Isolation of a sperm membrane sialoglycoprotein autoantigen from rabbit testes.

A single rabbit sperm membrane autoantigen has been isolated from rabbit testes by immunoadsorbent chromatography, agarose chromatography, and electrophoresis. This rabbit sperm autoantigen (RSA-1) is located in the sperm plasma membrane and blocks the sperm cytotoxic (immobilizing) activity of autoantisera. RSA-1 is a sialoglycoprotein, approximately 40% carbohydrate and containing 0.2 micrograms sialic acid/microgram protein. Its m.w. is estimated at 13,000 +/- 1200 by SDA-PAGE. RSA-1 can self-aggregate into higher m.w. forms. Approximately 0.6 mg of RSA-1 can be isolated from 100 rabbits testes. The isolated RSA-1 shows a 300-fold increase in specific cytotoxic (immobilizing) inhibitory activity over the starting testis pellet material.     

Localization of a single sperm membrane autoantigen (RSA-1) on spermatogenic cells and spermatozoa

The rabbit sperm membrane autoantigen RSA-1 is a sialoglycoprotein of 13,000 daltons which first appears on the surface of pachytene spermatocytes. Using specific antiserum to RSA-1 the antigen has been localized by immunofluorescence and immunoperoxidase staining. On testicular cells labeled at 37°C, RSA-1 is seen in patches on the surfaces of pachytene spermatocytes, round spermatids, and over the acrosomal area of later spermatids and spermatozoa. Over the postacrosomal and middle-piece regions of late spermatids and spermatozoa the labeling appears uniform. The uniformity can be seen to stop abruptly at the equatorial segment-postacrosomal border. Labeling cells after fixation gives a uniform distribution of label over the surface where patches were seen at 37°C. The polypeptides recognized by the antiserum used for labeling were identified by immunoadsorbent chromatography and subsequent SDS-PAGE. In testicular cells anti-RSA-1 recognizes the 13,000-dalton form and another component which migrates with the dye front. In ejaculated spermatozoa anti-RSA-1 recognizes a distinct ejaculate complex of higher-molecular-weight proteins containing an 84,000-dalton major band and five minor components.     

The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.     

Histochemical Specificity of β-Galactose Binding Lectins from Arachis Hypogaea (Peanut) And Ricinus Communis (Castor Bean)

Previous histochemical studies have demonstrated disparities in the binding of two lectins with a nominal specificity for terminal β-D-galactose. Biochemical studies have shown that the most complementary structure for binding peanut agglutinin (PNA) is the terminal disaccharide Gal-(β1 → 3)-GalNAc, whereas the most complementary structure for binding Ricinus communis agglutinin I (RCA I) is the terminal disaccharide Gal-(β1 → 4)-GlcN Ac. However, it is not known if only these differences in affinity account for the different histochemical staining reactions observed on tissue sections. In the present study we compared the staining patterns of PNA and RCA I by inhibiting in situ the binding of each lectin conjugated to horseradish peroxidase (HRP) with increasing concentrations of unlabeled PNA or RCA I. The PN A-HRP conjugate did not stain most tissue sites suspected of containing an abundance of glycoconjugates with terminal Gal-(β → 4)-GlcNAc. Moreover, unlabeled PNA failed to significantly inhibit strong RCA 1-HRP staining in these sites. In loci thought to contain variable amounts of glycoconjugates with terminal Gal-(β1 → 3)-GalNAc, unlabeled RCA I decreased PNA-HRP reactivity only slightly or not at all, whereas weak to strong RCA I-HRP staining was diminished or abolished by unlabeled PNA. The results suggest that PNA staining is restricted to glycoconjugates with terminal Gal-(β1 → 3)-GalN Ac. RCA I apparently reacts most strongly with glycoconjugates having the terminal disaccharide Gal-(β1 → 4)-GlcNAc, but also stains sites containing a moderate to abundant amount of glycoconjugates with the terminal Gal-(β→ 3)-GaINAc sequence.     

