Development of high-throughput quantitative analytical method for L-cysteine-containing dipeptides by LC-MS/MS toward its fermentative production.

L-Cysteine (Cys) is metabolically fundamental sulfur compound and important components in various cellular factors. Interestingly, free-form Cys itself as a simple monomeric amino acid was recently shown to function in a novel antioxidative system (cysteine/cystine shuttle system) in Escherichia coli. However, as for Cys-containing dipeptides, the biological functions, effects, and even contents have still remained largely elusive. The potential functions should be a part of cellular redox system and important in basic and applied biology. For its progress, establishment of reliable quantitation method is the first. However, such accurate analysis is unexpectedly difficult even in Cys, because thiol compounds convert through disulfide-exchange and air oxidation during sample preparation. Addressing this problem, in this study, thiol molecules like Cys-containing dipeptides were derivatized by using monobromobimane (thiol-specific alkylating reagent) and detected as S-bimanyl derivatives by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sample separation was processed with a C18 column (2.1 mm × 150 mm, 1.7 µm) and with water-acetonitrile gradient mobile phase containing 0.1% (v/v) formic acid at flow rate of 0.25 ml/min. The mass spectrometer was operated in the multiple reaction monitoring in positive/negative mode with electrospray ionization. The derivatization could indeed avoid the unfavorable reactions, namely, developed the method reflecting their correct contents on sampling. Furthermore, the method was successfully applied to monitoring Cys-containing dipeptides in E. coli Cys producer overexpressing bacD gene. This is the first report of the quantitative analysis of Cys-containing dipeptides, which should be useful for further study of fermentative production of Cys-containing dipeptides.

Lactoferrin promotes murine C2C12 myoblast proliferation and differentiation and myotube hypertrophy.

Lactoferrin (Lf) is a multifunctional glycoprotein, which promotes the proliferation of murine C2C12 myoblasts. In the present study, it was investigated how Lf promotes myoblast proliferation and whether Lf promotes myoblast differentiation or induces myotube hypertrophy. Lf promoted the proliferation of myoblasts in a dose­dependent manner. Myoblast proliferation increased on day 3 when myoblasts were cultured in the presence of Lf for three days and also when myoblasts were cultured in the presence of Lf for the first day and in the absence of Lf for the subsequent two days. In addition, Lf induced the phosphorylation of extracellular signal­regulated kinase (ERK)1/2 in myoblasts. The mitogen­activated protein kinase kinase 1/2 inhibitor U0126 inhibited Lf­induced ERK1/2 phosphorylation and repressed Lf­promoted myoblast proliferation. C2C12 myoblasts, myotubes and skeletal muscle expressed low­density lipoprotein receptor­related protein (LRP)1 mRNA and Lf­promoted myoblast proliferation was attenuated by an LRP1 antagonist or LRP1 gene silencing. The knockdown of LRP1 repressed Lf­induced phosphorylation of ERK1/2. Furthermore, when myoblasts were induced to differentiate, Lf increased the expression of the myotube­specific structural protein, myosin heavy chain (MyHC) and promoted myotube formation. Knockdown of LRP1 repressed Lf­induced MyHC expression. Lf also increased myotube size following differentiation. These results indicate that Lf promotes myoblast proliferation and differentiation, at least partially through LRP1 and also stimulates myotube hypertrophy.

Lactoferrin induces tropoelastin expression by activating the lipoprotein receptor-related protein 1-mediated phosphatidylinositol 3-kinase/Akt pathway in human dermal fibroblasts.

Dermal fibroblasts generate the extracellular matrix component elastin, which is synthesized as tropoelastin (TE) and play a critical role in maintaining skin elasticity. Lactoferrin (Lf), an 80-kDa iron-binding glycoprotein, has biological functions such as anti-bacterial, -inflammatory, and -cancer activities. We previously reported that bovine Lf increases TE mRNA expression in human dermal fibroblasts. However, it remains unclear how Lf up-regulates TE expression. Here, we investigated molecular mechanisms underlying this effect. Lf promoted the phosphorylation of Akt1 and extracellular signal-regulated protein kinase (ERK)1/2. As expected, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the MAPK inhibitor U0126 inhibited Lf-induced phosphorylation of Akt1 and ERK1/2, respectively. In contrast, LY294002, but not U0126, inhibited Lf-induced TE expression. Human dermal fibroblasts expressed lipoprotein receptor-related protein 1 (LRP-1) mRNA, and the LRP1 inhibitor receptor-associated protein attenuated Lf-induced increases in TE expression. Furthermore, siRNA-mediated knockdown of LRP-1 significantly suppressed Lf-increased TE expression and Lf-induced Akt1 phosphorylation. Iron-saturated Lf (holo-Lf) increased TE expression and promoted Akt1 phosphorylation, when compared to those parameters in cells treated with iron-free Lf (apo-Lf). Transforming growth factor (TGF)-ß1 also increased TE expression. LY294002 inhibited TGF-ß1-mediated TE upregulation, whereas TGF-ß1 activated Akt2, but not Akt1, phosphorylation. These results indicate that holo-Lf, but not apo-Lf, increases TE expression through LRP-1 in human dermal fibroblasts and suggest that holo-Lf and TGF-ß1 enhance TE expression by activating the PI3K/Akt1 and PI3K/Akt2 pathways, respectively.

