Surface sugar binding components of bovine spermatozoa as evidence by fluorescent neoglycoproteins

In the present study, the topographical distribution of carbohydrate binding sites on the plasma membrane of bovine epididymal spermatozoa was investigated using 15 different fluorescent neoglycoproteins and asialoglycoproteins. With mannose-bovine serum albumin (BSA)-fluoresceinthiocarbamyl (FTC), mannose-6-phosphate-BSA-FTC, lactose-BSA-FTC, maltose-BSA-FTC, asialolactoferrin-FTC and asialotransferrin-FTC a marked fluorescence was observed in the postacrosomal area. These results further substantiate the concept that carbohydrate binding sites of the spermatozoan plasma membrane and corresponding carbohydrates of the zona pellucida play a significant role in gamete interactions.     

Characterization of the cryptogein binding sites on plant plasma membranes

Cryptogein is a 98-amino acid proteinaceous elicitor of tobacco defense reactions. Specific binding of cryptogein to high affinity binding sites on tobacco plasma membranes has been previously reported (K(d) = 2 nM; number of binding sites: 220 fmol/mg of protein). In this study, biochemical characterization of cryptogein binding sites reveals that they correspond to a plasma membrane glycoprotein(s) with an N-linked carbohydrate moiety, which is involved in cryptogein binding. Radiation inactivation experiments performed on tobacco plasma membrane preparations indicated that cryptogein bound specifically to a plasma membrane component with an apparent functional molecular mass of 193 kDa. Moreover, using the homobifunctional cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes incubated with (125)I-cryptogein, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, two (125)I-cryptogein linked N-glycoproteins of about 162 and 50 kDa. Similar results were obtained using Arabidopsis thaliana and Acer pseudoplatanus plasma membrane preparations, whereas cryptogein did not induce any effects on the corresponding cell suspensions. These results suggest that either cryptogein binds to nonfunctional binding sites, homologues to those present in tobacco plasma membranes, or that a protein involved in signal transduction after cryptogein recognition is absent or inactive in both A. pseudoplatanus and A. thaliana.     

Reactions of hereophilic agglutinins with L-cells and different toxicity of anti-A HP to fibroblasts and L-cells in vitro]

The carbohydrate binding glycoprotein Anti-AHP binds to the plasma membrane of the (transformed) L-cell both in agglutination reactions and by the immunofluorescence technique. On the surface of (normal) mouse embryo fibroblast cells Anti-AHP receptors were not detectable. Anti-AHP is suitable as another marker for L cells. The random distribution of the binding sites at 4 degrees C changes with elevated temperatures (clustering, capping of sites). Anti-AHP inhibits the growth of L cells much more than that of fibroblasts. It does not act as a mitogen for the investigated cells.     

Interaction of human CBG with cell membranes

Specific binding sites for corticosteroid-binding globulin (CBG) and its pregnancy-associated variant (pCBG), having a modified carbohydrate moiety, were found in the plasma membranes of human liver, decidual endometrium and placental syncytiotrophoblast. The membrane binding was influenced by the conformation of the glycoprotein molecules and structure of their carbohydrate chains. CBG receptor was solubilized from the endometrium membrane and partially characterized. It was found to have a subunit structure, with a homooligomeric sialoglycoprotein consisting of four 20 kDa protomeric species being involved in the recognition of the CBG molecules complexed with progesterone or cortisol. A kinetic study using membrane microvesicles derived from the syncytiotrophoblast brush border revealed that neither CBG nor pCBG restricted cortisol accumulation in the intravesicular space, whereas only normal CBG could penetrate the syncytiotrophoblast membrane. Action of the CBG-cortisol complex on trophoblast cells resulted in the activation of membrane adenylate cyclase and growth of the cAMP accumulation within these cells. Collectively, these findings suggest that both normal CBG and pCBG are involved in the guided transport of steroid hormones to the target cells and transmembrane transfer of hormones and/or hormonal signals.     

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.     

Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold- particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin- binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.     

Hepatic Sinusoidal Endothelium Heterogeneity with Respect to the Recognition of Apoptotic Cells

The recognition and removal of human apoptotic peripheral lymphocytes in selected populations of periportal and perivenous endothelial cells was studied inin situandin vitroexperiments. Apoptotic peripheral blood lymphocytes once injected into the liver circulation were retained by the sinusoids showing a large heterogeneity of distribution: apoptotic cells are found in the periportal tract double the amount found in the perivenous region. Apoptotic PBL adhesion was lowered to a sixth of the control after preinjection with a sugar mixture (Mannose,N-acetylgalactosamine,N-acetylglucosamine,d-galactose), as suggested by the expression of modified surface glycoconjugates on the plasma membrane of apoptotic cells. A bimodal profile of the distribution of the hepatic sinusoidal cell population, regarding the number of galactose and mannose receptors and the porosity index, was found. Two endothelial cell subsets were present: low porosity cells (average index 14 ± 6%; periportal tract) with a high number of carbohydrate binding sites, and high porosity cells (average index 26 ± 7%; perivenous tract), with a low number of carbohydrate binding sites.     

