Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The size of a lysosomal cysteine proteinase from epimastigotes of Trypanosoma cruzi decreased from 60 to 54 kDa upon treatment with endo beta-N-acetylglucosaminidase H. A lower-molecular weight component (30-35 kDa), which usually accompanies the 60-kDa protein also increased its electrophoretic mobility, and seems to consist of a mixture of degradation products of the enzyme, since both the larger and the smaller components had the same N-terminal sequence as the 60-kDa protein. The amino acid composition of the protein moiety and the composition of the oligosaccharide chains have been determined. The oligosaccharide chains are of the high-mannose type, and contain 6, 7, 8 or 9 mannose residues, as shown both by in vivo labeling with [U-14C]glucose, and by labeling the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of the purified enzyme with tritiated sodium borohydride. The oligosaccharide chains did not contain phosphate residues. Further studies with [U-14C]-labeled total glycoproteins of T. cruzi and enzyme assays, suggest that T. cruzi and other trypanosomatids do not target their lysosomal enzymes to the organelle through the mannose-6-phosphate marker pathway.
Mol Biochem Parasitol 1990 Jan 01
PMID:Amino acid and carbohydrate composition of a lysosomal cysteine proteinase from Trypanosoma cruzi. Absence of phosphorylated mannose residues. 218 4

Cholecystokinin (CCK) receptors reside on a large number of cell types along the digestive tract and in the nervous system. A human neuroblastoma cell line (CHP212) has recently been described to express a type A receptor, with structural specificity similar to that on pancreatic acinar cells and gall bladder smooth muscle cells but different from the predominant type of binding site found in brain (type B). In this work, we have performed photoaffinity labeling and protease peptide mapping of the CHP212 receptor and have compared it to other type A CCK receptors. 125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)-CCK-26-33], a probe that possesses a photolabile residue at position 33 within the theoretical receptor-binding domain of this hormone, specifically labeled a Mr = 80,000-90,000 glycoprotein on this cell line, while labeling larger proteins (Mr = 85,000-95,000) on rat pancreas and human gall bladder. Deglycosylation with endo-beta-N-acetylglucosaminidase F yielded bands of Mr = 43,000 from CHP212 and gall bladder and Mr = 42,000 from pancreas. Peptide mapping of the deglycosylated bands using Staphylococcus aureus V8 protease demonstrated identical patterns in CHP212 and gall bladder and a similar but different pattern in pancreas. Thus, although possessing heterogeneity in their carbohydrate domains, CCK receptors on human neuroblastoma cells (CHP212) and human gall bladder smooth muscle cells have highly similar or identical protein cores. The core protein on another type A CCK receptor, from rat pancreas, appears to differ from these, likely representing molecular heterogeneity between species.
Mol Pharmacol 1990 Aug
PMID:Biochemical characterization of the cholecystokinin receptor on CHP212 human neuroblastoma cells. 220 Sep 53

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.
Mol Cell Biol 1990 Sep
PMID:The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line. 220 96

Two Trypanosoma congolense stocks, 1/148 FLY and TREU 921, were cloned in A/J strain mice immunosuppressed with cyclophosphamide. The cloned populations, AmNat 1.1 and AmNat 3.1, each characterized by a different variant antigen type, were checked for homogeneity by the indirect fluorescent antibody test using 6-day antisera developed in rabbits. The variant surface glycoproteins (VSGs) from both AmNat clones were purified to homogeneity. Electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels revealed that the apparent Mr values of the two VSGs were 51 700 (AmNat 1.1) and 49 900 (AmNat 3.1). Monospecific antisera prepared in rabbits to each VSG were used to confirm the homogeneity of the clones by the indirect fluorescent antibody test. The VSGs were susceptible to endoglycosidase H digestion, indicating the presence of high-mannose type oligosaccharides in these glycoproteins. The apparent Mr values of the endoglycosidase H-digested VSGs were 48 800 and 46 900 for AmNat 1.1 and 3.1, respectively. Poly(A+)-enriched RNA isolated from each clone was assayed for template activity using a mRNA-dependent rabbit reticulocyte lysate for in vitro protein synthesis. Radioactively labeled polypeptides were initially characterized by SDS-polyacrylamide gradient gel electrophoresis and visualized by fluorography. VSG-specific translation products were immunoprecipitated with IgGs isolated from the homologous monospecific antisera and analyzed on SDS-polyacrylamide gradient gels. The apparent Mr values for the AmNat 1.1 and 3.1 precursor VSGs synthesized in vitro were 39 000 and 43 000, respectively.
Mol Biochem Parasitol 1985 Jun
PMID:Isolation and cell-free synthesis of variant surface glycoproteins from Trypanosoma congolense. 241 15

