HAMLET: functional properties and therapeutic potential.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein-lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.

Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones.

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.

Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland.

Isografts of heterotypic recombinants of embryonic mammary epithelium with salivary mesenchyme undergo development morphogenetically resembling that of salivary gland. However, cytodifferentiation of the epithelium is like that of mammary gland. In lactating hosts these isografts respond to endogenous hormonal stimulation and synthesize a milk protein, alpha-lactalbumin.

Lactose synthase components in milk: concentrations of alpha-lactalbumin and beta1,4-galactosyltransferase in milk of cows from several breeds at various stages of lactation.

It is believed that milk production is determined by the number and activity of mammary secretory cells. Secretory activity, as assessed by milk volume, depends on secretion of the major osmole in milk, lactose, which is produced by lactose synthase. The amount of either of the two proteins in lactose synthase may regulate milk production. The objective of this study was to determine whether the concentrations in milk of the two components of lactose synthase, alpha-lactalbumin (alpha-LA) and beta1,4-galactosyltransferase (B4GALT), were related to genetic background, stage of lactation, breed or parity of dairy cows. alpha-Lactalbumin and B4GALT concentrations were measured by ELISA and by enzyme assays, respectively, from single milk samples. Two herds with a total of 279 cows were used in the analysis. One herd contained Ayrshire, Brown Swiss, Holstein and Jersey cows; the second herd contained two groups of cows; Holsteins selected for high milk production and Holsteins with 1960s genetics. The alpha-LA concentration in milk was greater in Jerseys and Ayrshires than in Holsteins and Brown Swiss. However, no difference in alpha-LA concentration was observed in milk from high and low genetic merit cows in the Minnesota herd or among different genetic backgrounds in the Illinois herd. beta1,4-Galactosyltransferase concentrations were similar for all groups that were analyzed. alpha-Lactalbumin concentrations were positively correlated with milk protein concentration, milk fat concentration and lactose concentration. beta1,4-Galactosyltransferase concentration in milk exhibited a strong positive correlation with number of days in milk. Although the concentration of B4GALT increased as lactation progressed, the values did not show any correlation with persistency of lactation or late lactation milk production. In conclusion, this survey shows that the two components of lactose synthase are each correlated to protein concentration and individually correlated to the concentration of other milk components and stage of lactation.

GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells.

The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane-associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.

Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

beta-1,4-Galactosyltransferase (GalTase) has two functionally distinct subcellular distributions. In the Golgi apparatus, GalTase participates in the glycosylation of secretory and membrane-bound glycoproteins, whereas on the cell surface it mediates specific aspects of intercellular adhesion. For this study, a murine GalTase clone was obtained by screening a lambda gt10 cDNA library made from F9 embryonal carcinoma cells with a heterologous bovine GalTase cDNA probe. The murine GalTase cDNA probe was used in conjunction with assays of GalTase activity to investigate the expression and distribution of GalTase during differentiation of F9 stem cells into secretory endodermal epithelium. During the initial phase of F9 cell differentiation, GalTase mRNA levels remained relatively constant; however, as differentiation progressed, as assayed by expression of the differentiation-specific marker laminin B1, GalTase mRNA levels and enzyme activity rose dramatically. Furthermore, subcellular fractionation of these cells showed that the increased GalTase levels were specifically associated with the Golgi apparatus, whereas GalTase specific activity on the plasma membrane remained constant. These results show that levels of cell surface and Golgi GalTase change relative to one another during F9 cell differentiation and suggest that these functionally distinct pools of GalTase are independently and differentially regulated.

Structure and catalytic cycle of beta-1,4-galactosyltransferase.

Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Crystal structures of guinea-pig, goat and bovine alpha-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase.

BACKGROUND: The regulation of milk lactose biosynthesis is highly dependent on the action of a specifier protein, alpha-lactalbumin (LA). Together with a glycosyltransferase, LA forms the enzyme complex lactose synthase. LA promotes the binding of glucose to the complex and facilitates the biosynthesis of lactose. To gain further insight into the molecular basis of LA function in lactose synthase we have determined the structures of three species variants of LA. RESULTS: The crystal structures of guinea-pig, goat and a recombinant from of bovine LA have been determined using molecular replacement techniques. Overall, the structures are very similar and reflect their high degree of amino acid sequence identity (66-94%). Nonetheless, the structures show that a portion of the molecule (residues 105-110), known to be important for function, exhibits a variety of distinct conformers. This region lies adjacent to two residues (Phe31 and His32) that have been implicated in monosaccharide binding by lactose synthase and its conformation has significant effects on the environments of these functional groups. The crystal structures also demonstrate that residues currently implicated in LA's modulatory properties are located in a region of the structure that has relatively high thermal parameters and is therefore probably flexible in vivo. CONCLUSIONS: LA's proposed interaction site for the catalytic component of the lactose synthase complex is primarily located in the flexible C-terminal portion of the molecule. This general observation implies that conformational adjustments may be important for the formation and function of lactose synthase.

