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A J Parodi

Publications and source records attributed to A J Parodi.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of the mannose 6-phosphate-dependent pathway of lysosomal enzyme routing in an invertebrate.

The mannose 6-phosphate (Man6P)-dependent pathway for routing lysosomal enzymes was characterized in the hepatopancreas of the estuary crab Chasmagnatus granulata: (a) an acid alpha-L-fucosidase was purified to homogeneity from the above-mentioned organ and was shown to contain mannose-linked phosphate residues; (b) high-mannose-type oligosaccharides isolated from a protein fraction enriched in acid hydrolases were found to contain acid-labile N-acetylglucosamine (GlcNAc) residues; (c) a membrane-bound UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase was detected that phosphorylated the estuary-crab alpha-L-fucosidase and bovine uteroferrin but not bovine pancreas ribonuclease B; (d) a GlcNAc-1-phosphodiester alpha-N-acetylglucosaminidase that released GlcNAc units from GlcNAc alpha 1-P6Man alpha 1-methyl was detected in microsomal membranes of the hepatopancreas; (e) two detergent-solubilized microsomal proteins having molecular masses of 205 and 215 kDa that were retained by a Man6P-rich mannan-Sepharose column, from where they were eluted with Man6P but not with glucose 6-phosphate, were recognized by antisera raised against bovine large (215 kDa) and small (46 kDa) Man6P receptors. This is the first description of all the components of the Man6P-dependent mechanism for routing lysosomal enzymes in an invertebrate.

Acetylglucosamine↗
PubMed1995

Structural characterization of the major glycosylphosphatidylinositol membrane-anchored glycoprotein from epimastigote forms of Trypanosoma cruzi Y-strain.

We have investigated the structure of the glycosylphosphatidylinositol (GPI) anchor and the O-linked glycan chains of the 40/45-kDa glycoprotein from the cell surface of the protozoan parasite Trypanosoma cruzi. This glycoconjugate is the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi Y-strain, epimastigote form. The GPI anchor was liberated by treatment with hot alkali, and the phosphoinositol-oligosaccharide moiety was characterized and shown to have the following structure. [formula: see text] Unusually the glucosamine was 6-O-substituted with 2-aminoethylphosphonate, and 2-aminoethylphosphonate was also present on the third mannose residue distal to glucosamine, partially replacing the ethanolamine phosphate. The beta-eliminated reduced oligosaccharide chains showed that two novel classes of O-linked N-acetylglucosamine oligosaccharide were present. The first series had the structures Galp beta 1-3GlcNAc-ol; Galp beta 1-6(Galp beta 1-3)GlcNAc-ol; and Galp beta 1-2Galp beta 1-6(Galp beta 1-3)GlcNAc-ol, whereas the other series had a 1-4 linkage to N-acetylglucosaminitol and had structures Galp beta 1-4GlcNAc-ol, Galp beta 1-6(Galp beta 1-4)GlcNAc-ol, and Galp beta 1-2Galp beta 1-6(Galp beta 1-4)GlcNAc-ol. We have also investigated the kinetics of in vitro sialylation of these O-linked oligosaccharides by the T. cruzi transsialidase and have shown that incorporation of one molecule of sialic acid hinders entry of a second molecule when two potential acceptor sites are present.

Animals↗
PubMed1995

Characterization and partial purification of a novel enzymatic activity. UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from the cellular slime mold Dictyostelium discoideum.

