Anthony M. Adamany

Two chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose

A biochemical basis for the pea and lentil lectin resistance of two Chinese hamster ovary (CHO) cell mutants, Lec13 and Lec13A, was investigated. Studies of the G glycopeptides of vesicular stomatitis virus grown in the mutants indicated that Lec13 cells essentially lack the ability to add fucose to complex carbohydrates while Lec13A cells synthesize significant proportions of fucosylated, complex moieties. However, both mutants were known to be reverted to lectin sensitivity by growth in L-fucose, making them similar to the mouse lymphoma mutant, PLR1.3, which is defective in the conversion of GDP-mannose to GPD-fucose [M. L. Reitman, I. S. Trowbridge, and S. Kornfeld (1980) J. Biol. Chem. 255, 9900-9906]. Optimal conditions for the production of GDP-fucose from GDP-mannose by CHO cytosol were found to occur at pH 8 in the presence of 7.5 microM GDP-mannose, 15 mM Mg2+, 0.2 mM NAD+, 0.2 mM NADPH, 10 mM niacinamide, 5 mM ATP, and 50 mM Tris-HCl. Under these conditions, Lec13 cytosol produced no detectable GDP-fucose nor GDP-sugar intermediates while Lec13A cytosol produced significant quantities of both. Mixing experiments with Lec13 cytosol identified the first enzyme of the conversion pathway (GDP-mannose 4,6-dehydratase, EC 4.2.1.47) as the site of the block. In addition to being markedly reduced, the Lec13A 4,6-dehydratase activity was relatively insensitive to changes in pH in comparison to the activity in parental cytosol, suggesting that Lec13A cells might possess a structurally altered GDP-mannose 4,6-dehydratase enzyme.

Extracellular proteins of the calf aortic media smooth muscle cells in culture

Abstract The calf aortic medial layer and confluent cultures of smooth muscle cells derived from this tissue were subjected to a non-destructive extraction scheme including 1.0% sodium dodecyl sulfate/0.33 M mercaptoethanol as the last step. The SDS/mercaptoethanol-soluble fraction obtained from both sources contained a heterogenous mixture of proteins of which two components were purified to near homogeneity. Actin was identified as the major component of this fraction by its subunit molecular weight of 45 000 and its content of N -methylhistidine (approx. 1 residue/45 000 subunit). Also a 200 000 dalton protein was partially purified and compared to the microfibrillar protein subunit and to the fibronectin class of proteins. Based on turnover studies and susceptibility to proteolysis, this protein appeared to be an extracellular component of the cultured cell. The SDS/mercaptoethanol residue from the medial layer contained the expected complement of collagen (22%) and elastin (78%). In contrast, the residue from confluent cultures grown in the absence of ascorbic acid contained proteins of high content of polar amino acids. Collagen and elastin were not detected. The SDS/mercaptoethanol-insoluble proteins accumulate extracellularly as a function of time in culture past cellular confluence but not as a function of number of population doublings. In addition to these proteins, these cultured cells deposit insoluble collagen, but not elastin, when grown in the presence of added ascorbic acid.

The chimpanzee M blood-group antigen is a variant of the human M-N glycoproteins

Chimpanzee erythrocytes express strong M but weak, occasional N blood-group activity, as detected by anti-M and anti-N reagents. We have found that the M activity is carried by a major membrane glycoprotein that is similar but not identical to the human MM glycoprotein (glycophorin A). We have isolated and characterized this glycoprotein from erythrocyte membranes of four individual chimpanzees. The purified glycoproteins strongly inhibited agglutination of M cells by rabbit anti-human M sera and only weakly inhibited the agglutination of N cells by rabbit anti-human N sera. They also displayed medium-to-strong inhibitory activity against chimpanzee iso- and crossimmune antisera tested with chimpanzee erythrocytes of various V-A-B-D and Wc specificities, which are known as chimpanzee extensions of the human type M-N system and the Miltenberger counterpart, respectively. Each glycoprotein was cleaved with CNBr into three fragments, whose size, solubility, and composition were analogous to those obtained by similar treatment of the human M-N antigens. The amino-terminal fragment was found to be a glycooctapeptide whose amino acid composition and partial sequence indicated that it is an intermediate form of the human M and N glycooctapeptides. Its carbohydrate content comprised two threonine-linked saccharide units that, although similar in composition to the human threonine-linked units, were fewer in number than the three units found in the corresponding human glycooctapeptides. Structural similarities to the human antigens strongly suggest that the amino terminus bears the major antigenic determinants of the molecule, and the occurrence in this region of numerous, albeit rare, variants among humans and in chimpanzees indicates that the corresponding coding sequence of the structural gene is particularly susceptible to mutational events. We conclude that the chimpanzee M gene product is a variant of the human type and that the chimpanzee gene is an allele of the human polymorphic M-N locus.

The Role of Phosphorylated Dolichols in Membrane Glycoprotein Biosynthesis: Relation to Cholesterol Biosynthesis

Publisher Summary The participation of certain phosphorylated long-chain polyisoprenols (dolichols) as saccharide carriers and donors in the biosynthesis of complex carbohydrates has been among the most actively pursued discoveries in the field of membrane biosynthesis and glycoprotein metabolism in the last decade. Dolichols are major long-chain polyisoprenols found in animal cells. They mediate the assembly of asparagine-linked oligosaccharide units of exported and intracellular glycoproteins. This chapter focuses on the animal cell, particularly the cultured vascular smooth muscle cell. The chapter reviews the aspects of glycoprotein structure, the participation of phosphorylated dolichols in glycoprotein biosynthesis, and the interdependent regulation of biosynthesis of phosphorylated dolichols and cholesterol. HMG-CoA reductase is an inducible enzyme that, under steady-state cultural conditions, is in the suppressed state and behaves as a classic nonequilibrium enzyme. It has a rapid turnover rate and its activity is barely detectable in vitro. In some tissues it undergoes covalent modification by phosphorylation/dephosphorylation as a means of modulating its activity. These characteristics of HMG-CoA contribute to its position as a rate-controlling enzyme in polyisoprenol biosynthesis. The increased flux of substrates through this enzyme is useful in discerning regulatory enzymatic steps secondary to the reductase and specific to the pathways of cholesterol or other polyisoprenols such as the dolichols.

