Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.
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PMID:Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues. 167 8

In an endeavor to further characterize human intercellular adhesion molecule-2 (ICAM-2), two murine monoclonal antibodies (mAb) were generated to ICAM-2 transfected COS cells, and designated CBR-IC2/1 and CBR-IC2/2. Immunoprecipitated, reduced ICAM-2 migrated as a broad band of Mr 60,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with N-glycanase revealed a peptide backbone of Mr 31,000, consistent with the size predicted from the cDNA. ICAM-2 had a broad distribution on hematopoietic cell lines and little expression on other cell lines, the sole exception being cultured endothelial cells which possess high levels of ICAM-2. Resting lymphocytes and monocytes expressed ICAM-2, while neutrophils did not. Staining of tissue sections with anti-ICAM-2 mAb confirmed their strong reactivity to vascular endothelium, but demonstrated a lack of ICAM-2 expression on other tissues. Small clusters of ICAM-2 positive cells were, however, seen in germinal centers. In contrast to ICAM-1 there was little or no induction of ICAM-2 expression on lymphocytes or cultured endothelium upon stimulation with inflammatory mediators. One of the two mAb, CBR-IC2/2, was found to totally inhibit binding of ICAM-2+ COS cells to purified lymphocyte function-associated antigen-1 (LFA-1). Using this mAb, LFA-1-dependent binding to both stimulated and unstimulated endothelium was found to be totally accounted for by ICAM-1 and ICAM-2. Homotypic aggregation of an Epstein-Barr virus-transformed B cell line, JY, was found to be solely ICAM-1 and ICAM-2-dependent, while in the case of the T cell lymphoma cell line, SKW3, anti- ICAM-2 mAb in conjunction with anti-ICAM-1 mAb could not inhibit the LFA-1-dependent aggregation. This suggests an additional LFA-1 ligand exists. Using a cell binding assay to purified LFA-1 in conjunction with anti-ICAM-1 and anti-ICAM-2 mAb, we have demonstrated that this putative third ligand for LFA-1 exists on SKW3 and other cell lines.
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PMID:Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1. 167 48

Apparently conflicting results have been reported regarding the role of env glycoprotein glycans in human immunodeficiency virus type 1 (HIV-1) infectivity and cytopathogenicity. Whereas we have shown that enzymic removal of carbohydrates from mature envelope glycoproteins has only limited effect on the ability of HIV-1 to bind to CD4 and to infect target cells, sugar analogues that interfere with the glycosylation process of the nascent molecule markedly reduce virus infectivity. Here we have investigated the effect of a glucosidase inhibitor, 1-deoxynojirimycin (dNM), on the bioactivity and immunoreactivity of precursor gp160 produced by recombinant vaccinia virus-infected BHK-21 cells (rgp160). dNM (4 mM) did not affect the amount of rgp160 recovered nor its secretion from the cells. As described by other authors the effect of dNM was incomplete, resulting in the production of rgp160, the glycosylation of which was heterogeneous with respect to apparent Mr distribution and to sensitivity to endoglycosidase H and endoglycosidase F, all the species being susceptible to N-glycanase. A major reduction of the binding to CD4+ cells was noted with rgp 160 produced by dNM-treated cells using a quantitative indirect immunofluorescence assay and labelling with polyclonal human anti-HIV IgG. Similarly, dNM treatment altered the accessibility to murine monoclonal antibody 110-4 of the exposed V3 loop of HIV-1 gp120 by at least 10-fold, as determined by either ELISA capture assay or immunoaffinity purification. Such bioactivity and conformation modifications, which result from the abnormal folding of the nascent glycoprotein due to aberrant glycosylation, may account for the impaired HIV-1 infectivity elicited by dNM.
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PMID:Effect of a glucosidase inhibitor on the bioactivity and immunoreactivity of human immunodeficiency virus type 1 envelope glycoprotein. 167 78

