N-ACETYLGALACTOSAMINE AND SIALIC ACID-CONTAINING PROTEINS IN CHINESE HAMSTER METAPHASE CHROMOSOMES

Cell Biology International, 1998, Vol. 22, No. 2, 85–89 Article No. cb970211

LOCALIZATION AND IDENTIFICATION OF GALACTOSE/N-ACETYLGALACTOSAMINE AND SIALIC ACID-CONTAINING PROTEINS IN CHINESE HAMSTER METAPHASE CHROMOSOMES JOHANNA MYLLYHARJU and SEPPO NOKKALA* Laboratory of Genetics, Department of Biology, University of Turku, Finland Received 27 May 1997; accepted 21 November 1997

Four lectins were used to recognize galactose/N-acetyl-galactosamine (Gal/GalNAc) and sialic acid residues in proteins of Chinese hamster metaphase chromosomes. In situ binding pattern of a fluorescein isothiocyanate-labelled (Gal/GalNAc)-specific lectin Sophora japonica agglutinin (SJA) showed that chromosomal SJA-binding proteins are primarily localized to the helically coiled substructure of chromatids. Numerous SJA-binding proteins were identified in Western blots of chromosomal proteins, their molecular weights ranging from 26 to 200 kDa. Another Gal/GalNAc-specific lectin, peanut agglutinin (PNA), with a slightly different sugar binding specificity, did not bind to Chinese hamster metaphase chromosomes, and in Western blots only two chromosomal protein bands were faintly stained. The in situ labelling patterns of two sialic acid-specific lectins, Maackia amurensis (MAA) and Sambucus nigra (SNA) agglutinins, both showed that the helically coiled substructure of chromatids is also enriched in sialylated proteins. In Western blot analysis 11 MAA-binding protein bands with molecular weights ranging from 54 to 215 kDa were identified, while SNA only bound to one protein band of 67 kDa. MAA and SNA are specific for á(2<3)- and á(2<6)-linked sialic acid residues, respectively. Thus, it is likely that á(2<3)-linked sialic acid residues are more common in chromosomal proteins than á(2<6)-linked sialic acid residues. These data suggest that Gal/GalNAc and sialic acid-containing glycoproteins exist in metaphase chromosomes and that these proteins may have a role in the formation of higher order metaphase chromosome  1998 Academic Press structures. K: glycoproteins; metaphase chromosomes; lectins; galactose; N-acetylgalactosamine; sialic acid

INTRODUCTION Glycosylation is traditionally regarded as an unlikely modification of nuclear and cytoplasmic proteins. The prevailing dogma of glycoprotein biosynthesis and transport has led to the concept that glycosylation is restricted to proteins at the cell surface or within lumenal compartments of intracellular organelles. However, numerous studies have suggested the presence of nuclear and cytoplasmic glycoproteins (for reviews, see Hart et al., 1988, 1989). Results from several biochemical and lectin binding studies indicate the presence of a variety of To whom correspondence should be addressed: Seppo Nokkala, Laboratory of Genetics, Department of Biology, University of Turku, FIN-20014 Turku, Finland. 1065–6995/98/020085+5 $30.00/0

carbohydrate residues, including glucose, mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid in chromatinassociated proteins of numerous species (for reviews, see Stoddart, 1979; Hart et al., 1988, 1989). We have shown previously that glycoproteins containing N-acetylglucosamine, mannose and fucose exist in Chinese hamster metaphase chromosomes, and they localize to distinct chromosomal substructures (Myllyharju and Nokkala, 1996a,b). In this study we have continued the identification and in situ localization of glycosylated Chinese hamster non-histone chromosomal proteins. Four lectins from two sugar specificity groups were used: Sophora japonica (SJA) and Peanut (PNA) agglutinins from the galactose/N-acetylgalactosamine  1998 Academic Press

