Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).
Mol Microbiol 1990 Feb
PMID:Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae. 214 Jan 48

Lymph node (LN) T cells from autoimmune MRL/MpJ-lpr/lpr (lpr) mice and control MRL/MpJ-(+)/+ (+/+) mice were compared as to glycosyl transferase activities involved in the biosynthesis of polylactosaminoglycans. The N-acetylglucosaminyl transferase (GlcNAc transferase) activity responsible for the extension of polylactosaminoglycans was assayed. The reaction products with this GlcNAc transferase were characterized by sequential glycosidase treatment and methylation analysis, and the enzyme was found to be classifiable as an UDP-GlcNAc:N-acetyllactosamine beta 1-3 GlcNAc transferase (polylactosamine extension enzyme). The activity of this GlcNAc transferase in T cells from enlarged LN of lpr mice was 3-6 times higher than that in T cells from +/+ mice. On the other hand, activated T cells from +/+ mice only showed about a 2-fold increase in the activity of the transferase, compared with that in resting T cells. B cells from +/+ mice also showed a significantly higher activity of the transferase than +/+ T cells, the enzyme activity being comparable to or slightly lower than that in lpr T cells. Furthermore, when the reaction mixture contained both UDP-GlcNAc and UDP-Gal as donors, extension of the Gal-GlcNAc residue was observed. These results indicated the biosynthetic basis for the abundance of polylactosaminoglycans in lpr T cells and normal B cells. We also found that lpr T cells exhibited significant UDP-GlcNAc:asialo-bovine submaxillary mucin GlcNAc transferase activity. Only weak activity of this enzyme was detected in +/+ resting and activated T cells, and B cells. This enzyme activity suggested the potential for polylactosaminoglycan formation on the mucin-type sugar chains on the surface of lpr T cells.
Mol Immunol 1990 Apr
PMID:Elevation of the activities of glycosyl transferases involved in polylactosaminoglycan biosynthesis in autoimmune MRL lpr/lpr mouse T cells. 214 66

3'-Azido-2',3'-dideoxyuridine (AzdU, CS-87) is a potent inhibitor of human immunodeficiency virus replication in human peripheral blood mononuclear cells (PBMC) with limited toxicity for human bone marrow cells (BMC). In the present study, metabolism of AzdU was investigated in human PBMC and BMC after exposure of cells to 2 or 10 microM [3H]AzdU. 3'-Azido-2',3'-dideoxyuridine-5'-monophosphate (AzdU-MP) was the predominant metabolite, representing approximately 55 to 65% of intracellular radioactivity in both PBMC and BMC at all times. The AzdU-5'-diphosphate and -5'-triphosphate intracellular levels were 10- to 100-fold lower than the AzdU-MP levels and, of note, AzdU-5'-triphosphate was not detected in human BMC. Using anion exchange chromatography, a new peak of radioactivity, distinct from any known anabolites, was detected. This chromatographic peak was found to be resistant to alkaline phosphatase but was hydrolyzed by 5'-phosphodiesterase, yielding AzdU-MP. Incubation of [3H]AzdU and D-[1-14C]glucose in PBMC and BMC produced a double-labeled peak with the same retention time as the anabolite, suggesting formation of a hexose derivative of AzdU. A novel high performance liquid chromatography method was developed that allowed for the separation of nucleosides, nucleotides, and carbohydrate derivatives thereof. Using this highly specific method, the putative AzdU-hexose actually was separated into two chromatographic peaks. These novel metabolites were identified as 3'-azido-2',3'-dideoxyuridine-5'-O-diphosphoglucose and 3'-azido-2',3'-dideoxyuridine-5'-O-diphospho-N-acetylglucosamine. Following 48 hr of incubation with [3H] AzdU, as much as 20 and 30% of these AzdU metabolites accumulated in PBMC and BMC, respectively. When AzdU was removed from the cell cultures, intracellular AzdU diphosphohexose concentrations decayed in a monophasic manner, with an elimination half-life of 14.3 hr. By 48 hr, levels of 0.3 pmol/10(6) cells were still detected, reflecting a gradual anabolism of these metabolites. Elimination of AzdU-MP and AzdU-5'-diphosphate was characterized by a two-phase process, with a short initial half-life of 0.83 and 0.24 hr and a long terminal half-life of 14.10 and 8.24 hr, respectively. Similar diphosphohexoses of deoxyuridine (dUrd) were also detected in human PBMC and BMC after exposure to [3H]dUrd, suggesting that dUrd derivatives are metabolized in a similar manner. In summary, the discovery of novel metabolic pathways for dUrd analogs demonstrates that AzdU has unique metabolic features that may contribute to the low toxicity of this anti-HIV agent in human BMC and also affect its mechanism of action.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1990 Dec
PMID:Cellular metabolism of 3'-azido-2',3'-dideoxyuridine with formation of 5'-O-diphosphohexose derivatives by previously unrecognized metabolic pathways for 2'-deoxyuridine analogs. 225 Jun 66