Lactoferrin and the lactoferrin-sophorolipids-assembly can be internalized by dermal fibroblasts and regulate gene expression.

Lactoferrin (Lf) is an iron-binding multifunctional protein, mainly present in external secretions. Lf is known to penetrate skin and may thus exert its multiple functions in skin. Sophorolipids (SLs) are glycolipid biosurfactants, which have been shown to enhance absorption of commercial bovine Lf (CbLf) in model skin via forming an assembly with CbLf. In this study, uptake and post-internalization localization of bovine Lf (bLf), CbLf, and human Lf (hLf) with or without forming assemblies with SLs in human dermal fibroblasts (HDFn) were determined using 125I-labeled Lfs and confocal microscopy, respectively. Our results show that all 3 Lfs were internalized by HDFn; although SLs did not significantly affect the uptake of Lfs, it changed Lf localization by accumulating Lfs in the perinuclear region. Furthermore, microarrays were used to investigate transcriptional profiling in HDFn in response to CbLf, SLs, or CbLf-SLs-assembly treatments. Transcriptome profiling indicates that CbLf may play roles in the protection of skin from oxidative stress, immunomodulatory activities, and enhancement of wound healing. The assembly had similar effects but dramatically modulated the transcription of some genes. SLs alone modified signaling pathways related to lipid metabolism, as well as synthesis of sex hormones and vitamins. Thus, CbLf may exert beneficial effects on skin, and these effects may be modulated by SLs.

Lactoferrin inhibits melanogenesis by down-regulating MITF in melanoma cells and normal melanocytes.

The aim of this study was to evaluate the effect of bovine lactoferrin (bLf) on melanin-producing cells and to elucidate its mechanism of action. We tested the anti-melanogenic effect of bLf on a 3-dimensional cultured pigmentation skin model and confirmed a 20% reduction in pigmentation, suggesting that bLf was transdermally absorbed and it suppressed melanin production. Treatment of human melanoma cells with bLf resulted in a significant, dose-dependent suppression of melanin production. Apo-bLf and holo-bLf suppressed melanogenesis to the same degree as bLf. The key feature behind this anti-melanogenic effect of bLf was the down-regulation of the microphthalmia-associated transcription factor (MITF), leading to the suppression of tyrosinase activity. Treatment with bLf resulted in both decreased expression of MITF mRNA and enhanced degradation of MITF protein. However, the primary effector was enhanced phosphorylation of extracellular signal-regulated kinase (ERK), leading to the phosphorylation and degradation of MITF. Our finding that bLf suppresses melanin production in melanocytes indicates that bLf is a possible candidate for application as a skin-whitening agent.

Protein-surfactant interactions between bovine lactoferrin and sophorolipids under neutral and acidic conditions.

To understand the protein-surfactant interactions between naturally derived sophorolipids (SLs) and bovine lactoferrin (bLf), we carried out spectroscopic, microscopic, and biochemical experiments under weakly acidic and neutral pH conditions. Particle size analysis, microscopy, and enzymatic digestion indicated that bLf and SLs interact with each other to form sheet-like and small aggregated structures reflecting the original self-organization of SLs at pH 5.0 and 7.0, respectively. Circular dichroism (CD) showed that SLs did not significantly affect the secondary structure of bLf.

Mogrol Derived from Siraitia grosvenorii Mogrosides Suppresses 3T3-L1 Adipocyte Differentiation by Reducing cAMP-Response Element-Binding Protein Phosphorylation and Increasing AMP-Activated Protein Kinase Phosphorylation.

This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride accumulation by affecting early (days 0-2) and late (days 4-8), but not middle (days 2-4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein ß (C/EBPß; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPß expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process.

Transdermal administration of lactoferrin with sophorolipid.

