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Biomedical subjects

M D Snider

Publications and source records attributed to M D Snider.

29 records · Page 2Linked to original sources
PubMed1982

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly.

The formation of N-glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.

Asparagine↗
PubMed1982

Transmembrane organization of protein glycosylation. Mature oligosaccharide-lipid is located on the luminal side of microsomes from Chinese hamster ovary cells.

The transmembrane orientation of Glc3Man9GlcNAc2-pyrophosphoryl-dolichol, which is the oligosaccharide donor in the glycosylation of asparagine residues of eukaryotic glycoproteins has been investigated. The lectin concanavalin A was used as a nonpenetrating probe to study the location of this oligosaccharide-lipid in microsomal vesicles prepared from cultured fibroblasts. Lectin treatment of intact vesicles, and vesicles made leaky with low concentrations of detergent showed that this oligosaccharide-lipid is on the luminal side of membrane. The oligosaccharide-lipid was bound by lectin only if the permeability barrier of the membrane had been destroyed by detergent; very little binding was seen in intact vesicles. This result suggests that glycosylation of nascent secretory and membrane glycoproteins occurs on the luminal side of the membrane. It also implies that sugar residues derived from cytoplasmic sugar nucleotides must be transported across the membrane at some point during the synthesis and accumulation of mature, luminal oligosaccharide-lipid, although the identity of the transported species remains unknown.

Animals↗
PubMed1980

Transmembrane location of oligosaccharide-lipid synthesis in microsomal vesicles.

The oligosaccharide-lipid which is the precursor of asparagine-linked oligosaccharides of eucaryotic glycoproteins is synthesized from sugar nucleotides in the endoplasmic reticulum. The transmembrane location of the assembly of this oligosaccharide-lipid has been studied in vitro in rat liver microsomes. Protease treatment of these sealed vesicles which are derived from the endoplasmic reticulum resulted in the inactivation of a number of enzymes of oligosaccharide-lipid synthesis. Three early steps, the synthesis of dolichol--phosphate--mannose, of dolichol--phosphate--glucose and of dolichol--pyrophosphoryl--di--N--acetylchitobiose, as well as the final steps, the addition of glucose residues to oligosaccharide-lipid, were inactivated under conditions where only the cytoplasmic side of the membrane was accessible to protease. This finding, and the fact that no activities were latent to protease in intact microsomal vesicles, suggest that oligosaccharide-lipid is assembled on the cytoplasmic side of the microsomal membrane. However, the possibility of enzymes spanning the bilayer with their active sites facing the lumen cannot be ruled out. These results are discussed in relation to the segregation of newly made glycoprotein products within the lumen of the endoplasmic reticulum.

Animals↗
PubMed1980

Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein.

Maturation of the vesicular stomatitis virus (VSV) glycoprotein (G) to the cell surface is blocked at the nonpermissive temperature in cells infected with temperature-sensitive mutants in the structural gene encoding for G. We show here that these mutants fall into two discrete classes with respect to the stage of post-translational processing at which the block occurs. In all cases the mutant glycoproteins are inserted normally into the endoplasmic reticulum membrane, receive the two-high-mannose oligosaccharides, and apparently lose the NH2-terminal signal sequence of 16 amino acids. In cells infected with one class of mutants, no further processing of the glycoprotein occurs, and we conclude that the mutant protein is blocked at a pre-Golgi stage. In cells infected with ts L511(V), however, addition of the terminal sugars galactose and sialic acid occurs normally. Thus the maturation of G proceeds through several Golgi functions but is blocked before its appearance on the cell surface. The oligosaccharide chain of ts L511(V) G, accumulated at either the permissive (where surface maturation occurs) or the nonpermissive temperature, lacks one saccharide residue, probably fucose. In addition, no fatty acid residues are added to the ts L511(V) G protein at the nonpermissive temperature, although addition does occur under permissive conditions.

Animals↗
PubMed1979

Control of membrane lipid synthesis in Escherichia coli during growth and during the stringent response.

The regulation of phospholipid synthesis in cells of Escherichia coli was studied in vivo during growth and during the stringent response to amino acid starvation. Strains harboring the hybrid plasmid pLC44-14 (Clark, L., and Carbon, J. (1976) Cell 9, 91-99), which had increased levels of glycerophosphate acyltransferase, were used to study the involvement of this enzyme in the control of phospholipid synthesis. In addition, regulation was studied by measuring the levels of three early intermediates of phospholipid synthesis:phosphatidic acid, CDP-diglyceride, and dCDP-diglyceride. The liponucleotides were measured by a new enzymatic method which allows determinations to be made on crude lipid extracts. Results from experiments on growing cells are consistent with regulation of membrane lipid synthesis occurring in fatty acid synthesis or at the level of glycerophosphate acylation, but not at any later step. Experiments on the inhibition of lipid synthesis during the stringent response make it possible to rule out explanations which involve the inhibition of a single enzyme; enzymes both before and after the liponucleotides in phospholipid synthesis must be affected.

Amino Acids↗
PubMed1977

Partial purification of glycerophosphate acyltransferase from Escherichia coli.

Glycerophosphate acyltransferase, a membrane-bound enzyme catalyzing the initial step of phospholipid biosynthesis in Escherichia coli, has been extracted with Triton X-100, a nonionic detergent, and purified 20- to 40-fold. This preparation is free from lysophosphatidate acyltransferase. Glycerophosphate acyltransferase is inactive in detergent extracts, but can be reconstituted by the addition of phospholipid. Under such conditions, the enzyme is associated with phospholipid. The sole product of the reaction with acyl coenzyme A as substrate is 1-acyl-sn-glycero-3-phosphate. Furthermore, the enzyme shows a marked preference for saturated fatty acyl conenzyme A, implying that this enzyme is responsible for the predominance of saturated moieties in position 1 of E. coli phospholipids. Acyltransferase from two mutants, plsA and plsB, was partially purified and characterized. Results support the view that plsB is a structural gene for the acyltransferase, but suggest that the plsA gene product is not directly involved in phospholipid biosynthesis.

Acyltransferases↗
PubMedDate not supplied

Adverse reaction to prednisone in a patient with systemic lupus erythematosus.

Oral corticosteroids are the main therapeutic choice for systemic lupus erythematosus (SLE). Adverse reactions to systemic corticosteroids rarely occur and the etiology is unclear in most cases. A 14-year-old girl with newly diagnosed SLE developed a pruritic bullous eruption while on prednisone. The patient had been treated successfully in the hospital with intravenous methylprednisolone. In preparation for discharge, the steroid preparation was changed to prednisone to which the patient reacted with a development of new crops of bullous lesions. Skin biopsy specimens of lesional areas showed a bullous eruption consistent with erythema multiforme. The patient underwent immediate and delayed hypersensitivity tests. Intradermal and patch tests to liquid prednisone were positive. The patient was discharged on oral methylprednisolone and has not had recurrence of the skin lesions. In conclusion, a case of prednisone sensitivity in a patient with SLE is presented here. An alternative preparation, methylprednisolone, was used to successfully treat her underlying condition.

Adolescent↗