Glycosylated forms of nuclear lamins.

Chromatin and pore complex-lamina preparations were obtained from pig and chicken tissues, and their proteins were analysed by mono- and bidimensional electrophoresis. A glycosylated form of lamin A, recognized by concanavalin A, was shown to be present in at least 3 of the tissues examined. Glycosylation is suggested to be a further postsynthetic modification, besides phosphorylation and methylation, which can modify the properties of lamins.

Crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum in intact cells. Involvement of nuclear matrix proteins.

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei, proteins were crosslinked to DNA by the action of cis-diamminedichloroplatinum on intact chicken liver cells and analyzed by two-dimensional gel electrophoresis. At least eleven species of crosslinked proteins were found to derive from the nuclear matrix prepared from the same cell type, and five of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig. This subset of common proteins, conserved in different animal species, is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.

Glycosylation of RNA polymerase II from wheat germ.

RNA polymerase II from wheat germ was analyzed for the presence of sugars. The two largest subunits and the 27 and 25 kDa subunits were found to be glycosylated by a variety of sugars. However, no N-acetylglucosamine was detected, which was found by Kelly et al. (J. Biol. Chem. (1993) 268, 10416-10424) in the largest subunit of RNA polymerase II from calf thymus. Thus it appears that the regulatory function of this sugar, postulated by Kelly et al., is performed in the wheat germ enzyme by other monosaccharides. Carbohydrate analysis of the two largest subunits of the calf thymus enzyme also revealed the presence, beside N-acetylglucosamine, of other sugars. Some similarities in the features of glycosylation of the two polymerases, isolated from very different organisms, suggest that the sugar moieties have an important role in the structure and/or function of these enzymes.

DNA-nuclear matrix interactions analyzed by cross-linking reactions in intact nuclei from avian liver.

To detect the interactions of DNA with the nuclear matrix proteins, DNA-protein cross-linkages were induced in intact nuclei from chicken liver by the use of cis-diammine dichloroplatinum. Methods have been devised for fast purification both of the proteins and of the DNA fragments involved in the cross-linked complexes. By Southern-Western blotting a number of matrix proteins isolated from the complexes have been shown to recognize specifically DNA sequences present in the cross-linked DNA fragments. This experimental approach not only allows to identify the nuclear matrix-DNA interactions existing in the nucleus before its disruption, but also provides a preparation of matrix proteins enriched in those species which are involved in such interactions and which can therefore be detected with high sensitivity.

Thiol proteins in chromatin.

Total half-cystine residues in proteins of pig liver chromatin have been measured. About half of them are present in the reduced state. Thiol groups of non-histone chromatin proteins, which amount to about 40 nmol/mg of protein, are preferentially located in chromatin fragments which are more easily solubilised either by DNAse I or by DNAse II. The data obtained are compatible with an involvement of SH and SS groups in chromatin structure and function.

Fucose-carrying nuclear glycoproteins: distribution and tissue specificity.

Glycoproteins recognized by Ulex europaeus lectin I, specific for fucose residues, have been identified in chromatin prepared from pig liver, heart and kidney nuclei. They are present among the proteins dissociated from DNA with salt solutions and, in higher amount, among the proteins dissociated with urea and guanidine. In any case these fucose-containing glycoproteins appear to have a marked tissue specificity, which suggests that they have specific regulatory roles in the processes taking place in the nuclei.

3'-5' cAMP phosphodiesterase in pig liver nuclei.

cAMP phosphodiesterase was partially purified from pig liver nuclei and some of its properties were investigated. The enzyme is a low km PDE. Inhibition by xanthine derivatives is in line with the behaviour of PDE from other mammalian sources, while small differences are observed towards nucleotides inhibitors. The enzyme is a chromatin-bound protein although no specific location related to transcriptional function could be demonstrated.

Glycoprotein distribution in non-histone chromatin proteins from pig liver.

Carbohydrate content of non-histone proteins from pig liver chromatin has been measured in different groups of chromatin fractions and does not seem to be related to the affinity of the proteins for DNA. Glycoproteins are preferentially located in the nuclease-sensitive fractions of chromatin. A 59,000 dalton glycoprotein has been identified as a characteristic components of a chromatin fraction solubilized by DNAase II.

Calorimetric analysis of sodium dodecylsulfate-chromatin interaction.

