Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.     

ACTH binding to phospholipid bilayers: A 1H-NMR study of the 5-14, 1-14, and 1-24 derivatives

The interaction of the 5-14, 1-14, and 1-24 fragments of ACTH with sonicated phospholipid bilayers containing egg yolk phosphatidylcholine (EPC) either pure or mixed with 10 mole % phosphatidic acid (EPA), was investigated by proton nuclear magnetic resonance (¹H-nmr). The effects observed with zwitterionic EPC vesicles were small, indicating a low binding of the ACTH derivatives. The N-terminal aromatic resonances of the ACTH peptides were markedly broadened in the presence of negatively charged vesicles (EPC/EPA 9:1 M/M), while those of the C-terminal end were barely affected, showing that ACTH interacts with its N-terminal fragment. The choline resonance of the EPC molecules of the outer monolayer was shifted and broadened upon ACTH binding to the lipid vesicles, while that of the inner layer was not affected, suggesting that the peptide molecules interact only with the external leaflet of the lipid bilayer. The C²H and C⁴H resonances of the histidine-6 side chain were both shifted downfield upon peptide binding to the negatively charged lipid interface. In the case of the 1–24 derivative, these resonances were also split into two signals reflecting two different species of membrane-bound ACTH 1–24. Analysis of the line width and chemical shift variations of the ACTH and lipid resonances observed upon peptide binding shows that the membrane-binding potency of the shorter 5–14⁺¹ fragment, which presents a +1 net charge, is roughly similar to that of the highly cationic 1–24⁺⁶ (net charge +6) derivative, implying that the 15–24⁺⁵ segment is not essential for membrane binding. The nmr measurements at a fixed lipid-to-peptide ratio in the presence of increasing amounts of spin-labeled lipids demonstrate that the N-terminal fragment of ACTH does not penetrate the hydrophobic core of the bilayer, and should lie parallel to the membrane surface. © 1997 John Wiley & Sons, Inc. Biopoly 42: 731–744, 1997     

Gerbich blood group deficiency of the Ge:-1,-2,-3 and Ge:-1,-2,3 types. Immunochemical study and genomic analysis with cDNA probes

Human erythrocytes carry several transmembrane glycoproteins, among which the two minor species associated with the blood group Gerbich (Ge) antigens, GP C and GP D, play pivotal role since they interact with the membrane cytoskeleton and contribute to maintain the normal red cell shape. On the red cells from two categories of homozygous donors lacking the Ge determinants (Ge:-1,-2,-3 and Ge:-1,-2,3), GP C and GP D are missing but instead there is a new glycoprotein, easily detected by SDS/polyacrylamide gel electrophoresis, which exhibits some properties shared by GP C and GP D. This was shown by immunochemical analyses with a murine monoclonal antibody, extraction of the glycoproteins by organic solvents and binding studies with the 125I-labelled Lens culinaris lectin. The red cells from obligate heterozygotes for the Ge:-1,-2,-3 condition also carry this new glycoprotein component but in a much lesser amount than expected on the basis of one gene dose response. Using a cDNA probe containing the coding sequence of human GP C and the entire 3' untranslated region of its mRNA, we have demonstrated by Southern analyses that the Ge:-1,-2,-3 and the Ge:-1,-2,3 conditions are associated with a constant 3-kbp deletion within the GP C gene. Similar studies indicated that this gene is present as a unique copy per haploid genome of Ge-positive control donors (Ge:1,2,3). To account for these data and for the glycoprotein profile of Ge-negative erythrocytes, it is proposed that a unique Gerbich gene encodes for GP C and GP D, either by alternative RNA splicing or by different post-translational events, and that, following a 3-kbp deletion within this gene, a new glycoprotein having properties common to GP C and GP D can be produced.     

9-1-1: PCNA's specialized cousin

All living organisms are vulnerable to DNA damage. Cells respond to this hazard by activating a complex network of checkpoint and repair proteins to preserve genomic integrity. The DNA-encircling, ring-shaped heterotrimeric 9-1-1 complex, a relative of the replication protein PCNA, is a central coordinator of these events. 9-1-1 is loaded to damaged sites where it serves as a platform for the selective recruitment of checkpoint and repair proteins. In this Opinion article, 9-1-1 and proliferating cell nuclear antigen (PCNA) are compared and discussed in light of their respective structures and functions. We propose that the interaction partners of 9-1-1 possess specific 9-1-1-interaction boxes, which discriminate between 9-1-1 and PCNA thereby enabling specific interactions with individual 9-1-1 subunits.     

Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.     

IL-2-dependent generation of natural killer cells from bone marrow: role of MAC-1-, NK1-1- precursors.

We have previously shown that interleukin-2 (IL-2) is able to induce the generation of natural killer (NK) activity in bone marrow (BM) cell cultures from mice pretreated with 5-fluorouracil (5-FU). Cell fractionation experiments to analyze the nature of BM precursors indicate that MAC-1-, NK1-1- noncytotoxic precursors are induced by IL-2 to proliferate and generate cytolytic NK cells. These data demonstrate that the phenotype and functional characteristics of the IL-2-responsive cells in the FUBM are different from those of mature NK cells in that they are MAC-1+, NK1.1+, CD3- and susceptible to boosting by IFN-alpha.     

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 from human plasma

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 human plasma is described. Plasma is fractionated by affinity chromatography on chicken hemoglobin-Sepharose using the full capacity of the column; then after washing the column thoroughly, haptoglobin is eluted with 8 M urea and the eluate is collected in fractions to separate active and denatured haptoglobin. The urea-free, active fractions of haptoglobin are fractionated by affinity chromatography on Affi-Gel Con A to remove nonglycoproteins, principally apolipoprotein A-I, and the haptoglobin is eluted with 0.5 M glucose. Then the haptoglobin-containing fractions are fractionated by negative immunoadsorption chromatography on anti-chicken hemoglobin-protein A-Sepharose to remove chicken hemoglobin-human haptoglobin complexes. Haptoglobin prepared by this three-step procedure is biologically active and nearly homogeneous. The recovery is approximately 70%, irrespective of phenotype. The procedure can be completed in 3 days. A partial purification of apolipoprotein A-I is obtained simultaneously by this method.     

Characterization of Chrysanthemum ClSOC1-1 and ClSOC1-2, homologous genes of SOC1

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.     

The structural role of histone H1: properties of reconstituted chromatin with various H1 subfractions (H1-1, H1-2, and H1o).

A previous study on the distribution of histone H1 subfractions in chromatin suggested that these proteins differ in the protection they confer to DNA. To elucidate further this suggestion, reconstitution experiments were carried out with purified H1 subfractions (H1-1, H1-2, H1o) and H1-depleted chromatin. We have studied the structural properties of H1o as compared to those of other H1 fractions by electrophoretic analysis of DNA and mononucleosomes obtained after micrococcal nuclease digestion, thermal denaturation, and electron microscopy. The three fractions studied reassociate to H1-depleted chromatin. However, differences in the extent of DNA protection are observed between H1o and the other fractions: H1o induces a more rapid degradation of long oligomers into mononucleosomes; these mononucleosomes bearing H1o only, have a greater electrophoretic mobility; furthermore, thermal denaturation shows that a small fraction of DNA is less efficiently protected by H1o than by the other fractions. Electron microscopy, on the other hand, shows that these differences are not due to areas of chromatin devoid of H1o in the reconstitute and that the reconstituted samples are able, under proper ionic conditions, to refold in a higher-order structure.