Lipid-deprived diet perturbs O-glycosylation of secretory proteins in rat mammary epithelial cells.

Nutrition modulates both production and composition of milk. Milk composition was studied in rats chronically fed a diet without additional lipids, and therefore eating only traces of the recommended supply of essential polyunsaturated fatty acid. Despite a large decrease in milk-protein synthesis, only protein composition, but not protein concentration, was found to change in the milk of rats following a lipid-deprived diet. Correlatively, we observed a substantial increase in the lactose concentration of milk. Analysis of milk proteins by two-dimensional electrophoresis demonstrated that the relative proportion of the various molecular forms of κ-casein, an O-glycosylated protein, was modified in the milk of rats receiving the lipid-deprived diet. In tissues, differences in the two-dimensional pattern of κ-casein between control and lipid-deprived rats were similar, if not identical. In contrast to κ-casein, the molecular forms of α-lactalbumin, an N-glycosylated protein, were not affected by the diet. These data provide evidence that O-glycosylation of milk proteins in the secretory pathway of mammary epithelial cells is modulated by the lipid content of experimental diets.

Targeting of PKA in mammary epithelial cells. Mechanisms and functional consequences.

Targeting of protein kinases, promoting association with specific partner-molecules and localisation to particular sites within the cell, has come to be recognised as a key mechanism for attributing specificity to these enzymes. In mammary epithelial cells, the repertoire of acute regulatory roles played by cyclic AMP-dependent protein kinase (PKA) differs from that in other lipogenic cell-types. Furthermore, PKA is implicated in the regulation of mammary-specific function, mediating a tonic stimulation of the flux of newly-synthesised casein through its basal secretory pathway. Both these observations imply mammary-specific properties of either PKA targeting systems or of PKA itself. Evidence for the latter is currently lacking. Pulse-chase labelling experiments in the presence and absence of selective effectors of PKA have enabled the site(s) of action of this protein kinase on casein secretion to be localised to the early stages of the secretory pathway. Possible mechanisms are considered for the physical targeting of PKA to the membrane-enclosed components of the secretory pathway and evidence for their occurrence in mammary epithelial cells is presented.

Establishment of plasma membrane polarity in mammary epithelial cells correlates with changes in prolactin trafficking and in annexin VI recruitment to membranes.

Mammary epithelial cells (MEC) of lactating animals ferry large amounts of milk constituents in vesicular structures which have mostly been characterized by morphological approaches (Ollivier-Bousquet, 1998). Recently, we have shown that under conditions of lipid deprivation, perturbed prolactin traffic paralleled changes in the membrane phospholipid composition and in the cytosol versus membrane distribution of annexin VI (Ollivier-Bousquet et al., 1997). To obtain additional information on the membrane events involved in the vesicular transport of the hormone to the apical pole of the cell, we conducted a biochemical study on prolactin-containing vesicles in MEC at two different stages of differentiation. We first showed that MEC of pregnant and lactating rabbits exhibited membrane characteristics of non-polarized and polarized cells respectively, using annexin IV and the alpha-6 subunit of integrin as membrane markers. Incubation of both cell types with biotinylated prolactin for 1 h at 15 degrees C, followed by a 10-min chase at 37 degrees C revealed that prolactin transport was activated upon MEC membrane polarization. This was confirmed by subcellular fractionation of prolactin-containing vesicles on discontinuous density gradients. In non-polarized MEC, (125)I-prolactin was mainly recovered in gradient fractions enriched with endocytotic vesicles either after incubation at 15 degrees C or after a 10-min chase at 37 degrees C. In contrast, in polarized MEC, the hormone switched from endocytotic compartments to a fraction enriched in exocytotic clathrin-coated vesicles during the 10-min chase at 37 degrees C. Association of annexin VI to prolactin carriers was next studied in both non-polarized and polarized cells. Membrane compartments collected at each gradient interface were solubilized under mild conditions by Triton X-100 (TX100) and the distribution of annexin VI in TX100-insoluble and TX100-soluble fractions was analyzed by Western blotting. Upon MEC polarization, the amount of annexin VI recovered in TX100-insoluble fractions changed. Quite interestingly, it increased in a membrane fraction enriched with endocytotic clathrin-coated vesicles, suggesting that annexin VI may act as a sorting signal in prolactin transport.