An enzymatic activity that transfers N-acetylglucosamine-1-phosphate residues from UDP-GlcNAc to serine units in proteins (UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase) was detected in membranes of the cellular slime mold Dictyostelium discoideum. The enzyme was partially purified by affinity chromatography in concanavalin A-Sepharose and ion exchange chromatography in a Mono Q column. The enzyme showed an absolute requirement for bivalent cations, Mn2+ being more effective than Mg2+. It had a broad optimum pH value (6.5-9.0). The Km for UDP-GlcNAc was 18 microM. In cell free assays it used apomucin and native or 8 M urea-denatured thyroglobulin but neither bovine serum albumin nor native or denatured uteroferrin as exogenous acceptors. Analysis of proteins isolated from cells grown in the presence of [32P]phosphate and from the culture medium showed that the majority of proteins bearing the structure Glc-NAc-1-P-Ser were secreted. In equilibrium density centrifugations of microsomes, the enzyme appeared in membranes having lighter densities than the enzyme that phosphorylates high mannose-type oligosaccharides. This showed that the activity that phosphorylates serine residues in proteins (UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase) is different from that phosphorylating protein-linked high mannose-type oligosaccharides (UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase).

Animals↗
PubMed1995

The presence of galactofuranose and ribose units in asparagine-linked oligosaccharides of the digenetic trypanosomatid Endotrypanum schaudinni.

It was found that the digenetic trypanosomatid Endotrypanum schaudinni transferred Man7GlcNAc2 in protein N-glycosylation. Endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were identified as Man7GlcNAc2, Man6GlcNAc2, Rib1Man6GlcNAc2 and/or Gal1Man6GlcNAc2, Man5GlcNAc containing two galactose or ribose units or one of each residues, Rib1Man5GlcNAc2 and Gal1Man5GlcNAc2. The galactoses were in the furanose configuration. Endo-beta-N-acetylglucosaminidase H-resistant glycopeptides that were retained by concanavalin A-Sepharose and eluted with alpha-methylmannoside were found to contain mannose, galactofuranose and ribose units. The presence of galactofuranoses in N-glycoproteins has been reported previously in several monogenetic trypanosomatids but only in one digenetic parasite (Trypanosoma cruzi). This and a recent publication on the structure of Blastocrithidia culicis N-linked oligosaccharides are the first reports on the presence of ribose in eukaryotic glycoconjugates.

Animals↗
PubMed1995

The presence of complex-type oligosaccharides at the C-terminal domain glycosylation site of some molecules of cruzipain.

Cruzipain is a lysosomal enzyme of the flagellate Trypanosoma cruzi. It has three potential asparagine-glycosylation sites, two in the catalytic domain and one in the C-terminal domain. The latter appeared to have both high mannose- and complex-type oligosaccharides, whereas the catalytic domain only had compounds of the former type. The partial susceptibility of the complex-type compounds to endo-beta-N-acetylglucosaminidase H and their relative mannose and galactose content indicate that they had hybrid/monoantennary and biantennary structures. The same pattern of high mannose-type compounds was found at both domains, thus indicating that in cruzipain molecules having only high mannose-type compounds, all oligosaccharides were equally exposed to processing glycosidases and glycosyltransferases. As heterogenity of the protein C-terminal domain has already been detected, it is suggested that this feature might elicit an increased accessibility to processing enzymes responsible for complex-type oligosaccharide formation in certain cruzipain molecules or, alternatively, that a second glycosylation site with increased accessibility might be present in certain cruzipain molecules. Furthermore, the presence of complex-type oligosaccharides strongly suggests that, as in mammalian cells, T. cruzi lysosomal enzymes traverse the entire Golgi apparatus up to the trans-Golgi cisternae and the trans-Golgi network before reaching lysosomes.

Animals↗
PubMed1995

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.

It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin-like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP-Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides.

1-Deoxynojirimycin↗
PubMed1994

Purification to homogeneity of UDP-glucose:glycoprotein glucosyltransferase from Schizosaccharomyces pombe and apparent absence of the enzyme fro Saccharomyces cerevisiae.

