SEARCH

SEARCH BY CITATION

Keywords:

  • chylomicrons;
  • fatty acid;
  • peptides;
  • postprandial lipemia;
  • triglycerides

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

The hepatic removal of triglyceride-rich chylomicrons during the postprandial phase represents an important step towards determining the bioavailability of dietary lipids amongst the peripheral tissues. Indeed, elevated postprandial lipemia is often associated with obesity and increased risk of coronary heart disease. The milk protein, lactoferrin, has been shown to inhibit hepatic chylomicron remnant removal by the liver, resulting in increased postprandial lipemia. Despite numerous studies on potential targets for lactoferrin, the molecular mechanisms underlying the effect of lactoferrin remain unclear. We recently demonstrated that the lipolysis stimulated lipoprotein receptor (LSR) contributes to the removal of triglyceride-rich lipoproteins during the postprandial phase. Here, we report that while lactoferrin does not have any significant effect on LSR protein levels in mouse Hepa1–6 cells, this protein colocalizes with LSR in cells but only in the presence of oleate, which is needed to obtain LSR in its active form as lipoprotein receptor. Ligand blotting using purified LSR revealed that lactoferrin binds directly to the receptor in the presence of oleate and prevents the binding of triglyceride-rich lipoproteins. Both C- and N-lobes of lactoferrin as well as a mixture of peptides derived from its hydrolysis retained the ability to bind LSR in its active form. We propose then that the elevated postprandial lipemia observed upon lactoferrin treatment in vivo is mediated in part by its direct interaction with free fatty acid activated LSR, thus preventing clearance of chylomicrons and their remnants through the LSR pathway.


Abbreviations
apoE

apolipoprotein E

FFA

free fatty acid

HPRT

hypoxanthine guanine phosphoribosyltransferase

LDL

low-density lipoprotein

LDL-R

LDL receptor

Lf

lactoferrin

LRP

LDL-R-related protein

LSR

lipolysis stimulated lipoprotein receptor

TG

triglyceride

VLDL

very-low-density lipoprotein

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

During the postprandial phase after ingestion of a meal, triglycerides (TGs) circulate in the plasma in the form of intestinally derived chylomicrons. These TG-rich lipoproteins distribute fatty acids to different tissues via the lipoprotein lipase system and are ultimately removed from the circulation through receptor-mediated processes in the liver [1]. Elevated postprandial lipemia is often associated with obesity [2] and has been reported in patients with cardiovascular disease [3]. Lactoferrin (Lf) is a milk protein that when injected intravenously has been demonstrated to increase postprandial lipemia by inhibiting chylomicron remnant removal from the liver [4].

Lf is a non-heme iron-binding glycoprotein belonging to the transferrin class. This monomeric protein of 689 residues folds into two domains of high sequence similarity, called the N- and C-lobes [5], each of which binds one ferric ion [6].

On the N-lobe, Lf contains an Arg-rich region similar to that of apolipoprotein E (apoE), which is the ligand for the uptake of chylomicron remnants [7]. Studies on parenchymal liver cells demonstrated that Lf inhibits the association of apoE-containing lipoproteins to a receptor which is distinct from the low-density lipoprotein (LDL) receptor (LDL-R) and LDL-R-related protein (LRP) but was unidentified [8].

It was at this time that Bihain and Yen [9] reported that fibroblasts from a patient deficient in LDL-R were able to internalize significant amounts of LDL via a previously unidentified receptor. It was demonstrated that this new pathway involves a receptor that mediates the binding of apoB- and apoE-containing lipoproteins, leading to their subsequent internalization and degradation [10]. This receptor displays highest affinity for TG-rich lipoproteins [chylomicrons and very-low-density lipoprotein (VLDL)] and is referred to as the lipolysis stimulated lipoprotein receptor (LSR) since it can only bind and internalize lipoproteins in the presence of free fatty acids (FFAs). Since LSR is activated by these lipolytic products, it is thus primarily active during the postprandial phase when the influx of chylomicrons into the circulation is high, which leads to increased lipase-mediated hydrolysis of the chylomicron TGs, leading to high FFA levels in the space of Disse of the liver [10-12]. LSR was identified as a multimeric complex consisting of three subunits derived by alternative splicing from the same gene [13]. In vivo data now demonstrate that LSR plays an important role in the clearance of TG-rich lipoproteins during the postprandial phase [14]. It was observed that Lf inhibited oleate-induced lipoprotein binding to rat hepatocyte plasma membranes [11, 12]. This inhibition was shown to occur without a significant decrease of the amount of membrane-associated oleate and thus differs from the inhibition caused by albumin that is achieved through oleate sequestration [11], suggesting that LSR is the molecular target of Lf. Even though this hypothesis was based on experiments done on liver membranes under conditions optimal for LSR activity, interaction between LSR and Lf remained to be demonstrated.

The aim of this study was to determine if the inhibition of LSR by Lf is due to direct interaction of these two proteins. Here, we show that bovine Lf and its hydrolysis products display the ability to bind to the FFA-activated form of the purified receptor and compete for binding of VLDL to LSR.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

In order to study the direct interaction of LSR with Lf, it was necessary to use Lf with high purity. Since SDS/gel analysis revealed that commercially available Lf contained a number of contaminating proteins, including albumin (Fig. 1A), we purified Lf from raw bovine milk to > 95% purity (Fig. 1A). Since Lf inhibits postprandial lipemia when injected intravenously into rats [4], we next verified that the purified Lf exhibited this same biological effect. C57BL/6RJ male mice were injected intravenously with Lf and then immediately received olive oil by gavage. Plasma TGs measured 1 h after the gavage were found to be significantly increased (= 0.04) by a factor of 4.3 in the group of mice that received purified Lf (Fig. 1B), while the 1.9-fold increase in plasma TGs in the group of mice that received commercial Lf only tended towards significance (= 0.08). We verified that Lf did not have any effect on free glycerol levels (increase in free glycerol 1 h after gavage: controls 16 ± 4.5 μg·mL−1 and Lf group 18.8 ± 2.3 μg·mL−1). The difference between purified and commercial Lf could be due to impurities detected in the latter, including albumin and immunoglobulin (Fig. 1A). These results therefore indicate that pure Lf retains its in vivo effect on postprandial lipemia, confirming that the effect derives purely from Lf and not from other contaminants such as albumin.

image

Figure 1. Effect of Lf on plasma TG level during the postprandial phase. (A) SDS/PAGE showing the Lf used for this study. Lane 1, molecular weight marker (M); lane 2, bovine Lf from Sigma; lane 3, bovine Lf purified from whey proteins (Lf lab); lane 4, whey proteins obtained after casein precipitation. LC-IgG, light chain immunoglobulin; HC-IgG, heavy chain immunoglobulin; LG, lactoglobulin. (B) Plasma TG increase 1 h after Lf injection. C57BL6/RJ male mice were injected intravenously (tail vein) with 100 μL NaCl/Pi (open bars) or NaCl/Pi containing 2 mg bovine Lf (closed bars) obtained from Sigma or purified from raw milk. After gavage with 300 μL olive oil, blood samples were removed and plasma TG levels were measured as described in Materials and methods. Results are shown as mean ± SEM (= 3 or 4 per condition) of the increase in plasma TG relative to control. Statistical significance is indicated by P values (compared to the corresponding NaCl/Pi group).