Casein secretion in mammary tissue: tonic regulation of basal secretion by protein kinase A.

Despite its quantitative importance in the secretion of lactoproteins, little is known about the triggering and control mechanisms that initiate, regulate and terminate the operation of the basal pathway of lactoprotein secretion throughout the lactation cycle. This study investigated the possible modulation by cAMP-mediated mechanisms, of cellular transit of newly-synthesised caseins and their basal secretion in explants of mammary tissue from lactating rats and rabbits. Enhancement of the rate of secretion of newly-synthesised caseins occurs when mammary explants are challenged in vitro with agents that activate protein kinase A (PKA). Inhibition of PKA slows casein secretion. The PKA-sensitive step(s) in casein secretion is early in the exocytosis pathway but inhibition of PKA does not impair casein maturation. Ultrastructural, immunochemical and biochemical methods locate PKA on membranes of vesicles situated in the Golgi region. Exposure of tissue to a cell-permeant PKA inhibitor results in morphological modification of these vesicular structures. We conclude that PKA mediates tonic positive regulation of the basal secretory pathway for lactoproteins in the mammary epithelial cell.

Lipid-depleted diet perturbs membrane composition and intracellular transport in lactating mammary cells.

When rats were fed a control or a lipid-depleted diet for five generations, reproduction was not disturbed but pup growth was affected. The membrane organization and the secretory activity of mammary epithelial cells from these lactating rats were investigated. This diet induced a large decrease in the level of polyunsaturated fatty acids of membrane phospholipids (26.6% versus 44.0%). The level of 20:4 (n-6) was strongly decreased, mainly in phosphatidylethanolamine. Annexin VI, which interacts preferentially with this phospholipid, accumulated at the periphery of the cell and was largely associated to the hydrophobic region of the bilayer as compared to control membranes. Casein synthesis and casein secretion measured in incubated explants, after pulse-chase metabolic labeling, were both reduced by about 60% in lipid-deprived cells. The secretory ratio (radioactive secreted caseins in %) was not modified, suggesting that the mechanism of basal secretion was not mainly affected. On the contrary, the secretagogue effect of prolactin disappeared. The intracellular transport of the hormone was considerably slowed down by the diet and prolactin did not reach the lumen of the acini after 1 h of chase, in contrast to what occurred in control cells. Addition of 20:4 (n-6), in vitro, to mammary fragments from lipid-deprived rats restored the localization of annexin VI, increased synthesis and secretion of caseins as well as intracellular transport of PRI. Together, these data underline the importance of the level of 20:4 (n-6) in membrane phospholipids for exocytic and endocytic transport in lactating mammary epithelial cells.

Brefeldin A differently affects basal and prolactin-stimulated milk protein secretion in lactating rabbit mammary epithelial cells.

When lactating mammary epithelial cells were treated with prolactin in vitro, numerous small vesicles rapidly accumulated in the Golgi area, and secretion of milk proteins increased. The effects of brefeldin A on these intracellular events were investigated. As observed by electron microscopy, stacks of the median Golgi were not altered after incubation in the presence of 50 nM brefeldin A but were dissociated when the drug concentration was > or = 500 nM. Small vesicles did not accumulate in the Golgi area when mammary cells were incubated in medium containing both prolactin and brefeldin A, whatever the concentration of the latter. Immunofluorescence experiments showed that 50 nM brefeldin A did not modify the localization of the CTR 433 median Golgi protein, but it induced redistribution of trans-Golgi network-associated proteins such as TGN38, AP-1 adaptor and clathrin. These effects occurred in the presence of brefeldin A plus prolactin. Pulse-chase experiments showed that brefeldin A concentrations > or = 100 nM induced the intracellular accumulation of milk proteins, provoked the appearance of immature forms of caseins, and inhibited milk protein secretion. In contrast, concentrations of brefeldin A of < or = 50 nM did not affect basal casein secretion but inhibited the secretagogue effect of prolactin. These data show not only that several biochemical events in the transport of milk proteins which are sensitive to different brefeldin A concentrations occur in lactating mammary epithelial cells, but also that it is possible to inhibit a hormonal stimulus in a selective manner, while the machinery responsible for basal secretion is still active.

