Lectins in the Cell Nucleus

Glycoblotogy vol. 1 no. 3 pp. 243-252, 1991 MINI REVIEW Lectins in the cell nucleus John L.Wang, James G.Laing and Richard L.Anderson Nuclear binding of neoglycoproteins implicates lectins Department of Biochemistry, Michigan State University, East Lansing, The existence of lectin molecules in the cell nucleus was first MI 48824, USA inferred from the binding of neoglycoproteins (see Hubert et al., 1989). These neoglycoproteins were derived by coupling specific saccharide structures onto a polypeptide backbone that normally bears no carbohydrate moiety [e.g. bovine serum albumin, Downloaded from https://academic.oup.com/glycob/article/1/3/243/610598 by guest on 27 September 2021 Key words: carbohydrate recognition/neoglycoproteins/ (BSA)]. Fluorescence microscopy and quantitative flow micro- nuclear glycoconjugates/S-type lectins/subcellular localization fluorometry were carried out with fluorescein-labelled neoglycoproteins and, in certain instances, ultrastructural analyses were performed with mannose (Man)-conjugated ferritin. When Triton-permeabilized baby hamster kidney (BHK) cells Introduction were incubated with fluorescein-labelled neoglycoprotein, fluorescence was observed in both the cytoplasm and the There is a general consensus that the potential to encode nucleus (Seve et al., 1985, 1986). Strongest binding was observed information in carbohydrate structures is enormous. Many of the for neoglycoproteins bearing a-L-rhamnose, whose structure possibilities of carbohydrate recognition in cellular function have resembles that of /S-D-galactose (Gal) (Monsigny et al., 1983). been explored for extracellular molecules and events such as Significant binding (> 3-fold over fluorescein-conjugated BSA) specific cell-cell recognition and cell adhesion to the extracellular was also observed for BSA bearing glucose (Glc), A'-acetyl- matrix. Our interest in these possibilities, however, can now be glucosamine (GlcNAc), lactose (Lac), mannose 6-phosphate and extended to the intracellular compartment as well, mainly as a fucose residues. Similarly, cryostat sections of calf tissues result of the demonstrations that carbohydrate structures such incubated with fluorescein-conjugated Lac-BSA showed cyto- as glycoproteins and glycosaminoglycans can be found in the plasmic and nuclear staining (Childs and Feizi, 1980). This cytoplasm and the nucleus (for a review, see Hart et al., 1989). staining could be competed with non-fluorescent Lac-BSA, but These advances in nuclear and cytoplasmic glycosylation have, not with Man-BSA. These results implicate the existence of in turn, stimulated interest in the intracellular localization of specific carbohydrate-binding proteins in both the cytoplasm and lectins. Defined as non-enzymatic and non-immune proteins the nucleus. which can selectively bind specific carbohydrate structures, these Karyoplasts obtained by enucleation of BHK cells also bound molecules appear to be good candidates for recognizing variations the same neoglycoproteins after permeabilization (Seve et al., in isomers, alternative linkages, sequence of saccharide units and 1986). Karyoplasts from exponentially growing cells bound a branched structures. much greater number of any given neoglycoprotein than did There are excellent reviews on glycosylation in the nucleus nuclei from density-inhibited cells. In the promyelocytic cell line and cytoplasm (Hart et al., 1989) and on the organization and HL60, in vitro differentiation into monocytes or granulocytes is functional implications of carbohydrate recognition domains in accompanied by a decrease in the binding of neoglycoproteins animal lectins (Barondes, 1988; Drickamer, 1988). The purpose to the nucleoplasm (Facy et al., 1990). These results suggest a of the present review is to summarize updated information in correlation between the nuclear localization of the lectin(s) and these areas and to discuss critically the available data pertaining the proliferation or differentiation state of the cells. Such a notion to the subcellular localization of those carbohydrate-binding is consistent with the results of comparing the level of the nuclear proteins that have been observed in the cell nucleus. lectin, carbohydrate binding protein 35 (CBP35), in mouse 3T3 As will be detailed in our discussion, all of the lectins located fibroblasts in quiescent versus proliferating cultures (Moutsatsos in the nucleus are also found in the cytoplasm. The nucleus and etai, 1987) (see below). Moreover, CBP35 has been found the cytosol, which communicate through the nuclear pores, are to be associated with the ribonucleoprotein elements (RNPs) of topologically continuous although functionally distinct. There the nucleoplasm (Laing and Wang, 1988), consistent with the are well-known examples of