Download figure to PowerPoint

As Lf has been shown to enter the nucleus and induce gene expression [15, 16], we first sought to determine whether Lf demonstrated any effect on LSR mRNA or protein levels in the mouse hepatoma cell line, Hepa1–6. Since LSR activity as a lipoprotein receptor is dependent upon the presence of FFAs, we incubated cells for 3 h at 37 °C in the absence or presence of oleate and Lf. The concentrations of Lf used were those shown previously to inhibit lipoprotein binding to liver membranes in the presence of oleate [11]. Incubation of cells in the presence of Lf appeared to slightly decrease cell LSR protein levels relative to actin as a loading control (Fig. 2A). However, because of the variations in expression, this difference was not statistically significant. Parallel experiments were conducted to determine whether LSR mRNA levels were affected. Only a small decrease was observed in cells incubated in the presence of 0.8 mm oleate and Lf compared with 0.8 mm oleate alone (Fig. 2B). No significant changes were observed when cells were incubated with or without Lf in the absence or presence of 0.4 mm oleate (Fig. 2B). Therefore, at least in short-term experiments, Lf did not have any detectable effect on LSR protein levels but did have a small inhibitory effect on LSR mRNA levels in the presence of oleate. We then initiated a study on the interaction between LSR and Lf both at the cellular and molecular levels. Intact cells were used, in which Alexafluor-488-labelled Lf was incubated with Hepa1–6 cells with or without oleate. Figure 3 shows strong labeling of permeabilized cells with both Lf and LSR, with little apparent colocalization in the merge picture in the absence of oleate (Fig. 3, top panels). However, when cells were incubated in the presence of oleate, colocalization of Lf and LSR labeling increased dramatically, as evidenced by the increased orange intensity in the merged picture (Fig. 3, lower right panel). This suggests that Lf interacts directly with LSR but only in the presence of oleate, thus when LSR is in its active conformation.

image

Figure 2. Effect of lactoferrin on LSR mRNA and protein levels in Hepa1–6 cells. Hepa1–6 cells were incubated for 3 h with 2 mg·mL−1 Lf with increasing concentrations of oleate as indicated. (A) Cell lysates were then prepared from which immunoblots were performed as described in Materials and methods to detect LSR. Actin served as loading control. A representative blot is shown and an analysis of three experiments with three different preparations of cells is shown in the bar graph as mean ± SEM ratio of LSR/actin. The values of LSR/actin for cells incubated in the absence of oleate and Lf were normalized to 100%, allowing us to compare the variations between different experiments. (B) Experiments were conducted in which mRNA was directly extracted from cells and then used in qPCR analysis. Data are shown as the relative expression of LSR versus the housekeeping gene, HPRT (mean ± SEM,= 3).

Download figure to PowerPoint

image

Figure 3. Colocalization of Lf and LSR in Hepa1–6 cells. Hepa1–6 cells were incubated with 2 mg·mL−1 Lf labeled with Alexafluor 488 in the absence (top panels) or presence (bottom panels) of oleate for 20 min at 37 °C. The cells were then washed, fixed, permeabilized and labeled with anti-LSR IgG as described in Materials and methods. Visualization of Lf is shown in the left panels and LSR in the middle panels. Merged pictures are shown in the right panels.

Download figure to PowerPoint

In order to confirm this possible interaction, ligand blot experiments were performed using purified LSR and Lf. LSR was purified from rat hepatocyte plasma membranes using anion exchange chromatography. The protein appeared to be at least 95% pure, as judged by SDS/PAGE analysis (Fig. 4A). Three bands were obtained under reducing conditions, with apparent molecular mass ranging from 54 to 70 kDa, which is consistent with the masses of the subunits that compose the receptor. Under non-reducing conditions, one single band was observed at an apparent molecular weight of ~ 240 kDa, representing the LSR complex (Fig. 4A) [13]. The identity of LSR was confirmed by immunoblot analysis (Fig. 4B).

image

Figure 4. SDS/PAGE and western blot of purified LSR. The purified LSR was loaded on 10% SDS/PAGE under non-reducing (NRC) or reducing (RC) conditions. (A) Silver staining. (B) Western blot. Revelation was made as described in Materials and methods. MM, molecular weight marker.

Download figure to PowerPoint

Ligand blots were then carried out in which LSR was migrated on SDS/PAGE under non-reducing conditions and, after transfer on nitrocellulose, incubated with the pure Lf. It was previously shown that FFAs were necessary for LSR binding to LDL [11, 12]. In view of the results of the colocalization experiments, the assay was conducted both in the absence and in the presence of oleate. As shown in Fig. 5A, lanes 1 and 2, Lf was revealed to bind to a band corresponding to the LSR complex (apparent molecular mass 240 kDa) [13] but only when oleate was present (Fig. 5A, lane 2), suggesting that FFAs are also required for Lf binding to this receptor. Control experiments in the absence of Lf demonstrate that this is not due to binding of anti-Lf IgG to LSR (data not shown).

image

Figure 5. Ligand blots showing the interaction between LSR and bovine Lf and its hydrolysis products. (A) Ligand blots of LSR with entire Lf or its hydrolysis products. LSR was loaded on SDS/PAGE under non-reducing conditions, and then transferred onto a nitrocellulose membrane and incubated with full-length Lf (2 mg·mL−1, lanes 1 and 2), its N-terminal lobe (2 mg·mL−1, lanes 3 and 4) and C-terminal lobe (2 mg·mL−1, lanes 5 and 6), and peptides derived from tryptic/chymotryptic hydrolysis (2 mg·mL−1, lanes 7 and 8). Strips were incubated without oleate (lanes 1, 3, 5 and 7) or with 0.8 mm oleate prior to incubation with Lf (lanes 2, 4, 6 and 8). Lf was then detected by immunoblotting with anti-Lf IgG as described in Materials and methods. (B) Ligand blots of LSR with VLDL in the presence of increasing concentrations of Lf. Purified LSR on nitrocellulose membranes was incubated with 0.8 mm oleate and 20 μg·mL−1 VLDL in the presence of the indicated concentrations of Lf. The VLDL was detected with anti-apoB IgG as described in Materials and methods. (C) SDS/PAGE of Lf and its mild hydrolysis products used for the ligand blot shown in (A). lane 1, M, molecular weight marker; lane 2, pure bovine Lf; lane 3, peptide representative of the C-domain; lane 4, peptides of the N-domain.

