Covalent structure of the minor monomeric beta-lactoglobulin II component from donkey milk

The complete primary structure of the minor beta-lactoglobulin II component from donkey milk is presented. It has been established by amino-acid sequencing and mass-spectrometry analysis of intact protein and peptides obtained after enzymatic and chemical cleavages. The molecular mass and the pI of the protein are calculated to be 18,261 Da and 4.5 respectively. Despite the close structural similarity of the donkey and horse major beta-lactoglobulin I components, their minor beta-lactoglobulin II components show substantial differences in sequence. Most observed exchanges are clustered at residues 78-106 where only 6 amino-acid residues are conserved. The primary structure of donkey beta-lactoglobulin II reveals some unusual features of minor beta-lactoglobulins II and gives new light to the evolution of beta-lactoglobulins and other lipocalins involved in retinol binding or reproductive functions.     

beta-Lactoglobulin identified in marsupial milk. The primary structure, binding site and possible function of beta-lactoglobulin from eastern grey kangaroo (Macropus giganteus)

beta-Lactoglobulin has been isolated in the milk of the Eastern Grey Kangaroo (Macropus giganteus). This is the first time this protein has been reported to be in the milk of marsupials. The complete amino-acid sequence has been determined by spinning cup and pulsed liquid phase microsequencing of the protein and peptides after enzymatic or cyanogen bromide cleavages. The 155-residue protein is the shortest beta-lactoglobulin so far sequenced. When the kangaroo protein is included in a comparison of the members of the beta-lactoglobulin family, the percentage of residues common to all members is reduced from 33% to 13%. Despite the large number of accumulated amino-acid exchanges the protein exists as a dimer and shows higher homology to the usually very conservative dimeric, ruminant beta-lactoglobulins than to the monomeric protein from monogastrics. Half-cystine residues that form disulphide bridges are conserved. The Eastern Grey Kangaroo beta-lactoglobulin possesses significant homology in several characteristic segments thought to be important for a functional trait common to the beta-lactoglobulin family and retinol-binding proteins. Structural similarity to the retinol-binding protein is indicated by 22% of identical residues. Homology to the beta-lactoglobulins and retinol-binding proteins, the binding site and possible function based on comparative structural studies are discussed.     

Isolation and complete primary sequence of a new ovine wild-type beta-lactoglobulin C

A new wild type of beta-lactoglobulin has been identified in the milk of sheep. It has been designated as ovine beta-lactoglobulin C. Its primary structure has been determined by direct protein microsequencing of intact protein and RP-HPLC-derived tryptic peptides. The new beta-lactoglobulin C is a subtype of ovine beta-lactoglobulin A with a single exchange Arg-Gln at position 148. This exchange may influence polymerisation of beta-lactoglobulin since in the crystal structure of orthorhombic bovine beta-lactoglobulin, residues 145-150 constitute a short beta-sheet region involved in dimer formation by pairing of dyad-related strands.     

The amino-acid sequence of beta-lactoglobulin II from horse colostrum (Equus caballus, Perissodactyla): beta-lactoglobulins are retinol-binding proteins

beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse beta-lactoglobulin II is unique in that it contains 166 amino acids. The additional four amino acids represent an insertion between positions 116 and 117 of other beta-lactoglobulins so far sequenced, including horse beta-lactoglobulin I. Sequence comparison of beta-lactoglobulins I and II from horse colostrum reveals 48 amino acid substitutions (30%). Such a diversity between members of the beta-lactoglobulin gene family has not been encountered before. Sequence comparison with bovine beta-lactoglobulin A shows 85 amino acid replacements accounting for 53% of the residues. The structural homology with human retinol-binding protein may reveal similar biological functions and clues to the origin of milk proteins.     

