OGT: a short overview of an enzyme standing out from usual glycosyltransferases.

O-GlcNAcylation is a highly dynamic post-translational modification whose level depends on nutrient status. Only two enzymes regulate O-GlcNAcylation cycling, the glycosyltransferase OGT (O-GlcNAc transferase) and the glycoside hydrolase OGA (O-GlcNAcase), that add and remove the GlcNAc moiety to and from acceptor proteins, respectively. During the last 30 years, OGT has emerged as a master regulator of cell life with O-GlcNAcylation being found in viruses, bacteria, insects, protists and metazoans. The study of OGT in different biological systems opens new perspectives for understanding this enzyme in many kingdoms of life. In this review, we summarize recent and older findings regarding the distribution of OGT in living organisms.

Detection and identification of O-GlcNAcylated proteins by proteomic approaches.

O-GlcNAcylation (O-linked beta-N-acetylglucosaminylation) is a widespread PTM confined within the nuclear, the cytosolic, and the mitochondrial compartments of eukaryotes. Recently, O-GlcNAcylation has been also detected in the close vicinity of plasma membranes particularly in lipid microdomains. The detection of this PTM can be easily done if appropriate controls and precautions are taken using a wide variety of tools including lectins, antibodies, or click-chemistry-based methods. In contrast, the identification of the proteins bearing O-GlcNAc moieties and the localization of the precise sites of O-GlcNAcylation remain challenging. This is due to the lability of the glycosidic bond between hydroxyl group of serine or threonine and N-acetylglucosamine using conventional fragmentation techniques such as CID. To tentatively overcome this technical limitation, electron-capture dissociation, or electron-transfer dissociation MS/MS are now used. Thanks to these breakthroughs, a large number of O-GlcNAc sites have been identified to date but these methodologies remain far from being used in routine.

Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating bax expression.

Delta-lactoferrin (∆Lf) is a transcription factor belonging to the lactoferrin family, the expression of which inhibits cell proliferation and leads to Skp1 and DcpS gene transactivation. In this study, we showed that ∆Lf expression also induces cell death via apoptosis in HEK 293 and MCF7 cells using a cell viability assay and DNA fragmentation. Western blot analyses showed that apoptosis was caspase-9, 7 and 8 dependent. Proteolytic cleavage of the endonuclease PARP was significantly increased. The levels of expression of Bcl family members were detected by immunochemistry and showed that the Bcl-xl/Bax and Bcl-2/Bax protein ratios were decreased. We determined that the pro-apoptotic effects of ∆Lf are mainly mediated by the activation of the mitochondria-dependent death-signaling pathway. Apoptosis induction by ∆Lf is concomitant with increased cellular levels of Bax protein. Analysis of the Bax promoter region detected a ∆Lf response element located at -155 bp from the transcription start site. Both luciferase reporter gene and chromatin immunoprecipitation assays confirmed that ∆Lf interacts in vitro and in vivo specifically with this sequence. Its deletion, realized using directed mutagenesis, totally abolished ∆Lf transcriptional activity, identifying it as a ∆Lf-responsive element. These results indicate that the Bax gene is a novel ∆Lf target. Moreover we also showed that the O-GlcNAc/P interplay, which controls ∆Lf transcriptional activity, modulates Bax transactivation.

Delta-lactoferrin, an intracellular lactoferrin isoform that acts as a transcription factor.

