Binding of beta-galactosidase from rat epididymal fluid to the sperm surface by high-affinity sites different from phosphomannosyl receptors.

beta-Galactosidase, known to be secreted by epithelial cells lining the rat epididymal duct, binds to the surface of spermatozoa from the caudal region with high affinity and in a saturable form. The binding was not inhibited by mannose-6-phosphate, but was inhibited by fructose phosphate derivatives, a peculiarity previously demonstrated for the membranes of epididymal tissue. Fructose phosphate derivatives released 55% of beta-galactosidase activity from the spermatozoa. These results suggest that in the epididymis there is a special transport system for hydrolases, which could be involved in the secretion of enzymes destined for spermatozoa. This transport would require receptors that recognize sugar ligands other than mannose-6-phosphate. These receptors were present in the epididymal tissue and on the sperm surface.     

Isolation and characterization of phosphorylated oligosaccharides from alpha-N-acetylglucosaminidase that are recognized by cell-surface receptors.

Adsorptive endocytosis of lysosomal enzymes by fibroblasts and hepatocytes involves binding to cell surface receptors that recognize on lysosomal enzymes a phosphorylated carbohydrate, most likely a mannose 6-phosphate residue [Kaplan et al. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 2026-2030; Ullrich et al. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1591-1598]. Loss of alpha-N-acetylglucosaminidase endocytosis after treatment with endoglucosaminidase H indicated that the recognition site of alpha-N-acetylglucosaminidase is located on N-glycosidically linked oligosaccharides of the high mannose type. Acidic oligosaccharides with an average molecular weight of 2200 were liberated from alpha-N-acetylglucosaminidase by endoglucosaminidase H. These oligosaccharides were susceptible to degradation by alkaline phosphatase, alpha-mannosidase and beta-N-acetylglucosaminidase. At the non-reducing terminal these oligosaccharides bear phosphorylated mannose and/or N-acetylglucosamine residues.     

Binding of small phosphorylated chromatin peptides to DNA

Low-molecular-weight peptides involved in gene expression and cell growth have been isolated from DNA preparation from eukaryotic cells. After phosphorylation with protein kinase CKII (pCKII) these peptides are able to bind to DNA in presence of divalent cations and salt/ethanol. This finding may explain the mechanism by which the peptides exert their activity.     

Binding of prolactin and monoclonal antibody to prolactin receptors immobilized on a nitrocellulose membrane filter

Mammary prolaction (PRL) receptors in 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps) extract were immobilized on a nitrocellulose (NC) filter, and a binding assay using the filter-bound receptors was carried out in the absence of detergent. PRL binding to the receptors was dependent on the quantity of the receptors and the PRL added to the filters. The filter-bound receptors retained the specificity of binding to peptide hormones. Scatchard analysis showed that the number of PRL receptors and the dissociation constant for PRL binding are essentially unchanged after immobilization on a NC filter, indicating that the PRL binding site does not participate in the binding to the NC filter and is equally available for PRL binding. The monoclonal antibody (MAb) against the PRL receptor was able to bind specifically to the Chaps-solubilized and filter-bound PRL receptors, as shown by curvilinear Scatchard plots. Immobilization on NC filters permits direct detection and characterization of the soluble PRL receptor using labeled PRL or MAb.     

Binding of phosphorylated histone H1 to DNA.

A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of 1 mole of histone H1. The DNA binding properties of this in vitro phosphorylated H1 were compared with those of unmodified H1. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 20mM-40mM NaCl by phosphorylated H1 than by unmodified H1. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified H1 are bound to DNA. It is therefore concluded that phosphorylated H1 binds distributively to many or all DNA molecules available (depending on the histone/DNA ratio) while unmodified H1 binds cooperatively to a fraction of the DNA molecules in the reaction mixture.     

Binding of phosphorylated and dephosphorylated heavy meromyosin to F-actin

The effect of myosin light chain phosphorylation in skeletal muscle was investigated with respect to the binding affinity of phosphorylated and dephosphorylated heavy meromyosin (HMM) for F-actin in the absence of ATP. For phosphorylated HMM the affinity was 2.5-times weaker in the presence of Ca²⁺ as in its absence (HMM divalent binding sites saturated only with Mg). For dephosphorylated HMM the reverse was true, the binding being 2.4-times higher in the presence of Ca²⁺.     

Binding of platelet factor 4 to heparin oligosaccharides.

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.     

Binding of Clostridium difficile toxins to human milk oligosaccharides

The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(β1-4)Glc, Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc and Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc. However, the binding of the HMOs is uniformly weak, with apparent affinities ≤10(3?)M(-1). The results of molecular docking simulations, taken together with the experimental binding data, suggest that a disaccharide moiety (lactose or lactosamine) represents the core HMO recognition element for both toxin fragments. The results of a Verocytotoxicity neutralization assay reveal that HMOs do not significantly inhibit the cytotoxic effects of TcdA or TcdB. The absence of protection is attributed to the very weak intrinsic affinities that the toxins exhibit towards the HMOs.     

Changes in distribution of phosphomannosyl receptors during maturation of rat spermatozoa

The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.     

