant kappa a value (10(3)-10(5) times higher than that of native Domain II) was exhibited between pH 5 and 8. At the same time, the dissociation constant at pH 8.0 became about 10(-2) times smaller, and thus the affinity between TLE-Se and amidated Domain II became stronger. These observations suggest that the electrostatic effect derived from the repulsion between the minus charges which are located at contact area sites of both molecules control the reactivity in the interaction between protease and a protein protease inhibitor.

Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 Å resolution: Structure solution, crystallographic refinement and preliminary structural interpretation

The three-dimensional structure of the proteic complex formed by bovine trypsinogen and the porcine pancreatic secretory trypsin inhibitor (Kazal type) has been solved by means of Patterson search techniques, using a predicted model of the trypsin-ovomucoid complex ( Papamokos et al., 1982 ). The structure of the complex, including 162 solvent molecules, has been refined at 1.8 Å resolution (26,341 unique reflections) to a conventional crystallographic R factor of 0.195. The inhibitor molecule binds to trypsinogen via hydrogen bonds and/or apolar interactions at sites P9, P7, P6, P5, P3, P1, P1′, P2′ and P3′ of the contact area. The structure of the inhibitor itself resembles closely that of the third domain of Japanese quail ovomucoid inhibitor, recently reported by Weber et al. (1981) . The trypsinogen part of the complex resembles trypsin, as is the case in the trypsinogen-basic pancreatic trypsin inhibitor complex, but two segments of the activation domain adopt a different conformation. Most notably in the N-terminal region the Ile16-Gly19 loop, which is disordered in free trypsinogen and in the trypsinogen-basic pancreatic trypsin inhibitor complex ( Huber & Bode, 1978), assumes a regular structure and the polypeptide chain can be traced as far as residue Asp14. This new and fixed structure allows the formation of a buried salt link between the side-chains of Lys156 and Asp194. Conformations differing from those of trypsin are also found for residues 20 to 28 and residues 141 to 155. Some structural perturbation is observed in other parts of the molecule, including the calcium loop.

Crystallographic refinement of Japanese quail ovomucoid, a Kazal-type inhibitor, and model building studies of complexes with serine proteases

Japanese quail ovomucoid third domain (OMJPQ3), a Kazal-type inhibitor, was crystallographically refined with energy constraints. The final R-value is 0.20 at 1.9 Å resolution. The four molecules in the asymmetric unit are very similar, with deviations of main-chain atoms between 0.2 and 0.3 Å. An analysis of the side-chain hydrogen-bonding pattern and amino acid variability in the Kazal family shows a high correlation between hydrogen-bonding and conservation. The conformation of the reactive site loop (P2-P2′) of OMJPQ3 is similar to those of basic pancreatic trypsin inhibitor, Streptomyces subtilisin inhibitor, and soybean trypsin inhibitor. This suggests a common binding mode and justifies model-building studies of complexes. Complexes of OMJPQ3 with trypsin, chymotrypsin and elastase were modelled on the basis of the trypsin-basic pancreatic trypsin inhibitor complex structure and inspected by use of a computer graphics system. Stereochemically satisfying models were constructed in each case and detailed interactions are proposed. The complex with elastase is of particular interest, showing that leucine and methionine are good P1 residues. A good correlation is observed between functional properties of ovomucoid variants and the position of the exchanged residues with respect to the modelled inhibitor-protease contact.

Identification of the trimannosyl-chitobiose structure in sugar moieties of Japanese quail ovomucoid.

Ovomucoid isolated from egg white of japanese quail contained mannose, galactose, glucosamine, and sialic acid (8.6 : 1.3 : 6.6 : 0.5 mol/mol) as sugar components. Sugar chains were liberated from the polypeptide portion by hydrazinolysis, N-acetylated and fractionated on Bio-Gel P-2. The reducing ends of the thus obtained sugar moieties were pyridylaminated. Chemical and exoglycosidase digestion studies of the fluorescent pyridylamino derivative of a major sugar fraction indicated the following structure for one of the major sugar chains of japanese quail ovomucoid; Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.

Crystallization, crystal structure analysis and molecular model of the third domain of Japanese quail ovomucoid, a Kazal type inhibitor

The third domain of Japanese quail ovomucoid, a Kazal type inhibitor, has been crystallized and its crystal structure determined at 2.5 Å resolution using multiple isomorphous replacement techniques. The asymmetric unit contains four molecules. In the crystal the molecules are arranged in two slightly different octamers with approximate D4 symmetry. The molecules are held together mainly by interactions of the N-terminal residues, which form a novel secondary structural element, a β-channel. The molecule is globular with approximate dimensions 35 Å × 27 Å × 19 Å. The secondary structural elements are a double-stranded anti-parallel β-sheet of residues Pro22 to Gly32 and an α-helix from Asn33 to Ser44. The reactive site Lys18-Asp19 is located in an exposed loop. It is close to Asn33 at the N terminus of the helical segment. The polypeptide chain folding of ovomucoid bears some resemblance to other inhibitors in the existence of an anti-parallel double strand following the reactive site loop.

A Ser162/Gly162 polymorphism in Japanese quail ovomucoid.

Japanese quail ovomucoid exists in two polymorphic forms. One has serine, the other glycine at position 162. The tryptic peptide corresponding to positions 160 to 164 was purified from ovomucoids isolated from egg whites of eggs laid by 11 different hens and subjected to amino acid analysis. The quantitative distribution of serine and glycine in this pentapeptide is consistent with the interpretation that the ovomucoid gene exists in two codominant allelic forms at one locus. Even though the gene product is apparently expressed only in the female, these results indicate that the ovomucoid structural gene is transmitted as a simple Mendelian character which is neither sex-linked nor shows dominance. Intact third domains (positions 131 to 186) isolated from the two allelic forms of ovomucoid interact with bovine beta-trypsin in a similar but not identical manner; the complex with the glycine form dissociates more rapidly. Evidence is presented which suggests that glycine is the ancestral residue at position 162; yet, the serine form is the more frequent phenotype.