Biomedical subjects

Armando J Parodi

Publications and source records attributed to Armando J Parodi.

13 recordsLinked to original sources

PubMed2006

Preferential transfer of the complete glycan is determined by the oligosaccharyltransferase complex and not by the catalytic subunit.

Most eukaryotic cells show a strong preference for the transfer in vivo and in vitro of the largest dolichol-P-P-linked glycan (Glc(3)Man(9)GlcNAc(2)) to protein chains over that of biosynthetic intermediates that lack the full complement of glucose units. The oligosaccharyltransferase (OST) is a multimeric complex containing eight different proteins, one of which (Stt3p) is the catalytic subunit. Trypanosomatid protozoa lack an OST complex and express only this last protein. Contrary to the OST complex from most eukaryotic cells, the Stt3p subunit of these parasites transfers in cell-free assays glycans with Man(7-9)GlcNAc(2) and Glc(1-3)Man(9)GlcNAc(2) compositions at the same rate. We have replaced Saccharomyces cerevisiae Stt3p by the Trypanosoma cruzi homologue and found that the complex that is formed preferentially transfers the complete glycan both in vivo and in vitro. Thus, preference for Glc(3)Man(9)GlcNAc(2) is a feature that is determined by the complex and not by the catalytic subunit.

Animals↗

PubMed2005

Absence of nucleoside diphosphatase activities in the yeast secretory pathway does not abolish nucleotide sugar-dependent protein glycosylation.

It is accepted that glycosyltransferase-generated nucleoside diphosphates are converted to monophosphates in the secretory pathway by nucleoside diphosphatases (NDPases) to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the lumen is coupled to exit of nucleoside monophosphates. Working with Saccharomyces cerevisiae mutants affected in anterograde and/or retrograde endoplasmic reticulum (ER)-Golgi vesicular traffic and/or defective in one or both secretory pathway (Golgi) NDPases, we show that UDP-Glc: glycoprotein glucosyltransferase-mediated glucosylation is not dependent on the presence of NDPases or on ER-Golgi vesicular traffic and that GDP-Man-dependent N- and O-mannosylations are reduced but not abolished in the absence of NDPases in the secretory pathway. Further, the absence of the main Man-1-P transferase (a Golgi GMP-generating enzyme) does not modify the limited mannosylation observed in the absence of NDPases. Based on these results and on available additional information, we suggest that in the absence of NDPases, the already characterized nucleotide sugar transporters allow entrance of nucleotide sugars into the luminal compartments and that resulting nucleoside diphosphates exit to the cytosol by a still unknown mechanism. Further, an unexpected side result suggests that formation of Ser/Thr-Man(2) may occur in the ER and not exclusively in the Golgi.

Acid Anhydride Hydrolases↗

PubMed2005

Characterization of Schizosaccharomyces pombe ER alpha-mannosidase: a reevaluation of the role of the enzyme on ER-associated degradation.

It has been postulated that creation of Man8GlcNAc2 isomer B (M8B) by endoplasmic reticulum (ER) alpha-mannosidase I constitutes a signal for driving irreparably misfolded glycoproteins to proteasomal degradation. Contrary to a previous report, we were able to detect in vivo (but not in vitro) an extremely feeble ER alpha-mannosidase activity in Schizosaccharomyces pombe. The enzyme yielded M8B on degradation of Man9GlcNAc2 and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man9GlcNAc2 present in misfolded glycoproteins even after a long residence in the ER. In addition, no preferential degradation of M8B-bearing species was detected. Nevertheless, disruption of the alpha-mannosidase encoding gene almost totally prevented degradation of a misfolded glycoprotein. This and other conflicting reports may be best explained by assuming that the role of ER mannosidase on glycoprotein degradation is independent of its enzymatic activity. The enzyme, behaving as a lectin binding polymannose glycans of varied structures, would belong together with its enzymatically inactive homologue Htm1p/Mnl1p/EDEM, to a transport chain responsible for delivering irreparably misfolded glycoproteins to proteasomes. Kifunensin and 1-deoxymannojirimycin, being mannose homologues, would behave as inhibitors of the ER mannosidase or/and Htm1p/Mnl1p/EDEM putative lectin properties.