Glycophorins of human erythroleukemic K562 cells

Glycophorins related to alpha glycophorin, of the human erythrocyte membrane, were isolated from human erythroleukemic K562 cells. The glycophorins were purified using sodium dodecyl sulfate (SDS)/trichloroacetic acid fractionation and Folch and hot phenol extractions. 0.1-0.2 micrograms was obtained/10(8) cells, or approximately a 15% yield. SDS-gel electrophoresis revealed a pattern similar to erythrocyte alpha glycophorin except for the slower mobility of the glycophorin monomer. Two populations of K562 glycophorins, present in nearly equivalent amounts, were distinguished by their binding to Lens culinaris lectin agarose. The two populations exhibited similar gel electrophoretic patterns except for the presence of delta-like glycophorin exclusively in the population that did not bind to L. culinaris lectin. Immunoblotting revealed a lack of reaction of the major alpha and delta-like glycophorin bands in all K562 glycophorins with M or N erythrocyte glycophorin-specific monoclonal antibodies. Only minor species of intermediate electrophoretic mobility in glycophorins not binding to L. culinaris showed a reaction with these antibodies. Both populations of glycophorins incorporated radiolabeled glucosamine, mannose, and fucose and contained O-glycosidically linked tri- and tetrasaccharides, present in a ratio of approximately 1:1 indicating a significant degree of hyposialylation when compared to erythrocyte alpha glycophorin. No precursor/product relationship was demonstrated between the major forms of two populations. K562 cell surface labeling with lactoperoxidase revealed that only the glycophorins that exhibited binding to L. culinaris were accessible to iodination and could be the only species expressed at the cell surface.

Collagen glucosyl- and galactosyltransferases of cultured human fetal lung fibroblasts☆

Abstract Collagen-galactosyltransferase and collagen-glucosyltransferase activities have been studied in cultured human fetal lung WI-38 and IMR-90 diploid fibroblasts. These enzymes functioned in concert to synthesize glucosylgalactosylhydroxylysine units as found naturally in collagens, basement membranes, and certain serum glycoproteins. The transferases used UDP-Gal and UDP-Glc as glycose donors, collagens and collagen-derived peptides or glycopeptides as glycose acceptors, and worked best in the presence of manganese as a required divalent cation. Two pH optima, between pH 6 and 6.5 and between pH 7.5 and 8, were noted for each type of transferase, and these optima, particularly in the case of glucosyltransferase, were evident regardless of size of acceptor employed in the assay. About 35% of the total activity of each enzyme was found in the soluble fractions of cell homogenates, and, of the particulate fraction activities, about 50% could be released by mild sonication or by treatment with Triton X-100. Assessment of transferase activities as a function of cellular aging in culture revealed that significant decreases in enzyme levels occurred as the cell approached senescence (late Phase II), and these effects were reversed when cells attained senescence (Phase III). Addition of ascorbic acid to young cultures, under conditions known to increase endogenous collagenpeptide hydroxylation, caused no effects on the activities of the glycosyltransferases toward exogenous acceptors. These results suggested that the activities of collagen-hydroxylases and glycosyltransferase might not be coordinately regulated, and that, regardless of the hydroxylation events, glycosylation of the peptide might be limited to a specific fraction of hydroxylysine residues during the post-translational modification of collagen.

Isolation and characterization of the internal elastic lamina from calf thoracic aorta

Abstract A procedure is described for the mechanical isolation of the internal elastic lamina (IEL) of calf thoracic aorta. Chemical properties of the isolated IEL are examined and compared to the underlying aortic medial layer. The IEL is an acellular structure consisting of elastin (79%), insoluble collagen (17%), and about 4% of a heterogenous mixture of proteins soluble in 1% sodium dodecyl sulfate, 0.33 M mercaptoethanol. IEL contains less than 1% carbohydrate. Closely similar distribution of collagen and elastin is found in the adjacent medial layer, suggesting a common biosynthetic origin for the extracellular matrix of both structures.

Glycosylation in Livers of Newborn Mice Homozygous for a Lethal Deletion

Abstract Endogenous glycoprotein and lipid biosynthesis have been examined in slices of liver and other organs from normal and mutant mice homozygous for a perinatally lethal deletion in chromosome 7. Pronase digests of total glycoproteins, radioactively labeled with glucosamine, followed by Bio-Gel P-6 column chromatography of the resultant glycopeptides, indicate that glycosylation in mutant mouse liver is dramatically reduced compared to that of normal littermates. Pulse-chase experiments suggest that this reduction is not due to a processing event, but rather to reduced biosynthesis. In addition, a quantitative reduction of glycopeptides was observed in mutant livers, when the radioactive peaks from the Bio-Gel P-6 fractionation were pooled and analyzed on a Dowex 50 column, followed by separation on DE-52 columns. Analysis, by affinity chromatography, of radioactively labeled total lipids indicated that homozygous mutant and normal littermate livers have similar quantities of neutral and acidic lipids, including phosphatidylserine, phosphatidylinositol, cerebrosides, and phospholipids. Furthermore, the analysis of other organs indicates that the reduction of glycoprotein synthesis observed in the mutant liver is specific to this organ.