Joel H. Shaper

Considerations in the isolation of rat liver nuclear matrix, nuclear envelope, and pore complex lamina☆

Abstract A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.

Handbook of glycosyltransferases and related genes

The CHST14 gene, localized at 15q14, is a single exon gene with an open reading frame of 1131 base pairs, encoding a 43 kDa protein dermatan-4-Osulfotransferase-1 (D4ST1) that catalyzes the 4-O-sulfation of N-acetyl-D-galactosamine residues in dermatan sulfate (DS). Both nearly exhaustively desulfated DS and partially desulfated DS serve as excellent substrates for the enzyme. Chst14/D4st1-deficient mice showed growth retardation as well asmultiple system abnormalities including neurology such as decreased neurogenesis and diminished T. Kosho (*) School of Medicine, Department of Medical Genetics, Shinshu University, Matsumoto, Japan e-mail: ktomoki@shinshu-u.ac.jp S. Mizumoto • K. Sugahara Laboratory of Proteoglycan Signaling and Therapeutics, Hokkaido University Graduate School of Life Science, Kita-ku, Sapporo, Japan e-mail: mizumoto@sci.hokudai.ac.jp; k-sugar@sci.hokudai.ac.jp N. Taniguchi et al. (eds.), Handbook of Glycosyltransferases and Related Genes, DOI 10.1007/978-4-431-54240-7_156, # Springer Japan 2014 1135 proliferation of neural stem cells. Recently, recessive loss-of-function mutations in the CHST14 gene were found to cause a specific form of Ehlers-Danlos syndrome (EDS) designated as D4ST1-deficient EDS (DD-EDS). The disorder is characterized by progressive multisystem fragility-related manifestations (skin hyperextensibilty and fragility, progressive spinal and foot deformities, large subcutaneous hematoma) and various malformations (facial features, congenital eye/heart/gastrointestinal defects, congenital multiple contractures). Glycosaminoglycan (GAG) chains from the affected skin fibroblasts were composed of a negligible amount of DS and excess chondroitin sulfate (CS), which was suggested to result from an impaired lock by 4-O-sulfation due to D4ST1 deficiency followed by back epimerization from L-iduronic acid to D-glucuronic acid. GAG chains of decorin from the affected skin fibroblasts were composed exclusively of CS and no DS, the opposite features observed in normal controls. Thus, skin fragility in the disorder was supposed to be caused by impaired assembly of collagen fibrils mediated by decorin bearing a CS chain that replaced a DS chain. The disorder stresses the importance of the role of CHST14/ D4ST1 and DS in human development and maintenance of extracellular matrices.

Bovine α1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases

α1,3‐galactosyltransferase (α3GalT, EC 2.4.1.151) is a Golgi‐resident, type II transmembrane protein that transfers galactose from UDP‐α‐galactose to the terminal N‐acetyllactosamine unit of glycoconjugate glycans, producing the Galα1,3Galβ1,4GlcNAc oligosaccharide structure present in most mammalian glycoproteins. Unlike most other mammals, humans and Old World primates do not possess α3GalT activity, which is relevant for the hyperacute rejection observed in pig‐to‐human xenotransplantation. The crystal structure of the catalytic domain of substrate‐free bovine α3GalT, solved and refined to 2.3 Å resolution, has a globular shape with an α/β fold containing a narrow cleft on one face, and shares a UDP‐binding domain (UBD) with the recently solved inverting glycosyltransferases. The substrate‐bound complex, solved and refined to 2.5 Å, allows the description of residues interacting directly with UDP‐galactose. These structural data suggest that the strictly conserved residue E317 is likely to be the catalytic nucleophile involved in galactose transfer with retention of anomeric configuration as accomplished by this enzyme. Moreover, the α3GalT structure helps to identify amino acid residues that determine the specificities of the highly homologous ABO histo‐blood group and glycosphingolipid glycosyltransferases.

A subset of non-histone nuclear proteins reversibly stabilized by the sulfhydryl cross-linking reagent tetrathionate. Polypeptides of the internal nuclear matrix.

When rat liver nuclei are isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, digested with DNase I and RNase A, and extracted with 1.6 M NaCl, nuclear envelope (NE) spheres depleted of intranuclear material, as analysed by thin-section electron microscopy, are obtained. Two-dimensional isoelectric focusing (IEF)/SDS-PAGE and non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE reveal that the predominant polypeptides are lamins A, B and C. Nuclei isolated in the absence of sulfhydryl blocking reagents yield salt- and nuclease-resistant structures which contain sparse but demonstrable intranuclear material. A number of non-histone polypeptides are seen in addition to the lamins. Nuclei treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) yield, after exposure to nucleases and 1.6 M NaCl, nuclear matrix-like structures containing an extensive intranuclear network and components of the nucleolus in addition to the NE. Increased amounts of the non-lamin, non-histone polypeptides are recovered with these structures. Subsequent treatment of these NaTT-cross-linked structures with reducing agents in 1.0 M NaCl selectively solubilizes the intranuclear components but leaves the nuclear envelope apparently intact. The lamins remain sedimentable and are virtually absent from the soluble (intranuclear) material. Instead, the major solubilized polypeptides are (a) 68 and 63 kD polypeptides which migrate in the vicinity of lamins B and C, respectively, but are distinguishable from the lamins by immunoblotting and by uni-dimensional peptide mapping; (b) a series of basic 60-70 kD polypeptides (pI greater than 8.0) which are not recognized by anti-lamin antisera; (c) an acidic (pI 5.3) 38 kD polypeptide; and (d) a number of high molecular mass (greater than 100 kD) polypeptides. These observations not only suggest a convenient method for fractionating matrix structures from rat liver nuclei into biochemically and morphologically discrete components, but also identify a subset of major non-lamin, non-histone nuclear polypeptides (comprising approx. 20% of the total nuclear protein) whose intermolecular interactions can be reversibly stabilized apparently by intermolecular disulfide bond formation by NaTT.