Carboxypeptidases↗

PubMed2005

Glycoprotein tertiary and quaternary structures are monitored by the same quality control mechanism.

Folding of glycoproteins entering the secretory pathway is strictly surveyed in the endoplasmic reticulum by a quality control system. Folding intermediates and proteins irreparably misfolded are marked via glucosylation by the UDPglucose:glycoprotein glucosyltransferase, an enzyme that acts as a folding sensor by exclusively labeling glycoproteins not displaying their native structures. Here we show that this sensing mechanism also applies to the oligomerization of protein complexes, as the glucosyltransferase appeared to be able to glucosylate folded complex subunits lacking the full complement of oligomer components.

Glycoproteins↗

PubMed2005

Glycoprotein reglucosylation.

Proteins following the secretory pathway acquire their proper tertiary and in certain cases also quaternary structures in the endoplasmic reticulum (ER). Incompletely folded species are retained in the ER and eventually degraded. One of the molecular mechanisms by which cells achieve this conformational sorting is based on monoglucosylated N-glycans (Glc1Man5-9GlcNAc2) present on nascent glycoproteins in the ER. This chapter discusses two of the steps that regulate the abundance of such N-glycan structures, including glycoprotein deglucosylation (by glucosidase II) and reglucosylation (by the UDP-Glc:glycoprotein glucosyltransferase), as well as an overview of methods to evaluate the N-glycans prevalent during glycoprotein biogenesis in the ER.

Animals↗

PubMed2004

The endoplasmic reticulum glucosyltransferase recognizes nearly native glycoprotein folding intermediates.

The UDP-Glc:glycoprotein glucosyltransferase (GT), a key player in the endoplasmic reticulum (ER) quality control of glycoprotein folding, only glucosylates glycoproteins displaying non-native conformations. To determine whether GT recognizes folding intermediates or irreparably misfolded species with nearly native structures, we generated and tested as GT substrates neoglycoprotein fragments derived from chymotrypsin inhibitor 2 (GCI2) bearing from 53 to 64 (full-length) amino acids. Fragment conformations mimicked the last stage-folding structures adopted by a glycoprotein entering the ER lumen. GT catalytic efficiency (V(max)/K(m)) remained constant from GCI2-(1-53) to GCI2-(1-58) and then steadily declined to reach a minimal value with GCI2-(1-64). The same parameter showed a direct hyperbolic relationship with solvent accessibility of the single Trp residue but only in fragments exposing hydrophobic amino acid patches. Mutations introduced (GCI2-(1-63)V63S and GCI2-(1-64)V63S) produced slight structural destabilizations but increased GT catalytic efficiency. This parameter presented an inverse exponential relationship with the free energy of unfolding of canonical and mutant fragments. Moreover, the catalytic efficiency showed a linear relationship with the fraction of unfolded species in water. It was concluded that the GT-derived quality control may be operative with nearly native conformers and that no alternative ER-retaining mechanisms are required when glycoproteins approach their proper folding.

Animals↗

PubMed2003

De novo synthesis of bacterial glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation.

Evidence is presented indicating that initiation of glycogen synthesis in Agrobacterium tumefaciens does not require the presence of alpha(1,4)-linked glucans. Crude cell extracts incubated with ADP-glucose (Glc) were able to form alpha(1,4)-linked glucans despite the fact that cells used for extract preparation displayed a genotype that prevented synthesis of Glc-containing sugar nucleotides and thus preformation of alpha(1,4)-linked glucans and that the defined growth medium used contained glycerol as carbon source. A. tumefaciens glycogen synthase (GS) purified to homogeneity from the above-mentioned cells was able to build its own primer by transferring Glc residues from ADP-Glc to an amino acid(s) in the same protein. Primed GS then became the substrate for further GS-catalyzed glucan elongation. It was concluded that, contrary to what happens in mammalian and yeast cells in which two different proteins are required for linear alpha(1,4)-linked glucan formation (glycogenin for initiation and GS for further elongation), in A. tumefaciens and probably in all other bacteria, the same protein is involved in both glycogen initiation and elongation.