Download figure to PowerPoint

We have previously reported that Lf can inhibit LSR activity as a lipoprotein receptor in liver cell and membrane studies as well as ligand blots using membrane extracts [11, 12]. Ligand blots were performed by incubating immobilized purified LSR with TG-rich VLDL in the presence of oleate and increasing concentrations of Lf. VLDL binding was revealed by detection of apoB on the 240-kDa LSR complex (Fig. 5B). This binding was decreased in intensity with increasing concentrations of Lf, thus demonstrating that direct binding of Lf to LSR can prevent binding of its lipoprotein ligand.

In order to determine which part of Lf is recognized by LSR, Lf was hydrolyzed by trypsin under mild conditions to obtain the N- and C-lobes separately. Four fragments with apparent molecular masses of 24, 34, 44 and 51 kDa were obtained on SDS/PAGE profile (data not shown). N-terminal sequencing and mass spectrometry analysis (Table 1) allowed the identification of the first two fragments as deriving from the N-terminal lobe and the second two fragments as belonging to the C-lobe (with or without the peptide connecting the two lobes of Lf). Precise identification of the C-terminus of the peptides corresponding to the N-lobe (a tryptic fragment) was difficult to obtain because of the presence of two Lys residues (at positions 280 and 282) and one Arg residue (at position 284) in this region. Indeed, it has been reported that two N-lobe fragments of apparent mass 34 kDa correspond either to the 1–284 Lf region [17] or to the 1–280 region [18], showing that identification of the C-terminal extremity remains difficult to determine. Moreover, determination of the mass of the entire N- and C-lobe peptides did not allow precise identification of the C-terminus because of the microheterogeneity of the glycan structures of Lf.

Table 1. Identification of the peptides obtained after mild tryptic hydrolysis of Lf. X corresponds to a residue that was not identified by Edman sequencing
Fragment nameN-ter sequence (Edman)Peptide (MS-MS)Domain
  1. a

    Uncertainties remain for the identification of the C-terminal end of the fragment.

PP51SFQLFGXPP-Ser285-Arg689C-domain
PP44VVWCAVGPEE-Val335-Arg689C domain
PP34APRKNV-Ala1-Lys280/282/Arg284aN-domain
PP24SAGXIIPMG-Ser122-Lys280/282/Arg284aN-domain

After isolation of the fragments accounting for each lobe by liquid chromatography (Fig. 5C), ligand blots were performed once again in the same conditions as above except that incubation was performed with fragments of the N-lobe (mixture of PP24 and PP34) or of the C-lobe (P51) rather than with the whole Lf molecule. As shown in Fig. 5A (lanes 3–6), both lobes of Lf are able to bind to LSR but, again, only if oleate is present. This also suggests that there are structural motifs or sequences located not only at the N-lobe but also the C-lobe of Lf that are recognized by LSR.

We pursued this line of thought by subjecting the trypsin hydrolysate of Lf to further hydrolysis by chymotrypsin in order to determine whether small peptides derived from Lf hydrolysis possess the ability to bind to the receptor. Non-digested N- or C-lobes or intermediate peptides having apparent molecular masses > 10 kDa were removed by ultrafiltration. Mass spectrometry analysis confirmed that peptides contained in the ultrafiltrate were small (Table 2), except for four peptides displaying masses of 2726, 2564, 2402 and 2240 Da. Fragmentation by tandem mass spectrometry showed that these peptides actually correspond to four glycopeptides with the same aglycoform VKNDTVW(543–549) produced by chymotrypsin action on the C-lobe. These glycopeptides carry on the Asn545 residue an oligomannosidic-type glycan structure (data not shown). The mass spectrometry analysis showed that the glycan moiety possesses a variable number of mannose residues, up to nine units, and this result is in accordance with the N-glycan structures of Lf reported by Spik et al. [19] and Coddeville et al. [20]. Furthermore, the experimental molecular mass of the deglycosylated peptide chain produced by MS2 fragmentation was 860 ± 1 Da, which is very close to the theoretical mass of the apoheptapeptide of 860.44 Da. The hydrolysate does not contain any other N-glycopeptide, as all the other molecular masses found in this fraction are lower than the usual masses of N-acetyllactosamine-type and oligomannosidic-type structures. Finally, the glycopeptides were discarded by cold ethanol precipitation in order to determine whether the interaction between the mixture of Lf peptides and LSR was due to the amino acid sequences rather than to the glycan moiety. Ligand blots performed on the mixture of these peptides (Fig. 5A) or on the mixture of peptides after elimination of the glycosylated fractions (data not shown) highlighted their ability to bind to LSR in the presence of oleate.

Table 2. Identification of the peptides obtained after hydrolysis of Lf by trypsin and chymotrypsin. The Mr values correspond to monoisotopic masses. nd, not determined; Man, mannose; GlcNAc, N-acetylglucosamine; RT, retention time
RT (min)Mr (experimental)Mr (calculated)SequenceResidues
  1. a

    Peptide identified as a Cys-containing peptide after reduction and alkylation. It was also observed in its protonated alkylated form [M + 57 + H]+.

13.52a959.5 (+57)959.427 (+57)CAVGPEEQK (+57)348–356
15.19721.5721.33RPTEGY428–433
15.30649.4649.32GGRPTY652–657
15.35344.2344.14YY523–524
15.80585.6585.37AVAVVK435–440
17.70204.2204.09Wnd
17.89702.5702.34KGEADAL386–392
18.12581.4581.27LGTEY661–665
18.48637.3637.27NLDGGY393–398
18.892726.0860.44 (aglycoform)VKNDTVW (9 Man + 2 GlcNAc)543–549
19.002564.0860.44 (aglycoform)VKNDTVW (8 Man + 2 GlcNAc)543–549
19.23800.4800.32PTYEEY655–660
19.272402.0860.44 (aglycoform)VKNDTVW (7 Man + 2 GlcNAc)543–549
19.492240.0860.44 (aglycoform)VKNDTVW (6 Man + 2 GlcNAc)543–550
20.38419.2419.18SAGW122–125
20.79746.5746.40APVDAFK237–243
20.801222.51222.47ENTNGESTADW550–560
21.57666.4666.32VDSALY314–319
22.73674.3674.43RMKKL25–29
23.30878.4878.38QDGAGDVAF200–208
23.68595.3595.28ETTVF211–215
25.13787.4787.40TESLEPL139–145
26.73402.3402.23VVW345–347
27.12404.2404.19LSW136–138
27.50802.5802.45LSKAQEK271–277
28.28779.3779.37DQYELL225–230
31.411060.41060.51SWTESLEPL137–145
32.90674.3674.33EDLIW264–268