Intracellular routing and release of caseins and growth hormone produced into milk from transgenic mice.

Growth hormone (GH) secretion, in mammary tissue from transgenic mice, containing a chimeric gene composed of the regulatory region of whey acidic protein gene and the structural region of GH gene, was compared to casein secretion. GH was expressed in milk and for a small percentage (1:1000) in blood as revealed by SDS-polyacrylamide gel electrophoresis and radioimmunoassay. As attested by immunofluorescence and immunogold electron microscopy, caseins and GH followed the same secretory pathway. However, contrary to caseins, which are essentially in micellar form, GH was detected in a nonaggregated form in secretory vesicles and in the lumen of the acini. Newly synthesized caseins and GH were carried simultaneously, mainly to the lumen of the acini, but also to the base of the cell. Secretion of newly synthesized proteins was increased by prolactin (PRL). As shown by immunoblotting, the proportion of GH versus other proteins, secreted in the presence of PRL was not modified, suggesting that GH secretion is subjected to the same hormonal regulation by PRL as other milk proteins. These results show that, in lactating mammary epithelial cells from transgenic mice, a recombinant GH and the caseins are carried simultaneously to the lumen and suggest that secretion of both proteins is increased by PRL during the same time course. Transport of these newly synthesized proteins occurs also to the base of the cell.

Annexins from bovine adrenal cortex exhibit specific cytosol/membrane solvation.

Cytosol/membrane localization of annexins I to VI was analyzed in tissue extracts from bovine adrenal cortex. Based on their solubility in either aqueous or detergents solutions, they were subfractionated in three groups named cytosolic (C), membrane-bound (MB) and membrane-inserted (MI). Less than 1% of the total annexins present in the tissue were recovered in the C fraction when as much as 76.5 and 22.5% were obtained respectively in the MB and the MI fractions. By immunoblotting after SDS-PAGE, it was shown that the various members of the annexin family were not equally recovered in the different fractions. A-V and A-VI were found present in the three fractions whereas the distribution of A-I, A-II, A-III and A-IV was distinct, suggesting different cellular functions.

Immunological evidence of a P-glycoprotein in the microorganism Dictyostelium.

Starting from the observation of an efficient resistance to the vital dye Hoechst 33342, we postulated the presence of a P-glycoprotein in Dictyostelium discoïdeum cells. This hypothesis is supported by the present data obtained by immunoblotting, using the murine JSB-1 monoclonal antibody. Two components of 170 and 115 kDa were recognized in lysates and crude plasma membrane of Dictyostelium cells grown in the presence of Hoechst 33342. Control experiments conducted without Hoechst 33342 demonstrated in these cells a constitutive expression of the P-glycoprotein, which was compared to its anthracycline-induced expression in a resistant human leukemic cell line, K562.

Spectroscopical characteristics of galactosylceramide-pyrene and ceramide-pyrene incorporated in model and in clathrin coated vesicles.