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(Gal/GalNAc) specificity group, and Maackia amurensis (MAA) and Sambucus nigra (SNA= Elderberry bark lectin, EBL) agglutinins from the sialic acid specificity group. Fluorescein isothiocyanate (FITC) or digoxigenin (DIG)-conjugated lectins were used for the determination of in situ distribution of chromosomal glycoproteins specifically bound by these lectins. The corresponding chromosomal proteins were identified in Western blot analysis with digoxigenin or biotin-conjugated lectins. MATERIALS AND METHODS Cell culture and isolation of metaphase chromosomes Chinese hamster Don cells were grown in Earle’s minimum essential medium (Flow) supplemented with 10% fetal bovine serum (Gibco), 1% nonessential amino acids (Flow), 2 m -glutamine (Flow), and 50 IU/ml penicillin–50 ìg/ml streptomycin (Flow). At a semiconfluent stage, colcemid was added to the cell cultures at a concentration of 0.1 ìg/ml. After 90 min, mitotic cells were selectively detached from the monolayer by washing with Earle’s balanced salt solution and gentle shaking. Metaphase chromosomes were isolated according to the procedure described by Myllyharju and Nokkala (1996a). Only preparations judged by phase-contrast microscopy to have well preserved chromosome morphologies and to be free of cell debris and interphase nuclei were used. For in situ lectin labeling a drop of chromosome suspension was spread on a dry glass slide and fixed with methanol: acetic acid (3:1) for 5 min. For gel electrophoresis chromosomes were isolated from seven 75-cm2 colcemid treated T-flasks, washed in 10 m Tris–HCl pH 8.0, 2 m CaCl2, 1% V/V hexylene glycol, 0.1% Nonidet P-40 (Jeppesen et al., 1978) and solubilized into 100 ìl of SDSsample buffer (Laemmli, 1970). 10 ìl of the sample was loaded into each well. In situ lectin labellings Chromosome preparations were incubated with 200–500 ìg/ml FITC-labelled lectins Sophora japonica agglutinin (SJA), Peanut agglutin (PNA) and Elderberry bark lectin (EBL=Sambucus nigra agglutinin, SNA) (Vector Laboratories), and digoxigenin labeled Maackia amurensis agglutinin (MAA) (Boehringer Mannheim) according to the method in Myllyharju and Nokkala (1996a).

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Specific binding of lectins was ascertained with the inclusion of competing sugars in the lectin incubation step. SDS-PAGE and Western blotting Gel electrophoresis was performed with Mini-Protean II Dual Slab Cell (Bio-Rad) using 7.5% gels. Electrophoresis was carried out as described by Laemmli (1970). Western blotting was performed with Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) at constant 100 V for 1 h. Transfer buffer was 25 m Tris, 192 m glycine, 20% V/V methanol, pH 8.3 Identification of chromosomal glycoproteins in Western blots Identification of PNA, MAA and SNA-specific chromosomal glycoproteins in Western blots was performed by using the DIG-Glycan Differentiation Kit (Boehringer Mannheim) (Myllyharju and Nokkala, 1996a). DIG-labelled lectins and control glycoproteins carboxypeptidase Y, transferrin, fetuin and asialofetuin were included in the kit. To ascertain the specific binding of the lectins used, appropriate control glycoproteins were run together with chromosomal proteins in Western blot analyses. For the identification of SJA-specific chromosomal proteins, 10 ìg/ml biotinylated SJA (Vector Laboratories) was used in the lectin incubation step. The bound biotinylated SJA was detected by incubation with 1 U/ml streptavidin– alkaline phosphatase (AP) (Boehringer Mannheim) and staining with NBT/BCIP solution (Myllyharju and Nokkala, 1996b). RESULTS In situ localization of Gal/GalNAc-containing proteins To visualize the distribution of Gal/GalNAccontaining proteins, preparations of isolated Chinese hamster metaphase chromosomes were incubated with FITC-conjugated Sophora japonica (SJA) and peanut (PNA) agglutinins. Binding of FITC–SJA resulted in a faint and uniform labeling of the surface of sister chromatids under which a more heavily labelled helically coiled substructure could be discerned (Fig. 1). The fluorescence intensity along the coil appeared to be uniform. The observed binding pattern was specific since inclusion of 200 m -galactose in the lectin

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Fig. 1. In situ FITC–SJA labelling of isolated Chinese hamster metaphase chromosomes. FITC–SJA binds to the surface and to the helically coiled substructure of chromatids. The helical coil is most clearly seen in the regions bordered by arrows.

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Fig. 3. In situ localization of MAA and EBL (=SNA)-specific Chinese hamster chromosomal proteins. The helically coiled substructure of chromatids is specifically labeled by both DIG–MAA (a) and FITC–EBL (b). The helical coil is most clearly stained in the regions bordered by arrows.