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Elevated postprandial lipemia following intravenous injection of Lf was shown to be due to accumulation of chylomicron remnants in the circulation and reduced clearance of these particles by the liver [4]. This led to numerous investigations of the molecular mechanisms involved with the objective of identifying the Lf-sensitive liver receptor(s) for these TG-rich lipoproteins. However, Lf has many other biological activities such as in host defense against infection or immunomodulation and, as a result, displays various affinities for a number of proteins on the cell surface [21, 22], which rendered the task of identifying the liver receptor difficult. Indeed, at the hepatic level, there have been studies reporting association of Lf with LRP1, the asialoglycoprotein receptor, as well as heparan sulfate proteoglycans [23, 24]. Of these different proteins, LRP1 was a potential candidate as chylomicron remnant receptor, but it was later demonstrated to play a back-up role in clearance of these particles in the absence of the LDL-R [25]. Indeed, the presence of a third pathway other than LRP1 and LDL-R for chylomicron remnants was postulated [25, 26]. Furthermore, GST-RAP, a specific inhibitor of LRP, did not appear to modify Lf's effect on β-VLDL binding to liver cells, leading the authors to conclude that the Lf effect was not mediated through LRP nor through the LDL-R [27]. While Lf has been shown to inhibit the binding and uptake of apoE containing lipoproteins, including chylomicron remnants, in the liver [28, 29], these studies used cell-based models or tissue extracts without identifying the actual target receptor or performing direct binding studies of Lf with the receptor in a purified state.

We identified LSR as an apoB,E receptor that plays an important role in the removal of TG-rich lipoproteins during the postprandial phase [14]. It serves as lipoprotein receptor only in the presence of FFA, which induces a conformational change to expose a binding site on the receptor for apoB or apoE [11]. We have previously shown that Lf can prevent the binding of lipoproteins to FFA-activated LSR [11]. In this study, we now demonstrate that Lf colocalizes with LSR in Hepa1–6 cells and that Lf binds to purified LSR but, in both cases, only in the presence of oleate and thus only when LSR is in its active form. Furthermore, Lf is able to directly compete with the TG-rich VLDL for binding to LSR. Taken together, this suggests that the previously observed inhibitory effect of Lf on oleate-induced binding of lipoproteins to hepatocytes or hepatic membranes is mediated by the direct interaction of Lf with the receptor. Since during the postprandial phase chylomicron influx into the liver is high leading to increased FFA production by lipolysis, LSR would be in its active form. We would propose then that the elevated postprandial lipemia observed upon Lf treatment in vivo is mediated in part by its direct interaction with FFA-activated LSR, thus preventing clearance of chylomicrons and their remnants through the LSR pathway.

We tested whether Lf demonstrated any effect on LSR mRNA or protein levels. Although protein levels did not change significantly, we did observe a small but significant decrease in LSR mRNA in cells incubated with Lf and oleate. Lf has previously been reported to enter the nucleus and induce gene expression via the transcription factor AP-1 [30]. Bioinformatics analysis revealed potential AP-1 response elements in the sequence upstream of the mouse lsr gene. Taking into account that AP-1 is involved in the control of genes related to cell survival and apoptosis [31, 32] and that a previous study has also reported that LSR expression can be induced by P53 [33], it may be that LSR is in some manner involved in the regulation of the cell cycle. Further investigation is required to examine this question. Nevertheless, because of the rapid effect of Lf on chylomicron remnant clearance, it seems unlikely that this was due to Lf's effect on LSR mRNA or protein levels.

Ligand blot studies using purified LSR revealed that both the C- and N-lobes of Lf were able to bind directly to LSR in the presence of oleate. Furthermore, a peptide mixture resulting from further hydrolysis of both lobes retained the capacity for recognizing the active form of LSR. Since the removal of glycopeptides did not affect the ability of Lf to interact with activated LSR, this suggests that the recognition is based purely on the amino acid sequence. The original hypothesis of the Lf effect was based on the fact that Lf possesses a number of Arg and Arg-Lys clusters on its N-terminal side that resemble the binding site of apoE, which is considered the ligand for the chylomicron remnant receptor. On the basis of chemical modification experiments, previous studies using liver parenchymal cells proposed that Arg residues were crucial for the recognition of Lf and its inhibitory effects on lipoprotein remnants [28, 34]. These studies were carried out on human Lf, which possesses a cluster of four Arg residues (positions 2–5) and an Arg/Lys-rich sequence at positions 25–31 (R-X-X-R-K-X-R), similar to that of apoE [4]. However, conflicting results were reported when Huettinger et al. [28] observed that digestion of Lf by trypsin that cleaved through the Arg (positions 2–5) cluster but left the helical region near position 35 intact suppressed the inhibitory effect of Lf on the internalization of remnants. On the other hand, Ziere et al. [29] demonstrated that truncated Lf lacking the first 14 residues and thus the Arg (positions 2–5) cluster was a more effective competitor than Lf for liver uptake of lipoprotein remnants. Therefore, the importance of the four Arg cluster in Lf's effect on chylomicron remnant clearance remains unclear.

Here, we use purified bovine Lf which, when injected into mice, leads to elevated postprandial plasma TG levels (Fig. 1B). This is in agreement with previous reports using bovine Lf that have demonstrated inhibition of chylomicron remnant clearance during the postprandial phase [35]. Interestingly, bioinformatics analysis revealed that bovine Lf possesses only an RK sequence instead of the Arg (2–5) cluster described in human Lf, thus clearly excluding a predominant role of this cluster in the binding to LSR. Moreover, the Arg/Lys-rich sequence at positions 25–31 is divergent in the bovine sequence (WRMKKLG) compared with that of human. This observation, along with the fact that both N- and C-terminal lobes of bovine Lf bind to the receptor, suggests that at least two sequences or structural motifs are recognized. Furthermore, given the size of the peptides present in the tryptic/chymotryptic hydrolysate that retain the ability to bind to LSR, our results suggest that this receptor recognizes one or several sequences on Lf rather than conformational features. For the N-lobe, the RMKKL sequence at positions 25–29 is found in the peptide mixture isolated from the tryptic/chymotryptic hydrolysate together with another basic peptide IVKLLSKAQEKFGK at positions 268–280, while no peptide possessing more than one basic residue and belonging to the C-lobe has been recovered in the hydrolysate, suggesting that determinants of the interaction might involve other features than basic sequences.