The UDP-Glc:glycoprotein glucosyltransferase was purified to homogeneity from the fission yeast Schizosaccharomyces pombe. The enzyme has been recently suggested to be involved in the mechanism by which unfolded, partially folded, or misfolded glycoproteins are retained in the endoplasmic reticulum. The pure yeast glucosyltransferase formed protein-linked Glc1-Man9GlcNAc2,Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 when incubated with UDP-Glc and denatured thyroglobulin. The same compounds were formed upon glucosylation of endogenous acceptors by crude microsomes. The enzyme was a soluble microsomal protein that required Ca2+ for activity, used UDP-Glc and not TDP-Glc, ADP-Glc, or UDP-Gal as sugar donor, had an almost neutral optimum pH value, and as the glucosyl-transferase obtained from rat liver, glucosylated denatured but not native glycoproteins or glycopeptides. A similar enzymatic activity could not be detected in Saccharomyces cerevisiae microsomes and transient glucosylation of glycoproteins (addition of a single glucose unit to glucose-free oligosaccharides by the glucosyltransferase followed by its removal by glucosidase II) could not be detected in intact S. cerevisiae cells. These are the only eukaryotic cells described so far in which these processing reactions of the endoplasmic reticulum do not occur. Availability of the pure S. pombe enzyme will eventually allow testing the possible involvement of the glucosyltransferase in sensing glycoprotein tertiary structures in the endoplasmic reticulum.

Carbohydrate Sequence↗
PubMed1994

Novel (rhamnosyl and ribosyl) and uncommon (xylosyl) monosaccharide residues are present in asparagine-linked oligosaccharides of the trypanosomatid Blastocrithidia culicis.

Blastocrithidia culicis is a trypanosomatid protozoon that transfers Man6GlcNAc2 in protein N-glycosylation. Compounds containing mannosyl, xylosyl, and rhamnosyl residues were found among the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of whole cell glycoproteins of this parasite. The compositions of some of them were as follows: Man5GlcNAc2, Man6GlcNAc2, Rha1Man5GlcNAc2, Rha2Man6GlcNAc2, Xyl1Rha2Man6-GlcNAc2, Xyl1Rha3Man6GlcNAc2, and Xyl2Rha3Man6-GlcNAc2. On the other hand, oligosaccharides containing mannosyl, xylosyl, rhamnosyl, and ribosyl units were liberated from endo-beta-N-acetylglucosaminidase-resistant glycopeptides upon treatment with N-glycanase. This is the first report on the presence of ribosyl units in eukaryote glycoconjugates, of rhamnosyl residues in asparagine-linked oligosaccharides, and of xylosyl units in high mannose-type compounds.

Animals↗
PubMed1994

The presence in Trypanosoma cruzi microsomes of alpha(1,2), alpha(1,3) and alpha(1,6) mannosidase activities not involved in protein-linked Man9GlcNAc2 processing.

Trypanosoma cruzi microsomes were found to possess membrane-bound alpha(1,2), alpha(1,3) and alpha(1,6) mannosidase activities that had an almost neutral optimum pH value, did not require CaCl2 for activity and were inhibited by swainsonine but not by deoxymannojirimycin. A mannosidase activity that degraded p-nitrophenylmannoside and that was inhibited by swainsonine was also present in the parasite microsomes. Experiments performed with intact cells showed that processing of protein-linked Man9GlcNAc2 was inhibited by deoxymannojirimycin but not by swainsonine. It was concluded that the activities detected were not involved in protein-linked Man9GlcNAc2 processing.

Animals↗
PubMed1993

The action of Trypanosoma cruzi trans-sialidase on glycolipids and glycoproteins.

