Molecular aspects of the synthesis and deposition of hens' egg yolk with special reference to low density lipoprotein.

Avian egg yolk contains three main macromolecular constituents: 1) the yolk granules; insoluble particles consisting largely of lipovitellin and phosvitin. They are synthesized in response to hormones in the liver as a precursor protein, vitellogenin, which is soluble in blood. It passes through the oolemma by receptor-mediated endocytosis and is split up enzymically to give fragments that precipitate as granules in the yolk. 2) The livetins; essentially blood serum proteins. The mechanism for their transfer to yolk is not clear. 3) The yolk low density lipoprotein; the major part of yolk (60% of the dry weight). It is synthesized and assembled in the liver as a modified blood very low density lipoprotein, whose main apoprotein is apo B. As with vitellogenin, it enters yolk by endocytosis. Furthermore it is split enzymically to give most of the yolk-lipoprotein apoproteins (apovitellenins III to VI). New evidence for the relationship between yolk apoproteins and apo B has been derived from a comparison of the N-terminal amino acid sequences of apovitellenin IV and apo B.

Separation of lipid-free egg yolk proteins by high-pressure liquid chromatography using solvents containing formic acid.

A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 M guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of "white yolk" and "yellow yolk."

New allergens from hen's egg white and egg yolk. In vitro study of ovomucin, apovitellenin I and VI, and phosvitin.

Three hen egg yolk proteins, apovitellenins I and VI and phosvitin, and one egg white protein, ovomucin, were purified and tested for their ability to bind IgE in the sera of patients hypersensitive to egg. All of the proteins bound IgE from the sera of egg-allergic individuals in the radioallergosorbent test, and they also inhibited binding of IgE to the parent fractions-either egg yolk (apovitellenins I and VI and phosvitin) or egg white (ovomucin). It appears that apovitellenins I and VI are major allergens for some of the individuals tested. This is the first report of the in vitro allergenicity of these proteins.

Proteolysis of apoprotein B during the transfer of very low density lipoprotein from hens' blood to egg yolk.

We report an example of the enzymic cleavage of an apoprotein B (apoB), the main apoprotein in the very low density lipoprotein (VLDL) of laying hens' blood, in a normal biological process, the formation of egg yolk. Plasma VLDL was labeled in vivo with 3H-amino acids, isolated by centrifuging, and injected into another laying hen. Yolk VLDL was isolated and its apoproteins were separated. ApoB was not detected in this lipoprotein. Most of the label originally in apoB was distributed among four smaller yolk apoproteins, apovitellenins III to VI, which are a large proportion of the apoproteins of VLDL in yolk. This distribution of 3H suggested that 80% of apoB was cleaved at three places. One yolk apoprotein, apovitellenin II, was not labeled, indicating that it did not originate from an apoprotein in plasma VLDL. The site for cleavage of apoB in the ovarian tissue has not been determined, but cleavage may occur during receptor-mediated endocytosis. The pattern of cleavage of apoB during transfer to yolk was not imitated by some known proteolytic enzymes.

Allergenic cross-reactivity of egg-white and egg-yolk proteins. An in vitro study.

The radioallergosorbent test (RAST) and RAST inhibition test were used to examine cross-allergenicity amongst the major hen's egg-white and egg-yolk proteins. Using ovalbumin as a reference allergen to compare cross-reactivity, it was apparent that the proteins conalbumin, ovomucoid and lysozyme substantially inhibited binding to ovalbumin discs of IgE in the sera of patients clinically hypersensitive to egg. The converse situation with conalbumin, ovomucoid and lysozyme on the discs and ovalbumin as the inhibitor also resulted in significantly decreased levels of IgE binding to the proteins on the discs. It was also demonstrated that cross-reactions occurred between ovalbumin and the yolk protein, apovitellenin I. Cross-reaction was also observed surprisingly when egg lysozyme was on the disc and the milk protein allergen alpha-lactalbumin was used as the inhibitor. The demonstration of cross-reaction between all of these proteins may signify that there are a number of common allergenic determinants on these egg proteins, thus providing a molecular basis for the phenomenon of cross-reactivity.