Agrobacterium tumefaciens↗

PubMed2003

The interplay between folding-facilitating mechanisms in Trypanosoma cruzi endoplasmic reticulum.

Lectin (calreticulin [CRT])-N-glycan-mediated quality control of glycoprotein folding is operative in trypanosomatid protozoa but protein-linked monoglucosylated N-glycans are exclusively formed in these microorganisms by UDP-Glc:glycoprotein glucosyltransferase (GT)-dependent glucosylation. The gene coding for this enzyme in the human pathogen Trypanosoma cruzi was identified and sequenced. Even though several of this parasite glycoproteins have been identified as essential components of differentiation and mammalian cell invasion processes, disruption of both GT-encoding alleles did not affect cell growth rate of epimastigote form parasites and only partially affected differentiation and mammalian cell invasion. The cellular content of one of the already identified T. cruzi glycoprotein virulence factors (cruzipain, a lysosomal proteinase) only showed a partial (5-20%) decrease in GT null mutants in spite of the fact that >90% of all cruzipain molecules interacted with CRT during their folding process in wild-type cells. Although extremely mild cell lysis and immunoprecipitation procedures were used, no CRT-cruzipain interaction was detected in GT null mutants but secretion of the proteinase was nevertheless delayed because of a lengthened interaction with Grp78/BiP probably caused by the detected induction of this chaperone in GT null mutants. This result provides a rationale for the absence of a more drastic consequence of GT absence. It was concluded that T. cruzi endoplasmic reticulum folding machinery presents an exquisite plasticity that allows the parasite to surmount the absence of the glycoprotein-specific folding facilitation mechanism.

Animals↗

PubMed2003

Nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments in Schizosaccharomyces pombe.

Nucleoside diphosphates generated by glycosyltransferases in the fungal, plant, and mammalian cell secretory pathways are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the secretory pathway lumen is coupled to the exit of nucleoside monophosphates. Analysis of the yeast Schizosaccharomyces pombe genome revealed that it encodes two enzymes with potential nucleoside diphosphatase activity, Spgda1p and Spynd1p. Characterization of the overexpressed enzymes showed that Spgda1p is a GDPase/UDPase, whereas Spynd1p is an apyrase because it hydrolyzed both nucleoside tri and diphosphates. Subcellular fractionation showed that both activities localize to the Golgi. Individual disruption of their encoding genes did not affect cell viability, but disruption of both genes was synthetically lethal. Disruption of Spgda1+ did not affect Golgi N- or O-glycosylation, whereas disruption of Spynd1+ affected Golgi N-mannosylation but not O-mannosylation. Although no nucleoside diphosphatase activity was detected in the endoplasmic reticulum (ER), N-glycosylation mediated by the UDP-Glc:glycoprotein glucosyltransferase (GT) was not severely impaired in mutants because first, no ER accumulation of misfolded glycoproteins occurred as revealed by the absence of induction of BiP mRNA, and second, in vivo GT-dependent glucosylation monitored by incorporation of labeled Glc into folding glycoproteins showed a partial (35-50%) decrease in Spgda1 but was not affected in Spynd1 mutants. Results show that, contrary to what has been assumed to date for eukaryotic cells, in S. pombe nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments. It is tentatively suggested that ER-Golgi vesicle transport might be involved in nucleoside diphosphate hydrolysis.

Acid Anhydride Hydrolases↗

PubMed2003

The UDP-glucose:glycoprotein glucosyltransferase is organized in at least two tightly bound domains from yeast to mammals.