In order to control the ability of two pyrene-sphingolipids (ceramide-pyrene (Cpyr) and galactosylceramide-pyrene (GCpyr)) to monitor the changes in the lipid bilayer dynamics of cellular membranes, their incorporation in three populations of clathrin coated vesicles which differ in their structural characteristics (Bomsel et al. (1988) Biochemistry 27, 6808-6812) was studied by both absorbance and fluorescence spectroscopy. The method of injection of an ethanolic solution of probe was used. The analysis of the spectra recorded after injection into a free-membrane buffer allowed to discriminate two dispersion states (micellar or aggregated) of the probes. The micellar state was identified as the one suitable for the incorporation within the bilayer. Rising the temperature up to 18 degrees C for a membrane labeling with GCpyr and to 37 degrees C for a membrane labeling with Cpyr was found to be necessary because it allowed to slow down the aggregation process which inhibited the incorporation within the lipid bilayer. The excimer/monomer (E/M) fluorescence intensities ratio of GCpyr was found to be characteristic of each population of coated vesicles. Cpyr could not be used as a diffusion probe because it partly aggregated during the cooling step necessary to establish the E/M versus temperature plot in the heating mode. An important point which arises from these data is that the use of absorbance spectroscopy can avoid misinterpretation of the pyrene derivatives fluorescence spectra in terms of diffusion.

Conformational changes of Robinia pseudoacacia lectin related to modifications of the environment: FTIR investigation.

The secondary structural characteristics of one of the Robinia pseudoacacia lectins (RPA3) have been investigated by FTIR spectroscopy and have been established from absorption measurements in the amide I,I' frequency range and from the quantitative estimation of the rate of NH----N2H exchange. In an anhydrous state the protein structure consists mainly of antiparallel and parallel beta-structures, which represent 60% of the overall secondary structure of RPA3. Data obtained in different polar media (KBr, 2-chloroethanol, 2H2O, NaCl-2H2O and/or DPPC) reveal that RPA3 is a highly flexible protein. In pure 2H2O a rapid solvation of free peptide units and weak peripheral hydrogen bonds occurs, followed by the solvation of more internal parts of the lectin. The protein precipitates before total unfolding is reached. Increasing the ionic strength modifies the rate of NH----N2H exchange. NaCl concentrations of less than or equal to 0.15 M stabilize RPA3 in a structure close to that of the lyophilized lectin and diminish the rate of exchange, whereas higher NaCl concentrations partially disrupt the original secondary structure and increase the rate of exchange. Furthermore RPA3 was shown to interact with DPPC through polar interactions between the polar heads of the phospholipid and specific peptide units. These interactions appear to favor the NH----N2H exchange.

In vitro insertion of the myelin proteolipid apoprotein into oligodendrocyte plasma membranes.

Uncoated vesicles (UCV) loaded with the myelin proteolipid apoprotein covalently tagged with fluorescein (PLPF) were found to interact with isolated oligodendrocytes from bovine brain at 4 degrees C as well as at 37 degrees C. After 1.5 hours of incubation, the labeled protein was localized in the cell membranes. After 2.5 hours the fluorescence intensity associated with the oligodendrocytes decreased and completely disappeared at t = 3.5 hours. Addition of KCl or EDTA in the incubation medium significantly hindered the interaction with cells. In contrast, the elimination of membrane proteins from UCV did not perturb cell labeling. A specific role of PLP was suggested since UCV loaded with a soluble protein (BSAF) led to a weak cell labeling.

Demonstration of cell-ricin interaction by electrophoretic light scattering.

Electrophoretic light scattering has been used to investigate the interaction of ricin, a vegetal toxin, with cells. This technique allowed measurements in the presence of free ligand and proved particularly useful for the study of a system with low affinity. The electrophoretic mobility of erythrocytes and oligodendrocytes was found equal to 2.08 x 10(-8) and 2.35 x 10(-8)m2s-1V-1, respectively. Upon ricin binding, these values decreased significantly. This change was related to the saturation of the binding sites. The specificity of the interaction was demonstrated by conducting the experiments in the presence of lactose. This specific inhibitor fully prevented the ricin-cell interaction.

Effects of calcium and calcium analogs on calmodulin: a Fourier transform infrared and electron spin resonance investigation.

Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopies have been used to monitor changes in the conformation of calmodulin induced by Ca2+ and Ca2+ analogs. Using FTIR spectroscopy we observe that Ca2+: (i) favors the alpha-helical conformation and decreases the flexibility of the molecule; (ii) multiplies the intramolecular hydrogen bonds (the ratio of freely vibrating NH/hydrogen bound NH groups decreases); (iii) induces changes in the C-terminal tyrosine environment; and (iv) increases compactness of the molecule (less NH groups in the peptide bonds can be deuterated). As proved by ESR, Ca2+ binding induces exposure of hydrophobic domains allowing binding of a spin-labelled phenothiazine on calmodulin. When the experiments are performed in the presence of increasing amounts of Ca2+, both ESR and FTIR provide evidence that major conformational changes result after the filling of only two Ca2+-binding sites. But achievement of the spectroscopical changes is only observed when the four binding sites are filled (Ca2+/calmodulin = 4). The effects of analogs are monitored with the same spectroscopical parameters. Zn+ does not induce structural modifications of calmodulin but all other analogs studied mimic the calcium effects to some extent. As regards the amplitude of the spectroscopical effects, analogs rank in the following order: Ca2+ greater than Cd2+ greater than Tb3+ = Eu3+ greater than Gd3+ greater than La3+ greater than Zn2+ = cation depleted. Except for Zn2+, ranking for their activating potency of MLCK, the analogs can be arranged in a similar order.

Comparative study of myelin proteolipid apoprotein solvation by multilayer membranes of synthetic DPPC and biological lipid extract from bovine brain. An FT-IR investigation.

The interaction between the aqueous form of the myelin proteolipid apoprotein (PLA) and model membranes prepared with either synthetic dipalmitoylphosphatidyl choline (DPPC) or biological lipids extracted from bovine brain (BE) has been investigated by Fourier-Transform IR spectroscopy. IR spectra obtained with lyophilized samples of PLA demonstrated 2 main peaks (amide I and amide II) culminating at 1656 cm-1 and 1545 cm-1, which we assigned to helical conformation. When PLA was solvated in DPPC or BE membranes, both the amide I and amide II features remained located at 1655 cm-1 and 1545 cm-1, although their half-width significantly decreased, demonstrating that the lipid environment favoured alpha helix structures. However differences between both mixtures were detected by measuring the amide I and amide II half-widths as a function of the L:P molar ratio. Moreover, analysis of the 1545/1515 peak intensity ratio brought evidence of different localization and/or molecular arrangement of the protein segments containing tyrosine residues, depending on the lipid composition of the membrane. According to previously published models, these data suggest that recombinants prepared with PLA and BE multilayers better mimic the biological membrane than do DPPC-PLA mixtures.

Membrane-damaging action of ricin on DPPC and DPPC-cerebrosides assemblies. A Raman and FTIR analysis.

Perturbations induced by a toxic lectin (ricin) on lipid organisation of model membranes prepared with DPPC and DPPC-cerebrosides mixtures have been analysed by Raman and infrared spectroscopy, two powerful and non-invasive methods. Our approach involves the observation of changes in the vibrational spectra of liquid multilayers in the PO2-, C = O and CH2 spectral regions for two lipid:ricin molar ratios (225:1, 75:1). The interfacial and polar regions of the multilayers, analysed by FTIR, appear to be perturbed by the protein. With both kinds of membranes, ricin mainly perturbs the C = O ester groups of the sn-2 acylchain of DPPC. In the PO2- stretching region, the frequency shifts are correlated with changes in polar group hydration. In the hydrophobic core of the multilayer membrane studied by Raman spectroscopy, the interaction of ricin is associated with changes in lipid packing. These perturbations depend upon the lipid composition of the membrane. With DPPC membranes, an affect is detected at temperatures lower than Tm. It corresponds to a decrease of the lipid ordering. With DPPC-cer membranes, the protein increases the acyl-chain packing order regardless of the temperature of the experiments (10 degrees C less than T less than 75 degrees C). No perturbation of Tm is observed after addition of ricin to either DPPC or DPPC-cer membranes. The different perturbations detected by Raman and FTIR suggest that ricin mainly interacts with the interfacial domains of the membranes.