proteins [e.g. Drosophila heat distribution of at least a portion of the neoglycoproteins bound shock protein hsp 70 (Velazquez and Undquist, 1984)] and RNAs to the nucleus. In all of the mammalian cells studied (BHK cells, [e.g. snRNAs (Mattaj and De Robertis, 1985)] that can shuttle HeLa cells, leukaemic murine L1210 cells and rat hepatocytes), between the nucleus and the cytoplasm. What is more curious, neoglycoproteins bearing rhamnose, fucose or glucose residues however, is the considerable amount of evidence that indicates bound mostly to interchromatin spaces of the nucleoplasm and intracellular lectins also occur at the cell surface and, in some to nucleoli (Seve etal., 1985, 1986; Bourgeois etai, 1987). cases, in the extracellular medium. These latter sites belong to Condensed chromatin, revealed by the DNA-specific dye Hoechst the extracellular compartment, which is topologically continuous 33342, showed little or no binding of the neoglycoproteins. with the lumen of the endoplasmic reticulum, the Golgi stack The localization of neoglycoproteins in two non-mammalian and the secretory vesicles, but is topologically distinct from the cell types has been mapped to specific substructures within the intracellular compartment. These fascinating observations form nucleus. Nuclei of lizard ovarian follicle cells were highly labelled the basis for much of the discussion to follow. when ultra-thin sections incubated in the presence of Man-ferritin © Oxford University Press 243 J.L.Wang et al. were observed with electron microscopy (Hubert et al., 1985). source exhibit slightly different carbohydrate-binding affinities This binding can be specifically competed with Man-BSA. (Roff and Wang, 1983). In general, the polypeptide of the Labelling over nucleoli, chromatin and the outer leaflet of the L-14 group consists of a single domain, the CRD (Figure 1). nuclear envelope was particularly strong. Due to the unique In non-denaturing solvents, the native L-14 molecules are often organization of the granular component of the nucleoli in the comprised of dimers of the constituent polypeptides. specialized granulosa cells, it was possible to conclude that the In the L-30 group fall all the S-type lectins whose polypeptide Man-binding sites were associated with the dense fibrillar mol. wts have been reported to be in the range of 29 000—35 000 component of nucleolus containing the transcribing 45S rRNA (Table I). These include proteins originally isolated from various genes and with the nucleolar granules related to ribosomal sources and designated different names, but all of which exhibit precursors to be exported into the cytoplasm. In a study carried saccharide-binding activity: (i) CBP35 (Jia and Wang, 1988) from out on isolated macronuclei from the ciliated protozoan Euplotes mouse 3T3 fibroblasts; (ii) L-34 (Raz et al., 1989) from mouse eurystomus, neoglycoprotein labelling was found over nucleoli, tumour cells; (iii) Mac-2 (Cherayil et al., 1989) from mouse interchromatin spaces and, most interestingly, the macronuclear macrophages; (iv) laminin binding protein (LBP) (Woo et al., replication bands (Olins et al., 1988). Inasmuch as these repli- 1990), also from mouse macrophages; (v) IgE-binding protein cation bands represent delimited areas of DNA synthesis, this (eBP) (Albrandt et al., 1987) from rat basophilic leukaemia Downloaded from https://academic.oup.com/glycob/article/1/3/243/610598 by guest on 27 September 2021 suggests a co-localization of saccharide-binding components and cells; (vi) RL-29 (Leffler etal., 1989) and HL-29 (Sparrow sites of DNA synthesis. et al., 1987) from rat and human lung tissue, respectively. The The identity of the lectin(s) responsible for the binding of the results of Southern blot analyses of genomic DNA suggest that various neoglycoproteins in the nucleus (and in the cytoplasm) there is only one gene coding for the polypeptides of the L-30 has not been determined. Thus, it is not clear how many different group (Raz et al., 1988; Cherayil et al., 1989). For any given lectins are present in the various regions of the nucleus or whether species, the few differences in the amino acid sequences report- the various neoglycoproteins can bind to different subsites of one ed for the different proteins (e.g. mouse CBP35, L-34 and particular lectin. Extraction of nuclei or subnuclear components, Mac-2) are most probably ascribable to minor sequencing errors. followed by affinity chromatography on specific neoglycoprotein Although alternative sites of transcription initiation and/or adsorbents, will need to be carried out to purify the putative alternative splicing of the primary transcripts may lead to multiple lectin(s), and correlation of the saccharide-binding specificities

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