Expression of pregnancy-specific genes in preneoplastic mouse mammary tissues from virgin mice.

Experimentally induced breast cancer is often preceded by the appearance of preneoplastic lesions which possess the attributes of hyperplastic normal tissue. These lesions can be isolated and carried as stably transplantable outgrowth lines which continue to morphologically resemble differentiating mammary tissue (Medina, D. Methods Cancer Res., 7: 3-53, 1973). We established seven serially transplantable hyperplastic alveolar nodule (HAN) outgrowth lines from virgin mouse mammary tissues following induction by mouse mammary tumor virus, dimethylbenz(alpha)-anthracene, and/or pituitary isografts. The expression of mammary differentiation-specific casein genes was measured in these hyperplastic outgrowths by immunocytochemistry, specific radioimmune precipitation, and blot hybridization of total RNA. All seven HAN outgrowth lines were immunologically positive for casein both in situ and upon explant culture. Unlike explants from normal virgin mouse mammary gland, exposure to insulin, hydrocortisone, and prolactin induced an increase in casein synthesis in HAN explant cultures which was independent of DNA synthesis. [35S]Methionine-labeled polypeptides synthesized in explant cultures of HAN outgrowths freshly isolated from virgin hosts were analyzed by radioimmune precipitation and gel electrophoresis. This analysis demonstrated that all major species of casein, alpha (Mr 46,000), beta (Mr 27,000), and gamma (Mr 25,000), were constitutively (i.e., in the absence of lactogenic stimuli) expressed in these preneoplastic alveolar mammary outgrowths. In support of this observation, RNA homologous to beta- and alpha-casein cDNA probes was often detectable in total RNA preparations from freshly isolated fragments of HAN outgrowths. A second mammary differentiation specific gene product, alpha-lactalbumin, was also detected in HAN outgrowths both in situ and following explant culture. Enzymatically active alpha-lactalbumin was present in extracts of freshly isolated HAN outgrowth tissues and was detectable in these same tissues by immunoperoxidase. In general, alpha-lactalbumin synthesis was increased during explant culture in the presence of lactogenic hormones; however, in contrast to casein synthesis, insulin-hydrocortisone-prolactin-induced increase in alpha-lactalbumin production in vitro was occasionally dependent upon DNA synthesis as it is in explants from normal virgin mouse mammary tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolactin-like activity of antiprolactin receptor antibodies in rat mammary tumor explants.

The effects of prolactin and a serum containing antiprolactin receptor antibodies on some lactogenic and mammogenic responses were investigated in nitrosomethylurea-induced mammary tumors in organ cultures. Prolactin was able to induce an increase in lactose synthetase activity, DNA synthesis, and prolactin-binding sites at moderate (i.e., physiological) concentrations of prolactin; at higher concentrations, desensitization of the tissues was observed for DNA synthesis and lactose synthetase activity, whereas the "down-regulation" of prolactin receptors occurred. Prolactin had no effect on casein synthesis. Antiprolactin receptor serum was capable of inducing an increase in lactose synthetase activity, DNA synthesis, and prolactin-binding sites, however, lower than that observed with prolactin, thus mimicking prolactin action. The antiserum did not induce any change in casein synthesis. These findings suggest that, in rat mammary tumors, as it has been observed in the normal mammary gland, the prolactin molecule is not required beyond the initial binding to its receptor for its action to be attained. It appears also that a differentiated function has lost its hormonal dependence in these tumors, although their hormonal dependence for other functions and growth is maintained.

Effect of hyperthermia on activity of three glycosyltransferases in Chinese hamster ovary cells.