Addition of sialic acid residues in the human pathogen Trypanosoma cruzi glycoconjugates is mediated by a trans-sialidase and not by a CMP-sialic acid:glycoconjugate sialyltransferase. Incubation of trans-sialidase with N-[galactose-14C]acetyllactosamine and O-linked oligosaccharides, N-linked glycopeptides (both obtained from fetuin) or sialyllactose showed that the last three compounds were donors of sialic acid residues to the first one. Moreover, N- and O-linked oligosaccharides in asialofetuin and asialomucin, respectively, served as acceptors of sialic acid units. Gangliosides GM3, GD1a and GT1b but not GM2, GM1a nor GD1b donated sialic acid units to N-acetyllactos amine when incubated with trans-sialidase. This showed that only sialic acid units bound to terminal galactosyl residues were transferred. GM1a was converted to GD1a, and GD1b to GT1b when incubated with the appropriate donor. The fact that asialo-GM1a was converted to a ganglioside migrating as GD1a on thin-layer chromatography suggested that sialic acid units may be transferred to internal galactosyl residues, although once linked to those residues they can not be further transferred to other glycoconjugates. Sialic acid residues linked alpha 2,3- but not alpha 2,6- or alpha 2,8- were transferred by the trans-sialidase. Methyl beta-galactoside but not methyl alpha-galactoside served as acceptor of sialic acid units, thus suggesting that terminal alpha-linked galactosyl units in T. cruzi and mammalian glycoproteins are not sialylated by the enzyme. As the trans-sialidase employed in these experiments has been shown to be located on the external surface of the parasite and to be shed to the medium, the relatively broad specificity shown by the enzyme with respect to protein- and lipid-linked oligosaccharides strongly suggests that infection by T. cruzi might alter the sialic acid distribution in glycoproteins and glycolipids of the mammalian host.

Animals↗
PubMed1993

N-glycosylation in trypanosomatid protozoa.

Trypanosomatid protozoa are parasites of considerable medical and economic importance in developing countries. The pathway leading to N-glycosylation in these microorganisms is characterized by the following features: (i) dolichols are composed of only 10-13 isoprene units; (ii) oligosaccharides transferred in N-glycosylation have the compositions Man(6,7,9)GlcNAc2, depending on the species; (iii) trypanosomatids are unable to synthesize dolichol-P-Glc and, in addition, some species lack certain dolichol-P-Man-dependent mannosyltransferases; (iv) the oligosaccharyltransferase does not require the presence of glucose units in the oligosaccharide in order to catalyse an efficient transfer reaction; (v) trypanosomatids have a glucosidase II-like enzyme, but lack glucosidase I; (vi) glucosidase II is required for deglucosylation of oligosaccharides glucosylated by the UDP-Glc:glycoprotein glucosyltransferase, an activity first detected in those parasites; (vii) the structures of polymannose-type compounds in these protozoa have no significant differences with those of their mammalian counterparts except for the presence, in certain species, of oligosaccharides having galactofuranose units linked to external mannose residues; (viii) biantennary complex-type oligosaccharides having in some cases terminal alpha-linked galactose units or poly-N-acetylactosamine extensions, but lacking sialic acid units, have been described in Trypanosoma brucei; (ix) complex-type oligosaccharides having alpha-linked galactose, fucose and sialic acid residues have been described in Trypanosoma cruzi. In this parasite, addition of sialic acid units to glycoproteins and glycolipids is mediated by a trans-sialidase located on the external surface of the parasite and not by an intracellular CMP-sialic acid-dependent sialyltransferase.

Animals↗
PubMed1992

On the post-translational modifications at the C-terminal domain of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, has a 130 amino acid-long C-terminal domain, which, although microheterogeneous in SDS-PAGE, has a single N-terminal amino acid sequence. Most of the Thr residues present at the beginning of this sequence are modified; the nature of this modification is still unknown, but O-glycosylation and phosphorylation seem both to be absent. The only potential site for N-glycosylation (Asn 254) is glycosylated in vivo. Most of the eight Cys residues are involved in disulfide bridges. The results are consistent with cruzipain being made of two well-defined domains, a catalytic one with high homology to cathepsin L, and a C-terminal domain, linked to the former by a 'hinge' corresponding to the Pro- and Thr-rich region at its N-terminus.

Amino Acid Sequence↗
PubMed1992

Purification to apparent homogeneity and partial characterization of rat liver UDP-glucose:glycoprotein glucosyltransferase.

