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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study examines the mechanism of endocytosis of testicular prosaposin by the nonciliated cells of the efferent ducts. Testicular prosaposin is secreted by Sertoli cells into the lumen of the seminiferous tubules as a 70 kDa isomer where it binds to the tail of spermatozoa. In the efferent ducts, after dissociating from the plasma membrane of the spermatozoa, prosaposin is endocytosed by the nonciliated cells, presumably by receptor-mediated endocytosis. The initial step of receptor-mediated endocytosis usually results from the binding of a ligand's terminal oligosaccharide to a receptor on the cell surface. Thus, in the present study, several monosaccharides were injected in the lumen of the efferent ducts to compete with the binding and endocytosis of prosaposin. A quantitative electron microscopic approach was utilized and the number of gold particles, indicating anti-prosaposin immunoreactive sites, were scored over the various cell compartments including the plasma membrane, endocytic vesicles, early endosomes, and late endosomes. The length of the plasma membrane and the areas of endocytic vesicles, early endosomes, and late endosomes were measured with an image analyzer and the number of grains expressed per microm (plasma membrane) and microm2 (endocytic vesicles/endosomes) respectively. The quantitative analysis was performed in untreated animals (controls) and animals treated with various sugars (i.e., glucose, galactose, mannose, mannose 6-phosphate, N-acetylglucosamine and N-acetylgalactosamine) injected into the lumen of the efferent ducts at a concentration of 20 mM. Sialic acid caused the greatest decrease in the labeling density of the endocytic elements. Mannose 6-phosphate also caused a decrease in labeling but to a lesser extent. Various amounts of sialic acid (0.02 mM, 0.2 mM, 2 mM, 20 mM, and 200 mM) showed that most of these concentrations produced a significant decrease in the labeling density of endocytic vesicles and endosomes. Moreover, Western blots of prosaposin isolated from seminiferous tubular fluids followed by glycan analysis with Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), revealed that this protein has sialic acid residues that are terminally linked to galactose and/or N-acetylgalactosamine (alpha-NeuNAc-[2->6]-Gal and alpha-NeuNAc-[2->6]-GalNAc). These data indicate that testicular prosaposin is removed from the lumen of the efferent ducts by the noncialiated cells via a receptor that recognizes prosaposin's terminal sialic acid residues.
Mol Reprod Dev 1998 Oct
PMID:Role of sialic acid in the endocytosis of prosaposin by the nonciliated cells of the rat efferent ducts. 974 Mar 23

UDP-GalNac: polypeptide N-acetylgalactosaminyltransferase from swine trachea epithelium was purified to homogeneity by procedures which included affinity chromatography on Sepharose 4B columns containing bound deglycosylated Cowper's gland mucin. The enzyme, purified 12,000-fold from microsomes with a yield of 40%, showed only a single band on dodecyl sulfate polyacrylamide gel electrophoresis. The homogenous enzyme has an apparent molecular mass of 70,000 Da, as determined by gel electrophoresis or gel filtration. The transferase has a broad pH optimum between 6.7-7.8 with maximal activity at pH 7.2, and required Mn2+ for activity with maximal activity at 5-7.5 mM. Higher concentrations of Mn2+, inhibited the enzyme. The purified transferase was specific for UDPGalNAc and glycosylated both threonine and serine residues in tryptic peptides prepared from deglycosylated Cowper's gland and swine and human trachea mucins. The apparent Km of the transferase for UDPGalNAc was 6.3 microM, and the Km values for deglycosylated Cowper's gland and human and swine trachea mucins were 0.83, 1.12 and 0.94 mg/ml, respectively. The Vmax of the purified enzyme was 2.1 micromol/min/mg with deglycosylated Cowper's gland mucin, as the glycosyl acceptor. However, the activities with peptides prepared from deglycosylated mucins by limited acid hydrolysis were 20-fold greater than the intact glycoprotein under identical conditions. The deglycosylated mucins and larger peptides aggregated with time of storage and precipitated from solution. Aggregation was accompanied by a corresponding loss of enzymatic activity even after dispersion of the aggregate by sonication. The deglycosylated mucins which were prepared by chemical treatment and periodate oxidation still contained about 20% of the N-acetylgalactosamine present in the intact mucin. When this residual amino sugar was removed by periodate oxidation the completely deglycosylated mucins became very poor substrates for the purified transferase. Data obtained in the current study indicate that the accessibility of serine and threonine in the polypeptide chains of mucin glycoproteins significantly influences the rate of glycosylation of these amino acids. The best substrates and affinity ligand for the enzyme were fragments of incompletely deglycosylated mucin polypeptide chains.
Mol Cell Biochem 1998 Aug
PMID:Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase from swine trachea epithelium. 974 19