Dolichol-linked oligosaccharides consisting of two N-acetylglucosamine, nine mannose, and three glucose residues (Glc3Man9GlcNAc2) are transferred to proteins that contain the consensus sequence Asn-X-Ser/Thr. This transfer occurs upon protein import into the lumen of the endoplasmic reticulum. An intermediate in the biosynthesis of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide contains two GlcNAc and five mannose residues. This intermediate serves as a substrate for further mannosylation and glucosylation before transfer to protein. The addition of the sixth mannose residue to this intermediate requires the enzyme mannosyltransferase VI and the mannose donor, mannose-P-dolichol. Several different CHO cell line mutants that fail to efficiently catalyze this transfer have been described. In this report, we examine seven independent mutant cell lines with various biochemical phenotypes and demonstrate that all can be assigned to one of three genetic complementation groups. One mutation affects mannose-P-dolichol biosynthesis (Lec15), three affect dolichol phosphate biosynthesis (Lec9), and three appear to affect the functional orientation of enzyme substrates (PIR).
Somat Cell Mol Genet 1990 Nov
PMID:Complementing mutant alleles define three loci involved in mannosylation of Man5-GlcNAc2-P-P-dolichol in Chinese hamster ovary cells. 226 28

Chicken heterophile antigenic determinant (CHAD-1) has been previously found in medullary lymphocytes of the bursa and thymus as well as in some non-lymphoid cells by the immunoperoxidase method, using rabbit antiserum to a complete Freund's adjuvant (CFA) as the first antibody. In this work we demonstrated that absorption of anti-CFA serum with highly purified preparations of hen egg white glycoproteins (ovomucoid, ovoinhibitor, ovalbumin) or chicken orosomucoid completely blocked immunoperoxidase staining for CHAD-1. Treatment of these glycoproteins with beta-N-acetylglucosaminidase suppressed their capacity to inhibit this staining. Absorption of anti-CFA serum with asparagine-linked glycopeptides which have the mannose alpha 1,3 arm disubstituted by GlcNAc residues and which have another GlcNAc residue linked beta 1,4 to the beta-linked mannose of the core also inhibited staining for CHAD-1. These data indicated that highly branched asparagine-linked oligosaccharides with terminal GlcNAc residues beta-linked to mannose represent immunoreactive domains of CHAD-1.
Mol Immunol 1987 Jul
PMID:Identification of terminal N-acetylglucosamine residues of highly branched asparagine-linked oligosaccharides as immunoreactive domains of a chicken heterophile antigenic determinant. 244 43

We had previously shown that the human colon produces at least two immunochemically distinct mucins, one neutral and the other a sialomucin [Gold et al. J. biol. Chem. 256, 6354-6358 (1981)]. In addition, the sialomucin was shown to contain an immunodeterminant restricted to colonic epithelium and may thus prove useful as a tissue-specific marker. In the current study we have shown that a specific linkage of sialic acid to the oligosaccharide backbone has a major role in the organ-specific immunodeterminant structure. Treatment of intact colonic mucin with sialidase (Cl. perfringens) cleaved 20-80% of the sialic acid as measured colorimetrically. Immunoreactivity was decreased by 0-42% with respect to the untreated material. Saponification (0.1 N KOH, 20 min at room temp) caused an approximate 90% decrease in immunoreactivity for each mucin. Subsequent to saponification, neuraminidase cleaved most of the sialic acid from the mucins. The majority of sialic acid was observed to be O-acetylated, thus making it sialidase-insensitive. Gas chromatography-mass spectrometric analyses of the trimethylsilyl sialic acid derivatives indicated the presence of NeuNAc; NeuNAc, 9-OAc; and NeuNAc, 7,9 diOAc as the major sialyl derivatives. The radioimmunoassay data appeared to indicate that O-acetylated sialic acid was necessary for immunoreactivity. It should be noted that jejunal mucin and bovine submaxillary mucin also contain O-acetylated sialic acid, but did not inhibit in our radioimmunoassay. This may have been due to differences in the O-acetylation pattern or the linkage of sialic acid to the core carbohydrate. Analyses of the partially methylated alditol acetate derivatives by gas chromatography-mass spectrometry of the untreated, as well as the saponified and neuraminidase treated, mucins revealed that sialic acid was attached to the carbohydrate core either to galactose, N-acetylglucosamine, and/or N-acetylgalactosamine. Linear regression analyses comparing immunoreactivity with specific epitope concns, in conjunction with RIA analyses of known structures, suggested that the organ-specific immunodeterminant was (or was dependent upon the presence of) the structure GlcNAc (1,3)[O-acetylated Neu5Ac(2,6)] GalNAc.
Mol Immunol 1989 Aug
PMID:Studies on the structure of the organ-specific determinant of human colonic mucin. 247 76