Delta-lactoferrin (ΔLf) is a transcription factor of which the expression is downregulated in cancer. It is a healthy tissue marker and a high expression level of its transcripts was correlated with a good prognosis in breast cancer. ΔLf results from alternative promoter usage of the hLf gene leading to the production of 2 isoforms with alternative N-termini: lactoferrin, which is secreted, and ΔLf, its nucleocytoplasmic counterpart. ΔLf possesses antiproliferative properties and induces cell cycle arrest. It is an efficient transcription factor interacting in vivo via a ΔLf response element found in the Skp1, Bax, DcpS, and SelH promoters. Since ΔLf possesses different target genes, modifications in its activity or concentration may have crucial effects on cell homeostasis. Posttranslational modifications modulate ΔLf transcription factor activity. Our earlier investigations showed that O-GlcNAcylation negatively regulates ΔLf transcriptional activity, whilst inhibiting its ubiquitination and increasing its half-life. On the other hand, phosphorylation potentiates ΔLf transcriptional activity. Recently, we showed that ΔLf is also modified by SUMOylation. Therefore, cooperation and (or) competition among SUMOylation, ubiquitination, phosphorylation, and O-GlcNAcylation may contribute to the establishment of a fine regulation of ΔLf transcriptional activity depending on the type of target gene and cellular homeostasis.

O-GlcNAcylation/phosphorylation cycling at Ser10 controls both transcriptional activity and stability of delta-lactoferrin.

Delta-lactoferrin (DeltaLf) is a transcription factor that up-regulates DcpS, Skp1, and Bax genes, provoking cell cycle arrest and apoptosis. It is post-translationally modified either by O-GlcNAc or phosphate, but the effects of the O-GlcNAc/phosphorylation interplay on DeltaLf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser(10) is O-GlcNAcylated and that this modification is associated with increased DeltaLf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the (391)KSQQSSDPDPNCVD(404) sequence as a functional PEST motif responsible for DeltaLf degradation and defined Lys(379) as the main polyubiquitin acceptor site. We next investigated the control of DeltaLf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a DeltaLf response element showed that O-GlcNAcylation at Ser(10) negatively regulates DeltaLf transcriptional activity, whereas phosphorylation activates it. Using a chromatin immunoprecipitation assay, we showed that O-GlcNAcylation inhibits DNA binding. Deglycosylation leads to DNA binding and transactivation of the Skp1 promoter at a basal level. Basal transactivation was markedly enhanced by 2-3-fold when phosphorylation was mimicked at Ser(10) by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the DeltaLf transcriptional complex binds to the DeltaLf response element and is phosphorylated and/or ubiquitinated, suggesting that DeltaLf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser(10) by either O-phosphate or O-GlcNAc coordinately regulates DeltaLf stability and transcriptional activity.

Discrimination and evaluation of lactoferrin and delta-lactoferrin gene expression levels in cancer cells and under inflammatory stimuli using TaqMan real-time PCR.

The lactoferrin gene is known to be expressed either constitutively or under inducible conditions such as hormonal stimuli or inflammation. Its transcription from alternative promoters leads to two products, lactoferrin (Lf) and delta-lactoferrin (DeltaLf) mRNAs the expressions of which are altered during oncogenesis. The comparison of the two enhancer/promoter regions revealed that the two isoforms might be differentially trans-activated. Nevertheless, concomitant expression of both transcripts has been found in some normal tissues and in a subset of breast cancer cell lines and biopsies. Moreover, we found putative inflammatory response elements in both P1 and P2 promoter regions suggesting that both Lf and DeltaLf might be upregulated under inflammatory stimuli. Therefore, a duplex Taqman gene expression assay has been developed and used to profile mRNA expression of the Lf gene in the case of cancer and under inflammatory conditions. Discrimination between the two transcripts is achieved by using a primer pairs/probe set within exon 1beta for DeltaLf and a primer pairs/probe set within exon 1 and exon 2 for Lf. In this study, we confirmed that Lf/DeltaLf Taqman gene expression assay is a powerful tool to investigate the expression of both Lf and DeltaLf transcripts. We also showed that lymphocytes and leukocytes isolated from fresh human blood expressed an extremely high level of DeltaLf messengers. An extensive series of cancer cell lines has been studied confirming that both P1 and P2 promoter regions of the Lf gene are downregulated or silenced in the case of cancer. Furthermore, using stimulation by bacterial lipopolysaccharides (LPS), we showed that in MDA-MB-231 and HT-29 epithelial cells, Lf expression is strongly increased with a higher expression level in MDA-MB-231 whereas DeltaLf expression is not. These results suggest that the NF-kappaB/cRel response elements present in the P1 promoter region are functional whereas those present in the P2 promoter region are not and show that DeltaLf is not regulated in inflammatory conditions.