Lipid solvation of the aqueous form of the myelin proteolipid apoprotein: evidence and characterization of two lipid populations by fluorescence polarization, differential calorimetry, and sucrose gradient centrifugation.

The interaction between dipalmitoylphosphatidylcholine (DPPC) and the aqueous form of the myelin proteolipid apoprotein (PLA) has been investigated. Lyophilization was found to be an efficient and nonperturbing method for membrane reconstitution. Mixtures of different lipid/protein ratios were analyzed by means of differential calorimetry, fluorescence polarization, and sucrose gradient centrifugation. The presence of two coexisting lipid populations, termed "bulk" and "interacting" lipids, was demonstrated by these three techniques. By differential calorimetry, 23 DPPC molecules per molecule of protein (30 kDa) were shown to be excluded from the lipid phase transition. By fluorescence polarization, we detected above the phase-transition temperature a large perturbation of the lipid acyl chain dynamics induced by the aqueous form of PLA. Increasing the protein content above 35% by weight within the recombinants caused drastic changes in both delta H values and the fluorescence anisotropy parameter, which could stem from protein aggregation.

Infrared spectroscopic study of the secondary structure of melittin in water, 2-chloroethanol, and phospholipid bilayer dispersions.

The conformations of melittin, an amphipathic polypeptide consisting of 26 amino acid residues, and its hydrophobic (residues 1--19) and hydrophilic (residues 20--26) fragments were examined in various solvent systems, including H2O, 2H2O, 2-chloroethanol, and 1,2-dimyristoylphosphatidylcholine (DMPC) multilayers, by infrared spectroscopy. Water and 2-chloroethanol were used as reference solvents for characterizing the amide I and II vibrational frequencies of the polypeptide in systems reflecting unordered, beta-structure, or alpha-helical forms. In DMPC bilayer assemblies both melittin and its hydrophobic fragment F1 exhibit alpha-helical conformations. In contrast, infrared spectra for the hydrophilic F2 fragment are suggestive of a beta conformation with perhaps spectral contributions from random-coil configurations. The alpha-helical conformation of intact melittin in DMPC multilayer dispersions remains unchanged as the bilayer passes from the gel to liquid-crystalline state. For melittin-water solutions the infrared spectra monitor changes in population of specific conformations as the temperature is varied. Thus, for melittin concentrations in which tetramers are dominant high temperatures (31 degrees C) favor the alpha-helical form, while low temperatures (8 degrees C) lead to populations of both beta and alpha-helical structures. At lower melittin concentrations for which monomers persist, high temperatures favor an unordered polypeptide form, while low temperatures induce an alpha-helical conformation. Although peak-height intensity ratios AII/AI for the amide I and II regions are difficult to interpret rigorously, values of this parameter for aqueous solutions of melittin suggest a sensitivity to structural changes involving the aggregation properties of the polypeptide.

Comparative effects of melittin and its hydrophobic and hydrophilic fragments on bilayer organization by Raman spectroscopy.

For bilayer systems consisting of 1,2-dimyristoyl phosphatidylcholine (DMPC) incubated with melittin, a polypeptide capable of integrating itself within the membrane, temperature profiles derived from Raman spectroscopic data indicate the existence of an immobilized lipid annulus surrounding the polypeptide. In particular, temperature profiles derived from C--H, C--D and C--C stretching mode parameters for 25:1, 14:1 and 10:1 lipid:protein mole ratios exhibit two order-disorder transitions. The primary (lower) gel to liquid crystalline phase transition is depressed when polypeptide concentration is increased. The concentration-independent higher temperature transition is associated with a fluidization of the immobilized boundary lipids present at the lipid-polypeptide interface within the bilayer. We estimate that five to seven lipids are involved in this discrete boundary layer around the inserted membrane component. The behavior of the intrinsic hydrophobic (residues 1-19) and of the extrinsic hydrophilic (residues 20-26) portions of melittin in the bilayer is compared with the properties of the intact polypeptide. We emphasize evidence that both intrinsic and extrinsic components immobilize lipids contiguous to the polypeptide.