Treatment of Mo-MuLV-infected cells with cytochalasin B (CB), a microfilament disrupting drug, caused a reduction in virus yield as judged by infectivity assay and reverse transcriptase activity. Pulse-chase experiments with [3H]leucine showed that the env precursor, gPr80env, was inefficiently processed in cells treated with CB. In the presence of monensin, an inhibitor of glycoprotein transport, gPr80env accumulated intracellularly and no gp70 was observed on the cell surface, indicating a complete block in the processing of gPr80env. Pulse-chase studies also showed that gPr80env was not processed in the presence of monensin. SDS-PAGE analysis of TX-100-extracted cell cytoskeletons (TX-insoluble fraction) iodinated and immunoprecipitated with goat anti-gp70 antiserum showed that CB or monensin treatment caused a marked increase of gPr80env in the cytoskeleton-rich fraction. However, the amount of gPr80env associated with the TX-soluble fraction in both CB or monensin-treated and untreated cells labeled with [3H]leucine was about the same. The gPr80env in the TX-100-soluble fraction of the cell was the endoglycosidase H (Endo-H) sensitive mannose-rich form, whereas the cytoskeleton-associated gPr80env was the partially Endo-H-resistant complex carbohydrate form. In the presence of CB or monensin, the complex carbohydrate form of gPr80env accumulated in the cytoskeleton-rich cell fraction. Examination of Mo-MuLV ts1 mutant, which is defective in the processing of env precursor polyprotein, also revealed an accumulation of the complex carbohydrate form of gPr80env in the cytoskeleton-rich fraction and an absence of gp70 on the surface of the cell at the restrictive temperature (39 degrees C). These studies suggest that the cytoskeleton plays a role in the transport and processing of MuLV gPr80env and that oligosaccharide conversion is an important factor in this process. Further, the accumulation of gPr80env on the cytoskeleton of ts1 infected cells at restrictive temperature may play a role in the neurological disorder caused by Mo-MuLV ts1 mutant.
Exp Mol Pathol 1987 Jun
PMID:Role of cell cytoskeleton in Mo-MuLV env transport and processing: implications in ts1 neuropathology. 243 68

The proto-oncogene c-kit encodes a transmembrane kinase which is related to the receptors for colony-stimulating factor type 1 and platelet-derived growth factor, as well as to the immunoglobulin superfamily. Antibodies specific for the kinase domain of the P80 gag-kit protein of the Hardy-Zuckerman 4 feline sarcoma virus were prepared. These kit-specific antibodies were used to identify and characterize the c-kit protein in cat brain tissue. The c-kit protein product displays an autophosphorylating activity in immune complex kinase assays, and, in turn, this activity was used to identify the c-kit protein in different tissues. In cat brain, a single 145-kilodalton (kDa) glycoprotein was detected. Its N-linked carbohydrates were found to be sensitive to digestion with the endoglycosidases (neuraminidase, endoglycosidase F, and endoglycosidase H), indicating hybrid and/or complex and high-mannose structures. A partial purification of the c-kit protein was achieved by wheat germ agglutinin affinity chromatography, and the autophosphorylating activity of the partially purified c-kit protein was characterized and found to be specific for tyrosine. The kit antibodies cross-react with the murine c-kit protein product, and variant c-kit proteins in different mouse tissues were identified, with sizes of about 145 kDa (brain), 160 kDa (spleen), and 150 kDa (testis).
Mol Cell Biol 1988 Nov
PMID:c-kit protein, a transmembrane kinase: identification in tissues and characterization. 246 68