In conclusion, this study clearly demonstrates a direct interaction of Lf with the FFA-activated form of hepatic LSR, which prevents the binding of TG-rich lipoproteins to this receptor and therefore could explain the elevated postprandial lipemia and reduced chylomicron remnant clearance observed in rodents treated with Lf. Further experiments are needed to identify the specific peptides of Lf that bind to LSR and to determine whether these peptides, when isolated, have the ability to modulate postprandial lipemia.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Chemicals and reagents

All chemicals and reagents were purchased from Sigma Aldrich Co. (St Quentin Fallavier, France) unless otherwise indicated. Cell culture media and supplements, Oregon Green 488 dye and ProLong Gold antifade fluormount reagents were obtained from Invitrogen (Alfortville, France). Mouse recombinant leptin was purchased from Merck Chemicals (Darmstadt, Germany). Secondary anti-rabbit horseradish peroxide (HRP) conjugated IgG were acquired from Cell Signaling Technology (Boston, MA, USA).

Purification of proteins

Purification of Lf

Bovine Lf was purified from raw skim milk. First, whey proteins were separated from caseins according to the procedure described in Egito et al. [36]. Lf was then purified from the whey proteins by ÄKTA-FPLC chromatography (GE Healthcare, Uppsala, Sweden) by passing sequentially through three Hitrap CM-FF 5/5 cation exchange columns (1.5 × 2.5 cm) equilibrated in 50 mm Tris/HCl buffer, pH 8.0. A linear gradient of NaCl in the same buffer was applied (1 mL·min−1) for the elution. For the colocalization experiments, Lf was conjugated with fluorescent probe Alexafluor 488 according to the manufacturer's instructions (Pierce, Thermo Fisher, Illkirch, France). The Lf : dye ratio was 1 : 16 for the reaction at room temperature for approximately 1 h in the dark. The mixture was dialyzed against NaCl/Pi buffer for 48 h and concentrated. Coupling efficiencies were determined to check the amount of fluorescent dye linked to protein (3.17 mol of dye per mol of protein).

Purification of LSR

LSR-enriched solubilized protein fractions were first prepared from rat hepatocyte plasma membrane according to Mann et al. [11]. Membrane proteins were solubilized in 20 mm Tris/HCl buffer, pH 8.0, containing 150 mm NaCl, 2 mm EDTA, 13 mm Chaps and inhibitor cocktail and fractionated by ÄKTA-FPLC on a Mono-Q HR 5/5 anion exchange column (GE Healthcare). The column was washed with 4 mL Tris/HCl buffer and proteins were eluted with a 13-mL linear gradient of 150–900 mm NaCl in the same buffer (flow rate 0.5 mL·min−1). Fractions were collected and placed on ice; the presence of LSR in fractions was verified by immunoblots. The purity of eluted fractions was analyzed by SDS/PAGE. Protein concentrations were determined using a Bradford reagent assay.

Animal studies – effect of Lf on plasma TG level during the postprandial phase

Adult male C57BL/6RJ mice (Janvier Breeding, Le Genest Saint Isle, France) were housed in certified animal facilities on a 12-h light/dark cycle with a mean temperature of 21–22 °C and relative humidity of 50 ± 20%, and were provided with rodent chow diet and water ad libitum. Animals were handled in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for the use and care of laboratory animals. Mice 14 weeks old were divided into two groups. Lf (2 mg) in NaCl/Pi was injected intravenously (tail vein) to one group of animals while NaCl/Pi was injected to the other group as a control. Immediately after injection of Lf (= 0), the mice were given 300 μL of olive oil by gavage, and samples of blood from the retro-orbital cavity were collected at = 0 and 1 h into paraoxon-treated tubes to inhibit lipase activity [37]. Blood samples were immediately centrifuged (10 000 g, 5 min at 4 °C) and the plasma was stored at −20 °C. Plasma TG concentrations were analyzed using a colorimetric enzymatic kit (Biomérieux, Craponne, France) according to the manufacturer's instructions.

Cell studies

The mouse hepatoma cell line (Hepa1–6) was obtained from DSMZ (Braunschweig, Germany) and was maintained in DMEM containing 10% fetal bovine serum and 1 mm glutamine as previously described [14]. Cells were seeded in six-well plates and used after 48 h (80–90% confluence). The cells were washed in NaCl/Pi, incubated for 1 h with 10 ng·mL−1 leptin in complete DMEM to optimize LSR expression [38] and then treated with 0.8 mm oleate, followed by incubation with Lf in DMEM at 37 °C in a 95% air, 5% CO2 environment. Cells were placed on ice and washed twice with NaCl/Pi containing 0.2% BSA, followed by two washes with NaCl/Pi.

qPCR analysis

Hepa1–6 cells treated with Lf were scraped in NaCl/Pi, collected and centrifuged to recover the cell pellets and stored at −80 °C. Cell pellets were homogenized in QIAzol lysis reagent according to the manufacturer's instructions. RNA extraction and qPCR experiments were conducted as described by Stenger et al. [38]. The housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) was used as a reference. Calculations of relative expression and statistical analysis were performed using relative expression software tool (rest) 2009. The results are expressed as the means of three independent experiments.

Western blot analysis

After incubation with Lf, the washed cells were recovered in radioimmunoprecipitation (RIPA) lysate buffer as described previously [14, 38]. Protein samples (25 μg) were separated by SDS/PAGE and transferred onto nitrocellulose membrane. Immunoblots were performed using anti-LSR (Sigma, 1/1000 dilution) followed by HRP-conjugated secondary IgG. The bands were revealed by chemiluminescence (GE Healthcare). Actin was used as loading control. Densitometric analysis of autoradiographs was performed using the software imagej (US National Institute of Health, Bethesda, MD, USA: http://rsbweb.nih.gov/ij/download.html).

Colocalization experiments

Hepa1–6 plated onto coverslips was incubated with recombinant mouse leptin (10 ng·mL−1) for 1 h at 37 °C to optimize LSR expression and then incubated with Alexafluor-488-labelled Lf (2 mg·mL−1) in the presence of 0.8 mm oleate for 20 min at 37 °C. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min and then permeabilized with NaCl/Pi containing 0.1% BSA and 0.1% Triton X-100. The presence of LSR was detected with rabbit anti-LSR IgG (1/100 dilution), followed by incubation with an IgG conjugated with Alexafluor 555 recognizing rabbit IgG (1/2000 dilution; Invitrogen). Finally, the nuclei of cells were labelled by incubating with DAPI (4′,6-diamidino-2-phenylindole, 1/500 dilution). The cells were then mounted onto slides using ProLong Gold antifade fluormount reagent (Invitrogen) and images were taken using a confocal microscope (FV10i Fluoview; Olympus Biotech, Lyon, France).

Ligand blots

Binding of bovine Lf and its hydrolysates to LSR

Purified LSR (20 μg·well−1) was subjected to electrophoresis on a 10% SDS/polyacrylamide gel under non-reducing conditions and transferred to nitrocellulose membrane. After blocking with 3% BSA, nitrocellulose strips were washed and then incubated for 5 min in the presence or absence of 0.8 mm oleate in NaCl/Pi at 37 °C. The strips were incubated for 1 h at 37 °C with Lf or its hydrolysates (2 mg·mL−1). The nitrocellulose strips were incubated with rabbit anti-Lf IgG PA1-40280 (Thermoscientific, Brebieres, France; 1/500 dilution) for 2 h at 37 °C. The LSR–Lf complex was detected after incubation with anti-rabbit IgG HRP-linked IgG (1/2000 dilution), and the protein bands were revealed by chemiluminescence.

