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Retention of Glucose Units Added by the UDP-GLC:Glycoprotein Glucosyltransferase Delays Exit of Glycoproteins from the Endoplasmic Reticulum Carlos Labriola, Juan J

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Retention of Glucose Units Added by the UDP-GLC:Glycoprotein Glucosyltransferase Delays Exit of Glycoproteins from the Endoplasmic Reticulum Carlos Labriola, Juan J Retention of Glucose Units Added by the UDP-GLC:Glycoprotein Glucosyltransferase Delays Exit of Glycoproteins from the Endoplasmic Reticulum Carlos Labriola, Juan J. Cazzulo, and Armando J. Parodi Instituto de Investigaciones Bioqufmicas, Fundaci6n Campomar, 1405 Buenos Aires, Argentina Abstract. It has been proposed that the UDP- three potential N-glycosylation sites, two at the cata- Glc:glycoprotein glucosyltransferase, an endoplas- lytic domain and one at the COOH-terminal domain, mic reticulum enzyme that only glucosylates improp- was isolated in a glucosylated form from cells grown in erly folded glycoproteins forming protein-linked the presence of the glucosidase II inhibitor 1-deoxy- GlclMan7.9- GlcNAc2 from the corresponding ungluco- nojirimycin. The oligosaccharides present at the single sylated species, participates together with lectin-like glycosylation site of the COOH-terminal domain were chaperones that recognize monoglucosylated oligosac- glucosylated in some cruzipain molecules but not in charides in the control mechanism by which cells only others, this result being consistent with an asynchro- allow passage of properly folded glycoproteins to the nous folding of glycoproteins in the endoplasmic reticu- Golgi apparatus. Trypanosoma cruzi cells were used to lum. In spite of not affecting cell growth rate or the test this model as in trypanosomatids addition of glu- cellular general metabolism in short and long term in- cosidase inhibitors leads to the accumulation of only cubations, 1-deoxynojirimycin caused a marked delay monoglucosylated oligosaccharides, their formation be- in the arrival of cruzipain to lysosomes. These results ing catalyzed by the UDP-Glc:glycoprotein glucosyl- are compatible with the model proposed by which transferase. In all other eukaryotic cells the inhibitors monoglucosylated glycoproteins may be transiently re- produce underglycosylation of proteins and/or accumu- tained in the endoplasmic reticulum by lectin-like an- lation of oliogosaccharides containing two or three glu- chors recognizing monoglucosylated oligosaccharides. cose units. Cruzipain, a lysosomal proteinase having RANSFER of the oligosaccharide Glc3Man9GlcNAc2 same mannose and with the same bond as in GlclMan9- from a dolichol-P-P derivative to nascent polypep- GlcNAc2-P-P-dolichol. The glucosylating enzyme (UDP- T tide chains is followed by the immediate removal of Glc:glycoprotein glucosyltransferase) has been detected in the glucose units. Two glucosidases have been described to mammalian, plant, fungal, and protozoan cells (38). The be involved in this process: glucosidase I, an et(1, 2) glu- enzyme appeared to be a soluble protein of the lumen of the cosidase that removes the more external residues and glu- endoplasmic reticulum and to have a remarkable property: cosidase II, an e~(1,3)glucosidase responsible for removal it glucosylated in cell-free assays denatured glycoproteins of the other two units. All reactions mentioned above have whereas native species or glycopeptides were not glucosy- been described to occur in the lumen of the endoplasmic lated (12, 36, 38--40). Recognition by the glucosyltrans- reticulum. One or two mannose units may be cleaved in ferase of a protein domain only exposed in denatured con- the same subcellular location (Fig. 1 a) (21). formations was required for the transfer reaction (36). The An additional processing reaction also occurring in the glucosyltransferase behaved, therefore, as a sensor of un- endoplasmic reticulum is the transient glucosylation of folded, partially folded and misfolded conformations. high mannose-type, protein-linked oligosaccharides: as de- Proteins entering the secretory pathway acquire their fi- scribed to occur in mammalian cells GlclMan9GlcNAc2, nal tertiary and in some cases also quaternary structures in GlclMansGlcNAc2 and GlclMan7GlcNAc2 are formed from the lumen of the endoplasmic reticulum. Species that fail the corresponding unglucosylated compounds (Fig. 1 a) to fold properly are retained in that subcellular location (32, 33). The newly added glucose units are immediately where they are proteolytically degraded (24). A very strin- removed by glucosidase II, as the glucoses are linked to the gent quality control is therefore required to prevent pas- sage of misfolded proteins to the Golgi cisternae. A model for such quality control applicable to glycoproteins has Please address all correspondence to A. J. Parodi, Instituto de Investiga- ciones Bioquimicas, Fundacion Carnpomar, Antonio Machado 151, 1405 been recently proposed (16, 18). According