Annie-Pierre Sève

The human HIP gene, overexpressed in primary liver cancer encodes for a C-type carbohydrate binding protein with lactose binding activity

HIP was originally identified as a gene expressed in primary liver cancers, and in normal tissues such as pancreas and small intestine. Based on gene data base homologies, the HIP protein should consist of a signal peptide linked to a single carbohydrate recognition domain. To test this hypothesis HIP and the putative carbohydrate recognition domain encoded by the last 138 C‐terminal amino acids, were expressed as glutathione‐S ‐transferase proteins (GST‐HIP and GST‐HIP‐142, respectively). Both recombinant proteins were purified by a single affinity purification step from bacterial lysates and their ability to bind saccharides coupled to trisacryl GF 2000M were tested. Our results show that HIP and HIP‐142 proteins bind to lactose, moreover the binding requires divalent cations. Thus the HIP protein is a lactose‐binding lectin with the characteristics of a C‐type carbohydrate recognition domain of 138 amino acids in the C‐terminal region.

The cellular prion protein: A new partner of the lectin CBP70 in the nucleus of NB4 human promyelocytic leukemia cells*

Prion diseases are characterized by the presence of an abnormal isoform of the cellular prion protein (PrPc) whose physiological role still remains elusive. To better understand the function of PrPc, it is important to identify the different subcellular localization(s) of the protein and the different partners with which it might be associated. In this context, the PrPc–lectins interactions are investigated because PrPc is a sialoglycoprotein which can react with lectins which are carbohydrate‐binding proteins. We have previously characterized a nuclear lectin CBP70 able to recognize N‐acetyl‐β‐d‐glucosamine residues in HL60 cells. Using confocal immunofluorescence, flow‐cytofluorometry, and Western‐blotting, we have found that PrPc is expressed in the nucleus of the NB4 human promyelocytic leukemia cell line. It was also found that the lectin CBP70 is localized in NB4 cell nuclei. Moreover, several approaches revealed that PrPc and CBP70 are colocalized in the nucleus. Immunoprecipitation experiments showed that these proteins are coprecipitated and interact via a sugar‐dependent binding moiety. In conclusion, PrPc and CBP70 are colocalized in the nuclear compartment of NB4 cells and this interaction may be important to better understand the biological function and possibly the conversion process of PrPc into its pathological form (PrPsc). J. Cell. Biochem. 84: 408–419, 2002.

CBP70, a glycosylated nuclear lectin

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N‐acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post‐translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N‐ and O‐glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by β‐elimination, hydrazinolysis, peptide‐N‐glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis‐I agglutinin (RCA‐I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin‐blotting analysis [Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated‐WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal βGal residues, Galβ1–3 GalNAc, Man α1–3 Man, sialic acid α2–6 linked to Gal or GalNAc; and sialic acid α2–3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and α‐L‐fucose residues.

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin‐1, galectin‐3, galectin‐10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well‐known lectin, galectin‐3, a lactose‐binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin‐3 interacts in the cytoplasm with Bcl‐2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N‐acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur? J. Cell. Biochem. 78:638–649, 2000.

HnRNP CBP35-CBP67 interaction during stress response and ageing

Previous studies have demonstrated the existence of nuclear carbohydrate binding proteins in a variety of mammalian cells with molecular masses of 35,000, 67,000, and 70,000 (CBP35, CBP67, and CBP70), which are associated with nuclear ribonucleoprotein (RNP) complexes. CBP35 consists of two domains, an amino-terminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear RNP complex, and a carboxyl-terminal portion homologous to beta-galactoside-specific lectins. CBP35 it has been proposed, like the glucose-specific lectin, CBP67, to guide RNP complexes through the nuclear pore. Here we show that the exposure of mature rats to stress induces an increase in nuclear CBP35 bound to CBP67 and retained on immobilized glucose. Nuclear extracts from the livers of old rats displayed no detectable stress response. This CBP35.CBP67 association detected in rat liver is considered with respect to the CBP35.CBP70 association recently observed in HL60 cell nuclear extracts.

Nuclear and cytoplasmic expressions of the carbohydrate-binding protein CBP70 in tumoral or healthy cells of the macrophagic lineage.