Female BALB/c mice were immunized with a whole-hyphal-cell extract obtained from Candida albicans wild-type strain 4918 grown in Lee medium. Monoclonal antibody (MAb)-producing hybridomas were prepared by fusing immune splenocytes with NS-1 myeloma cells. One of the hybrid cell clones (1.183) secreted an immunoglobulin G1 antibody that reacted with C. albicans hyphae in an indirect immunofluorescence assay but not with yeast cells and pseudohyphal segments directly originating from parent blastoconidia. In the same assay eight of nine recent clinical C. albicans isolates and Candida stellatoidea tested positive for hyphal cell-specific reactivity with MAb 1.183. The recognized antigen on hyphal cells was sensitive to heat treatment, beta-mercaptoethanol reduction, and proteolysis with pronase, trypsin, and subtilisin. Western blot (immunoblot) analysis of hyphal whole-cell and dithiothreitol extracts with MAb 1.183 revealed two major proteins with approximate molecular masses of 55 and 60 kilodaltons (kDa) under reducing conditions. Endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment reduced the molecular mass of the 60-kDa protein slightly but did not affect recognition by MAb 1.183, whereas peptide:N-glycosidase F (N-glycanase) had no effect on either protein. When exponentially growing yeast cells were treated sequentially with EDTA, beta-mercaptoethanol, and Zymolase to form protoplasts, a specific immunofluorescence signal was obtained with MAb 1.183. In a Western blot, MAb 1.183 showed reactivity with a 20-kDa protein in the sodium dodecyl sulfate extract from protoplasts, whereas no reactivity was found with cell wall material obtained from yeast cells. In summary, these experiments indicated that specific cell surface components from C. albicans hyphae are related to antigens which are present in yeast cells but are not detectable on the surface of the latter.
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PMID:A monoclonal antibody that defines a surface antigen on Candida albicans hyphae cross-reacts with yeast cell protoplasts. 168 99

Immunochemical methods have been used to detect and characterize two classes of polypeptide-associated keratan sulphate (KS) in epithelial secretions from human endometrium. Monoclonal antibody D9B1 binds to a hormonally regulated sialylated epitope associated with KS in a high relative molecular mass (250,000-350,000) component that bands as a doublet in SDS/PAGE. These KS chain(s) are sensitive to keratanase, endo-beta-galactosidase and N-glycanase. A second, more highly sulphated, type of KS is also present, that is resistant to all three enzymes. This can be detected using monoclonal antibody 5D4. It is present throughout the menstrual cycle and is associated principally with a component of Mr 140,000. Thus secretory KS contributes to the environment of the implanting embryo, may be used as a molecular index of endometrial function and could be important in the establishment of pregnancy.
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PMID:Menstrual-cycle-dependent expression of keratan sulphate in human endometrium. 169 31

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.
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PMID:Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells. 169 20

A hybridoma (3B2-F7) has been established which secretes a monoclonal antibody (Mab) directed against a peptide determinant of human seminal plasma glycoprotein (HSP-gP). The deglycosylation of HSP-gP was performed chemically with TFMS hydrolysis and enzymatically in the presence of detergent and further treated with periodic acid after fixing deglycosylated HSP on plastic wells. The Mab 3B2-F7 (IgM, kappa) exhibited sperm immobilization activity (256 units of SI50) and inhibited sperm binding to human zona pellucida. Human epididymis, pancreatic islets of Langerhan's and distal tubulus of kidney were strongly labelled whilst other tissues were essentially negative by avidin-biotin complex tissue staining with this Mab. The antigen epitope to the Mab was in the 36 kDa molecule of human HSP-gP. The antigenic determinant recognized by Mab 3B2-F7 was destroyed by six different proteases, but was resistant to N-glycanase and other carbohydrate splitting enzymes. This epitope is therefore likely to be composed of a polypeptide chain. Peptide fragments after proteolysis of the HSP molecule with Staph. aureus V8 protease and trypsin retained antigenicity, hence the epitope corresponding to the Mab may be a peptide chain and not dependent on the conformational structure of the polypeptide.
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PMID:Monoclonal antibody recognizing an apparent peptide epitope of human seminal plasma glycoprotein and exhibiting sperm immobilizing activity. 169 87