Formation of complexes between lecithin and apovitellenin I, an avian egg-yolk apoprotein.

In a study of lipid-protein interactions in egg yolk, it was found that L-alpha-dipalmitoyl lecithin gave two distinct noncovalent complexes (A and B) with apovitellenin I, an apoprotein in the major yolk lipoprotein. Interaction took place under widely varied conditions, and yolk lecithin gave similar complexes. Complex A, which was formed within minutes, consisted of round particles of about 9 nm diameter. Complex B, which was formed more slowly, consisted of larger particles, possibly resembling curved discs, with diameter of 30-40 nm. The preparation and some properties of these complexes are described. It is suggested that they may be suitable for an extensive study of phospholipid-protein interactions in yolk.

Allergens in the white and yolk of hen's egg. A study of IgE binding by egg proteins.

The radioallergosorbent test (RAST) was used to compare the IgE binding of egg white and yolk, and allergenic proteins were detected by immunoelectrotransfer ('Western blotting'). The main allergens were found in egg white, but for a large proportion of the egg-sensitive patients, yolk contained specific IgE-binding constituents. For blood sera from 36 patients, there was a positive correlation between the results of RAST for egg white and for yolk. Lysozyme was found to be an allergen for some patients. The effect of heating on the allergenicity of egg white was examined and the allergenicity of hen egg white was compared with that of a duck egg. The allergens in yolk were associated with each of the three yolk fractions, and several of the proteins in the low-density lipoprotein fraction bound IgE.

Lipoproteins from the blood and egg yolk of the hen. The transfer of very-low-density lipoprotein to egg yolk and possible changes to apoprotein B.

As part of a study of the transfer of proteins and lipids from avian blood to egg yolk, some properties of lipoproteins from the blood of laying hens were compared with those of the low-density lipoprotein (YLP) from egg yolk of the same hens. Although it is known from previous work that particles of the very-low-density lipoprotein (VLDL) of blood are the most likely precursors of YLP, their apoprotein patterns are different, according to electrophoresis and chromatography, with only one protein in common. YLP has the more complicated pattern which does not, however, include apoprotein B (ApoB) the main apoprotein of VLDL. It is suggested that during the transfer of VLDL to yolk, ApoB is cleaved to give smaller yolk apoproteins, especially apovitellenins IV and VI. Some evidence for this suggestion from the similarity of protein digests is presented.

Effect of particle size and lipid composition of bovine blood high density lipoprotein on its function as a carrier of beta-carotene.

As part of a study on the influence of dietary lipids on vitamin transport and metabolism in lactating cows, we have examined the beta-carotene content and other properties of fractions of the high-density lipoprotein (HDL, density 1.05-1.16 g/ml) of bovine blood. Our purpose was primarily to explain previous results indicating that feeding cows polyunsaturated lipids alters the properties of the HDL and increases the concentration of beta-carotene in the blood but not in the milk. Fractions of HDL of different particle size were prepared by gel-filtration chromatography and the particle diameters measured by electron microscopy. We found that large HDL particles contain more beta-carotene per unit weight than small particles. Furthermore the HDL from cows fed lipid-rich diets with a high proportion of linoleic-acid residues, which had been protected against microbial degradation in the rumen, had a high percentage of HDL particles with large diameters. The blood from these cows had a higher concentration of beta-carotene than before feeding polyunsaturated lipids, but their milk had a lower concentration. We suggest that HDL is the main store of beta-carotene in bovine blood. Moreover the concentration of beta-carotene in blood is increased by feeding polyunsaturated lipids largely because of the increase in the percentage of large HDL particles, which contain more beta-carotene. The effect on the concentration of beta-carotene in milk implies that the transfer mechanism is less efficient as a result of feeding polyunsaturated lipids. This lower efficiency may be due in part to the higher percentage of large HDL particles.

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs. V. Protein sulfhydryl groups: accessibility in the lipoprotein and the effect of temperature.