Suitability of a cytotoxicity assay for detection of potentially harmful compounds produced by freshwater bloom-forming algae

Detecting harmful bioactive compounds produced by bloom-forming pelagic algae is important to assess potential risks to public health. We investigated the application of a cell-based bioassay: the rainbow trout gill-w1 cytotoxicity assay (RCA) that detects changes in cell metabolism. The RCA was used to evaluate the cytotoxic effects of (1) six natural freshwater lake samples from cyanobacteria-rich lakes in central Ontario, Canada; (2) analytical standards of toxins and noxious compounds likely to be produced by the algal communities in these lakes; and (3) complex mixtures of compounds produced by cyanobacterial and chrysophyte cultures. RCA provided a measure of lake water toxicity that could not be reproduced using toxin or noxious compound standards. RCA was not sensitive to toxins and only sensitive to noxious compounds at concentrations higher than reported environmental averages (EC₅₀ ≥ 10³ nM). Cultured algae produced bioactive compounds that had recognizable dose dependent and toxic effects as indicated by RCA. Toxicity of these bioactive compounds depended on taxa (cyanobacteria, not chrysophytes), growth stage (stationary phase more toxic than exponential phase), location (intracellular more toxic than extracellular) and iron status (cells in high-iron treatment more toxic than cells in low-iron treatment). The RCA provides a new avenue of exploration and potential for the detection of natural lake algal toxic and noxious compounds.     

Histochemical specificity of beta-galactose binding lectins from Arachis hypogaea (peanut) and Ricinus communis (castor bean)

Previous histochemical studies have demonstrated disparities in the binding of two lectins with a nominal specificity for terminal beta-D-galactose. Biochemical studies have shown that the most complementary structure for binding peanut agglutinin (PNA) is the terminal disaccharide Gal-(beta 1----3)-GalNAc, whereas the most complementary structure for binding Ricinus communis agglutinin I (RCA I) is the terminal disaccharide Gal-(beta 1----4)-GlcNAc. However, it is not known if only these differences in affinity account for the different histochemical staining reactions observed on tissue sections. In the present study we compared the staining patterns of PNA and RCA I by inhibiting in situ the binding of each lectin conjugated to horseradish peroxidase (HRP) with increasing concentrations of unlabeled PNA or RCA I. The PNA-HRP conjugate did not stain most tissue sites suspected of containing an abundance of glycoconjugates with terminal Gal-(beta 1----4)-GlcNAc. Moreover, unlabeled PNA failed to significantly inhibit strong RCA I-HRP staining in these sites. In loci thought to contain variable amounts of glycoconjugates with terminal Gal-(beta 1----3)-GalNAc, unlabeled RCA I decreased PNA-HRP reactivity only slightly or not at all, whereas weak to strong RCA I-HRP staining was diminished or abolished by unlabeled PNA. The results suggest that PNA staining is restricted to glycoconjugates with terminal Gal-(beta 1----3)-GalNAc. RCA I apparently reacts most strongly with glycoconjugates having the terminal disaccharide Gal-(beta 1----4)-GlcNAc, but also stains sites containing a moderate to abundant amount of glycoconjugates with the terminal Gal-(beta 1----3)-GalNAc sequence.     

Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg

Biochemical and antigenic similarities exist among members of what can be considered a family of low molecular weight rabbit sperm autoantigens. These autoantigens are intrinsic plasma membrane glycoproteins specific to spermatogenic cells and spermatozoa. The amino acid and carbohydrate compositions of rabbit sperm autoantigen-1 (RSA-1) and RSA-2 were compared and monoclonal antibodies (mAb) were raised in mice against rabbit sperm autoantigens. The epitopes recognized by the antibodies were present on RSA-1, 2 and 3. A monoclonal anti-RSA-1, 2 and 3 (designated A.F. 1) was used to localize the antigen on spermatozoa and testis cells and investigate the epitope's tissue specificity. This mAb inhibited in vitro fertilization but did not block the sperm from dispersing the cumulus cells surrounding the egg. The mAb also demonstrated the presence of RSA-1, 2 and 3 on the plasma membrane of the egg after fertilization. It is concluded that the RSA family plays a central role in zona penetration.     