Recent characterization of the human sequences encoding two lysosomal hydrolases, alpha-galactosidase A (alpha-Gal A) and alpha-N-acetylgalactosaminidase (alpha-GalNAc) revealed that these two enzymes with distinct enzymatic activities shared about 50% overall amino acid identity and that their genomic sequences had a conserved common gene structure. These findings suggested that these genes, which are located on different chromosomes, arose by duplication and divergence from a common ancestral gene. To further compare this alpha-galactosidase gene family, the murine alpha-GalNAc cDNA and genomic sequences were isolated and characterized. The full-length cDNA contained an open-reading frame of 1245 bp encoding a 415 amino acid polypeptide and had 5' and 3' untranslated regions of 94 and 333 bp, respectively. The coding region had 81% nucleotide and 81.9% amino acid identities with those of the corresponding human alpha-GalNAc sequence. Northern analysis revealed a single transcript of approximately 1.9 kb. The functional integrity of the cDNA was demonstrated by transient expression in COS-1 cells. The murine alpha-GalNAc genomic sequence spanned approximately 9 kb and was identical in structure with the human alpha-GalNAc gene with eight introns interrupting the coding sequence at identical positions. In addition, the deduced amino acid sequence of the murine alpha-GalNAc gene was highly homologous with alpha-GalNAc and alpha-Gal A genes from other species providing further support for a common evolutionary ancestor of the alpha-galactosidase gene family. The availability of the murine gene will permit additional evolutionary comparisons, structure/function analyses, and the generation of mice with alpha-GalNAc deficiency by gene targeting.
Mol Genet Metab 1998 Oct
PMID:Murine alpha-N-acetylgalactosaminidase: isolation and expression of a full-length cDNA and genomic organization: further evidence of an alpha-galactosidase gene family. 978 8

In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars.
Mol Cell Endocrinol 1998 Aug 25
PMID:Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor. 980 48

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.
Mol Microbiol 1999 Jan
PMID:Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N. 1002 64

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104. Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.
J Mol Biol 1999 Mar 05
PMID:Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus. 1004 89

Carbohydrates that terminate in beta-linked N-acetylgalactosamine (betaGalNAc) residues are concentrated in the postsynaptic apparatus of the skeletal neuromuscular junction and have been implicated in the differentiation of the postsynaptic membrane. We now report that distinct synapse-specific betaGalNAc-containing carbohydrates are associated with motor nerve terminals. Two monoclonal antibodies that recognize distinct betaGalNAc-containing epitopes, CT1 and CT2, both stain synaptic sites on skeletal muscle fibers. However, CT1 selectively stains nerve terminal, whereas CT2 selectively stains the postsynaptic apparatus. Likewise, CT1 and CT2 selectively stain motoneuron-like and muscle cell lines, respectively. Using the cell lines, we identify distinct CT1- and CT2-reactive glycolipids and glycoproteins. Finally, we show that GalNAc modulates the adhesion of motoneuron-like cells to recombinant fragments of a synaptic cleft component, laminin beta2. Together, these results show that pre- as well as postsynaptic membranes bear and are affected by distinct but related synapse-specific carbohydrates.
Mol Cell Neurosci 1999 Feb
PMID:Distinct structures and functions of related pre- and postsynaptic carbohydrates at the mammalian neuromuscular junction. 1019 69