120, 40, and 35 kDa stained fairly weakly, and there were also several very weakly stained bands in the 145–200 kDa range. The rest of the protein bands stained with intermediate intensities. Only two PNA-binding protein bands of 63 and 45 kDa were very weakly stained in Western blots incubated with DIG-PNA (Fig. 2). In situ localization of sialylated proteins

Fig. 2. Identification of SJA- and PNA-specific Chinese hamster chromosomal proteins. Western blot of chromosomal proteins incubated with biotinylated SJA (lane a) and DIG– PNA (lane c). Lane b is a control lane showing three protein bands bound by streptavidin–AP alone.

incubation step prevented SJA from binding to the chromosomes. Under the same incubation conditions FITC–PNA did not bind to any regions along the chromosomes even at raised lectin concentrations. The same result was obtained by using DIG-conjugated PNA and anti-DIG-AP. Identification of SJA- and PNA-specific chromosomal proteins SJA-binding proteins were identified by incubating Western blots of chromosomal proteins with biotinylated SJA and streptavidin–AP. Numerous (at least 29) SJA-binding non-histone protein bands were identified, and their molecular weights ranged from 26 to 200 kDa (Fig. 2). The 135, 97–100, 64–67, 42–44, and 38-kDa protein bands were most intensively stained. The bands migrating at 200,

In order to study the distribution of sialylated proteins in chromosomes, preparations of isolated metaphase chromosomes were incubated with DIG–MAA and FITC–EBL. Binding of both DIG–MAA and FITC–EBL to isolated chromosomes resulted in a faint staining of the helically coiled substructure of chromatids (Fig. 3). No longitudinal differences in the staining intensity of the helical coil were observed. The MAA and EBL-binding patterns were specific since addition of 100 m sialyllactose to the lectin incubation step prevented them from binding to metaphase chromosomes. Identification of sialylated chromosomal proteins Sialylated, proteins were identified by incubating Western blots of chromosomal proteins with DIG–MAA and DIG–SNA (=EBL). Several (at least 11) MAA-binding proteins were identified, the 65-kDa protein staining most intensively, while the other proteins with a molecular weight range of 54–215 kDa stained only weakly (Fig. 4). Only one SNA-specific protein band at 67 kDa was stained with moderate intensity in Western blots incubated with DIG-SNA (Fig. 4). DISCUSSION In the present study, we report that Chinese hamster chromosomes contain numerous

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Fig. 4. Identification of MAA- and SNA-specific Chinese hamster chromosomal proteins. Western blot of chromosomal proteins incubated with DIG–MAA (lane a) and DIG–SNA (lane b).

glycoproteins specifically bound by lectins belonging to the galactose/N-acetylgalactosamine and sialic acid specificity groups. The lectin binding patterns showed that these chromosomal glycoproteins are preferentially localized to the helically coiled substructure of chromatids. The labelling patterns of two Gal/GalNAcspecific lectins, SJA and PNA to metaphase chromosomes were conspicuously different. Binding of FITC-SJA resulted in a preferential labeling of the helically coiled substructure of chromatids, while FITC– or DIG–PNA did not detectably bind to chromosomes under the same incubation conditions. This observation was supported by the blotting analyses. SJA bound to numerous nonhistone protein bands with molecular weights ranging from 26 to 200 kDa, while PNA recognized only two very faintly staining protein bands of 63 and 45 kDa. It seems probable that the amount of PNA-binding proteins in single chromosomes is too low to be detected in in situ labelled chromosomes. Although SJA and PNA both belong to the same overall specificity group, they exhibit differences in the sugar structures preferentially bound by them. The disaccharide -Galâ(1<3)-GalNAc is the most potent inhibitor for both of these lectins (Lotan et al., 1975; Wu et al., 1981). However, a â-linked glycoside of -Galâ(1<3)-GalNAc may be an essential requirement for binding of SJA and a hydrophobic contribution to the site subterminal to the nonreducing moiety may be required (Wu et al., 1981). Of the monosaccharides tested, 28 ì and 100 m -galactose is required for 50% inhibition of SJA and PNA haemagglutination activity,