The UDP-Glc:glycoprotein glucosyltransferase is a soluble protein of the endoplasmic reticulum that catalyzes the glucosylation of protein-linked, glucose-free, high mannose-type oligosaccharides. In vivo, the newly glucosylated compounds are immediately deglucosylated, presumably by glucosidase II. The glucosyltransferase has been purified to apparent homogeneity from rat liver. The enzyme appears to have a molecular weight of 150,000 and 270,000 under denaturing and native conditions, respectively. The pure enzyme shows an almost absolute requirement for Ca2+ ions and for UDP-Glc as sugar donor. The same as crude preparations, the pure enzyme synthesized Glc1 Man7-9GlcNAc2-protein from Man7-9GlcNAc2-protein. Denatured glycoproteins are glucosylated much more efficiently than native ones by the apparently homogeneous glucosyltransferase. Availability of the pure enzyme will allow testing the possible involvement of transient glucosylation of glycoproteins in the folding of glycoproteins and/or in the mechanism by which cells dispose of malfolded glycoproteins in the endoplasmic reticulum.

Animals↗
PubMed1992

Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.

It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endoplasmic reticulum mannosidases decreases the extent of glycoprotein glucosylation. All five different glycoproteins tested (bovine and porcine thyroglobulins, phytohemagglutinin, soybean agglutinin, and bovine pancreas ribonuclease B) were found to be poorly glucosylated or not glucosylated unless they were subjected to treatments that modified their native conformations. The effect of denaturation was not to expose the oligosaccharides but to make protein determinants, required for enzymatic activity, accessible to the glucosyltransferase because (a) cleavage of denatured glycoproteins by unspecific (Pronase) or specific (trypsin) proteases abolished their glucose acceptor capacities almost completely except when the tryptic peptides were held together by disulfide bonds and (b) high mannose oligosaccharides in native glycoproteins, although poorly glucosylated or not glucosylated, were accessible to macromolecular probes as concanavalin A-Sepharose, endo-beta-N-acetylglucosaminidase H, and jack bean alpha-mannosidase. In addition, denatured, endo-beta-N-acetylglucosaminidase H deglycosylated glycoproteins were found to be potent inhibitors of the glucosylation of denatured glycoproteins. It is suggested that in vivo only unfolded, partially folded, and malfolded glycoproteins are glucosylated and that glucosylation stops upon adoption of the correct conformation, a process that hides the protein determinants (possibly hydrophobic amino acids) from the glucosyltransferase.

Animals↗
PubMed1992

Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi.

The gene(s) encoding the Trypanosoma cruzi shed-acute-phase-antigen (SAPA) has a 5' end encoding a region containing two totally and two partially conserved Ser-X-Asp-X-Gly-X-Thr-Trp motifs which are present in bacterial neuraminidases, and a 3' end encoding tandemly repeated units of 12 amino acids. It is now reported that 54-87% of the total neuraminidase activity present in the parasite could be immunoprecipitated with polyclonal or monoclonal antibodies against the repeated amino acid units of SAPA. These immunoprecipitates also had greater than 80% of the trans-sialidase activity of the parasite. SAPA used sialyllactose, fetuin and 4-methylumbelliferyl-sialic acid as substrate donors. In the presence of a suitable acceptor molecule (lactose) the sialic acid residues were transferred to the disaccharide, whereas in the absence of acceptors the residues were transferred to water. If relatively inefficient acceptors (maltose or cellobiose) were added to the incubation mixtures, the sialic acid units were transferred both to the disaccharides and to water. It is concluded that a major T. cruzi antigen has both the trans-sialidase and the neuraminidase activities of the parasite. Both activities are probably located on the N-terminus of SAPA since antibodies directed against the C-terminus, which contains the repeated amino acid units, do not affect the enzymatic activities.

Amino Acid Sequence↗