Differences in glycosylation and sperm-egg binding inhibition of pregnancy-related glycodelin

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.     

The contribution of D-mannose, L-fucose, N-acetylglucosamine, and selectin residues on the binding of glycodelin isoforms to human spermatozoa

Previous data showed that glycodelin-A from amniotic fluid and glycodelin-F from follicular fluid inhibited sperm-zona pellucida binding. Solubilized zona pellucida reduced the binding of glycodelin-F to sperm extract dose dependently. This study demonstrated that the zona pellucida proteins also reduced the binding of glycodelin-A to sperm extract. Ionophore-induced acrosome reaction reduced the binding of iodinated glycodelin-A and -F to sperm, indicating that the glycodelin-binding sites are on the outer acrosomal membrane or on the sperm plasma membrane overlying the acrosome. While the binding of glycodelin-A to sperm was suppressed by mannose and fucose neoglycoproteins, that of glycodelin-F was also reduced by acetylglucosamine neoglycoprotein. Pretreatment of sperm with inhibitors of mannosidase and acetylglucosaminidase reduced the binding of glycodelin-F to sperm. On the other hand, inhibitor of mannosidase but not of acetylglucosaminidase inhibited the binding of glycodelin-A. In a competition binding assay, mannosidase reduced both glycodelin-A and -F binding whereas acetylglucosaminidase reduced only glycodelin-F binding. While fucosidase reduced the binding of both glycodelins, fucosidase inhibitor was marginally active in suppressing the binding of glycodelins to human sperm. Among the selectins tested, only E-selectin had a slight inhibitory effect on the binding of glycodelin-A to sperm. The binding of glycodelin-F was unaffected by selectins and their antibodies. In conclusion, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E- selectin residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine but not the selectin residue.     

Glycodelin-S in human seminal plasma reduces cholesterol efflux and inhibits capacitation of spermatozoa

Tight control of sperm capacitation is important for successful fertilization. Glycodelin-S is one of the most abundant glycoproteins in the human seminal plasma. However, its function is unclear. We investigated the role of glycodelin-S on capacitation of human spermatozoa. Binding kinetics experiments demonstrated the presence of two saturable and reversible binding sites of glycodelin-S on human spermatozoa. Differently glycosylated other isoforms of glycodelin, glycodelin-A and -F, did not compete with glycodelin-S for these binding sites, suggesting that the glycodelin-S binding sites are different from those of the other isoforms. Indirect immunofluorescent staining revealed specific binding of glycodelin-S around the sperm head. This immunoreactivity was greatly reduced in spermatozoa that had migrated through the cervical mucus surrogates. Glycodelin-S at physiological concentrations significantly reduced the bovine serum albumin and cyclodextrin-induced cholesterol efflux and down-regulated the adenylyl cyclase/protein kinase A/tyrosine kinase signaling pathway, resulting in suppression of capacitation. Deglycosylation abolished glycodelin-S binding and the effect of glycodelin-S on bovine serum albumin-induced capacitation. This indicates that the carbohydrate moiety of glycodelin-S is critical for the function of the molecule. It is concluded that glycodelin-S in seminal plasma maintains the uncapacitated state of human spermatozoa.     

Cumulus oophorus-associated glycodelin-C displaces sperm-bound glycodelin-A and -F and stimulates spermatozoa-zona pellucida binding

Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-zona pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the zona pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced zona pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoazona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoazona binding inhibitory glycodelin isoforms and enhances the zona binding capacity of spermatozoa passing through the cumulus oophorus.     

Identification of alpha-lactalbumin and beta-lactoglobulin in cynomolgus monkey (Macaca fascicularis) milk.

1. An electrophoretic analysis of whey protein from cynomolgus monkey milk revealed that its constituents are more similar to bovine milk than human milk, i.e. cynomolgus monkey milk whey contains, besides alpha-lactalbumin-like protein (LaP), another predominant component similar to bovine beta-lactoglobulin (LgP), in its electrophoretic behavior on both disc- and SDS-polyacrylamide gel electrophoreses. 2. The amino acid composition of LaP shows close similarity to that of human alpha-lactalbumin, and LaP forms an immunoprecipitin line with anti-human alpha-lactalbumin rabbit antiserum. The homology between LaP and alpha-lactalbumin was further confirmed by an analysis of the N-terminal amino acid sequence. 3. LgP is not immunologically identical to bovine beta-lactoglobulin, but its amino acid composition is similar. The result of the N-terminal amino acid sequence analysis of LgP (up to the 26th residue) strongly suggests homology between this protein and beta-lactoglobulin.     

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.     

Zona-binding inhibitory factor-1 from human follicular fluid is an isoform of glycodelin

Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.     

Dimerization, stability and electrostatic properties of porcine beta-lactoglobulin.

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico-chemical characterization of porcine beta-lactoglobulin, purified from pooled milk: size-exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine beta-lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer-dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess alpha helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine beta-lactoglobulin in the crystal structure. Interaction studies revealed that porcine beta-lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.     

ESR study of aqueous dispersions of beta-lactoglobulin and spin-labelled glyceryl monostearate.