The complete amino-acid sequence of dimeric beta-lactoglobulin from mouflon (Ovis ammon musimon) milk

beta-Lactoglobulin from Mouflon (Ovis ammon musimon) milk has been isolated and its complete primary structure determined. This protein has been isolated in dimeric form and has a molecular mass of 37 kDa. The amino-acid sequence has been determined by microsequencing of the native protein and the peptides were obtained after tryptic cleavage. The tryptic peptides were isolated by reversed phase high-performance liquid chromatography. The primary structure of mouflon beta-lactoglobulin shows close similarity to ruminant beta-lactoglobulins. The presence of His at position 20 indicates that this protein belongs to the B-type of dimeric ovine beta-lactoglobulins. Mouflon beta-lactoglobulin is a 162 amino acid long polypeptide chain with two disulphide bridges and one free thiol group. Structural similarities to the bilin-binding protein, BG protein from olfactory epithelium and retinol-binding protein are discussed.     

Homology between the primary structures of beta-lactoglobulins and human retinol-binding protein: evidence for a similar biological function?

Two types of beta-lactoglobulins were identified and isolated from horse colostrum: beta-1g I and beta-1g II. The amino-acid sequence of some tryptic peptides from the new monomeric beta-lactoglobulin II was determined and aligned to the other beta-lactoglobulins of known sequence and to the human plasma retinol-binding protein. The comparison of the primary structures of beta-lactoglobulins and human retinol-binding protein shows an unexpectedly high homology of 25%. We found 37 identities among 149 possible homologous residues. Among them is a tryptophan residue at position 19 of beta-lg which might represent the binding site of beta-ionone. These data suggest a common origin of beta-lactoglobulin and human retinol-binding protein and imply that beta-lactoglobulins may be involved in the metabolism of retinol.     

Construction and characterization of beta-lactoglobulin chimeras

At neutral pH, equine beta-lactoglobulin (ELG) is monomeric, whereas bovine beta-lactoglobulin (BLG) exists as a dimer. To understand the difference in the oligomerization properties between ELG and BLG, three mutants of ELG (LP, I, and LPI) were constructed by substituting amino acids responsible for important interactions at the dimer interface of BLG into ELG. The mutant LP has an AB loop mutation (S34A/E35Q), the mutant I has an I strand mutation (G145M/R146H/V147I/Q148R/I149L/V150S/P151F/D152N/L153P) and the mutant LPI includes both the LP and I mutations. The far- and near-UV CD spectra of the three mutants are similar to that of the wild-type ELG, indicating that the secondary and the tertiary structures of ELG are not significantly affected by the mutations. Ultracentrifuge analysis shows that all three mutants are monomeric at neutral pH, suggesting that the protein sequences in the AB loop and I strand of BLG alone cannot support dimerization of ELG. Thus, structural differences must exist between ELG and BLG that prevent the ELG mutants from forming the same interactions as BLG at the dimer interface.     

Conformation and stability of thiol-modified bovine beta-lactoglobulin

Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.     

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.     

Structural and conformational changes of beta-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature

Infrared spectra of 2.5 mM solutions of beta-lactoglobulin B were recorded as a function of pH (from pH 2 to pH 13) and as a function of temperature (from -100 degrees C to +90 degrees C). An analysis of the pH- and temperature-induced changes in the secondary structure was performed based on changes in the conformation-sensitive amide I bands of beta-lactoglobulin. Whereas the total amount of beta-structure remains constant (56-59%) between pH 2 and pH 10, the proportions of the various beta-components do change. In particular, the dimerization of the monomeric protein, induced by raising the pH from 2 to 3 , leads to an increase in the intensity of the 1636 cm-1 band (associated with antiparallel beta-sheet), at the expense of the 1626 cm-1 band (associated with exposed beta-strands). Both the thermal and alkaline denaturation of beta-lactoglobulin occur in two distinct stages. Although the spectra (i.e., the structures) after complete thermal or alkaline denaturation are clearly different, the spectrum of the protein after the first stage of thermal denaturation (at about 60 degrees C) is the same as that after the first stage of alkaline denaturation (at pH 11), suggesting a common denaturation intermediate, which probably represents a crossover point in a complex potential hypersurface.     