[Lactoferrin: a multifunctional protein]. / La lactoferrine : une protéine multifonctionnelle.

Lactoferrin (Lf) is an iron-binding glycoprotein of the transferrin family that is expressed and secreted by glandular cells and found in the secondary granules of neutrophils from which it is released in infected tissues and blood during the inflammatory process. Initially described as an iron-binding molecule with bacteriostatic properties, Lf is now known to be a multifunctional or multi-tasking protein. It is a major component of the innate immune system of mammals. Its protective effects range from direct anti-microbial activities against a large panel of microorganisms including bacteria, viruses, fungi, and parasites, to anti-inflammatory and anti-cancer activities. While iron chelation is central to some of the biological functions of Lf, other activities involve interactions of Lf with molecular and cellular components of both hosts and pathogens. Its powerful antimicrobial activities, immunomodulatory properties and prevention of septic shock, anti-carcinogenic functions and its growing importance in iron delivery and bone growth, combined with the data obtained either by in vivo studies or clinical trials, make this molecule and its derivatives very promising tools for health or nutritional applications.

Lactoferrin structure and functions.

Lactoferrin (Lf) is an iron binding glycoprotein of the transferrin family that is expressed in most biological fluids and is a major component of mammals' innate immune system. Its protective effect ranges from direct antimicrobial activities against a large panel of microorganisms, including bacteria, viruses, fungi, and parasites, to anti-inflammatory and anticancer activities. This plethora of activities is made possible by mechanisms of action implementing not only the capacity of Lf to bind iron but also interactions of Lf with molecular and cellular components of both host and pathogens. This chapter summarizes our current understanding of the Lf structure-function relationships that explain the roles of Lf in host defense.

Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression.

Delta-lactoferrin is a cytoplasmic lactoferrin isoform that can locate to the nucleus, provoking antiproliferative effects and cell cycle arrest in S phase. Using macroarrays, the expression of genes involved in the G(1)/S transition was examined. Among these, Skp1 showed 2-3-fold increased expression at both the mRNA and protein levels. Skp1 (S-phase kinase-associated protein) belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination of cellular regulators leading to their proteolysis. Skp1 overexpression was also found after delta-lactoferrin transient transfection in other cell lines (HeLa, MDA-MB-231, HEK 293) at comparable levels. Analysis of the Skp1 promoter detected two sequences that were 90% identical to those previously known to interact with lactoferrin, the secretory isoform of delta-lactoferrin (GGCACTGTAC-S1(Skp1), located at - 1067 bp, and TAGAAGTCAA-S2(Skp1), at - 646 bp). Both gel shift and chromatin immunoprecipitation assays demonstrated that delta-lactoferrin interacts in vitro and in vivo specifically with these sequences. Reporter gene analysis confirmed that delta-lactoferrin recognizes both sequences within the Skp1 promoter, with a higher activity on S1(Skp1). Deletion of both sequences totally abolished delta-lactoferrin transcriptional activity, identifying them as delta-lactoferrin-responsive elements. Delta-lactoferrin enters the nucleus via a short bipartite RRSDTSLTWNSVKGKK(417-432) nuclear localization signal sequence, which was demonstrated to be functional using mutants. Our results show that delta-lactoferrin binds to the Skp1 promoter at two different sites, and that these interactions lead to its transcriptional activation. By increasing Skp1 gene expression, delta-lactoferrin may regulate cell cycle progression via control of the proteasomal degradation of S-phase actors.

Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin.

Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability.

SILAC-based proteomic profiling of the human MDA-MB-231 metastatic breast cancer cell line in response to the two antitumoral lactoferrin isoforms: the secreted lactoferrin and the intracellular delta-lactoferrin.