Isolation and purification of an organ-specific autoantigen from rabbit seminal plasma

A specific autoantigen of rabbit accessory glands was isolated and purified from seminal plasma using DEAE-Sephadex batch adsorption and Sephadex G-100 chromatographic fractionations. The autoantigen appeared homogeneous by polyacrylamide electrophoresis and by immunodiffusion. It has an extinction coefficient at 284 mμ (E_1cm^1%) of 6.42. It contains 87.73% of polypeptides. The carbohydrate moiety is composed of 9.21% hexose, 1.03% hexosamines, 0.33% fucose. From these chemical values the autoantigen has been identified as glycoprotein.     

Systematic comparison of oligosaccharide specificity of Ricinus communis agglutinin I and Erythrina lectins: a search by frontal affinity chromatography

Ricinus communis agglutinin I (RCA120) is considered a versatile tool for the detection of galactose-containing oligosaccharides. However, possible contamination by the highly toxic isolectin 'ricin' has become a critical issue for RCA120's continued use. From a practical viewpoint, it is necessary to find an effective substitute for RCA120. For this purpose, we examined by means of frontal affinity chromatography over 100 lectins which have similar sugar-binding specificities to that of RCA120. It was found that Erythrina cristagalli lectin (ECL) showed the closest similarity to RCA120. Both lectins prefer Gal beta1-4GlcNAc (type II) to Gal beta1-3GlcNAc (type I) structures, with increased affinity for highly branched N-acetyllactosamine-containing N-glycans. Their binding strength significantly decreased following modification of the 3-OH, 4-OH and 6-OH of the galactose moiety of the disaccharide, as well as the 3-OH of its N-acetylglucosamine residue. Several differences were also observed in the affinity of the two lectins for various other ligands, as well as effects of bisecting GlcNAc and terminal sialylation. Although six other Erythrina-derived lectins have been reported with different amino acid sequences, all showed quite similar profiles to that of ECL, and thus, to RCA120. Erythrina lectins can therefore serve as effective substitutes for RCA120, taking the above differences into consideration.     

Removing Selenate from Groundwater with a Vegetable Oil-Based Biobarrier

Vegetable oil–based permeable reactive biobarriers (PRBs) were evaluated as a method for remediating groundwater containing unacceptable amounts of selenate. PRBs formed by packing laboratory columns with sand coated with soybean oil were used. In an initial 24-week study a simulated groundwater containing 10 mg L⁻¹ selenate-Se was supplied to three soil columns and the selenate and selenite content of the effluent waters monitored. Two of the soil columns were effective at removing selenate and, during the final 21 weeks of the study, effluents from these columns contained almost no selenate or selenite. Almost all (95%) of the selenate removed was recovered as immobilized selenium sequestered in the solid matrix of the column. For unknown reasons, the third column failed to reduce selenate. A second study looked at the ability of PRBs to remove selenate when nitrate was present. As was done in the first study, three columns were evaluated but this time the water supplied to the columns contained 20 mg L⁻¹ nitrate-N and 10 mg L⁻¹ selenate-Se. Nitrate quickly disappeared from the effluents of these columns and during the final 23 weeks of the study, the nitrate content of the effluent water averaged less than 0.03 μg ml⁻¹ nitrate-N. Selenate was also removed by these columns but at a slower rate than observed with nitrate. In the final 6 weeks of the study, about 95% of the selenate applied to the columns was removed. In situ PRBs containing soybean oil might be used to remediate groundwater contaminated with both selenate and nitrate.     

