Biochimica et Biophysica Acta (BBA) - General Subjects
ReviewDynamics of galectin-3 in the nucleus and cytoplasm
Introduction
Galectin-3 (Gal3) belongs to a family of widely distributed proteins that (a) bind to β-galactoside-containing glycoconjugates and (b) contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide [1]. The protein consists of a single polypeptide (Mr ∼ 30,000) whose amino acid sequence suggests that it is a chimera of two distinct domains: an NH2-terminal domain containing repeats of a proline- and glycine-rich motif and a COOH-terminal CRD that shows sequence similarity with the corresponding CRDs of other members of the galectin family [1], [2]. Each CRD, ∼ 130 amino acids, contains highly conserved amino acid residues that interact with carbohydrate, as revealed by X-ray crystallography [3].
The carbohydrate-binding specificity of purified Gal3 has been studied extensively [2]. The protein binds type 1 or type 2 Galβ1→3(4)GlcNAc chains and the affinity for straight chain polylactosamine structures or complex-type branched glycans is increased over the simple disaccharide. Fucosylation (H-type 1 or H-type 2), or sialylation, or further substitution by (α1→3)–linked galactose or N-acetylgalactosamine on the terminal galactose residue of the lactosamine unit does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Using isothermal microcalorimetry, thermodynamic analysis of the binding of Gal3 to a series of saccharide ligands yielded the following order of Kd values: (a) lactose (Lac) ∼ 900 μM; (b) thiodigalactoside (TDG) ∼ 550 μM; (c) N-acetyllactosamine (LacNAc) ∼ 150 μM; and (d) blood group A tetrasaccharide, ∼ 60 μM [4]. Other physico-chemical measurements, such as equilibrium dialysis using radioactive Lac as the saccharide ligand or fluorescence polarization assays using the same series of carbohydrates, usually yielded Kd values 5–10 times lower, although the relative affinities were essentially identical [5], [6], [7], [8].
Since its initial isolation on the basis of carbohydrate-binding activity, a vast number of phenomenological observations have been reported on Gal3 [9], [10]. At least part of the reason for the large number of studies has to do with the fact that Gal3 exhibits a diverse range of subcellular localizations and molecular interactions. First, Gal3 (along with several other members of the galectin family) exhibits the striking phenomenon of dual localization [11], being found in both the intracellular as well as the extracellular compartments [12], [13]. The mechanism of externalization appears to be unusual inasmuch as the amino acid sequence of Gal3 does not reveal an obvious signal sequence for directing the polypeptide into the classical endomembrane pathway for secretion [13].
Human, rat, and murine Gal3 can be isolated, respectively, from human WiL-2 cells, rat basophilic leukemia cells, and mouse 3T3 fibroblasts in the absence of any reducing agents [14]. The purified recombinant proteins expressed from the rat and mouse cDNA constructs did not require reducing agents such as thiols to maintain carbohydrate-binding activity [14], [15]. Therefore, Gal3 does not appear to be sensitive to oxidative inactivation as might occur in the extracellular compartment. Nevertheless, the NH2-terminus of the polypeptide isolated from cell extracts is blocked by acetylation [16] and Gal3 is found predominantly as an intracellular protein in most cell types studied.
Second, intracellular Gal3 has been localized in both the nucleus and cytoplasm. Depending on the cell type and specific experimental conditions, the protein has been reported to be exclusively/predominantly cytoplasmic [17], [18], predominantly nuclear [17], [19] or distributed between the two subcellular compartments. Finally, even within the nucleus, Gal3 can be found diffusively in the nucleoplasm as well as being associated with a number of discrete punctate structures [20], [21]. These latter may correspond to subnuclear domains characterized by distinct ultrastructural features or by specific marker proteins under light microscopy: interchromatin granule clusters, speckles, Cajal bodies, etc. [22], [23].
On the basis of these observations, there appear to be exquisite mechanisms by which Gal3 localization, transport, and association with distinct subcellular components are regulated. In this special issue on nucleocytoplasmic glycosylation, it seems appropriate to review the studies that have shed new light on the dynamic behavior of a carbohydrate-binding protein in the nucleus and in the cytoplasm. Using Gal3 as a paradigm, the conditions that govern its nuclear versus cytoplasmic distribution, the signals for its nuclear import and export, its association with and dissociation from macromolecular complexes, and subnuclear domains will be discussed.
Section snippets
Intracellular distribution under different experimental conditions
A large number of observations on the nuclear versus cytoplasmic distribution of Gal3 have been reported, correlating the presence or absence of the protein in a particular compartment of the cell to various parameters such as source of the cells under study, specific cell type, culture conditions, proliferation status of the cell/culture, or neoplastic transformation. For example, it has been reported that adaptation of murine peritoneal macrophages to in vitro culture reduces the nuclear
Gal3 shuttles between the nucleus and the cytoplasm
Nucleo-cytoplasmic shuttling is typically defined as the repeated bidirectional movement of a protein across the nuclear pore complex. By this criterion, Gal3 shuttles between the nucleus and cytoplasm [43]. Human fibroblasts were fused with mouse fibroblasts and the localization of human Gal3 in the heterodikaryon was monitored using the monoclonal antibody NCL-GAL3, which recognizes human Gal3 but not the mouse homolog [44]. The human Gal3 protein localized to both nuclei in a large
General comments on Gal3 ligands
In addition to the transport receptors importin-α and exportin-1, many other proteins that interact with Gal3 have been reported (Table 1). In the context of the present article on dynamics of Gal3 inside cells, several general comments on these ligands might be illuminating. First, although some of these ligands have been documented to be compartment specific (e.g., the cysteine-rich, histidine-rich protein Chrp is exclusively cytoplasmic [62]), other ligands appear to coexist with Gal3 in
Dynamics of Gal3 association with the spliceosome
Several lines of evidence have now been accumulated to document that Gal3 and Gal1 are two of many proteins involved in the splicing of pre-mRNA, as assayed in a cell-free system [80], [83]: (a) NE derived from HeLa cells, capable of carrying out splicing of pre-mRNA, contained both Gal1 and Gal3; (b) depletion of both galectins from NE, either by Lac affinity chromatography or by antibody adsorption, resulted in the concomitant loss of splicing activity; (c) either recombinant Gal1 or
Concluding remarks
The studies reviewed in the present article have documented that Gal3, as an example of an intracellular carbohydrate-binding protein, exhibits dynamic behavior in the nucleus and cytoplasm of cells. A considerable amount of evidence has been accumulated that Gal3 is associated with RNP complexes and participates in splicing of pre-mRNA. Strikingly, it has recently been reported that at least three members of another family of nuclear splicing factors, characterized by an arginine- and
Acknowledgments
The work carried out in the authors' laboratories has been supported by a Calvin Research Fellowship from Calvin College (EJA), by grants Cottrell College Science Award from the Research Corporation (EJA), GM-38740 from the National Institutes of Health (JLW), 06-IRGP-858 from the Michigan State University Intramural Research Grant Program (JLW), and MCB-0092919 from the National Science Foundation (RJP).
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