BACKGROUND: Lactoferrins exhibit antitumoral activities either as a secretory lactoferrin or an intracellular delta-lactoferrin isoform. These activities involve processes such as regulation of the cell cycle and apoptosis. While lactoferrin has been shown to exert its function by activating different transduction pathways, delta-lactoferrin has been proven to act as a transcription factor. Like many tumor suppressors, these two proteins are under-expressed in several types of cancer, particularly in breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: In order to compare the differential effects of the re-introduction of lactoferrin isoforms in breast cancer cells we chose the cancerous mammary gland MDA-MB-231 cell line as a model. We produced a cell line stably expressing delta-lactoferrin. We also treated these cells with fresh purified human breast lactoferrin. We performed two quantitative proteomic studies in parallel using SILAC coupled to mass spectrometry in order to compare the effects of different doses of the two lactoferrin isoforms. The proteome of untreated, delta-lactoferrin expressing and human lactoferrin treated MDA-MB-231 cells were compared. Overall, around 5300 proteins were identified and quantified using the in-house developed MFPaQ software. Among these, expression was increased by 1.5-fold or more for around 300 proteins in delta-lactoferrin expressing cells and 190 proteins in lactoferrin treated cells. At the same time, about 200 and 40 proteins were found to be downregulated (0-0.7-fold) in response to delta-lactoferrin and lactoferrin, respectively. CONCLUSIONS/SIGNIFICANCE: Re-introduction of delta-lactoferrin and lactoferrin expression in MDA-MB-231 mainly leads to modifications of protein profiles involved in processes such as proliferation, apoptosis, oxidative stress, the ubiquitin pathway, translation and mRNA quality control. Moreover, this study identified new target genes of delta-lactoferrin transcriptional activity such as SelH, GTF2F2 and UBE2E1.

Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme.

The expression of the transcription factor DeltaLf is deregulated in cancer cells. Its overexpression provokes cell cycle arrest along with antiproliferative effects and we recently showed that the Skp1 gene promoter was a target of DeltaLf. Skp1 belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination and the proteosomal degradation of numerous cellular regulators. The transcriptional activity of DeltaLf is highly controlled and negatively regulated by O-GlcNAc, a dynamic post-translational modification known to regulate the functions of many intracellular proteins. We, therefore, constructed a DeltaLf-M4 mutant corresponding to a constitutively active DeltaLf isoform in which all the glycosylation sites were mutated. In order to discover novel targets of DeltaLf transcriptional activity and to investigate the impact of the O-GlcNAc regulation on this activity in situ we compared the proteome profiles of DeltaLf- and DeltaLf-M4-expressing HEK293 cells versus null plasmid transfected cells. A total of 14 differentially expressed proteins were visualized by 2D electrophoresis and silver staining and eight proteins were identified by mass spectrometry analyses (MALDI-TOF; LC-MS/MS), all of which were upregulated. The identified proteins are involved in several processes such as mRNA maturation and stability, cell viability, proteasomal degradation, protein and mRNA quality control. Among these proteins, only DcpS and TCPB were also upregulated at the mRNA level. Analysis of their respective promoters led to the detection of a cis-regulating element in the DcpS promoter. The S1(DcpS) is 80% identical to the S1 sequence previously described by He and Furmanski [Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA, Nature 373 (1995) 721-724]. Reporter gene analyses and ChIP assays demonstrated that DeltaLf interacts specifically with the DcpS promoter in vivo. These data established that DcpS, a key enzyme in mRNA decay, is a new target of DeltaLf transcriptional activity.

Construction of a multi-functional helper-dependent adenovirus based system for cancer gene therapy.