Sulfhydryl groups in the apoproteins of the major lipoprotein (density 0.95 g/ml) of hen's egg yolk have been studied by colorimetry, analysis for carboxymethylcysteine, and labelling with [14C]iodo-compounds and their distribution amongst the individual apoproteins (i.e. apovitellenins I-VI) has been determined. In the intact lipoprotein, reaction of sulfhydryl groups with aqueous reagents was slower than in the apoprotein mixture and there were large differences amongst different reagents in the extent of reaction. If the lipoprotein was heated for a short time in 1 mM EDTA above about 70 degrees C and then cooled, more sulfhydryl groups were accessible although the total number did not change. This increase in accessibility was accompanied by the disruption of the lipoprotein structure and an increase in the viscosity of its solutions. Furthermore, the isolated apoprotein mixture was less soluble in urea solutions and formed a gel, probably as a result of cross-linking by inter-protein disulfides. There was also a small change in the distribution of sulfhydryl groups so it is likely that sulfhydryl-disulfide interchanges occurred on heating. It is suggested that such interchanges help determine the physical properties of heated yolk and that in the intact lipoprotein sulfhydryl groups are concealed to prevent sulfhydryl-disulfide interchanges in the egg.

Hydrophobic chromatography of proteins in urea solutions. The separation of apoproteins from a lipoprotein of avian egg yolk.

A method is described for the chromatographic separation of mixtures of egg-yolk proteins of low solubility by using a hydrophobic column (phenyl-Sepharose) and eluting with increasing concentrations of aqueous urea at low pH. The resolving power of the method was established by tests on proteins and protein fragments of known sequence. The theoretical basis for the method remains, however, unclear. Factors such as the aggregation of the protein often appeared to be more important than its hydrophobicity in determining the urea concentration needed for elution. The method was applied to the mixture of apoproteins from the low-density lipoprotein (density about 0.95 g/ml) of avian egg yolk. For the previously studied apoproteins from egg yolk of the hen (Gallus domesticus), hydrophobic chromatography provided a new and convenient method for isolating the main apoproteins (hen apovitellenins I-VI). For the hitherto unexplored apoproteins from egg yolk of the duck (Anas platyrhynchos) the method has now been used to isolate three new proteins, two of which were not readily separated by methods based on molecular size. The elution pattern obtained with duck egg-yolk apoproteins is not the same as that of the hen egg-yolk apoproteins, although we suggest a relationship for the three new apoproteins based on their amino acid compositions and other properties. Possible roles for the apoproteins in avian egg yolk are described.

Proteins of the outer layer of the vitelline membrane of hen's eggs.

A study has been made of the proteins in the vitelline membrane of hen's eggs before and after mechanical separation into the inner and outer layers. The membranes were dissolved in detergent (sodium dodecyl sulphate) and chromatographic fractions were examined by gel electrophoresis. The separated inner and outer layers were compared by gel electrophoresis. The outer layer contained (i) enzymically active lysozyme (EC 3.2.1.17) (about 60% dry weight), (ii) an insoluble ovomucin complex and (iii) a new protein, VMOI (vitelline membrane outer I). These account for most of the protein. In addition, some minor constituents were detected by gel electrophoresis but were not isolated. Except for ovomucin, the constituents of the outer layer could be dissolved from the membrane at high ionic strength (greater than 0.5 M sodium chloride), resulting in a loss of its structure. On lowering the ionic strength the soluble proteins recombined with the membrane, partially regenerating the original structure. Ovomucin appears to form the skeleton of the outer layer, but the salt-soluble proteins, especially lysozyme, are responsible for its integrity. The function of the newly-recognized protein (VMOI) is not known. Its molecular weight is 17,500 according to gel electrophoresis in detergent and it contains no methionine. The inner layer consists largely of the proteins GPI, GPII and GPIII isolated by Kido et al. (Kido, S., Janado, M. and Nunoura, H. (1975) J. Biochem. 78, 261-268) from the whole membrane.

Effects of dietary supplements of protected lipids on the concentration and transport of beta-carotene and cholesterol in bovine blood and milk: unusual chromatographic behaviour of the high-density lipoprotein with high levels of beta-carotene.