Proteomic tools to characterize the protein fraction of Equidae milk

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.     

Characterization of the gene encoding ovine beta-lactoglobulin. Similarity to the genes for retinol binding protein and other secretory proteins

Beta-lactoglobulin is the major whey protein in the milk of ruminants and is expressed in the mammary gland during pregnancy and lactation. Here we describe the isolation and characterization of genomic clones encoding ovine beta-lactoglobulin. Two very similar but non-identical, types of beta-lactoglobulin clone were obtained. DNA sequence analysis of one of these showed that the gene is 4900 bases long and contains seven exons. It codes for a protein of 180 amino acid residues, containing an 18-residue signal peptide, within exons I to VI; exon VII is non-coding. We show that the genes encoding serum retinol binding protein, major urinary protein, alpha-1-acid glycoprotein and apolipoprotein D have a similar organization of exons and introns to beta-lactoglobulin. In particular, a comparison between beta-lactoglobulin and retinol binding protein shows that both genes encode equivalent elements of three-dimensional protein structure within analogous exons. These proteins are all members of a large, diverse family of secretory proteins, many of which function in binding small hydrophobic molecules.     

Characterisation of an amylase from a psychrotrophic Vibrio isolated from a deep-sea mud sample

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase.     

食品及饲料中马属动物源性成分的PCR检测研究

采用马和驴mtDNA中特异性片段引物 ,利用聚合酶链反应技术 (polymerasechainreaction ,PCR) ,建立了饲料中马属动物源性动物成分的快速检测方法。通过内切酶HaeⅢ、Sau3A和AluⅠ可对扩增结果进行验证并能够区分马成分和驴成分。扩增产物测序结果表明 :驴扩增产物序列与数据库序列完全一致 ,马样品扩增产物与数据库序列的同源性达 99%。该方法的检测低限分别为 0 5 %(w w)和 0 2 5 %(w w) ,可作为食品及饲料中马属动物成分鉴别检测的有效方法。     

Solution structure and dynamics of bovine beta-lactoglobulin A

Using heteronuclear NMR spectroscopy, we studied the solution structure and dynamics of bovine beta-lactoglobulin A at pH 2.0 and 45 degrees C, where the protein exists as a monomeric native state. The monomeric NMR structure, comprising an eight-stranded continuous antiparallel beta-barrel and one major alpha-helix, is similar to the X-ray dimeric structure obtained at pH 6.2, including betaI-strand that forms the dimer interface and loop EF that serves as a lid of the interior hydrophobic hole. [1H]-15N NOE revealed that betaF, betaG, and betaH strands buried under the major alpha-helix are rigid on a pico- to nanosecond time scale and also emphasized rapid fluctuations of loops and the N- and C-terminal regions.     

Ionic strength dependence of protein-polyelectrolyte interactions

The effect of univalent electrolyte concentration on protein-polyelectrolyte complex formation has been measured by frontal analysis continuous capillary electrophoresis (FACCE) and turbidimetry for the interaction of bovine serum albumin (BSA) with a synthetic hydrophobically modified polyacid, for BSA with (porcine mucosal) heparin (Hp), a highly charged polyanion, and for Hp and insulin. All three highly diverse systems display maxima or plateaus in complex formation in the range of ionic strength 5 < I < 30 mM, confirmed in the case of BSA-Hp by multiple techniques. Similar maxima are reported in the literature, but with little discussion, for BSA-poly(dimethyldiallylammonium chloride), lysozyme-hyaluronic acid, and lysozyme-chondroitin sulfate, always in the I range 5-30 mM. While inversion of salt effect has been discussed specifically for the interaction of gelatin and sodium polystyrenesulfonate with gelatin(28) and with beta-lactoglobulin,(10) the general nature of this phenomenon, regardless of polyelectrolyte origin, molecular weight, and charge sign has not been recognized. The position of the maxima and their occurrence when protein and polyelectrolyte have the same net charge imply that they arise when Debye lengths extend, at low I, beyond half the protein diameter so that addition of salt screens repulsions, as well as attractions. This appears to be a general effect caused by electrostatic repulsions that can coexist simultaneously with hydrophobic interactions. Modeling of protein electrostatics via Delphi is used to visualize this effect for BSA, lysozyme, insulin, and beta-lactoglobulin.     