Production of antisera specific to aldosterone: effect of hapten density and of carrier proteins

In order to investigate the influence of hapten density and of carrier proteins on the immunological characteristics of antisera, 4 groups of rabbits were injected with different aldosterone-carboxymethoxime protein conjugates. Six animals immunized with an aldosterone rabbit serum albumin (RSA) conjugate carrying 15 steroid molecules (RSA-2 conjugate) showed markedly higher antibody titers than rabbits injected with a RSA conjugate carrying 8 aldosterone molecules (RSA-1 conjugate). Low antibody titers were found in 8 animals immunized with an aldosterone bovine gamma globulin (BGG) conjugate showing a molar incorporation of 15. In a group of rabbits which was first injected with the RSA-1 conjugate and re-immunized with the RSA-2 conjugate the magnitude of antibody production was not enhanced. No differences in antibody sensitivity or specificity were observed between the 4 groups. It was concluded from these experiments a) that the density of haptenic groups depending on the molar incorporation of haptens and on the molecular weight of the carrier protein had influenced the magnitude of antibody production, b) that hapten density or carrier proteins had no effect on antibody sensitivity or specificity, c) that the magnitude of antibody production cannot be altered by re-immunizing with a more potent antigen.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10⁶-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

Structural analysis of the oligosaccharides of DR1 and DQw1 molecules

The major glycopeptide fractions of the alpha- and beta-chains of HLA-DR1 and DQw1 molecules were isolated on columns of immobilized concanavalin A (Con A), Lens culinaris (Lens), Ricinus communis agglutinin Type I (RCA), and leuko-phytohemagglutinin. Oligosaccharides were prepared from these fractions by enzymatic digestion with Endoglycosidases H or F and were analyzed on Bio-Gel P-6. The glycopeptides tightly bound to Con A (ConA III) were mostly associated with alpha-chains and were resolved as a single oligosaccharide peak (Kd = 0.72) on Bio-Gel P-6 after Endo H digestion. Man-5 is the minimal polymannosyl structure which can be deduced for the ConA III fractions of either DQw1 or DR1 oligosaccharides. The major component of the glycopeptides of the alpha-chains of either DR1 or DQw1 molecules which were weakly bound to Con A (ConA II fraction) did not interact with RCA before or after mild acid hydrolysis or neuraminidase treatment. This component represents a biantennary complex with neither terminal galactose nor sialic acid residues with a minimal structure terminating in N-acetyl glucosamine on the Mannose alpha 1----6 arm, referred to as GnM. The ConA II fractions, which constitute 10% of the total glycopeptides of beta-chains, are associated primarily with fucosylated, sialylated biantennary oligosaccharides not seen on the alpha-chains. The ConA I unbound fractions of either alpha- or beta-chains were mostly bound to RCA after mild acid hydrolysis, suggesting that the minimal structure was a sialylated triantennary structure. The major component associated with the beta-chains was bound to Lens such that a more definite structural assignment can be made, i.e., a triantennary structure with the Mannose on the alpha 1----6 arm substituted at C-2 and C-6. The oligosaccharides of alpha- and beta-chains were resolved as broad peaks on Bio-Gel P-6, suggesting that a mixture of tri- and tetraantennary structures with variable degrees of sialylation and galactosylation were present. The structural differences reported here between oligosaccharides of alpha- and beta-chains of DQw1 and of the two subsets of DR1 molecules could be responsible in part for the differential recognition properties expected of human class II molecules encoded by distinct loci.     

Sulfation of fucoidin in focus embryos: III. Required for localization in the rhizoid wall