The expression of the carbohydrate‐binding protein CBP70 was analyzed in undifferentiated HL60 cells, HL60 cells differentiated into monocytes/macrophages or granulocytes, healthy monocytes and in vitro monocyte‐derived macrophages (MDM) using an anti‐CBP70 serum. This study was performed by immunoblotting analysis of nuclear and cytoplasmic extracts before and after N‐acetylglucosamine affinity chromatography and by indirect immunofluorescence microscopy. The results of this study show, for the first time, that CBP70 is expressed in the nucleus and the cytoplasm of healthy or leukemic cells of the macrophagic lineage. However, striking differences were observed depending upon the leukemic or normal state of cells and cell differentiation. Indeed, the level of expression and the intracellular distribution of CBP70 were found to be different in undifferentiated HL60 cells and monocytes/macrophages differentiated from these cells. Major differences were also observed according to whether macrophages differentiated from leukemic HL60 cells or healthy monocytes. Thus, the total cellular expression of CBP70 was markedly lower in MDM than in HL60‐derived macrophages and the intracellular distribution of the protein was different. Nevertheless, in both cases, the total cellular expression of CBP70 was enhanced during cell differentiation. Another important result is the finding that CBP70 behaviour was totally different when HL60 cells were induced to differentiate into macrophages or granulocytes. These data could therefore suggest that CBP70 is involved in phagocytic cell differentiation. Moreover, we show that an additional 60 kDa polypeptide (p60), recognized by the anti‐CBP70 serum, is expressed in HL60 cells differentiated into macrophages or granulocytes as well as in healthy monocytes or MDM but not expressed in undifferentiated HL60 cells. Although CBP70 und p60 appeared to be closely related polypeptides, their relationship remains to be precised. These findings are discussed with regard to data available on galectin‐3.

Stable expression of functional CBP70 lectin during heat shock.

CBP70 is a glycoslylated lectin that interacts through either glycan‐lectin or protein‐protein interactions. In addition, depending on its cellular localization, this lectin has different partners, for example, galectin‐3, an 82‐kDa ligand in the nucleus, or Bcl‐2 in the cytoplasm. In this study, we observed the persistence of plurilocalized lectin CBP70 after two heat‐shock treatments conducted either under mild conditions, i.e., , incubating the cells for 1 h at 42°C then for 1, 3, 5, 7, or 9 h at 37°C, or harsh conditions, i.e., incubation at 42°C for 1, 2, 4, 6, 8, or 10 h. By combining the information collected from biochemical, fluorocytometric, confocal, and affinity‐chromatography analyses, we concluded that CBP70 persisted in HL60 cells and its N‐acetylglucosamine‐binding sites remained active after all the heat‐shock treatments tested. These data and the previously published findings reviewed in this report concur in supporting the hypothesis that CBP70 could function as an organizer of multimeric assembly, leading to the formation of various complexes in different cellular compartments, according to the needs of the cell. J. Cell. Biochem. 77:615–623, 2000.

Rev protein suppression of complex formation between nuclear proteins and rev-responsive element-containing RNA of human immunodeficiency virus-1.

The Rev protein from human immunodeficiency virus type 1 (HIV-1) is known to bind Rev responsive element (RRE) sequence of HIV-1 mRNA. This interaction is thought to enhance expression of viral structural proteins but the mechanism for this effect is uncertain. The aim of this study was to investigate (i) whether other cellular proteins also bind to the RRE sequence and (ii) whether binding of cellular proteins to RRE RNA is influenced by Rev protein. Our results revealed that a variety of RNA-protein complexes are formed when in vitro transcribed RRE-containing RNA is incubated with proteins present in HeLa nuclear extracts. The molecular masses of the most prominent bands in RNase protection assays were ∼90, 82, 67, 50, and 35–45 kDa. Addition of recombinant Rev protein suppressed the complex formation between RRE RNA and nuclear proteins. The strongest effect was observed when the RNA was preincubated with Rev before addition of HeLa proteins. Rev protein bound to the RRE-containing RNA under formation of oligomeric complexes with a Svedberg coefficient of 4.0–4.5 S. The β-galactosidespecific nuclear carbohydrate-binding protein 35 (CBP35), which is present in nuclear ribonucleoprotein (RNP) particles, was identified in the complexes formed between RRE RNA and HeLa nuclear protein. Binding of these complexes to a galactose affinity matrix was abolished by addition of Rev protein. We propose that binding of Rev protein to RRE-containing HIV transcripts prevents RNP complex formation between these transcripts and cellular proteins; this may inhibit spliceosome assembly allowing the transport of the unspliced or incompletely spliced HIV-1 mRNA into the cytoplasm. Such an effect could be expected to promote expression of viral structural proteins.