Microcalorimetric titrations of whole chromatin and histones with sodium dodecylsulfate were performed at pH 7 and 25 degrees C. Enthalpy variations at low detergent concentration (less than 0.02%) are much more negative for histones than for chromatin. At 0.065% sodium dodecylsulfate the difference between the two curves becomes constant and, after correction for monomerization effects, amounts to +130 kcal/mol of nucleosomal unit. Core particles show heat effects similar to those of histones. These findings suggest that the chromatin structure is not stabilized exclusively by electrostatic interactions and that hydrogen bonds responsible for the additional stability may be contributed by non histone chromatin proteins.

Protein-DNA interactions at the nuclear scaffold regions of DNA loops.

DNA in cell nuclei is organized in large loops, which are formed by the binding of DNA to proteins present in a nuclear structure called matrix or scaffold. There is ample evidence that these interactions have not only a structural role, but also a functional one. Studies of these interactions have so far been carried out mainly in vitro. Their relevance, therefore, for the in vivo situation is not proven. We have analyzed the DNA-protein interactions directly in the intact nucleus by means of cross-linking reactions. Cross-linking by UV irradiation and by cis-diamine dichloro platinum (II) have been performed on nuclei from chicken liver and compared. The platinum complex has been found to be more efficient. The proteins complexed to DNA have been isolated and analyzed, and have been found to be enriched in species present in the nuclear scaffold, independently from its method of preparation.

Effect of infliximab on the glycosylation of IgG of patients with rheumatoid arthritis.

In patients with rheumatoid arthritis (RA) a decrease in the terminal galactose content of N-linked glycans of the Fc region of agalactosyl immunoglobulin G (IgG) (G0) occurs. The aim of this study was to evaluate, for the first time, the effect of infliximab, a new monoclonal antibody for the treatment of RA, on this phenomenon. A total of 19 patients with active RA were treated with intravenous infliximab (3 mg/kg) in combination with methotrexate (MTX) (10-20 mg). IgG was purified from their serum by caprylic acid. Analysis of IgG glycosylation was performed by lectin blotting/immunoblotting and enzyme linked lectin assay (ELLA)/enzyme linked immunosorbent assay (ELISA) using the Griffonia (bandeiraea) simplicifolia lectin II and protein-A/alkaline phosphatase. The purity of IgG samples obtained was higher than 90%. The sensitivity of the lectin/immunoblotting method was of about 0.25 microg of IgG. The inter- and intraassay coefficients of variation (CV) were 1.3% and 9.0% for lectin blotting, and 4.6% and 8.3% for immunoblotting, respectively. The sensitivity of the ELLA/ELISA approach was 0.025 microg/microL and the inter- and intraassay CV were 6.2% and 7.7% for ELLA, and 5.1% and 14.1% for ELISA, respectively. A good linear correlation (r2=0.18, P<0.05) was obtained between the two different experimental approaches. A decrease of G0 was observed in patients who clinically improved (according to the American College of Rheumatology criteria) following the pharmacological treatment. Our data indicate that infliximab can reduce the concentration of G0 in patients with active RA.

Influence of the carbohydrate moiety on the stability of glycoproteins.

To study the role of oligosaccharides on the properties of glycoproteins, five glycoproteins (yeast external invertase, bovine serum fetuin, glucoamylase from Aspergillus niger, and chicken egg white ovotransferrin and avidin) of previously established glycan patterns were purified to homogeneity and deglycosylated with endo- and exo-glycosidases in native conditions. Thermal stability and conformational changes were measured by high-resolution differential scanning microcalorimetry and circular dicroism spectroscopy before and after they were deglycosylated. It was found that deglycosylation decreases protein thermal stability, as judged by the decrease in denaturation temperature and denaturation enthalpy, while it does not affect substantially the conformation as indicated by the CD spectra in the far UV range. The destabilization effect of deglycosylation seems to depend on the carbohydrate content, i.e., the maximum effect was observed for the most heavily glycosylated protein, irrespective of the types (N-linked or O-linked) or patterns (mono- or multi-branched) of the covalently attached carbohydrate chains. In addition, studies of the reversibility to heat denaturation revealed that deglycosylated proteins have a poorer thermal reversibility in calorimetric scans than their native counterparts and tend to aggregate during thermal inactivation at acidic pH. These results suggest that carbohydrate moieties, in addition to the apparent stabilizing effect, may prevent the unfolded or partially folded protein molecules from aggregation. Our results support the hypothesis that the general function of protein glycosylation is to aid in folding of the nascent polypeptide chain and in stabilization of the conformation of the mature glycoprotein.