Receptors for calcitonin gene-related peptide on the rat liver plasma membranes

Specific binding sites for calcitonin gene-related peptide (CGRP) were identified in the rat liver plasma membrane. The binding of 125I-[TyrO]rat CGRP to rat liver plasma membrane was time dependent, saturable and reversible. Scatchard analysis of the data revealed a single class of binding sites with apparent dissociation constant of 260.8 pM and a maximal binding capacity of 26.6 fmol/mg of protein. Rat, chick, and human CGRP and their synthetic analogues inhibited label binding in a dose-dependent manner with relative potencies as follows; chick greater than rat greater than human greater than [TyrO]rat CGRP. Salmon, human and [Asu1'7]eel calcitonin also inhibited label binding but only at higher concentrations. These results clearly indicate the presence of specific binding sites for CGRP in rat liver plasma membrane and suggest that CGRP has possible biological actions on the rat liver.     

Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).

Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres). The binding of fluorescein-labeled factor VIII to lipospheres with membranes of 15% phosphatidylserine was equivalent to binding to phospholipid vesicles (KD = 4.8 nM). Purified von Willebrand factor (vWf), a carrier protein for factor VIII, decreased membrane binding of factor VIII with a Ki of 10 micrograms/ml. Likewise, normal plasma decreased bound factor VIII by more than 90% whereas plasma lacking vWf decreased the binding of factor VIII by only 20%. Proteolytic activation of factor VIII by thrombin, which releases factor VIII from vWf, increased liposphere binding in the presence of vWf and in the presence of normal plasma. Although factor V is homologous to factor VIII and binds to lipospheres with the same affinity, purified factor V was not an efficient competitor for the membrane binding sites of factor VIII. These results indicate that phosphatidylserine-containing membrane sites have sufficient specificity to select thrombin-activated factor VIII from the range of phospholipid-binding proteins in plasma.     

Subcellular localization of binding sites for cytochalasin D: evidence from activation energies.

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at greater than 11.5 degrees C, 18 kcal/mol at less than 11.5 degrees C) was taken as evidence that [3H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [3H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).     

Selective loss of binding sites for the iodinated alpha-neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes

Removal of asparagine-linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated alpha-neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of alpha-neurotoxin-I-saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose-rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high-affinity binding sites for alpha-neurotoxin I (kd = 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites (Kd = 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin-receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of alpha-neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d-tubocurarine or alpha-bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine-linked carbohydrate chains.     

Variations in distribution of con A receptor sites and anionic groups during red blood cell differentiation in the rat

The distribution of receptors for concanavalin A (Con A) and anionic groups on plasma membranes of developing blood cells was investigated in the rat. Glutaraldehyde-fixed bone marrow and circulating blood cells were exposed to ferritin-conjugated Con A or positively chaged ferric oxide (CI) and processed for electro n microscopy. The frequency of Con A and CI binding sites varied during different erythroid developmental stages and amont different leukoid cell types. There was a constant inverse relationship between Con A and CI binding sites. Among leukoid cells, Con A binding was high on plasma cells and macrophages, lower on neutrophils and lymphocytes, and still lower on eosinophils and basophils; CI binding was highest in the latter and lowest in plasma cells and macrophages. Among erythroid cells, there was a progressive increase in Con A and a decrease in CI binding after successive divisions of erythroblasts, and a progressive decrease in Con A and an increase in CI binding upon maturation of the orthochromatic erythroblast to the reticulocyte. The most pronounced modification in distribution of these sites occurred during nuclear expulsion: that protion of the plasma membrane surrounding the extruded nucleus was heavily labeled by Con A (up to four times that of the orthochromatic erythroblast) whereas the reticulocyte had considerably fewer sites. The situation was reversed with CI. The results suggest that the concentration of nonsialated glycoproteins (to which Con A binds) varies inversely to that of sialoproteins (to which CI binds) in the membrane of the differentiating erythroid cell. The remarkable changes observed at the time of nuclear extrusion suggest that there is either local modification of glycosylated groups with removal of sialyl residues from the membrane surrounding the extruded nucleus of selective segregation of membrane glycoproteins leading to a high concentration of sialoproteins (glycophroin) in the membrane of the mature erythrocyte.     

The absence of a role for the carbohydrate moiety in the binding of apolipoprotein B to the low density lipoprotein receptor.

The binding of low density lipoprotein (LDL) to fibroblasts occurs through apolipoprotein B, a glycoprotein. The role of the carbohydrate in binding was assessed in two ways: (1) LDL, freed of sialic acid and most of the glucosamine and hexoses by digestion with a mixture of glycosidases, bound to fibroblasts as does native LDL. (2) The glycopeptides liberated from apoprotein B by trypsin and pronase failed to inhibit LDL binding to fibroblasts. Apparently the carbohydrate moiety of LDL does not interact with the plasma membrane receptor.     