Abnormal solubility behavior of beta-lactoglobulin: salting-in by glycine and NaCl

The causes of the salting-in of beta-lactoglobulin by glycine and NaCl, a solubility behavior contrary to expectations, were probed by a detailed study of the interactions between these solvent components and the protein. The preferential interactions of beta-lactoglobulin with solvent components in aqueous glycine and NaCl systems have been compared with those of bovine serum albumin and lysozyme. At neutral pH, beta-lactoglobulin exhibited insignificant preferential interactions in glycine and NaCl at low cosolvent concentrations and an increasing preferential hydration at higher concentrations, the levels approaching the values expected from the other two proteins. These results indicate considerable binding of the electrolytes to beta-lactoglobulin, sufficient to compensate for the exclusion due to perturbation of the solvent surface tension. The difference between the preferential interactions of beta-lactoglobulin and the other proteins with these two solvent additives was shown to be the cause of the increase of beta-lactoglobulin solubility even at high concentrations of the additives, at which they have salting-out effects on the other proteins. The preferential interactions of NaCl with the three proteins were examined as a function of pH. The results showed no pH dependence of the preferential hydration for bovine serum albumin and lysozyme, while this parameter increased significantly for beta-lactoglobulin at lower pH. This suggests that the binding of electrolytes to beta-lactoglobulin is due to a unique charge distribution on the surface of the protein around neutral pH, which imparts to this protein a large dipole moment.     

Relationships between conformation of beta-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of beta-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of beta-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon beta-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding beta-sheet structure as evidenced by the appearance of a strong band at 1614 cm(-1) at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of beta-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular beta-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of beta-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular beta-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Time-dependent polymerization of beta-lactoglobulin through disulphide bonds at the oil-water interface in emulsions

Time-dependent intermolecular sulphydryl-disulphide interchange involving beta-lactoglobulin adsorbed at the oil-water interface in n-tetradecane-in-water emulsions (10 wt% oil, 0.5 wt% protein, pH 7.0) has been investigated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). While only monomers are detected in the adsorbed protein immediately after emulsion formation with pure beta-lactoglobulin, on storing the emulsion the amount of polymerized beta-lactoglobulin and the sizes of the oligomers are found to increase with time. There is no polymerization of adsorbed protein in emulsions made with pure alpha-lactalbumin after 72 h, or in emulsions made with beta-lactoglobulin in the presence of a reagent (N-ethylmaleimide) for modifying sulphydryl groups. Analysis by two-dimensional SDS-PAGE of adsorbed protein from aged emulsions made with a mixture of alpha-lactalbumin + beta-lactoglobulin shows some linking by disulphide bonds between alpha-lactalbumin and beta-lactoglobulin at the interface. Taken together with earlier time-dependent surface viscosity measurements, the results indicate the important role of free sulphydryl groups in the development of the high surface viscoelasticity of adsorbed globular proteins at the oil-water interface.     

The amino acid sequence of goat beta-lactoglobulin

The isolation of beta-lactoglobulin from milk of the goat is described. The purified protein was checked for purity and has been characterized by its gross composition and end groups. The native or the modified protein was then degraded by tryptic and cyanogen bromide cleavage. The cleavage products were isolated and sequenced in the sequenator using a Quadrol and propyne program. These data provide the complete sequence of beta-lactoglobulin of the goat. The results are discussed and compared particularly with bovine beta-lactoglobulin components AB. Some biological aspects are described.