The effects of feeding lipids protected against microbial degradation in the rumen, on the metabolism of beta-carotene and cholesterol in the blood and milk of cows were studied. The diets fed to the cows consisted of a basal mixture of crushed oats and lucerne hay with a protected vitamin supplement containing a-tocopheryl acetate and beta-carotene fed in conjunction with either (i) protected sunflower oil-seed rich in linoleic acid (PO), (ii) protected tallow (PT), or (iii) formaldehyde-treated casein (C) as a control. Diets PO and PT raised the concentrations of beta-carotene and cholesterol in the blood plasma over that observed for diet C. Milk cholesterol concentrations were not affected by dietary supplements, but the level of beta-carotene in milk of cows on diet PO showed a tendency fo fall compared with milk from cows fed PT or C. The properties of the high density lipoprotein (HDLP) of the blood plasma which contained the beta-carotene were affected by the PO diet. As a result of feeding this diet, the fatty-acid composition of the HDLP was altered and it emerged from a gel-filtration chromatographic column earlier than the control. This change in chromatographic behaviour was used as a measure of the effect of the diet, which for some cows, was apparent long after the diet was changed. It is suggested that the altered lipid composition resulting from the PO diet affected the distribution of particle sizes of the HDLP and might interfere with the transfer of beta-carotene from plasma to milk.

Two low-molecular weight apoproteins (apovitellenins I and II) from a lipoprotein of goose's egg yolk: a comparison with related species.

As part of a comparative study of egg yolk from different avian species, the major lipoprotein and its mixed apoproteins from the egg yolk of the chinese goose (Anser cygnoides) have been prepared. From the apoprotein mixture, two new proteins, of molecular weight approximately 10000 and 22000 according to gel electrophoresis in detergent, have been isolated by gel-filtration chromatography in urea. The protein of lower molecular weight corresponds in amino acid sequence to apovitellenin I, a protein previously isolated from other avian species. As a comparison with other members of the same avian family (Anatidae), the amino acid sequence of apovitellenin I from the pekin duck (Anas platyrhynchos) was re-investigated and that of the muscovy duck (Cairina moschata) investigated. These were found to be identical to the sequence of goose's apovitellenin I. The second new protein is similar in composition, molecular weight, and solubility to apovitellenin II, a protein present in small amount in hen's egg yolk. A protein corresponding to apovitellenin II could not, however, be detected in the egg yolk of either species of duck.

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apo VLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences.

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellenin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs III. Influence of salt concentration during isolation on the amount and composition of the apoproteins.

When the major lipoprotein of hen's eggs was prepared by centrifuging yolk in salt solutions, the ionic strength affected the apoprotein mixture. At high salt concentrations (ionic strength above about 2) more protein was present in the lipoprotein than at lower ionic strength. The extra protein was not removed by subsequently lowering the ionic strength. This extra protein consisted largely of proteins of high molecular weight, including, according to electrophoresis, gamma-livetin from the aqueous phase of yolk.

Lipid-protein globules of avian egg yolk. Isolation and properties of globules stable in concentrated sodium chloride solution.

A new type of globular particle, the 'insoluble yolk globule', was isolated from the egg yolk of three avian species (hen, duck, and emu) by centrifugation or gel-filtration chromatography. These globules are stable in NaCl and urea solutions at concentrations that dissolve or disrupt other constituents of yolk, The isolated globules are about 1% of the dry yolk of hen's and duck's eggs but about 8% emu's-egg yolk. Most of these globules are less than 2 micrometer in diameter. Electron micrographs of sections show a preponderance of globules in the range 0.125-0.25 micrometer, each with a thick shell surrounding a feature-less anterior. Globules with the same appearance were seen in sections of unfractionated yolk. Two kinds of larger particles were also observed: (i) particles with a distinct outer membrane and a vesiculated interior; (ii) featureless spheres, possibly of lipid. The insoluble yolk globules comprise protein (8-11% by dry wt.), phospholipid (31-35% total lipid), triacylglycerols (49-53%), cholesterol (8%) and cholesteryl esters (2-3%); the variations being among species. The phospholipid is accessible to phospholipase C. The isolated protein is heterogeneous and resembles the apoprotein from the yolk low-density lipoprotein.