Binding of VLDL to LSR in the presence of Lf

VLDL (< 1.006 g·mL−1) was prepared from fresh human plasma as described previously [9]. Purified LSR on nitrocellulose was prepared as described above. After blocking with 3% BSA, the strips were incubated for 30 min at 37 °C with 0.8 mm oleate in the presence of 0.1 m phosphate buffer, 350 mm NaCl and 2 mm EDTA (pH 8.0) as described previously for optimal binding of lipoprotein to LSR [11]. The nitrocellulose strips were then incubated for 1 h at 37 °C with 20 μg·mL−1 VLDL protein, in the presence of increasing concentrations of Lf. Following washes with NaCl/Pi containing 0.5% Triton X-100, strips were incubated with rabbit anti-apoB IgG (Santa-Cruz Biotechnology, Heidelberg, Germany) to detect VLDL binding to LSR. Protein bands were detected as described above for the Lf ligand blot. Control experiments revealed no bands in the absence of oleate (data not shown).

Isolation of the N- and C-terminal lobes of Lf

Lyophilized Lf was solubilized in 100 mm Tris/HCl buffer, pH 8.2, containing 25 mm CaCl2, and subjected to mild hydrolysis by trypsin (EC 3.4.21.4) in the following conditions: 37 °C, 4 h, enzyme/peptide molar ratio 1 : 50. The reaction was stopped by addition of HCl until a pH of 3.0 was achieved. The N-terminal lobe was purified using a procedure adapted from Takayama et al. [39]. The Lf hydrolysate (2 mL, 8 mg·mL−1) was loaded onto a TSK SP-5PW column (75 mm × 7.5 mm; Interchim, Montluçon, France) equilibrated with a 50 mm sodium phosphate buffer, pH 7.0. Elution was carried out with a linear gradient of NaCl (0–0.5 m) and 250 mm NH4OH in the same buffer (flow rate 1 mL·min−1). The C-terminal lobe was purified on a Nucleosil C4 column (150 × 2 mm inner diameter, 5 μm particle size, 10 nm pore size; Cluzeau, Sainte-Foy-la-Grande, France) connected to an Alliance 2690 HPLC system (Waters, Milford, MA, USA). The hydrolysate (0.1 mg·mL−1) was solubilized in water containing 5% acetonitrile and 0.1% trifluoroacetic acid. Elution was carried out by a linear gradient of acetonitrile in water, in the presence of 0.1% trifluoroacetic acid. The fractions were separated by SDS/PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The protein bands were stained with 0.2% Ponceau S Red, excised from the PVDF membrane and stored in 1% acetic acid. Before enzymatic digestion, the band proteins on PVDF membrane were destained with ultra-pure H2O along with salts and detergents that passed through the PVDF membrane while the protein retained on the PVDF membrane were drawn through the PVDF by absorption forces (ProSorbTM system; Applied Biosystems, Foster City, CA, USA). The protein was digested by trypsin and peptides were submitted to microsequencing by Edman degradation [40] (Service Commun de Séquence des Protéines, Université de Lorraine). Identification of the corresponding protein bands was performed using the blast program for protein sequencing http://blast.ncbi.nlm.nih.gov/Blast.cgi.

Mass spectrometry analysis of the N- and C-lobes

Mass spectrometry analysis of the N- and C-lobes was performed as previously described [41]. Electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) analysis was performed in the Laboratoire de Spectrométrie de Masse Biologique et Protéomique of ESPCI-ParisTech (École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, France). The Lf tryptic hydrolysate was separated by SDS/PAGE and revealed by 0.1% Coomassie Blue R-250 in 2% acetic acid and 50% ethanol. The bands corresponding to the N-lobe and C-lobe were cut and conserved in 1% acetic acid at −20 °C. The excised bands corresponding to C-lobe and N-lobe were digested with trypsin and chymotrypsin, respectively. The generated peptides were applied to an UltiMate® 3000 Nano LC system (Dionex, Amsterdam, Netherlands) coupled to a hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT; Thermofisher, San Jose, CA, USA). The sample (8 μL injected) was desalted and preconcentrated using a PepMap C18 pre-column (5 × 0.3 mm inner diameter, 3 μm particle size, 10 nm pore size; Dionex) before eluting and separating on a reversed phase analytical column (PepMap C18, 150 × 0.075 mm inner diameter, 3 μm particle size, 10 nm pore size; Dionex). The peptides were eluted with 1% solvent B (90% acetonitrile and 0.1% formic acid in water) in solvent A (2% acetonitrile and 0.1% formic acid in water) for 5 min at a flow rate of 15 μL·min−1 in the first step, 5% solvent B in solvent A for 3 min at a flow rate of 300 nL·min−1 in the second step and then a linear gradient of 5–25% of solvent B in solvent A for 32 min at a flow rate of 220 nL·min−1. Data were acquired by the LTQ-FT with automatic alternation between MS and MS/MS modes according to Hesse et al. [42], with a modified m/z range of 400–2000. Mass accuracy tolerance was set to 10 p.p.m. in MS mode and 0.1 Da in MS/MS mode. Data were processed using myproms 2.7 (Unit 900 INSERM-Mines ParisTech, Paris, France). Two missed cleavages for trypsin, chymotrypsin and fixed modifications such as carbamidomethylation of cysteine residues, oxidation of methionine residues were allowed.

Hydrolysis of the N- and C-terminal lobes of Lf by chymotrypsin: production of Lf smaller fragments

The peptides obtained following tryptic hydrolysis of Lf and accounting for the N- and C-terminal lobes were incubated with chymotrypsin (EC 3.4.21.1) for 15 h at 37 °C (enzyme : peptides = 1 : 200). Reaction was stopped by addition of HCl. Amicon centrifuge filters (Millipore, St-Quentin-en-Yvelines, France) were successively used to isolate peptides of apparent molecular mass lower than 10 and 3 kDa. The samples were then loaded onto a PD-10 column for desalting and lyophilized. To eliminate glycopeptides, the sample was dissolved in ultra-pure water and the glycopeptides were extracted by precipitation with nine volumes of cold ethanol (0 °C).