Binding of the insecticidal Bacillus thuringiensis Cry1Ac toxin to the putative receptor aminopeptidase N is specifically inhibited by N-acetylgalactosamine (GalNAc), suggesting that this toxin recognises GalNAc on the receptor. A possible structural basis for involvement of domain III of the toxin in carbohydrate-mediated receptor recognition was noted in the similarity between the domain III fold of the related toxin Cry3A and a carbohydrate-binding domain in the 1,4-beta-glucanase from Cellulomonas fimi. This possibility was investigated by making selected mutations in domain III of the Cry1Ac delta-endotoxin. Mutagenesis of residues Asn506, Gln509 or Tyr513 resulted in toxins with reduced binding and a slower rate of pore formation in Manduca sexta midgut membrane vesicles compared to the wild-type Cry1Ac. These mutants also showed reduced binding to the 120 kDa Cry1Ac putative receptor aminopeptidase N. Unlike the wild-type toxin, binding of the triple mutant N506D,Q509E,Y513A (Tmut) to M. sexta midgut membrane vesicles could not be inhibited by GalNAc. These data indicate that GalNAc binding is located on domain III of Cry1Ac and therefore support a lectin-like role for this domain. A preliminary analysis of the Cry1Ac crystal structure locates Asn506, Gln509 and Tyr513 in a region on and adjacent to beta-16 in domain III, which has a unique conformation compared to the other known Cry structures. These residues are in a favourable position to interact with either soluble or protein-bound carbohydrate.
J Mol Biol 1999 Apr 16
PMID:N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin. 1022 7

The neoglycoproteins alpha-D-mannose-bovine serum albumin (mannose-BSA) and N-acetyl-alpha-D-glucosamine-BSA (glucNAc-BSA) were shown to rapidly increase intracellular free calcium ([Ca2+]i) in human spermatozoa. The increase in [Ca2+]i induced by these neoglycoproteins accounts for the known ability of these compounds to induce the acrosome reaction in human spermatozoa. Our data support the hypothesis that mannose-BSA, but not progesterone, activates T-type Ca2+ channels in human spermatozoa for the following reasons: (i) the capacity of mannose-BSA to increase [Ca2+]i was inhibited by the specific T-type Ca2+ channel blocker mibefradil (IC50 = 10(-6) mol/l) while progesterone was not inhibited by 10(-5) M mibefradil; (ii) the effect of mannose-BSA to elevate [Ca2+]i was inhibited more potently by Ni2+ (IC50 = 0.1 mmol/l) than Cd2+ (IC50 = 0.5 mmol/l), whereas the effect of progesterone to elevate [Ca2+]i was inhibited equally by Ni2+ and Cd2+ (IC50 = 0.25 mmol/l); (iii) the effects of mannose-BSA and progesterone to increase [Ca2+]i were greater than additive. These data support the idea that mannose-BSA and progesterone were activating distinct Ca2+ channels, one of which was a T-type Ca2+ channel activated by mannose-BSA whereas the Ca2+ channel that was activated by progesterone has yet to be defined at the molecular level.
Mol Hum Reprod 1999 Jun
PMID:The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa. 1034 Sep 95

A mistletoe lectin was isolated from water extracts of Korean mistletoe, a subspecies of Viscum album, grown on Quercus mongolica using CM-Sepharose chromatography followed by an affinity chromatography on a concanavalin A-Sepharose column. The compound proved to be a mistletoe lectin II with D-galactose and N-acetyl-D-galactosamine specificity. Matrix-assisted laser desorption time-of-flight mass spectroscopy showed it to have an average molecular mass of 62.7 kDa and to consist of two subunits of 30.6 kDa and 32.5 kDa. It was a basic protein with isoelectric points of 9.4 and 9.6 by capillary isoelectric focusing and was cytotoxic to Molt4 cell.
Cell Mol Life Sci 1999 Apr
PMID:Isolation and characterization of biologically active lectin from Korean mistletoe, Viscum album var. Coloratum. 1035 36


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