Adenovirus holds great promise as a gene delivery system; it can hold large amounts of exogenous DNA and can be chemically and genetically modified to improve targeting to specific cells and tissues. A recombinant adenovirus construct expressing p53 is currently in clinical use as a cancer therapy in China. However, the use of adenovirus constructs in therapy is limited due to patients' strong immune response against these viruses and their gene products. To overcome this problem helper-dependent adenoviruses which do not express any viral gene products have been developed. Because the helper-dependent viruses do not express any viral gene products, a helper virus is required for their replication and encapsidation into infectious particles. This manuscript describes the construction of a prototype helper-dependent adenovirus system built such that it can be easily modified. The helper-dependent virus described here is built of a series of four cassettes, each with its own function. Furthermore, each individual cassette can be removed and replaced with a cassette with a different function. In this way, different helper-dependent viruses can be readily created. This type of system could be very useful in cancer therapy: For example, libraries of different cassettes could be maintained, allowing rapid assembly of constructs able to provide therapy for individual tumor types.

Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer.

We investigated the expression levels of human lactoferrin (Lf), a steroid hormone-inducible gene product the expression of which is often altered during oncogenesis, and of Delta-lactoferrin (DeltaLf), its alternative isoform, which has been shown to be absent from tumor cell lines in commonly used human breast epithelial cell lines, using semiquantitative RT-PCR. Both mRNAs were detected but with levels of expression lower than those found in normal breast epithelial cells. This downregulation was much more visible for DeltaLf since its expression was either significantly diminished (BT-20, MCF-7 cell lines) or practically absent (MDA-MB-231, T-47D, HBL 100 cell lines). In order to determine whether Lf gene products are useful prognosic tools, we further analyzed their expression levels in 99 primary breast cancer biopsies. DeltaLf transcripts were found in all of the samples, whereas Lf transcripts were found in 88% of them. Lf and DeltaLf expression levels were positively correlated (p = 0.003). Lf expression was related to tumor type with a higher recovery in lobular-type tumors (p = 0.04). DeltaLf expression was related to the histoprognostic grading (p = 0.02). In univariate analyses, DeltaLf and Lf expressions were prognosis parameters, high concentrations being associated with a longer overall survival.

Lactoferrin and host defence: an overview of its immuno-modulating and anti-inflammatory properties.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins that is abundantly expressed and secreted from glandular epithelial cells. In secretions, such as milk and fluids of the intestinal tract, lactoferrin is an important component of the first line of host defence. During the inflammatory process, lactoferrin, a prominent component of the secondary granules of neutrophils (PMNs), is released in infected tissues and in blood and then it is rapidly cleared by the liver. In addition to the antimicrobial properties of lactoferrin, a set of studies has focused on its ability to modulate the inflammatory process and the overall immune response. Though many in vitro and in vivo studies report clear regulation of the immune response and protective effect against infection and septic shock by lactoferrin, elucidation of all the cellular and molecular mechanisms of action is far from being achieved. At the cellular level, lactoferrin modulates the migration, maturation and function of immune cells. At the molecular level and in addition to iron binding, interactions of lactoferrin with a plethora of compounds, either soluble or membrane molecules, account for its modulatory properties. This paper reviews our current understanding of the cellular and molecular mechanisms that explain the regulatory properties of lactoferrin in host defence.

Expression of delta-lactoferrin induces cell cycle arrest.

Delta-lactoferrin (deltaLf) mRNA is the product of alternative splicing of the Lf gene. It has been found in normal tissues and was reported to be absent from their malignant counterparts. Our recent investigations have shown that deltaLf expression is a good prognostic indicator in human breast cancer. However, deltaLf has up till now only been identified as a transcript, and in order to characterize the deltaLf protein and determine its function we have used a deltaLf cDNA construct to produce the protein in vitro and in vivo. A 73 kDa protein was immunoprecipitated from in vitro translation products and this molecular weight is in accordance with the use of the first in frame AUG start codon located in exon 2. We also produced a cell line expressing deltaLf under doxycycline induction. Using this model we have been able to show that deltaLf is mainly distributed in the cytoplasm. Its expression induces cell cycle arrest and inhibits cell proliferation. Our results suggest that deltaLf may play an important role in the regulation of normal cell growth.