Role of N-Linked Oligosaccharide Recognition, Glucose Trimming, And
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Proc. Nati. Acad. Sci. USA Vol. 91, pp. 913-917, February 1994 Cell Biology Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control (Ifluenza hemagglutinio/vesicular stomafitis virus glycoproten/glucosidasc lnhlbitors/chaperones/endoplasmic reticulum) CRAIG HAMMOND, INEKE BRAAKMAN*, AND AmI HELENIUS Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510 Communicated by Richard D. Klausner, September 24, 1993 ABSTRACT Using a pulse-chase approach combined with brane-bound ER chaperone called calnexin (or p88 or 1P90). immunoprecipitation, we showed that newly synthesized influ- Calnexin, a 64-kDa type I membrane protein, has been shown enza virus hemagglutinin (HA) and vesicular stomatitis virus G to interact transiently with a variety ofmembrane and soluble protein ssociate transiently during their folding with calnexin, glycoproteins in the ER, and it is thought to have a chaper- a membranebound endoplasmic reticulum (ER) chaperone. one-like function in the retention and oligomeric assembly of Inhibitors ofN-linked glycosylation (tunicamycin) and glucosi- several important membrane glycoproteins (10-15). The re- dases I and II (castanospermine and 1-deoxynojirimycin) prew sults reported here lead us to propose a hypothesis which vented the association, whereas inhibitors of ER a-mannosi- includes calnexin as one of several key players in a folding dases did not. Our results indicated that binding of these viral and quality control system. The model helps explain why glycoproteins to calnexin correlated closely with the composi- proteins need carbohydrates for maturation and why tion oftheir N-linked oligosaccharide side chains. Proteins with N-linked oligosaccharides undergo trimming in the ER. monoglucosylated oligosaccharides were the most likely bind- ing species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on MATERIALS AND METHODS glycoproteins, we propose that the ER contains a unique folding Cell Lines, Viruses, and Reagents. The X31 strain of influ- and quality control machinery in which calnein acts as a enza virus, wild-type VSV, and the temperature-sensitive chaperone that binds proteins with partially glucose-timmed ts045 mutant of VSV were all propagated and used as carbohydrate side chains. In this model glucosidases I and H previously described (16, 17). Chinese hamster ovary (CHO) serve as signal modifiers and UDP-glucose:glycoprotein gluc- 15B cells are defective in N-acetylglucosaminyltransferase I osyltransferase, as a folding sensor. and thus allow trimming of N-linked carbohydrates to Man5GlcNAc2 without further modification (17, 18). All The lumen of the endoplasmic reticulum (ER) provides a reagents were purchased from Sigma with the exceptions of highly specialized compartment for the folding and oligo- 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfo- meric assembly of secretory proteins, plasma membrane nate (CHAPS) from Pierce and 1-deoxynojirimycin and proteins, and proteins destined for the various organelles of 1-deoxymannojirimycin from Boehringer Mannheim. A mix- the vacuolar system. Their conformational maturation is a ture of [35S]methionine and [35S]cysteine used for metabolic complex process determined not only by the amino acid labeling was purchased from ICN. The rabbit anti-calnexin sequence but also by post- and cotranslational modifications, antibody, raised against a C-terminal peptide, was kindly by the intralumenal milieu, and by a variety of chaperones provided by John J. M. Bergeron (McGill University). and folding enzymes present (for recent reviews, see refs. 1 Metabolic Labeling and Immunoprecipitation. Infection of and 2). The ER possesses an efficient but still poorly under- CHO and CHO 15B cells with X31 influenza virus or VSV stood "quality control" system to ensure that transport is and subsequent metabolic labeling have previously been limited to properly folded and assembled proteins (3). described in detail (16, 17). With the exception of the exper- Of the covalent modifications that occur in the ER, the iment shown in Fig. 5, cells were metabolically labeled for 1 cotranslational addition of N-linked oligosaccharides in the hr prior to infection to allow detection of calnexin, which is form of a 14-saccharide core unit (Glc3Man9GlcNAc2) is one the short times used for viral ofthe most common (4). Glycosylation inhibitors, mutant cell not efficiently labeled with pulse lines, and site-specific mutagenesis of consensus glycosyla- proteins. Cells were lysed and precipitated with anti-calnexin tion sequences have shown that it is crucial for the folding of antibodies as