The endoplasmic reticulum UDP-Glc:glycoprotein glucosyltransferase (GT) exclusively glucosylates nonnative glycoprotein conformers. GT sequence analysis suggests that it is composed of at least two domains: the N-terminal domain, which composes 80% of the molecule, has no significant similarity to other known proteins and was proposed to be involved in the recognition of non-native conformers and the C-terminal or catalytic domain, which displays a similar size and significant similarity to members of glycosyltransferase family 8. Here, we show that N- and C-terminal domains from Rattus norvegicus and Schizosaccharomyces pombe GTs remained tightly but not covalently bound upon a mild proteolytic treatment and could not be separated without loss of enzymatic activity. The notion of a two-domain protein was reinforced by the synthesis of an active enzyme upon transfection of S. pombe GT null mutants with two expression vectors, each of them encoding one of both domains. Transfection with the C-terminal domain-encoding vector alone yielded an inactive, rapidly degraded protein, thus indicating that the N-terminal domain is required for proper folding of the C-terminal catalytic portion. If, indeed, the N-terminal domain is, as proposed, also involved in glycoprotein conformation recognition, the tight association between N- and C-terminal domains may explain why only N-glycans in close proximity to protein structural perturbations are glucosylated by the enzyme. Although S. pombe and Drosophila melanogaster GT N-terminal domains display an extremely poor similarity (16.3%), chimeras containing either yeast N-terminal and fly C-terminal domains or the inverse construction were enzymatically and functionally active in vivo, thus indicating that the N-terminal domains of both GTs shared three-dimensional features.

Animals↗

PubMed2003

Quality control and protein folding in the secretory pathway.

The biosynthesis of secretory and membrane proteins in the endoplasmic reticulum (ER) yields mostly properly folded and assembled structures with full biological activity. Such fidelity is maintained by quality control (QC) mechanisms that avoid the production of nonnative structures. QC relies on chaperone systems in the ER that monitor and assist in the folding process. When folding promotion is not sufficient, proteins are retained in the ER and eventually retranslocated to the cytosol for degradation by the ubiquitin proteasome pathway. Retention of proteins that fail QC can sometimes occur beyond the ER, and degradation can take place in lysosomes. Several diseases are associated with proteins that do not pass QC, fail to be degraded efficiently, and accumulate as aggregates. In other cases, pathology arises from the downregulation of mutated but potentially functional proteins that are retained and degraded by the QC system.

Animals↗

PubMed2002

UDP-Glc:glycoprotein glucosyltransferase recognizes structured and solvent accessible hydrophobic patches in molten globule-like folding intermediates.

Protein folding in the cell involves the action of different molecular chaperones and folding-facilitating enzymes. In the endoplasmic reticulum (ER), the folding status of glycoproteins is stringently controlled by a glucosyltranferase enzyme (GT) that creates monoglucosylated structures recognized by ER resident lectins (calnexincalreticulin, CNXCRT). GT serves as a folding sensor because it only glucosylates misfolded or partly folded glycoproteins. Nevertheless, the molecular mechanism behind this recognition process remains largely unknown. In this paper we explore the structural determinants for GT recognition by using a single domain model protein. For this purpose we used a family of chemically glycosylated proteins derived from chymotrypsin inhibitor-2 as GT substrates. Structural characterization of species showing higher glucose acceptor capacity suggests that GT recognizes solvent accessible hydrophobic patches in molten globule-like conformers mimicking intermediate folding stages of nascent glycoproteins. It was further confirmed that BiP (binding protein, a chaperone of the heat shock protein 70 family) preferentially recognized neoglycoproteins displaying extended conformations, thus providing a molecular rationale for the sequential BiP-CNXCRT interaction with folding glycoproteins observed in vivo.

Amino Acid Sequence↗

PubMed2002

Protein sweetener.

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Endoplasmic Reticulum↗