Lactoferrin (Lf), a multifunctional glycoprotein, is known to activate dermal fibroblasts. Enhancing percutaneous absorption without decreasing the activity of Lf is critical in making the dermal administration of Lf beneficial. Sophorolipid (SL), a glycolipid-type biosurfactant, is known to form assemblies that may elevate the efficiency of the transdermal delivery of active ingredients. Here, we investigated the role of SL in the transdermal absorption of bovine Lf (bLf) and the effect of SL on the bLf activity on dermal fibroblasts. Transdermal absorption of bLf through a model skin was enhanced by 1.3-fold to 1.7-fold when SL was added. The effects of SL on the bLf activities on dermal fibroblasts were examined by cell proliferation activities and by gene expression levels of elastic fiber components, collagen IV, and hyaluronan synthases, revealing that SL did not depress the effect of bLf to any extent. Instead, the tropoelastin gene expression was upregulated ~60-fold by bLf alone, which was further increased to ~160-fold by bLf and SL together, suggesting a significant synergism between bLf and SL. Protein levels of elastin, assessed by immunohistochemistry, correlated well with the results of gene expressions. These results indicate the feasibility of the transdermal administration of bLf with SL.

Digestion and absorption of Siraitia grosvenori triterpenoids in the rat.

When administered to rats, mogroside V (a pentaglucose-conjugated mogroside), the main sweetening component of Siraitia grosvenori, was mostly degraded by digestive enzymes and intestinal microflora, and was excreted in the feces as mogrol (aglycone) and its mono- and diglucosides. However, trace amounts of mogrol and its monoglucoside were found in the portal blood as sulfates and/or glucuronide conjugates.

The N1 domain of human lactoferrin is required for internalization by caco-2 cells and targeting to the nucleus.

Human lactoferrin (hLf) has been shown to interact with cells from the Caco-2 human small intestinal cell line. There currently is little information about the molecular details of its interaction. As a first step toward detailed characterization of this interaction, we used a series of Lf chimeras to analyze which part of Lf is responsible for the interaction with Caco-2 cells. Recombinant chimeric proteins consisting of segments of hLf and bovine transferrin (bTf) were produced in a baculovirus-insect cell system and purified by a combination of cation exchange chromatography and immobilized bTf antibody affinity chromatography. Each chimera was labeled with a green fluorescent dye to monitor its interaction with Caco-2 cells. Similarly, the intestinal Lf receptor (LfR), also known as intelectin, was probed with an anti-LfR antibody that was detected with a secondary antibody conjugated with a red-color fluorescent dye. The results demonstrated that chimeric proteins containing the N-lobe or the N1.1 subdomain of Lf bound as well as intact Lf to Caco-2 cells. Confocal microscopy analysis revealed that these proteins, along with the LfR, were internalized and targeted to the nucleus. These results indicate that the N1.1 subdomain of hLf is sufficient for binding, internalization, and targeting to the nucleus of Caco-2 cells.

Antidiabetic effect of long-term supplementation with Siraitia grosvenori on the spontaneously diabetic Goto-Kakizaki rat.

Siraitia grosvenori Swingle (SG) is a traditional Chinese fruit used as a folk medicine. Its extract (SG-ex) contains potent sweet elements with a sweetness several hundred times higher than table sugar. We investigated the antidiabetic effect of SG-ex in the type 2 diabetic Goto-Kakizaki (GK) rat. Diabetic 7-week-old GK rats were fed a diet supplemented with 0.4 % of the SG-ex for 13 weeks, and its antidiabetic effects were evaluated. SG-ex had no effect on food intake or body weight. In oral glucose tolerance tests (OGTT), SG-ex supplementation improved the insulin response at 15 min (control, 63 (sem 6) pm; SG-ex, 107 (sem 20) pm; P < 0.05) and reduced the plasma glucose level at 120 min after the glucose administration (control, 18.5 (sem 0.8) mm; SG-ex, 14.8 (sem 0.7) mm; P < 0.05). The total amount of insulin in whole pancreas taken from fasting rats was higher in the SG-ex-supplemented group, which may explain the greater capacity to secrete insulin during the OGTT. Thiobarbituric acid-reactive substances in both the liver and the plasma were lower in the SG-ex-supplemented group, suggesting that an absorbable component in SG-ex has an antioxidative effect on lipid peroxidation, thereby counteracting the oxidative stress caused by a diabetic state. Excreted urine volume and urinary albumin level for 24 h were both reduced in the SG-ex-supplemented group, suggesting the attenuation of kidney damage that is caused by diabetes. These data indicate that SG-ex supplementation may prevent complications and attenuate pathological conditions for type 2 diabetes, along with its sweet characteristics.

Ontogenic changes in lactoferrin receptor and DMT1 in mouse small intestine: implications for iron absorption during early life.