The erasable Western blot

A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a 10-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70 degrees C in 2% (w/v) sodium dodecyl sulfate containing 100 mM beta-mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed.

Murine Sperm-Zona Binding, A Fucosyl Residue Is Required for a High Affinity Sperm-binding Ligand A SECOND SITE ON SPERM BINDS A NONFUCOSYLATED, β-GALACTOSYL-CAPPED OLIGOSACCHARIDE

An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specificO-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role ofO-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminalN-acetylglucosaminyl or, alternatively, a terminal α-galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit thein vitro binding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAcβ1,4GlcNAcβ1,4GlcNAc,was inactive (1–72 μm range). In contrast, a β4-galactosyl-capped trisaccharide (Galβ1,4GlcNAcβ1, 4GlcNAc) and an α3-galactosyl-capped trisaccharide (Galα1,3Galβ1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50 = 42 μm and 5.3 μm, respectively). The addition of an α3-fucosyl residue to each of these two competitive inhibitors, forming Galβ1,4[Fucα1,3] GlcNAcβ1,4GlcNAc or Galα1,3Galβ1, 4[Fucα1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50 = 500 and 430 nm). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the α3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated β-galactosyl-capped trisaccharide.

Temporally specific involvement of cell surface β-1,4 galactosyltransferase during mouse embryo morula compaction

Cell surface beta-1,4 galactosyltransferase (GalTase) is shown to mediate intercellular adhesions between embryonal carcinoma (EC) cells and specifically during late morula compaction in the preimplantation mouse embryo. Monospecific anti-GalTase IgG raised against affinity-purified bovine beta-1,4 GalTase recognizes F9 EC cell GalTase as judged by immunoprecipitation and inhibition of GalTase activity, as well as by immunoprecipitation of a single 52 kd metabolically labeled membrane protein. Anti-GalTase IgG inhibits cell adhesions between EC cells, dissociates compacted mouse morulae, and inhibits blastocyst formation. Anti-GalTase IgG specifically inhibits cell adhesions during late morula compaction, coincident with a peak of surface GalTase activity as determined by direct enzyme assay. On EC cells, GalTase activity can be proteolytically released from intact cells, and is localized by indirect immunofluorescence to areas of intercellular contact, consistent with its proposed role in cell adhesion. Beta-1,4 GalTase is the first cell adhesion molecule identified that participates during late morula compaction, subsequent to uvomorulin function.

Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation

We have previously shown that sperm-egg recognition in the mouse is mediated by the binding of galactosyltransferase (GalTase) on the sperm surface to its appropriate glycoside substrate in the egg zona pellucida [L. C. Lopez, E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur (1985) J. Cell Biol. 101, 1501-1510]. In the present study, we have defined the spatial and temporal expression of surface GalTase during spermatogenesis and epididymal maturation. Purified populations of spermatogenic cells were isolated by unit gravity sedimentation, and surface GalTase expression was determined by indirect immunofluorescence and by direct enzymatic assay. GalTase is present on the surface of all spermatogenic cells assayed. During differentiation, there is a progressive redistribution of GalTase from an initially diffuse and uniform localization on the surface of primary spermatocytes to a restricted plasma membrane domain overlying the dorsal aspect of the mature acrosome. This apparent redistribution of surface GalTase was confirmed by direct enzymatic assays, which show that surface GalTase activity, normalized per cell, remains relatively constant throughout spermatogenesis, despite a drastic reduction in cell surface area. When normalized to the relevant cell surface area, the GalTase concentration per square micrometer increases 77-fold from pachytene spermatocytes to cauda epididymal sperm. Cell surface GalTase is thought to be a cytoskeletally associated transmembrane protein [N. L. Shaper, P. L. Mann, and J. H. Shaper (1985) J. Cell Biochem. 28, 229-239]; consequently we examined whether cytoskeletal components may be involved in the redistribution of GalTase during spermatogenesis. beta-Tubulin, monomeric actin, and filamentous actin were found to be present during spermatogenesis, as assayed by indirect immunofluorescence and by Western immunoblotting. alpha-Actinin and vinculin were not detectable under these conditions and served as negative controls. During spermatogenesis, the distribution of tubulin coincides with the appearance of the mitotic spindle, flagellum, and manchette. On the other hand, the distribution of filamentous actin coincides with surface GalTase, suggesting that actin-containing microfilaments may participate in the redistribution of surface GalTase during spermatogenesis.

Transcriptional Regulation of Murine 1,4-Galactosyltransferase in Somatic Cells ANALYSIS OF A GENE THAT SERVES BOTH A HOUSEKEEPING AND A MAMMARY GLAND-SPECIFIC FUNCTION

β1,4-Galactosyltransferase (β4-GT) is a constitutively expressed enzyme that synthesizes the β4-N-acetyllactosamine structure in glycoconjugates. In mammals, β4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine β4-GT gene specifies two mRNAs of 4.1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L.(1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian β4-GT gene to accommodate the recruited role of β4-GT in lactose biosynthesis.

Association of topoisomerase II with the hepatoma cell nuclear matrix: The role of intermolecular disulfide bond formation

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.