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respectively. DGalNAc alone does not inhibit PNA haemagglutination activity, while 50 ì DGalNAc is sufficient for 50% inhibition of SJA activity (Lotan and Sharon, 1978; Wu et al., 1981). In previous studies it has been shown that the high-mobility group non-histone proteins 14 and 17 of mouse and bovine cells contain galactose moieties among other sugar residues (Reeves et al., 1981). Yeoman et al., (1976) have identified a 26-kDa galactosamine-rich protein in the chromatin of Walker 256 carcinosarcoma and Novikoff hepatoma cells. Kelly and Hart (1989) have investigated the glycosylation of Drosophila chromatin proteins, and they did not observe any specific binding of a galactose-specific lectin Ricinus communis agglutinin to any regions along the chromosomes. However, the SJA and PNA binding patterns observed in this study suggest that the structure of Gal/GalNAc-containing side chains in chromosomal proteins can be very specific and not recognized by all the lectins belonging to the Gal/ GalNAc-specificity group. Interestingly, it has been demonstrated that numerous galactoside binding lectins, for which no substrate or role has been defined, exist in the nucleus (Carding et al., 1985; Moutsatsos et al., 1986). Also, it has been shown that a fraction of sea urchin non-histone chromosomal proteins are able to agglutinate erythrocytes, and this agglutinating ability is inhibited by high concentrations of free -galactose (Sevaljevic et al., 1977). Kelly and Hart (1989) suggested that galactosylation of chromosomal proteins would create a substrate for these lectins, and thus the possibilities for function become numerous. Spangler et al. (1975) have shown that sialic acid residues are present in chromatin-rich fractions of rat liver and Morris hepatoma nuclei. The in situ binding patterns of sialic acid-specific lectins MAA and SNA observed in this study show that in Chinese hamster metaphase chromosomes sialylated proteins are localized primarily to the helically coiled substructure of chromatids. MAA and SNA bind specifically to á(2<3)- and á(2<6)linked sialic acid residues, respectively (Shibuya et al., 1987; Wang and Cummings, 1988). In Western blots of chromosomal proteins MAA bound to several protein bands ranging from 54 to 215 kDa while SNA recognized only one protein band of 67 kDa. The most intensively labelled band in the blots incubated with MAA had an apparent molecular weight of 65 kDa. As the molecular weights of this and the SNA-specific protein band (65 and 67 kDa, respectively) are so close to each other, it may well be that they represent the same protein containing both á(2<3)- and

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á(2<6)-linked sialic acid residues. In general, á(2<3)-linked sialic acid residues seem to be more common in chromosomal proteins than á(2<6)-linked sialic acid residues. In spite of well-established existence of nuclear glycoproteins, their function has remained obscure. Many roles for the glycosyl moieties in nuclear proteins have been suggested. These include roles in nucleosytoplasmic transport, formation or stabilization of multiprotein complexes, targeting proteins to the nucleus, regulation of transcription, blocking sites of phosphorylation on proteins, protection from proteolysis, and the structural organization of chromatin (for reviews see Hart et al., 1988, 1989). The results obtained in the present and our previous studies (Myllyharju and Nokkala, 1996a, 1997b) suggest firstly, that numerous Chinese hamster chromosomal proteins are modified by glycosylation, and secondly, that these glycosylated non-histone proteins probably have a role in the formation of higher order metaphase chromosome structures. Chromosomal glycoproteins are found at three distinct structural domains of Chinese hamster metaphase chromosomes and each of the three structural domains is characterized by proteins modified by specific sugar residues. The surface domain contains proteins with GlcNAc and/or mannose residues, but is devoid of proteins with fucose residues. The next structural level below the surface domain, the helically coiled substructure of chromatids, seems to be characterized by proteins with complex saccharide side chains composed of GlcNAc, fucose, galactose/ GalNAc and/or sialic acid residues. The third structural domain, the Q-band domain, is enriched in proteins with mannose and/or fucose residues. ACKNOWLEDGEMENTS This study was supported by the Academy of Finland. REFERENCES C SR, T SJ, T R, F T, 1985. Multiple proteins related to the soluble galactose-binding animal lectin revealed by a monoclonal anti-lectin antibody. Biochem Biophys Res Commun 127: 680–686. H GW, H GD, H RS, 1988. Nuclear and cytoplasmic glycosylation: novel saccharide linkages in unexpected places. TIBS 13: 380–384.

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