It has been proposed that lactoferrin receptor (LfR) may be involved in intestinal iron transport during early life. However, it is known that iron homeostasis is regulated by divalent metal transporter 1 (DMT1; Nramp2/DCT1) in the adult small intestine. To address the hypothesis that LfR may play a role as an alternative iron transport pathway during early life, we used immunohistochemistry (IHC) to examine the localization of mouse LfR (mLfR) and DMT1. In addition to studying the localization and abundance of LfR and DMT1 on the apical membrane, intestinal brush-border membrane vesicles (BBMV) were isolated during the first 3 postnatal weeks (postnatal day (PD) 0, 5, 10, and 20). We found that mLfR is expressed in fetal mice as early as gestational days (GD) 13.5, 15.5, and 18.5. A 34 kD band for mLfR was detected at PD 0 through PD 20 in total intestine homogenate. However, mLfR protein did not appear in the BBMV preparations until PD 5 and was highly expressed at PD 10. By IHC, DMT1 protein was minimally observed at PD 0 and PD 5, but by PD 10 DMT1 was predominantly localized in the apical membrane of the maturing intestine. BBMV fractionation revealed 50-120 kD protein bands for DMT1. In these BBMV preparations, the apical-membrane-associated 120 kD band for DMT1 increased in abundance with age. However, in the corresponding total homogenates, only the deglycosylated form of DMT1 (50 kD) was identified. These results indicate that DMT1 is mislocalized during late gestation, minimally expressed during early life, and predominantly expressed in its deglycosylated form until PD 20. The immunolocalization and abundant protein expression of mLfR suggest that accrual of iron from Lf may be the principal iron uptake pathway at this age. In conclusion, our findings support the notion that until the development-dependent expression of DMT1 in the intestine is induced, mLfR may serve as an alternative iron uptake pathway.

Triterpene glycosides of Siraitia grosvenori inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration of maltose in rats.

The effect of the crude extract from Siraitia grosvenori Swingle (SG-ex) on the postprandial rise in blood glucose level was investigated. The increase in plasma glucose level in response to the oral administration of maltose was significantly suppressed in rats when SG-ex was given orally 3 min before the maltose administration. There was, however, no effect when glucose was administered instead, suggesting that the antihyperglycemic effect of SG-ex is elicited by inhibition of maltase in the small intestinal epithelium. In vitro, SG-ex inhibited rat small intestinal maltase. Similar effects were also observed both in vivo and in vitro when the concentrate of the sweet elements (triterpene glycosides) prepared from SG-ex was used. Furthermore, the main sweet element of SG-ex, mogroside V, and some minor elements such as mogroside IV, siamenoside I, and mogroside III also exhibited maltase inhibitory effect with IC50 values of 14, 12, 10, and 1.6 mM, respectively. These results suggest that SG-ex exerts anti-hyperglycemic effects in rats by inhibiting maltase activity and that these effects are at least partially exerted by its sweet elements, triterpene glycosides.

Baculovirus expression of mouse lactoferrin receptor and tissue distribution in the mouse.

Lactoferrin (Lf) has been shown to have a role in the immune system and in early development of the mouse embryo. A specific receptor for Lf has been suggested to mediate the functions of Lf. We have recently identified a Lf receptor (LfR) in human fetal small intestine. We therefore hypothesized that the mouse homologue of this protein functions as a LfR. We expressed mouse Lf (MLf) and the mouse homologue (MLfR) in a baculovirus-insect cell system. The recombinant MLfR (rMLfR) was purified by immobilized recombinant MLf (rMLf) affinity chromatography, demonstrating an interaction between rMLf and the rMLfR. RT-PCR revealed that MLfR was expressed in various tissues and during embryonic development. Immunohistochemical analysis revealed that the MLfR was localized in various tissues including small intestinal epithelium, stomach, kidney, ovary, and various regions of brain. In summary, the MLfR functions as a receptor for MLf, is expressed and localized in various tissues, and may be involved in the indispensable function of MLf during early embryonic development.

Cellular internalization of lactoferrin in intestinal epithelial cells.