described (15). Viral proteins were precipitated many, but not all, glycoproteins (5, 6). Without added sugars, as described (16). SDS/PAGE, fluorography, and quantita- proteins misfold, aggregate, and get degraded without trans- tion was performed as described (16). port to the Golgi complex. For optimal folding and secretion, Glycosidase Digestion. After immunoprecipitation of sam- some glycoproteins must undergo a series of early trimming ples, the wash buffer was removed by washing the staphy- steps involving the removal of the three terminal glucose lococcal protein A-Sepharose beads once with deionized residues (7-9). Trimming, catalyzed by glucosidases I and II, water. Then 20 Al of0.05 M sodium citrate buffer, pH 4.4, and begins on the nascent chain and is followed by a series of ER 0.28 unit of jack bean a-mannosidase were added to each a-mannosidase cleavages (4). sample. Samples were incubated at 370C for 24 hr before As part of our studies on the folding of influenza hemag- analysis by SDS/PAGE. glutinin (HA) and vesicular stomatitis virus (VSV) G protein in living cells, we have analyzed the interaction between Abbreviations: ER, endoplasmic reticulum; dNM, 1-deoxynojirimy- these well-characterized viral glycoproteins and a mem- cin; HA, influenza virus hemagglutinin; VSV, vesicular stomatitis virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-pro- panesulfonate. The publication costs of this article were defrayed in part by page charge *Present address: University of Amsterdam, Department of Bio- payment. This article must therefore be hereby marked "advertisement" chemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Amsterdam, The Netherlands. 913 Downloaded by guest on September 23, 2021 914 Cell Biology: Hammond et al. Proc. Natl. Acad Sci. USA 91 (1994) RESULTS with anti-calnexin. Precipitation ofNT was only partial (25% at 0-min chase). As already shown for other glycoproteins To analyze the interaction between the viral glycoproteins (10, 15, 21, 22), it was apparent that HA associated tran- and ER chaperones, we used a pulse-chase approach already siently with calnexin. Quantitation by densitometry (Fig. ic) extensively utilized in our laboratory to study the folding and indicated that the til2 for calnexin binding of HA was 5 min, oligomerization of HA and G protein (16, 17). Both are type with minimal binding observed after 15 min. For G I membrane proteins with the majority of their mass in the protein, lumen ofthe ER. HA has seven N-linked carbohydrates and the binding peaked about 5 min after the pulse and dropped six intrachain disulfides (19), whereas G protein has two rapidly thereafter. These results showed that the viral pro- N-linked carbohydrates and, most likely, seven intrachain teins do not undergo initial folding as free monomers in disulfides (20). CHO 15B cells were first labeled with P5S]me- solution, but they do so in association with calnexin. thionine for 60 min to incorporate radioactivity into resident Recent studies by Bergeron and co-workers (15) have lumenal proteins. The cells were then infected with virus and, shown that in HepG2 cells only glycoproteins associate with 5 to 6 hr after infection, pulsed with [35Simethionine for 2-3 calnexin. This conclusion was supported by our observation min and chased in medium containing 5 mM unlabeled me- that tunicamycin, a glycosylation inhibitor, blocked the as- thiomne and0.5 mM cycloheximide. At different chase times, sociation of HA and G protein with calnexin (Fig. 2). Since the cells were treated with a membrane-penetrating alkylat- both proteins are unable to fold properly when synthesized in ing agent, N-ethylmaleimide, to block any free sulfhydryl the presence of tunicamycin (23, 24), it was apparent that groups, and lysed with a zwitterionic detergent, CHAPS. The incomplete folding alone is not sufficient for their attachment postnuclear supernatant was immunoprecipitated by using to calnexin; the proteins must also have N-linked carbohy- antibodies to the viral glycoproteins or calnexin. drates. When analyzed under nonreducing conditions, the anti-HA This raised the possibility that calnexin binds to the precipitates gave a series of heterogeneous bands on SDS/ N-linked oligosaccharides of newly synthesized proteins. To PAGE faster than the fully reduced proteins (Fig. la). explore it further, we tested the effects of castanospermine According to the previously established nomenclature (16), and 1-deoxynojirimycin (dNM), which inhibit glucosidases I the incompletely oxidized folding intermediates were labeled and II and thus prevent the trimming of the three glucoses IT1 and IT2, while the band corresponding to the fully from the core oligosaccharide (7). The