to it, high Buenos Aires, Argentina. Tel.: 54 1 88 4015. Fax: 54 1 865 2246. mannose-type oligosaccharides in the endoplasmic reticu- © The Rockefeller University Press, 0021-95251951081"17119 $2.00 The Journal of Cell Biology, Volume 130, Number 4, August 1995 771-779 771 0 b moval of glucose units exclusively added by the UDP-GIc: glycoprotein glucosyltransferase on the time required by %~.GNac~-P-p-D newly synthesized cruzipain, a cysteine proteinase having high mannose-type oligosaccharides, to reach the lyso- G~M,TA~-P' somes (2, 8). This is practically the only known trypanoso- G~MoGNAc e- Pr matid glycoprotein that fulfills the above mentioned requi- G~Ms)GNAc. z- Pr Mi GNAca-P-P-D sites. It is currently accepted that differences in the time ¢ ¢ required for arrival to their final destination among glyco- M~GNAcz-Pr GI M9 GNAc;~-Pr MI; GiAcfPr , ~' G~ MjGNAca-Pr proteins having the same localization (external milieu, MaGNAcEPr . Gl Me GNAc2 - Pr Me GiAca- Pr . G1 Me GNAcz- Pr plasma membrane, lysosomes, etc.) reflect differences in the x time required for leaving the endoplasmic reticulum (24). M~,GNA;2-Pr ~' GI M7 GNAcz- Pr M7 GiAcz- Pr ~ G~M.;, GNAc;(Pr M6GNAca-Pr Ms GiAc z- Pr Materials and Methods Figure 1. Initial glycoprotein processing reactions occurring in Materials mammalian (a) and T. cruzi (b) cells. G stands for glucose; M for Jack bean (x-mannosidase, 1-deoxynojirirnycin (DNJ), 1 monosaccharide mannose; GNAc for N-acetylglucosamine; D for dolichol; and Pr standards, bovine thyroglobulin, p-nitrophenyl-a-o-mannoside, endo-13- for protein. N-acetylglucosaminidase H (Endo H), trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane (E-64) and Streptomyces griseus protease type XIV (Pronase) were from Sigma Chem. Co. (St. Louis, MO). Ham's F-12 me- lum shuttle between monoglucosylated and unglucosy- dium (methionine, proline, and glycine free) was purchased from Bio- lated structures, their formation catalyzed by the UDP- chrom KG (Berlin, Germany). [14C]Glucose (320 Ci/mol) was from ARC (St. Louis, MO) and [35S]methionine was from New England Nuclear Glc:glycoprotein glucosyltransferase and glucosidase II. A (Wilmington , DE). Con A-Sepharose and Sephadex G-25 prepacked membrane-bound chaperone, calnexin, that has a lectin- (NAP 10) columns were from Pharmacia (Uppsala, Sweden). like activity that recognizes the monoglucosylated oli- gosaccharides (16), would bind the monoglucosylated Standards structures, and thus retain glycoproteins in the endoplas- [14C]Man6.9GlcNAe standards from hen oviduct and [glucose-~4C]Glcl - mic reticulum as long as the protein moieties are not prop- ManT_9GlcNAc from rat liver microsomes were prepared as described pre- erly folded. On attaining the correct native conformations, viously (30, 38). Treatment of [14C]Man6.gGlcNAc with ct-mannosidase glycoproteins would become substrates for the glucosidase produced ManGlcNAc and that of GlczMan7.9GlcNAc with the same en- but not for the glucosyltransferase and thus be liberated zyme yielded GlclMana,sGlcNAc. from the calnexin anchor. Glycoproteins would then be Cells able to be transported to the Golgi apparatus. This model predicts that inhibition of removal of the T. cruzi cells of the Tulahuen strain (Tul 2 stock) were grown as described before (7). For the purification of cruzipain, cells from a 20-ml culture glucos e units added by the glucosyltransferase would de- containing 1-2 mCi of [z4C]glucose and where indicated also 6 mM DNJ lay or abolish (depending on how tightly the lectin binds were harvested at a density of 4-6 × 107 cells/ml and mixed with those ob- monoglucosylated oligosaccharides) exit of glycoproteins tained from an unlabeled culture. from the endoplasmic reticulum. This prediction cannot be tested, however, in most eukaryotic cells as glucosidase II Purification and Self-Proteolysis of Cruzipain is responsible for removal of both a(1,3)-linked glucose The proteinase was purified to homogeneity from the mixture of labeled units. Moreover, known inhibitors of glucosidase II also and unlabeled ceils as previously described (6). It was submitted to self- inhibit glucosidase I (21). Addition of those drugs to cells proteolysis as before (19). leads to the accumulation of oligosaccharides having two Preparation of the COOH-Terminal Domain and of or three glucoses that are not recognized by calnexin (16, Glycopeptides from the Catalytic Domain 17, 20). The prediction can be tested, however, in trypanosoma- The autolysis mixture was dialyzed against 5 mM triethylamine-acetate tid protozoa. These parasites are the only known wild-type buffer, pH 7.2. The dialysate was lyophilized and the glycopeptides arising from the catalytic domain were separated from amino acids and small cells in which unglucosylated olgiosaccharides are trans- peptides by gel filtration chromatography through a 57 × 1.2 cm Sepha- ferred in vivo to nascent polypeptide chains and, there- dex G-10 column equilibrated against
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