LEC10 is a dominant glycosylation mutant of Chinese hamster ovary (CHO) cells that expresses a developmentally regulated glycosyltransferase (GlcNAc-TIII) not detectable in parental CHO cells. Several mutagens were found to increase the frequency of LEC10 mutants up to 10-fold over the spontaneous frequency of less than or equal to 10(-7), while 5azaC treatment had no effect. Revertants were obtained at high frequency (approximately 10(-4)) and were found to belong to two classes. Three independent revertants gave rise to new LEC10 mutants at high frequency (approximately 10(-4)) while seven others gave new LEC10 mutants at the low frequency typical of unmutagenized parental CHO cells. No evidence of a general mutator phenotype was found in the revertant lines with a high rereversion frequency. The combined data suggest a novel form of genomic instability at the LEC10 locus in CHO cells. Genetic events that affect the expression of developmentally regulated glycosyltransferases may be identified by further studies of LEC10 and other dominant CHO glycosylation mutants.
Somat Cell Mol Genet 1989 Sep
PMID:Novel genetic instability associated with a developmentally regulated glycosyltransferase locus in Chinese hamster ovary cells. 252 29

Chinese hamster ovary (CHO) glycosylation mutants provide an approach to cloning mammalian glycosyltransferases by transfection and gene rescue. In this paper, complementation of the lec1 CHO mutation by human DNA is described. Lec1 transfectants expressed human N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and possessed common human DNA fragments. Cloning of GlcNAc-TI should therefore be possible.
Mol Cell Biol 1989 Dec
PMID:Transfection of a human gene that corrects the Lec1 glycosylation defect: evidence for transfer of the structural gene for N-acetylglucosaminyltransferase I. 253 Dec 85

Low density lipoprotein (LDL) is a spherical particle with a diameter of 22 nm. It consists of an apolipoprotein and a lipid moiety, in which a variety of lipophilic drugs and prodrugs can be incorporated. In the present study, lactose was coupled to the apolipoprotein of LDL by reductive amination (398 +/- 40 residues/LDL particle). After injection into rats, radioactively labeled lactosylated LDL was cleared rapidly from the plasma (half-life, less than 2 min). Ten minutes after injection, the liver contained about 90% of the dose, whereas only small amounts of radioactivity were found in other tissues. Preinjection of N-acetylgalactosamine completely blocked liver uptake, whereas N-acetylglucosamine was ineffective. This indicates that the hepatic recognition site is galactose specific. Subcellular fractionation of liver indicated that the recognition of lactosylated LDL is followed by internalization and degradation of the apolipoprotein in the lysosomes. In the liver, Kupffer cells are mainly responsible for uptake. At 10 min after injection, these cells contained a 70 and 7 times higher amount of lactosylated LDL per mg of cell protein than parenchymal and endothelial cells, respectively. After galactose-specific uptake in parenchymal cells was blocked with asialofetuin, the relative concentration in Kupffer cells was even higher. The hepatic uptake of the lipid moiety of lactosylated LDL, labeled with [3H]cholesteryl oleoyl ether, was identical to that of the 125I-labeled apolipoporotein, which indicates that the particle is taken up as a unit. Thus, lactosylated LDL is taken up rapidly and selectively by Kupffer cells, and it appears that it might be a very effective vehicle for the specific delivery of lipophilic drugs, e.g., immunomodulators, to these cells.
Mol Pharmacol 1989 Sep
PMID:Lactosylated low density lipoprotein: a potential carrier for the site-specific delivery of drugs to Kupffer cells. 255 Jul 81

Human chorionic gonadotropin (hCG) behaves as an antagonist upon chemical deglycosylation with hydrogen fluoride. In this study it was found that the alpha- and beta-subunits of deglycosylated hCG (DGhCG) still retained their ability to bind to wheat germ agglutinin (WGA) but not to concanavalin A. The antagonism of DGhCG against activation of adenylate cyclase and steroidogenesis was reversed, when WGA was bound to the N-linked carbohydrate moieties of hCG alpha- and beta-subunits followed by addition of purified Leydig cells. The complex formed by incubation at an approximately equimolar ratio induced the maximal reversal of antagonism. This reversal of antagonism was diminished by addition of N-acetylglucosamine. No increase of steric hindrance at the receptor sites was seen in binding studies of the [125I]DGhCG-WGA complex, indicating that the receptor-binding domain of hCG may not be adjacent to the carbohydrate moieties. Kinetic studies of the hormonal response showed that the DGhCG-WGA complex terminated cAMP accumulation after 30 min of incubation, but not testosterone production. Our results suggest that tetravalent WGA can also reverse the antagonism of DGhCG, as in bivalent antibodies to hCG beta.
Mol Cell Endocrinol 1989 Oct
PMID:Reversal of the antagonism of deglycosylated human chorionic gonadotropin by aggregation with wheat germ agglutinin. 255 27


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