We studied the cellular internalization of lactoferrin (Lf) in an intestinal epithelial cell line, Caco-2, to investigate the mechanism of biological actions of ingested Lf. RT-PCR and Western blotting analyses revealed that differentiated Caco-2 cells express LfR mRNA and its protein with a 34 kD molecular weight under reducing conditions. Biotin-labeled Lf showed specific binding to the cellular membrane of differentiated Caco-2 cells with a dissociation constant (Kd) of 0.16 microM. The cellular internalization of Lf was studied in differentiated Caco-2 cells grown as monolayers on Transwell inserts, and compared to that of human transferrin (Tf). After labeling with fluorescent dye, either Lf or Tf was added to Caco-2 cells from the apical side or the basolateral one. Laser scanning confocal microscopy showed that labeled Lf was internalized only from the apical side and localized to the nuclei. On the other hand, labeled Tf was internalized from the basolateral side, not from the apical side, and localized in the cytoplasm. The internalization of labeled Lf was inhibited by excess of unlabeled Lf, but not of Tf. The internalization of labeled Lf, but not of labeled Tf, was also suppressed by heparin. This indicates that a heparin-binding site in the N-terminal region of Lf could be important for the internalization of Lf. These findings suggest that ingested Lf might be internalized by the intestinal epithelium in a manner different from Tf and might function in the nucleus.

Expression, characterization, and biologic activity of recombinant human lactoferrin in rice.

BACKGROUND: Lactoferrin has been suggested to have many biologic activities, such as facilitating iron absorption and having antimicrobial and antiinflammatory effects. In humans, several of these activities are likely to only be facilitated by human lactoferrin because they depend on the binding of human lactoferrin to specific receptors. Rice may be a useful vehicle to introduce recombinant human lactoferrin to infant foods because it has low allergenicity and is likely to be safer than using microorganisms or transgenic animals. METHODS: Recombinant human lactoferrin was expressed in the rice cell culture system, and its biologic activity was assessed by iron-binding and -releasing properties, antimicrobial activity, and binding and uptake to Caco-2 cells. The authors also compared the stability of recombinant and native human lactoferrins against heat, low pH, and in vitro digestion. RESULTS: Biologic activity of rice-expressed recombinant human lactoferrin was similar to that of native human lactoferrin. Heat-treated proteins retained their functional activities except with severe treatment at 100 degrees C for 8 seconds, which disturbed the iron-binding capacity of recombinant human lactoferrin. Both types of proteins retained their functional activities between pH 2 and 7.4. After in vitro digestion, 50% of both proteins were detectable by enzyme linked immunosorbent assay. The remaining native and recombinant lactoferrins retained antimicrobial and Caco-2 binding and uptake activities. CONCLUSIONS: The results indicate recombinant human lactoferrin has stability similar to native human lactoferrin when exposed to thermal treatment, pH treatment, and in vitro digestion, suggesting it may be active when added to infant formula.

High specific activity heme-Fe and its application for studying heme-Fe metabolism in Caco-2 cell monolayers.

Heme-Fe is an important source of dietary iron in humans. Caco-2 cells have been used extensively to study human iron absorption with an emphasis on factors affecting nonheme iron absorption. Therefore, we examined several factors known to affect heme iron absorption. Cells grown in bicameral chambers were incubated with high specific activity [59Fe]heme alone or with 1% globin, BSA, or fatty acid-free BSA (BSA-FA) to examine the effect of protein source on absorption. Heme iron absorption was enhanced by globin and inhibited by BSA and BSA-FA. Absorption of heme iron in cells pretreated for 7 days with serum-free medium containing 1, 25, 50, or 100 microM Fe was higher in the 1-microM-Fe pretreatment group than in all other groups (P < 0.05), showing an effect of iron status. Increased heme concentrations resulted in decreased percent absorbed but increased total heme iron absorption and increased transport rate across the basolateral membrane. Finally, cells treated with 10 microM CdCl2, which induces heme oxygenase, demonstrated higher absorption of [59Fe]heme than control cells (P < 0.05). Our results from Caco-2 cells are in agreement with human studies and make this a promising model for examining intestinal heme iron absorption.

Characterization of mammalian receptors for lactoferrin.

Lactoferrin (Lf) has been suggested to have several physiological functions. Specific binding of Lf, indicating the presence of Lf receptors (LfRs), has been observed in various types of mammalian cells such as lymphocytes, hepatocytes, and enterocytes. These LfRs are considered to function as a mediator for some of the functions of Lf. We here review current knowledge of mammalian LfRs characterized in different tissues. We also briefly present evidence for the existence of an LfR provided by our cloning of a human intestinal LfR (HLfR). The entire coding region of the HLfR was cloned by polymerase chain reaction (PCR), and a recombinant HLfR (rHLfR) was expressed in a baculovirus system. The rHLfR was purified by immobilized human Lf (HLf) affinity chromatography, indicating that the rHLfR retained the capacity to bind HLf. The gene was expressed at high levels in fetal small intestine and in adult heart but at lower levels in Caco-2 cells. In summary, we demonstrate the presence of a unique receptor-mediated mechanism for Lf, functioning in the small intestine of the newborn infant and possibly in other tissues of human adults.