Tissue specificity of chromatin glycoproteins recognized by concanavalin A.

Chromatin glycoproteins recognized by Concanavalin A have been isolated from pig liver, kidney and heart by the use of immobilized lectin. Two groups of proteins differing in affinity for DNA have been analysed. Glycoproteins are mainly present in the group of proteins which are tightly bound to DNA. Mono and bidimensional electrophoretic patterns of total tightly bound proteins reveal a similarity among the three organs examined, while the corresponding patterns of the glycoproteins are typical for each organ. The tissue specificity of chromatin glycoproteins, together with their capability to interact not only with DNA but possibly also with other nuclear components, suggest a role for these proteins in the mechanism of genome expression.

Specific recognition sites for oligosaccharides in avian liver nuclei.

The presence of glycoproteins and sugar-binding sites in the nucleus is well ascertained. In order to verify the existence of specific nuclear protein-carbohydrate interactions, oligosaccharides were released from nuclear or non-nuclear glycoproteins by Peptide-N-glycosidase F, labeled by reduction with NaB3H4 and added to whole chicken liver nuclei and to subnuclear insoluble fractions (nucleoli, nuclear matrix and nuclear envelope). The analysis of the binding of the oligosaccharides to the nuclear fractions, performed in the presence or absence of competitor sugars, suggest that some rare oligosaccharides species, present only among the nuclear carbohydrates, are specifically recognized by the nuclear matrix.

Isolation of a novel nuclear glycoprotein from pig kidney.

A nuclear glycoprotein with an apparent Mr of 66,000 Da has been isolated from pig kidney chromatin after extraction with urea, guanidine-HCl and 2 M NaCl, and some of its structural features have been characterized. It belongs to the group of N-glycosylated proteins, which in the nucleus has so far received little attention. From its monosaccharide composition and recognition by lectins its oligosaccharides appear to be of high mannose and/or hybrid types. Some properties of its protein moiety suggest that it has a role in the packing of the DNA loops in the condensed chromatin.

The presence of N-glycosylated proteins in cell nuclei.

The protein-DNA crosslinking capability of cis-dichloro diammineplatinum has been exploited to check the intranuclear location of N-glycosylated proteins. When intact liver cells were treated with this reagent, a number of glycoproteins, recognized by Concanavalin A, have been shown to become crosslinked to DNA; many of them have been recognized as nuclear matrix components. The recognition by this lectin was abolished by treatment with N-glycosidase F, showing the presence of N-glycosidic bonds between the sugar moiety and the protein. Most of the glycoproteins appeared to have high mannose oligosaccharide chains, but sialic acid containing oligosaccharides were also identified.

Nuclear glycoproteins in higher vertebrates.

Nuclear glycoproteins recognized by Concanavalin A have been isolated from pig, rabbit and chicken tissues. Mono and bidimensional electrophoresis patterns of proteins loosely and tightly bound to DNA have been examined. The tissue specificity rather than species-specificity appears to be a quite general property of these proteins, suggesting for them a role in the mechanism of regulation of chromatin functions.

Cross-linked telomere-protein complexes from chicken erythrocyte nuclei: isolation by a new procedure.

DNA-protein cross-linkages were produced in intact nuclei of chicken erythrocytes by the action of cis-diammine dichloroplatinum. The telomeric DNA-protein cross-linked complexes were then isolated by hybridization with a biotinylated oligonucleotide and selective binding on immobilized streptavidin. Two main nonhistone proteins were present in the purified complexes, migrating in SDS-gel electrophoresis with apparent molecular masses of 66 and 58 kDa, respectively. Although the identity of these two proteins is still unknown, it is significant that two proteins with similar electrophoretic behavior have been described as constituents of the human telomeric complexes. This procedure could also be applied to the isolation of DNA-protein cross-linked complexes containing any chosen DNA sequence.

Glycoproteins of the nuclear matrix from chicken liver cells.

The nuclear matrix from chicken liver cells contains a small amount of glycoproteins recognized by Concanavalin A. These proteins are present not only in the peripheral matrix, but also in the internal one. In this latter localization many glycoprotein species appear, by cross-linking experiments, to be placed in the proximity of DNA. The effect of a partial enzymatic deglycosylation of matrix preparations suggests that these proteins contribute to the stabilization of the native matrix structure.