Reduction and alkylation of peptides containing disulfide bonds

After tryptic and chymotryptic hydrolysis, reduction of disulfide bonds and alkylation of cysteine residues were carried out on a fraction of peptide hydrolysate. A volume of 10 μL of 100 mm NH4CO3 was added to 15 μL of hydrolysate solution followed by addition of Tris/HCl buffer (pH 8) to reach a pH of 8.5. Reduction was then achieved by adding 100 mm dl-dithiothreitol (100-fold molar excess over the cysteine content) to the peptide solution and incubated at 37 °C for 1 h. Alkylation was performed by adding 200 mm iodoacetamide (2.5-fold molar excess over dithiothreitol) and incubating at room temperature in the dark for 45 min.

Mass spectrometry analysis of peptides

Liquid chromatography coupled to mass spectrometry (LC-MS) was performed using a system (ThermoFisher Scientific, San Jose, CA, USA) equipped with an LTQ ion trap as mass analyzer. Chromatographic separation was performed on a C18 Alltima reverse phase column (150 × 2.1 mm inner diameter, 5 μm porosity – Grace/Alltech, Darmstadt, Germany) equipped with a C18 Alltima pre-column (7.5 × 2.1 mm inner diameter, 5 μm porosity – Grace/Alltech) at 25 °C and with mobile phases consisting of water modified with trifluoroacetic acid (0.1%) for A and acetonitrile modified with trifluoroacetic acid (0.1%) for B. The peptides were eluted using a linear gradient from 5% to 60% of B phase for 60 min at a flow rate of 0.2 mL·min−1. A double photodiode array and mass detection was performed during the entire run. The mass spectrometric conditions were as follows: electrospray positive ionization mode was used; source gases were set (in arbitrary units per minute) for sheath gas, auxiliary gas and sweep gas at 20, 5 and 5, respectively; capillary temperature was set at 250 °C; capillary voltage at 26 V; and tube lens, split lens and front lens voltages at 90 V, −42 V and −5.75 V, respectively. The ion optics parameters were optimized by automatic tuning using a standard solution of the dipeptide Val-Trp at 0.1 g·L−1 infused in mobile phase (A/B 50 : 50) at a flow rate of 5 μL·min−1. Full scan MS spectra were repeatedly performed from 100 to 2000 m/z during the time of the run and, also, specific MS2 scans were realized in order to obtain structural information for peptides of interest. First, LC-MS analysis was carried out on tryptic and chymotryptic hydrolysate which allows the characterization of peptides without disulfide bonds. In this case, main peptides were searched on the basis of a UV 215 nm chromatogram and were also observed on MS spectra as their protonated form [M + H]+. In a second step, LC-MS analysis focused on the reduced and alkylated peptide hydrolysate fraction and allowed the characterization of peptides containing disulfide bridges. In this case, alkylated peptides were searched by comparison between UV 215 nm chromatograms of non-treated hydrolysate and reduced/alkylated hydrolysate. Peptides were also observed on MS spectra as their protonated alkylated form [M + 57 + H]+, the additional mass of 57 amu corresponding to the acetamide group carried on the sulfur atom of cysteine.

Statistical analysis

Results are shown as mean ± SD or SEM as indicated in the figure captions. The non-parametric Mann–Whitney U test was used for Fig. 1B, and Student's t test for Fig. 2A. Statistical analysis of the qPCR results was performed using the rest software as described above.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