Identification and characterization of parathyroid hormone receptors on dog kidney, human kidney, chick bone and human dermal fibroblast. A comparative study of functional and structural properties.

To evaluate the functional and structural characteristics of the parathyroid hormone (PTH) receptors on different tissues and the possible heterogeneity in structure and function, PTH receptors on dog kidney membrane, human kidney membrane, chick bone cell membrane and human dermal fibroblast membrane were evaluated. The results showed that human kidney plasma membrane, canine kidney plasma membrane and chick bone cell membrane possess one single class of PTH receptor with a Kd (dissociation constant) of 1-5 nM and an IC50 also of 1-5 nM. The number of binding sites was 800 fmol per mg of protein for chick bone cell particulate membrane, 1-5 pmol per mg of protein for human kidney plasma membrane and 2.2 pmol per mg of protein for dog kidney plasma membrane. Photoaffinity labelling identified a major binding component with a molecular mass of 70 kDa in all three types of membrane. The plasma membrane fraction from human dermal fibroblast contained two different binding sites for PTH with high (Kd = 2 nM) and low (Kd = 580 nM) affinities respectively. The IC50 for the adenylate cyclase is about 2 nM, which is similar to the Kd of the high-affinity site. Photoaffinity labelling also demonstrated a major binding component with a molecular weight of 70 kDa. We conclude that structural and functional similarity exists among the PTH receptors present on chick bone cell membrane, dog kidney membrane and human kidney membrane. The human dermal fibroblast possesses two different binding sites, one of which is coupled to adenylate cyclase.     

Effect of the nature of the surface of disperse silica on its interaction with the reproductive cells and seminal plasma of cattle]

The interaction of various disperse silica of I, II, III kind possessing various structure of surface groups (-OH; -O-CH2-CH2-O-CH2-CH2OH; -O-CH2-CH2-NH2 respectively) was investigated with some above membrane matrix polymers of bovine reproductive cells and seminal plasma (namely the surface proteins and carbohydrate polymers containing the N-acetyl-neuraminic acid (NANA) as terminal residue). Protein binding was preferentially observed for silica surface modified by aminoethoxy--and ethylene glycol groups and depended on concentration of silica in the mixture. It was found that biopolymers containing carbohydrate groups had larger affinity to I than to II or III. The binding value of I-III was 12-16% with respect to plasma proteins. Silicas I and II with -OH-groups on the surface absorb 17-21% N-ANA-containing polymers of bovine seminal plasma.     

Changes in binding activity of luteinizing hormone receptors by site directed mutagenesis of potential glycosylation sites.

Site directed mutagenesis of the rat ovarian luteinizing hormone (LH) receptor cDNA was performed at each of the six potential N-linked glycosylation sites to determine the effect of putative carbohydrate chains on the activity of the membrane receptor. The conversion of Asn173 to Gln resulted in the total loss of hormone binding to the surface of the transfected cell. Mutant receptors synthesized with substitutions at the remaining potential N-linked glycosylation positions of 77, 152, 269, 277 and 291 revealed no significant change in the hormone affinity. However Asn77Gln and Asn152Gln exhibited significant decreases (approximately 80%) in the number of high affinity hormone binding sites. The changes in hormone binding activity upon elimination of the potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.     

Purification and Molecular Characterization of the Brain Synaptic Membrane Glutamate-Binding Protein

A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.     

Prediction of carbohydrate binding sites on protein surfaces with 3-dimensional probability density distributions of interacting atoms

Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall prediction MCC was 0.49. Independent tests on anti-carbohydrate antibodies showed that the carbohydrate antigen binding sites were predicted with comparable accuracy. These results demonstrate that the predictors are among the best in carbohydrate binding site predictions to date.     

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

Enzymological, morphological, and immunological methods were used to characterize further the interaction of noncapacitated boar spermatozoa with the porcine zona pellucida. Transmission electron microscopy showed that sperm usually bind to the zona over the head region of the cell. Only the plasma membrane is involved in this binding. Bound sperm will undergo the acrosome reaction when treated with calcium and the ionophore A23187. The ability of intact sperm to bind to porcine eggs in vitro and the ability of sperm plasma membrane vesicles to absorb univalent antibody to the sperm binding site for the zona were used to determine the effects of various physical, chemical, and enzymological treatments on the sperm binding sites. These sites were resistant to a number of enzymes including proteases and polysaccharidases, but were inactivated by heat and trichloroacetic acid. Binding sites on the zona were inactivated by extracts from small quantities of sperm. Binding was also blocked by Fab antibody to whole zonae absorbed to other swine tissue and by similarly absorbed Fab antibody to sperm plasma membranes. These data provide further support for the presence of zona recognition sites on the plasma membrane of noncapacitated boar sperm. The binding sites on the sperm plasma membrane do not appear to be peripheral membrane proteins nor major constituents of a surface glycocalyx.     

Subcellular localization of binding sites for cytochalasin D: Evidence from activation energies

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at > 11.5 °C, 18 kcal/mol at < 11.5 °C) was taken as evidence that [³H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [³H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).