We measured activities of three glycosyltransferases at various times during heat-induced thermotolerance development. Glycosyltransferases are normally located in the Golgi apparatus and catalyze cellular glycosylation reactions. UDP-Gal:N-acetylglucosamine beta 1,4-galactosyltransferase (beta 1,4-GalT) is known to participate in the formation of N-linked glycoproteins; when compared to cell survival, beta 1,4-GalT activity was significantly more heat resistant (50% loss of activity: 80 min, 45 degrees C) and showed little elevation at a time when thermotolerance was fully expressed. However, beta 1,4-GalT activity increased twofold by 24-h postheating when thermotolerance had begun to decay. Activity of beta 1,4-GalT was compared with glycosyltransferase activities that are considered to be specific for O-linked glycoproteins: UDP-Gal:N-acetylgalactosamine-beta 1,3-galactosyltransferase (beta 1,3-GalT), and UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (Gal-NAcT). Heat-inactivation experiments with heating times up to 60 min at 45 degrees C failed to reduce either activity below that of unheated control cells. Instead both beta 1,3-GalT and GalNAcT activity increased approximately twofold immediately after 10 min at 45 degrees C. Activity of beta 1,3-GalT rapidly decreased with time after heating and returned to control levels by 6-h postheating. In contrast, GalNAcT activity continued to increase with time after 10 min at 45 degrees C, and was 4.5-fold above unheated controls by 6-h postheating. GalNAcT activity returned to control levels 24- to 48-h postheating. A comparison with the cellular survival response showed that GalNAcT activity preceded thermotolerance expression by 2-4 h and also decayed more rapidly than heat resistance in thermotolerant cells. These data, together with other published results, suggest that expression of thermotolerance may be associated with enhanced glycosylation of intracellular proteins.

Difference between mammary epithelial cells from mature virgin and primiparous mice.

Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.

Immobilized bovine lactose synthase. A method of topographical analysis of the active site.

Bovine galactosyltransferase (UDPgalactose: D-glucose 4beta-galactosyltransferase, EC 2.4.1.22) was covalently coupled to Sepharose 4B by reaction at pH 5.0 with the activated mixed disulfide Sepharose-glutathione-2(5-nitropyridyl)-disulfide. The Sepharose-protein conjugate was presumably coupled via the unique highly reactive cysteine of those thiols on the bovine enzyme. The gel-bound N-acetyllactosamine and lactose synthase activity of about 0.4% was consistent with the affects of diffusion and the 90% activity reduction noted upon thiol modification of the dissolved enzyme. The residual lactose biosynthetic activity of the bound enzyme appeared possible only if the reactive thiol were physically distinct from the active site since the bulky Sepharose-glutathione group must not obscure the alpha-lactalbumin binding region.

Purification by affinity chromatography and some properties of microsomal galactosyltransferase from pig thyroid.

Membrane-bound 4-beta-galactosyltransferase (lactose synthase; UDP galactose: D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) was purified 1500-fold to near homogeneity from pig thyroid microsomes with about 30% yield. The purified enzyme behaved as a lipophilic protein, rapidly losing activity and aggregating if not supplemented with either Triton X-100 or serum albumin (both of these were equally effective for long-term stabilization). The enzyme preparation showed an absolute requirement for Mn2+, which could not be replaced by other cations. Catalytic properties were very similar to those reported for soluble forms of the enzyme in biological fluids. The purified galactosyltransferase showed a major protein band of approx. 74,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity was eluted at approx. 70,000 daltons. It is concluded that the membrane-bound thyroid galactosyltransferase is a monomeric protein significantly larger than the soluble forms of this enzyme described earlier; but it resembles recently reported galactosyltransferases from sheep mammary Golgi membranes and liver microsomes.

Lactose synthase: effect of alpha-lactalbumin on substrate activity of N-acylglucosamines.

N-Acetyl-, N-propionyl-, N-butyryl- and N-valerylglucosamines were synthesized as topographical probes to localize further the interaction site of alpha-lactalbumin on galactosyltransferase. All these compounds were found to be substrates for galactosyltransferase with Km values in the millimolar range. In the presence of alpha-lactalbumin, the Michaelis-Menten constants were diminished. However, the effect on the initial rates of these reactions varied. Thus, at low N-acylglucosamine concentrations, alpha-lactalbumin activated the enzyme activity, but at high concentrations, alpha-lactalbumin became inhibitory. This mixed-type inhibition kinetics indicated that a quaternary complex between galactosyltransferase, alpha-lactalbumin, Mn2+-UDPgalactose and N-acylglucosamine existed during the catalytic process. The ability of these N-acylglucosamine substrates to bind to lactose synthase complex was further substantiated by the physical association of galactosyltransferase onto the solid-bound alpha-lactalbumin in the presence of any one of these compounds. The data revealed that the presence of the N-acyl group up to five carbons in length did not interfere with the interaction between alpha-lactalbumin and galactosyltransferase, suggesting that alpha-lactalbumin was not bound in the vicinity of the C-2 region of the monosaccharide site. The inhibitory effect of alpha-lactalbumin on N-acyllactosamine formation is probably a consequence of conformational changes of galactosyltransferase.