The authors gratefully acknowledge Dr Christophe Stenger for his helpful advice in the cell culture colocalization studies, Franck Saulnier (Service Commun de Séquence des Protéines, Université de Lorraine) for N-terminal microsequencing and Dr Joëlle Vinh (ESPCI CNRS USR3149 Biological Mass Spectrometry and Proteomics Lab) for identification of N- and C-lobe peptides by mass spectrometry. The authors also thank Erwan Magueur of Genclis SAS for his excellent technical assistance for the in vivo study. The rats for the LSR purification were kindly provided by Dr Henri Schroeder. This work was supported by a grant from the Région Lorraine – Université Henri Poincaré (BQR). NA and SA both received overseas thesis fellowships from the Higher Education Commission of Pakistan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  • 1
    Attie AD, Pittman RC & Steinberg D (1982) Hepatic catabolism of low density lipoprotein: mechanisms and metabolic consequences. Hepatology 2, 269281.
  • 2
    Ginsberg HN, Zhang YL & Hernandez-Ono A (2006) Metabolic syndrome: focus on dyslipidemia. Obesity (Silver Spring) 14 (Suppl 1), 41S49S.
  • 3
    Lopez-Miranda J, Perez-Martinez P, Marin C, Moreno JA, Gomez P & Perez-Jimenez F (2006) Postprandial lipoprotein metabolism, genes and risk of cardiovascular disease. Curr Opin Lipidol 17, 132138.
  • 4
    Huettinger M, Retzek H, Eder M & Goldenberg H (1988) Characteristics of chylomicron remnant uptake into rat liver. Clin Biochem 21, 8792.
  • 5
    Anderson BF, Baker HM, Norris GE, Rice DW & Baker EN (1989) Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J Mol Biol 209, 711734.
  • 6
    Bluard-Deconinck JM, Williams J, Evans RW, van Snick J, Osinski PA & Masson PL (1978) Iron-binding fragments from the N-terminal and C-terminal regions of human lactoferrin. Biochem J 171, 321327.
  • 7
    Mahley RW & Rall SCJ (1995) Type III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS & Valle D, eds), pp. 19531980. McGraw-Hill, New York.
  • 8
    van Dijk MC, Ziere GJ, Boers W, Linthorst C, Bijsterbosch MK & van Berkel TJ (1991) Recognition of chylomicron remnants and beta-migrating very-low-density lipoproteins by the remnant receptor of parenchymal liver cells is distinct from the liver alpha 2-macroglobulin-recognition site. Biochem J 279 (Pt 3), 863870.
  • 9
    Bihain BE & Yen FT (1992) Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 31, 46284636.
  • 10
    Stenger C, Corbier C & Yen FT (2012) Structure and function of the lipolysis stimulated lipoprotein receptor. In Chemical Biology (Ekinci D, ed.), pp. 267292. InTech, Rijeka.
  • 11
    Mann CJ, Khallou J, Chevreuil O, Troussard AA, Guermani LM, Launay K, Delplanque B, Yen FT & Bihain BE (1995) Mechanism of activation and functional significance of the lipolysis-stimulated receptor. Evidence for a role as chylomicron remnant receptor. Biochemistry 34, 1042110431.
  • 12
    Yen FT, Mann CJ, Guermani LM, Hannouche NF, Hubert N, Hornick CA, Bordeau VN, Agnani G & Bihain BE (1994) Identification of a lipolysis-stimulated receptor that is distinct from the LDL receptor and the LDL receptor-related protein. Biochemistry 33, 11721180.
  • 13
    Yen FT, Masson M, Clossais-Besnard N, Andre P, Grosset JM, Bougueleret L, Dumas JB, Guerassimenko O & Bihain BE (1999) Molecular cloning of a lipolysis-stimulated remnant receptor expressed in the liver. J Biol Chem 274, 1339013398.
  • 14
    Yen FT, Roitel O, Bonnard L, Notet V, Pratte D, Stenger C, Magueur E & Bihain BE (2008) Lipolysis stimulated lipoprotein receptor: a novel molecular link between hyperlipidemia, weight gain, and atherosclerosis in mice. J Biol Chem 283, 2565025659.
  • 15
    Garre C, Bianchi-Scarra G, Sirito M, Musso M & Ravazzolo R (1992) Lactoferrin binding sites and nuclear localization in K562(S) cells. J Cell Physiol 153, 477482.
  • 16
    He J & Furmanski P (1995) Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA. Nature 373, 721724.
  • 17
    Sitaram MP & McAbee DD (1997) Isolated rat hepatocytes differentially bind and internalize bovine lactoferrin N- and C-lobes. Biochem J 323 (Pt 3), 815822.
  • 18
    Superti F, Siciliano R, Rega B, Giansanti F, Valenti P & Antonini G (2001) Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection. Biochim Biophys Acta 1528, 107115.
  • 19
    Spik G, Coddeville B & Montreuil J (1988) Comparative study of the primary structures of sero-, lacto- and ovotransferrin glycans from different species. Biochimie 70, 14591469.
  • 20
    Coddeville B, Strecker G, Wieruszeski JM, Vliegenthart JF, van Halbeek H, Peter-Katalinic J, Egge H & Spik G (1992) Heterogeneity of bovine lactotransferrin glycans. Characterization of alpha-D-Galp-(1[RIGHTWARDS ARROW]3)-beta-D-Gal- and alpha-NeuAc-(2[RIGHTWARDS ARROW]6)-beta-D-GalpNAc-(1[RIGHTWARDS ARROW]4)- beta-D-GlcNAc-substituted N-linked glycans. Carbohydr Res 236, 145164.
  • 21
    Suzuki YA & Lonnerdal B (2002) Characterization of mammalian receptors for lactoferrin. Biochem Cell Biol 80, 7580.
  • 22
    Suzuki YA, Lopez V & Lonnerdal B (2005) Mammalian lactoferrin receptors: structure and function. Cell Mol Life Sci 62, 25602575.
  • 23
    Ji ZS & Mahley RW (1994) Lactoferrin binding to heparan sulfate proteoglycans and the LDL receptor-related protein. Further evidence supporting the importance of direct binding of remnant lipoproteins to HSPG. Arterioscler Thromb 14, 20252031.
  • 24
    McAbee DD, Jiang X & Walsh KB (2000) Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor's lectin properties. Biochem J 348 (Pt 1), 113117.
  • 25
    Rohlmann A, Gotthardt M, Hammer RE & Herz J (1998) Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants. J Clin Invest 101, 689695.
  • 26
    Mahley RW & Huang Y (2007) Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing. J Clin Invest 117, 9498.
  • 27
    Ziere GJ, Kruijt JK, Bijsterbosch MK & van Berkel TJ (1996) Recognition of lactoferrin and aminopeptidase M-modified lactoferrin by the liver: involvement of proteoglycans and the remnant receptor. Biochem J 313 (Pt 1), 289295.
  • 28
    Huettinger M, Retzek H, Hermann M & Goldenberg H (1992) Lactoferrin specifically inhibits endocytosis of chylomicron remnants but not alpha-macroglobulin. J Biol Chem 267, 1855118557.
  • 29
    Ziere GJ, Bijsterbosch MK & van Berkel TJ (1993) Removal of 14 N-terminal amino acids of lactoferrin enhances its affinity for parenchymal liver cells and potentiates the inhibition of beta-very low density lipoprotein binding. J Biol Chem 268, 2706927075.
  • 30
    Oh SM, Hahm DH, Kim IH & Choi SY (2001) Human neutrophil lactoferrin trans-activates the matrix metalloproteinase 1 gene through stress-activated MAPK signaling modules. J Biol Chem 276, 4257542579.
  • 31
    Eferl R & Wagner EF (2003) AP-1: a double-edged sword in tumorigenesis. Nat Rev Cancer 3, 859868.
  • 32
    Whitmarsh AJ & Davis RJ (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med (Berl) 74, 589607.
  • 33
    Kannan K, Amariglio N, Rechavi G, Jakob-Hirsch J, Kela I, Kaminski N, Getz G, Domany E & Givol D (2001) DNA microarrays identification of primary and secondary target genes regulated by p53. Oncogene 20, 22252234.
  • 34
    Ziere GJ, van Dijk MC, Bijsterbosch MK & van Berkel TJ (1992) Lactoferrin uptake by the rat liver. Characterization of the recognition site and effect of selective modification of arginine residues. J Biol Chem 267, 1122911235.
  • 35
    Chang S, Maeda N & Borensztajn J (1996) The role of lipoprotein lipase and apoprotein E in the recognition of chylomicrons and chylomicron remnants by cultured isolated mouse hepatocytes. Biochem J 318 (Pt 1), 2934.
  • 36
    Egito AS, Miclo L, Lopez C, Adam A, Girardet JM & Gaillard JL (2002) Separation and characterization of mares' milk alpha(s1)-, beta-, kappa-caseins, gamma-casein-like, and proteose peptone component 5-like peptides. J Dairy Sci 85, 697706.
  • 37
    Zambon A, Hashimoto SI & Brunzell JD (1993) Analysis of techniques to obtain plasma for measurement of levels of free fatty acids. J Lipid Res 34, 10211028.
  • 38
    Stenger C, Hanse M, Pratte D, Mbala ML, Akbar S, Koziel V, Escanye MC, Kriem B, Malaplate-Armand C, Olivier JL et al. (2010) Up-regulation of hepatic lipolysis stimulated lipoprotein receptor by leptin: a potential lever for controlling lipid clearance during the postprandial phase. FASEB J 24, 42184228.
  • 39
    Takayama Y, Mizumachi K & Takezawa T (2002) The bovine lactoferrin region responsible for promoting the collagen gel contractile activity of human fibroblasts. Biochem Biophys Res Commun 299, 813817.
  • 40
    Gonzalez-Marquez H, Perrin C, Bracquart P, Guimont C & Linden G (1997) A 16 kDa protein family overexpressed by Streptococcus thermophilus PB18 in acid environments. Microbiology 143 (Pt 5), 15871594.
  • 41
    Chang OK, Perrin C, Galia W, Saulnier F, Miclo L, Roux E, Driou A, Humbert G & Dary A (2012) Release of the cell-envelope protease PrtS in the growth medium of Streptococcus thermophilus 4F44. Int Dairy J 23, 9198.
  • 42
    Hesse AM, Marcelo P, Rossier J & Vinh J (2008) Simple and universal tool to remove on-line impurities in mono- or two-dimensional liquid chromatography-mass spectrometry analysis. J Chromatogr A 1189, 175182.