We studied the oligosaccharide moieties of recombinant human thyroid peroxidase (hTPO) expressed in Chinese hamster ovary (CHO) cells, and the role of these glycans in hTPO antigenicity in Hashimoto's thyroiditis. To determine whether hTPO carbohydrate moieties were N-linked, O-linked, or both, and to obtain information about the characteristics of the carbohydrate component(s), we digested hTPO with deglycosylating enzymes of varying specificity. Proteins in CHO-TPO cells were labeled with [35S]methionine, and hTPO was immunoprecipitated with anti-hTPO antibodies present in Hashimoto's thyroiditis serum. Digestion with endoglycosidase (endo) F, which removes both complex and polymannose N-linked glycans, increased the electrophoretic mobility of the hTPO doublet from approximately 115 kD and 110 kD to 110 kD and 105 kD. Endo H, which acts similarly to endo F, but only on polymannose, and not complex, glycans, had a similar effect. In contrast, O-glycanase and neuraminidase, which remove O-linked glycans and terminal neuraminic acid, respectively, did not alter the mobility of radiolabeled hTPO. Radiolabeled recombinant hTPO was retained by concanavalin A, but not by wheat germ agglutinin, Ricinus communis agglutinin 1, peanut agglutinin and Ulex europaeus lectins. To determine whether or not the glycan moieties in hTPO play a role in the disease-associated epitopes in Hashimoto's thyroiditis, radiolabeled recombinant hTPO was immunoprecipitated after digestion with N-glycanase. Removal of the N-linked carbohydrate chains with endo F and endo H did not prevent antibody binding. In summary, the present data indicate that: i) hTPO expressed in CHO cells contains N-linked, but not O-linked glycan moieties; ii) the N-linked carbohydrate is primarily of the polymannose variety; and, iii) the glycan moieties do not contribute to the hTPO epitopes in Hashimoto's thyroiditis.
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PMID:Carbohydrate moieties in recombinant human thyroid peroxidase: role in recognition by antithyroid peroxidase antibodies in Hashimoto's thyroiditis. 169 64

We have isolated four insulin-like growth factor binding proteins (IGFBPs) from adult human serum by insulin-like growth factor (IGF) I affinity chromatography and high performance liquid chromatography. A 36-kDa binding protein (BP), not digestible with N-glycanase, is increased in patients with extrapancreatic tumor hypoglycemia and during IGF I administration in healthy adults. Its 38 NH2-terminal amino acids are identical to those of an IGFBP sequence derived from a human cDNA that cross-hybridizes with the rat IGFBP-2 cDNA. With probes encoding a NH2-terminal, COOH-terminal, and a middle region of this protein we have obtained three cDNA clones from a Hep G2 cDNA library; one encodes human IGFBP-2, and the other two presumably represent unspliced heteronuclear and alternatively spliced mRNA, respectively. A 28-30-kDa IGFBP represents a novel BP species in human serum. Its 30 NH2-terminal amino acids are not homologous to IGFBP-1, -2, or -3. It is not digestible with N-glycanase and does not bind 125I-IGF I. The NH2-terminal sequences of a 42/45- and a 31-kDa IGFBP are identical to that of human IGFBP-3. The 42/45-kDa proteins are two glycosylation variants of BP-3. The 31-kDa protein presumably is a degradation product of BP-3 that lacks the COOH terminus. It is likely that the different IGFBPs modulate auto-/paracrine and endocrine effects of IGFs on growth and metabolism in a different and specific manner.
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PMID:Isolation from adult human serum of four insulin-like growth factor (IGF) binding proteins and molecular cloning of one of them that is increased by IGF I administration and in extrapancreatic tumor hypoglycemia. 169 83

The biochemical and immunochemical characteristics of T. spiralis molecules (group II antigens) sharing an immunodominant epitope were examined. Six major proteins, ranging from 43-68 kDa, and from pI 5.0-6.3, express the determinant. Together, they account for at least 3% by weight of the total protein in L1 larval homogenate. The antigens are glycosylated. Following periodate oxidation, they reacted with biotin aminocaproyl hydrazide, and treatment with trifluoromethanesulfonic acid decreased their Mr. Deglycosylated group II antigens lost immunoreactivity with a monoclonal antibody specific for the determinant, and oligosaccharides released by treatment with mild base blocked binding of the monoclonal antibody to native antigens. The determinant on one of the group II antigens (43 kDa) was removed by N-glycanase. Neither phosphorylcholine nor antibody to phosphorylcholine interfered with monoclonal antibody binding to native group II antigens. Together, these results suggest that the immunodominant group II antigen epitope is associated with N- and O-linked oligosaccharides, and that it is not phosphorylcholine.
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PMID:Characterization of Trichinella spiralis antigens sharing an immunodominant, carbohydrate-associated determinant distinct from phosphorylcholine. 169 36


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