Heterogeneity in alpha-lactalbumins. I. Human alpha-lactalbumin.

alpha-Lactalbumin from human milk shows an heterogeneous behaviour when subjected to ion exchange chromatography with DEAE-Sephadex. Two components have been separated, showing identical patterns in the following studies: amino acid compositions, fluorescence and circular dichroism spectra, transition temperature of denaturation, antigenicity, lactose synthase specifying activity and hydrodynamic properties. After rechromatography of either peak, these two components appeared to be in equilibrium. This equilibrium varies with the temperature and the pH of chromatography. Moreover, an increase of n-alcohol concentration in the eluting buffer also induces an increase of the second protein peak eluting at higher ionic strength. These two peaks seem to be the result of some conformational change induced upon the binding of the protein to the solid anionic matrix.

On the reactivities of the tryptophan residues of human alpha-lactalbumin to 2-hydroxy-5-nitrobenzyl bromide.

The reaction of human alpha-lactalbumin with the tryptophan reagent 2-hydroxy-5-nitrobenzyl bromide has been studied. This protein has 3 tryptophan residues (Trp-60, Trp-104 and Trp-118) all of which are accessible to the reagent at pH 2.7 or 7. Trp-60 of human alpha-lactalbumin is much more reactive than Trp-60 of bovine alpha-lactalbumin (Barman, T. E. (1972) Biochim. Biophys. Acta 257, 297-313). As with bovine alpha-lactalbumin, at pH 2.7, 2-hydroxy-5-nitrobenzyl bromide is specific for tryptophan but at pH 7 His-32 also reacts. When treated with the tryptophan reagent, both alpha-lactalbumins lose their specifier protein activities in the lactose synthase (UDPgalactose:D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) reaction.

Chemical modification of tyrosyl and lysyl residues in goat alpha lactablbumin and the effect on the interaction with the galactosyl transferase.

The effect of acylation of goat alpha-lactalbumin on lactose synthetase activity and the ability of alpha-lactalbumin to inhibit the transfer of galactose to N-acetylglucosamine is biphasic. Approx. 15% of the lactose synthase activity of goat alpha-lactalbumin and 10% of its inhibitory power is lost in the initial phase, with corresponding losses of 65 and 30% in the second phase. Deacylation of reacted tyrosyl groups with hydroxylamine restored inhibitory power completely in the initial phase and partially in the second phase. Removal of acyl groups in the initial phase decreased lactose synthase activity, but had no effect in the second phase. The differential effect of acylation of alpha-lactalbumin on lactose synthase and inhibitory properties appears to be the result of differential changes in the affinity of the UDP-Gal-galactosyl-transferase-alpha-lactalbumin ternary complex for monosaccharides.

Use of concanavalin A as a topographical probe for protein-protein interaction. Application to lactose synthase.

Galactosyltransferase (EC 2.4.1.38) has been shown to bind to Con A-Sepharose. Concentrations of methyl-alpha-mannoside greater than 0.7 M were required to release the enzyme from the immobilized lectin. Molecular weight determination by gel filtration revealed that galactosyltransferase formed a 1:1 complex with concanavalin A. Preincubation of the enzyme with excess concanavalin A did not affect its catalytic activity either in the presence or absence of alpha-lactalbumin. The galactosyltransferase-concanavalin A complex was retained on an alpha-lactalbumin-Sepharose column in the presence of N-acetylglucosamine and manganese chloride and was eluted from the column in their absence. Galactosyltransferase immobilized onto a Con A-Sepharose was still active either in the presence or absence of alpha-lactalbumin. Lactose synthase activity was also observed when the galactosyltransferase-concanavalin A complex was assayed with alpha-lactalbumin immobilized on Sepharose. These data indicate that the carbohydrate moiety of galactosyltransferase is involved in neither the catalytic process nor the binding of alpha-lactalbumin and must be linked to the enzyme at a location where it does not present any steric hindrance on the binding of concanavalin A, either free or immobilized on Sepharose.