From the ESR spectra of aqueous dispersions of synthetic glyceryl monostearate (spin labelled at C-12) a critical micelle concentration of 30 mumol/l at room temperature was obtained, which agrees with that deduced from surface tension measurements. At monoglyceride concentrations smaller or larger than the critical micelle concentration, the monomers show increased motional restriction with increasing molar ratio of beta-lactoglobulin to monoglyceride up to a value of 10, as determined from calculated rotational correlation times; A similar progressive interaction was deduced from spectral changes observed on equimolar dispersions of beta-lactoglobulin and monoglyceride on raising the temperature to 55 degrees C at which the protein and monoglyceride coprecipitate. The relevance of these finding for non-labelled monoglyceride dispersions is indicated by the similarity of the pH-dependent flocculation behaviour of labelled and non-labelled monoglycerides, both in the absence and presence of beta-lactoglobulin; In addition, proton magnetic resonance and mechanical stability measurements suggest that spin-labelled glyceryl monosterate behaves analogously to non-labelled glyceryl monooleate.     

Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid.

The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.     

Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA),⁴ glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2–5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complex-type structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc), NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc), NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc), Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3–5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5).Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10). For example, both GdA and GdF inhibit the spermatozoa-zona pellucida binding (11) via fucosyltransferase-5 (12), but only the latter inhibits progesterone-induced acrosome reaction, thus preventing a premature acrosome reaction of the spermatozoa. There is evidence that cumulus cells can convert exogenous GdA and -F to GdC, the physicochemical properties of which suggest that it is differently glycosylated compared with GdA/F (5). Moreover, GdC stimulated spermatozoa-zona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound GdA and -F (4, 5). GdS suppresses capacitation probably via its inhibitory activity on cholesterol efflux from spermatozoa (13).Except for the effects on fertilization, GdA is involved in fetomaternal defense. This glycodelin isoform suppresses proliferation and induces apoptosis of T cells (2) and inhibits natural killer cell (14) and B-cell (15) activities. Glycosylation is involved in the binding of GdA to receptors on T cells (16). The sialic acid of GdA contributes to the apoptotic activity in T cells (17, 18) and binding to CD45, a potential GdA receptor (16). The importance of glycosylation in glycodelin is further shown by the absence of immunosuppressive activities in GdS with different glycosylation (18). The immunomodulating activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different glycodelins to exhibit their binding activities and biological effects (13, 19, 20). The present study aims to identify the effect of all four glycodelin isoforms on lymphocyte viability, cell death, and interleukin-2 (IL-2) secretion and to correlate these bioactivities with their glycosylation patterns determined by mass spectrometry.     

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.     

The core lipocalin, bovine beta-lactoglobulin

The lipocalin family became established shortly after the structural similarity was noted between plasma retinol binding protein and the bovine milk protein, beta-lactoglobulin. During the past 60 years, beta-lactoglobulin has been studied by essentially every biochemical technique available and so there is a huge literature upon its properties. Despite all of these studies, no specific biological function has been ascribed definitively to the protein, although several possibilities have been suggested. During the processing of milk on an industrial scale, the unpredictable nature of the process has been put down to the presence of beta-lactoglobulin and certainly the whey protein has been implicated in the initiation of aggregation that leads to the fouling of heat exchangers. This short review of the properties of the protein will concentrate mainly on studies carried out under essentially physiological conditions and will review briefly some of the possible functions for the protein that have been described.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

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.     

Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and 13C-labeled N-acetylneuraminic acid

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.     

Binding of retinoids and beta-carotene to beta-lactoglobulin. Influence of protein modifications

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.     

Characterization of pH-induced transitions of beta-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies.

Depending on solution conditions, beta-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of beta-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v degrees (-0.008(+/-0.003) cm3 x g(-1)), and adiabatic compressibility, k degrees (S) (-(0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v degrees (0.004(+/-0.003) cm3 x g(-1)), and compressibility, k degrees (S) ((0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v degrees (-0.006(+/-0.003) cm3 x g(-1)), and an increase in compressibility, k degrees (S) ((1.5(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of beta-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v degrees (-0.014(+/-0.003) cm3 x g(-1)) and k degrees (S) (-(7.0(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of beta-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.     

Effects of temperature, pH, and salt concentration on beta-lactoglobulin deposition kinetics studied by optical waveguide lightmode spectroscopy

Deposition kinetics of beta-lactoglobulin at a solid-liquid interface was studied with optical waveguide lightmode spectroscopy (OWLS) over a range of temperatures between 61 and 83 degrees C. A new temperature-controlled cell for OWLS measurements allows fast, on-line monitoring of the deposit formation at elevated temperatures. Primary protein layers were deposited at 25 degrees C in order to precondition and stabilize the waveguide surface. Sustained deposition lasting from a few minutes (around 80 degrees C) to hours (below 70 degrees C) resulted in multilayer deposits up to several tens of nanometers thick. The measured deposition rates were strongly influenced by temperature, pH, and NaCl concentration. Deposition rates decreased with increasing pH from 5.5. to 7.4, in a trend similar to that for noncovalent aggregation of beta-lactoglobulin in solution. Activation energies for deposition rates decreased with increasing pH, from 340 kJ/mol at pH 5.5 to 230 kJ/mol at pH 7.4 and were similar to the activation energies for denaturation of beta-lactoglobulin in solution.