Binding of insulin dimers to receptors

A synthesis of current views of the nature of the self-association of insulin in solution, of the identity of the amino acid residues in the insulin molecule constituting the receptor binding site, and of characteristics of the apparent 'negative cooperativity' of the binding leads to the seemingly contradictory conclusions that only the monomeric form of the protein is available and capable of binding, yet that other species must bind as well. A solution that has been proposed is that an alternative binding site exists, one which is not involved in insulin dimerization. Here, these conclusions are re-examined in the light of the characteristics of a new model for the polymerization of insulin. Firstly, it is found that the idea that dimers and higher polymers of insulin can bind to receptors is plausible, even necessary, secondly, that the postulate of an alternative binding site on insulin molecules is not required, and finally, that the characteristics of the insulin-receptor interaction warrant further investigation specifically in terms of the ability of some insulins to polymerize in solution and their potential to cross-link receptors.     

Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml−1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml-1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.     

Reactivation of phosphorylated cholinesterase immobilized in a gelatin membrane

The soluble and immobilized cholinesterases (acetyl cholinesterase of human blood erythrocytes (EC 3.1.1.7) and butyryl cholinesterase of equine blood serum, (EC 3.1.1.8] were inactivated by such irreversible inhibitors as diisopropyl fluorophosphate (DFP), O,O-dimethyl-O-(2,2)-dichlorovinyl) phosphate (DDVP), paraoxone, armine. The inactivated enzymes were reactivated under the effect of TMB-4 (1,1'-trimethylene-bis)-4-formyl-pyridine bromide (dioxime). The values of the reactivation rate constants proved to be equal both for the soluble and immobilized cholinesterases inactivated by the same irreversible inhibitor. The immobilized enzyme is simpler and more correct to study the reactivating action than the soluble one.     

Binding of oligosaccharides of hyaluronic acid to proteoglycans (Short Communication)

Oligosaccharides derived from hyaluronic acid were shown to inhibit proteoglycan-hyaluronic acid interaction, as measured in a viscometer. The relative inhibition increased with the size of the oligosaccharide and the results suggested that decasaccharides were the smallest fragments able to bind strongly to the proteoglycan.     

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.     

Human beta-glucuronidase pinocytosis and binding to the immobilized phosphomannosyl receptor. Effects of treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase

beta-D-Glucuronidase (EC 3.2.1.31) was purified to homogeneity from human spleen, and enzyme fractions from CM-Sephadex were examined for uptake by fibroblasts and retention by a column of immobilized phosphomannosyl receptor. Uptake and binding were enhanced by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase, greatly reduced by prior treatment with alkaline phosphatase, and restored by subsequent treatment with alpha-N-acetylglucosaminyl phosphodiesterase. Immobilized phosphomannosyl receptor was used to separate high and low uptake enzyme forms. About 25% of the total beta-glucuronidase was retained by the receptor column and eluted with mannose 6-phosphate. The rate of uptake of retained enzyme was 2.5-3.0-fold greater than that of the enzyme applied to the receptor column. The fraction retained by the column was reduced to 5-10% by prior treatment of the enzyme with alkaline phosphatase. This phosphatase-resistant, receptor-retained fraction was taken up at only 24% the rate of non-phosphatase-treated, receptor-retained enzyme. However, its uptake was increased 7-fold by treatment with alpha-N-acetylglucosaminyl phosphodiesterase. The enhanced rate of pinocytosis conferred by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase was destroyed by a subsequent treatment with alkaline phosphatase. These studies demonstrate that although most of the "high uptake" enzyme in beta-glucuronidase from human spleen binds to receptors through phosphomonoesters of mannose, a significant fraction can interact with immobilized phosphomannosyl receptor and be taken up by fibroblasts through interactions involving mannose 6-phosphate in diester linkage with N-acetyl-D-glucosamine.     

Varicella-zoster virus glycoprotein oligosaccharides are phosphorylated during posttranslational maturation.

Varicella-zoster virus (VZV)-infected human embryonic lung fibroblasts (HELF) do not release infectious virions into their growth medium. Extracellular virions are pleomorphic, suggesting that they are partially degraded before their release from cells. To examine the intracellular pathway of viral maturation, [2-3H]mannose-labeled virus-encoded glycoproteins were isolated from VZV-infected HELF. Oligosaccharides attached to the glycoproteins were processed to complex-type units, some of which were phosphorylated. The major intracellular site of accumulation of VZV gpI was found to be perinuclear and to correspond to that of the cation-independent mannose 6-phosphate (Man 6-P) receptor. Subsets of VZV-containing cytoplasmic vacuoles were coated, Golgi-associated, or accessible to endocytic tracers. Phosphorylated monosaccharides protected HELF from the cytopathic effect of VZV in proportion to their ability to block Man 6-P receptor-mediated endocytosis. These data suggest that the unusual phosphorylated oligosaccharides mediate an interaction between VZV and Man 6-P receptors of the host cell; this interaction may be responsible for withdrawal of newly synthesized virions from the secretory pathway and for their diversion to prelysosomal structures.