Zygotes of the brown alga Fucus distichus L. Powell accumulate a sulfated polysaccharide (fucoidin) in the cell wall at the site of rhizoid formation. Previous work indicated that zygotes grown in seawater minus sulfate do not sulfate the preformed fucan (an unsulfated fucoidin) but form rhizoids. Under these conditions, we determined whether sulfation of the fucan is required for its localization in the rhizoid wall. This was accomplished by developing a specific stain for both the fucan and fucoidin. Using a precipitin assay, we demonstrated in vitro that the lectin ricin (RCA(I)) specifically complexes with both the sulfated and desulfated polysaccharide. No precipitate is observed when either is incubated in 0.1 M D-galactose or when RCA(I) is mixed with laminarin or alginic acid, the other major polysaccharides in Fucus. RCA(I) conjugated with fluorescein isothiocyanate (FITC) is also shown to bind specifically to fucoidin using a filter paper (DE81) assay. When added to zygotes, RCA(I)-FITC binds only to the site of fucoidin localization, i.e., the rhizoid cell wall. However, RCA(I)-FITC is not observed in the rhizoid wall of zygotes grown in the absence of sulfate. This observation is not due to inability of RCA(I)-FITC to bind to the fucan in vivo. Chemically desulfated cell walls that contained fucoidin in the rhizoid wall bind RCA(I)-FITC only in the rhizoid region. Also, the concentration of fucose-containing polymers and polysaccharides that form precipitates with RCA(I) is the same in embryos grown in the presence or absence of sulfate. If sulfate is added back to cultures of zygotes grown without sulfate, fucoidin is detected at the rhizoid tip by RCA(I)-FITC several hours later. These results support the conclusion that the enzymatic sulfation of the fucan is a modification of the polysaccharide required for its localization and/or assembly into a specific region of the cell wall.     

Comparison of Chiral Separations of Aminophosphonic Acids and Their Aminocarboxylic Acid Analogs Using a Crown Ether Column

Crown ethers are capable of complexing with primary amines and have been utilized in chromatography to separate amino acid racemates. This application has been extended to resolve (1‐amino‐1‐phenylmethyl)phosphonic acid and (1‐aminoethyl)phosphonic acid racemates, along with their aminocarboxylic acid analogs (2‐phenylglycine and alanine, respectively), via a ChiroSil RCA crown ether based chiral stationary phase. Effects of the organic modifier, temperature, and acid type and concentration on retention and selectivity were also investigated. Trends in retention and selectivity varied between aminophosponic acids and their aminocarboxylic analogs. Computer modeling and ¹H NMR analyses were performed to potentially gain a better understanding of interactions of the aforementioned molecules with the ChiroSil RCA chiral stationary phase. Theoretical predictions of the most stable conformations for (R)‐ and (S)‐enantiomers were compared to elution order; it was found that the elution order agreed with molecular modeling such that the longest retention correlated with the predicted most stable complex between the enantiomer and crown ether. ¹H NMR demonstrated interactions of aminophosphonic and aminocarboxylic racemates with (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid in solution and was utilized to determine enantiomeric excess of (1‐amino‐1‐phenylmethyl)phosphonic acid after its enantioenrichment via crystallization through diastereomeric salt formation with the crown ether followed by filtration. Chirality 25:369–378, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

The autoantigen La/SSB is a calmodulinmbinding protein

The work reported here has been directed to the identification of new nuclear calmodulin-binding proteins. To achieve this goal, nuclei from rat hepatocytes were purified and a fraction enriched in DNA- and RNA-binding proteins was extracted using DNase I and RNase A. Calmodulin-binding proteins present in this nuclear subfraction were purified by chromatography using first a DEAE-Sephacel column and subsequently a calmodulin-Sepharose column. Four major polypeptides of 118, 107, 48 and 45 kDa were found to bind to the calmodulin column in a Ca²⁺-dependent way. [¹²⁵I]-calmodulin overlay analysis confirmed that the proteins of 118, 48 and 45 kDa are calmodulin-binding proteins. These proteins bind single-stranded and also double-stranded DNA. A partial amino acid sequence obtained from the 48 kDa protein revealed a 100% identity with the La/SSB protein, an autoantigen implicated in several autoimmune diseases, such as lupus erythematosus and Sjögren's syndrome. Two-dimensional gel electrophoresis, Western blot analysis and experiments of binding to poly(U), also supports the identity of p48 as La/SSB. CaM and La/SSB protein colocalize in the heterochromatinic regions within the nucleus of rat hepatocytes. Preincubation of La/SSB with calmodulin in the presence of Ca²⁺ resulted in an increase in the binding of ssDNA to La/SSB, suggesting that calmodulin can play a role in the regulation of the association of La/SSB with DNA.