The structural characteristics and glycosylation properties of the lactogenic receptor were examined in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized plasma membranes from female mouse liver. The specific binding of the radioiodinated human growth hormone [( 125I]hGH) was displaced with an equivalent potency by both hGH and prolactin. After a mild neuraminidase treatment, this binding was increased by 40%, as a result of an increase in receptor affinity. Affinity chromatography on immobilized lectins revealed that the [125I]hGH-receptor complexes were specifically retained and eluted from ricin lectin-agarose, concanavalin A and lentil lectin, indicating the presence of N-linked glycans. Covalent cross-linking of solubilized [125I]hGH-receptor complexes with disuccinimidyl suberate, followed by analysis by sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) under reducing conditions, and autoradiography resulted in the appearance of two bands with apparent Mr approximately 62,000 and approximately 100,000. The labelling of these bands was prevented by unlabelled hGH or ovine prolactin (oPrl) but not by bovine growth hormone (bGH). Neuraminidase treatment of the two receptor forms resulted in increased electrophoretic mobility which was inhibited by simultaneous addition of sialyl-lactose, a neuraminidase substrate. The both cross-linked forms were unaffected by endoglycosidase H, while endoglycosidase F decreased the molecular weight of each of the forms by about 8000 Da, yielding bands at Mr approximately 54,000 and approximately 92,000. In conclusion, taking into account that hGH is a Mr 22,000 polypeptide, the two forms of the receptor correspond to glycoproteins of Mr approximately 40,000 and approximately 78,000, respectively. They contain polypeptide backbones of Mr approximately 32,000 and approximately 70,000, and complex N-linked oligosaccharide chains with terminal sialic acid residues which could be involved in receptor binding affinity.
Mol Cell Endocrinol 1989 Aug
PMID:Glycosylation characteristics of the mouse liver lactogenic receptor. 250 88

A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol-oligosaccharides might alter the degree of beta-1,6 branching in N-linked carbohydrates are discussed.
Mol Cell Biol 1989 Mar
PMID:Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis. 272 6

The 43 kDa human chorionic gonadotropin (hCG) (SP-hCG) was purified from human placenta and analyzed for sugar moieties. The low hexosamine content suggests that SP-hCG probably lacks O-linked sugar chains in the beta-subunit and incompletely formed N-linked sugar chains in the alpha- and beta-subunits. In the present study SP-hCG was hydrolyzed with various glycosidases. Treatment of hCG or SP-hCG with O-glycan peptide hydrolase increased the mobility of asialo-hCG beta in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) while that of SP-hCG beta was unaffected, indicating that SP-hCG beta does not contain NeuNAc-Gal-GalNAc unit. Alpha-Mannosidase and endoglycosidase H hydrolyzed mannose and the high mannose-GlcNAc moieties, respectively, from alpha- and beta-subunits of SP-hCG, but not from the subunits of authentic hCG. Glycopeptidase F hydrolyzed completely the N-linked sugar chains from SP-hCG subunits, producing alpha- and beta-subunits with estimated Mr of 15,000 and 18,500, respectively. The biological activity of purified SP-hCG is about 50-80% of highly purified authentic hCG. In an in vitro system SP-hCG increased cAMP accumulation and testosterone production by rat Leydig cells to the same levels as that induced by hCG. However, the biological activity of SP-hCG was markedly reduced, following treatment with endoglycosidase H or alpha-mannosidase. To attain the level of testosterone production equivalent to that induced with untreated SP-hCG, 10-20 times higher dose of treated SP-hCG was required. On the other hand, cAMP accumulation induced with treated SP-hCG even at a very high concentration was substantially lower than that attained with untreated SP-hCG. In conclusion, the mannose moieties are essential structural components of the hormone in stimulating cAMP accumulation and steroidogenesis by rat Leydig cells.
Mol Cell Endocrinol 1989 Mar
PMID:Carbohydrate moieties of small placental hCG: requirement of mannose structure for biological activity. 274 19

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment.
Mol Endocrinol 1987 Aug
PMID:Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary. 285 9


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