Calnexin and Calreticulin Bind to Enzymically Active Tissue-Type
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Biochem. J. (1997) 328, 113–119 (Printed in Great Britain) 113 Calnexin and calreticulin bind to enzymically active tissue-type plasminogen activator during biosynthesis and are not required for folding to the native conformation Simon ALLEN and Neil J. BULLEID1 University of Manchester, School of Biological Sciences, 2.205, Stopford Building, Oxford Road, Manchester M13 9PT, U.K. The roles of the endoplasmic-reticulum lectins calnexin and cells under conditions preventing complex formation with cal- calreticulin in the folding of tissue-type plasminogen activator nexin and calreticulin, the translation product had a specific (tPA) have been investigated using an in itro translation system plasminogenolytic activity identical with that when synthesized that reconstitutes these processes as they would occur in the under conditions permitting interactions with both lectins. Fur- intact cell. Using co-immunoprecipitation of newly synthesized thermore, complexes of tPA bound to calnexin and calreticulin tPA with antibodies to calnexin and calreticulin, it was demon- were shown to be enzymically active. These results demonstrate strated that the interaction of tPA with both lectins was that calnexin and calreticulin can form a stable interaction with dependent upon tPA glycosylation and glucosidase trimming. correctly folded tPA; however, such interactions are not required When tPA was synthesized in the presence of semi-permeabilized for the synthesis of enzymically active tPA. INTRODUCTION that deglucosylated glycoproteins are reglucosylated by UDP- glucose:glycoprotein glucosyltransferase. This glucosyltrans- Calnexin, a 64 kDa transmembrane protein, and calreticulin, a ferase had been shown to be specific for oligosaccharides on 45 kDa soluble lumenal homologue of calnexin [1], are thought non-native proteins with no activity towards oligosaccharides on to be involved in protein folding, retention and quality control in native proteins [19]. These observations have led Helenius and the endoplasmic reticulum (ER). Calnexin was initially demon- co-workers to propose that as long as a protein is non-native it strated to interact with assembling MHC class I molecules [2] in is a target for reglucosylation and hence a ligand for calnexin and the ER, and has since been shown to interact with a diverse set calreticulin, and thus retained in the ER [1,5,7,16]. Additionally, of glycoproteins [3]. Several lines of evidence suggest calnexin is there has been a number of non-glycosylated proteins shown to a component of the ER quality-control system. Dissociation of co-precipitate with calnexin antibodies [12,20,21], and some calnexin from proteins correlates well with maturation events. glycoproteins have been shown to remain calnexin bound on During the folding of MHC class I [4], influenza haemagglutinin enzymic deglycosylation [15]. These observations led to the (HA) [5–7] and transferrin [3], calnexin dissociation correlates notion that the lectin interaction is a prelude to a stronger with completion of disulphide-bond formation. During MHC protein–protein interaction [15]. This concept is now losing class I assembly, calnexin dissociates on formation of heavy- favour for a number of reasons. For example, glucosidase- chain-β#-microglobulin dimers [8,9]. Exit of MHC class I [2] and deficient cells are devoid of substrates binding to calnexin [22]. A MHC class II [10] from the ER closely follows calnexin dis- study with vesicular stomatitis virus G protein has demonstrated sociation. Evidence that calnexin can prevent export of unas- that the protein will co-immunoprecipitate with calnexin in sembled oligomeric complexes was provided by co-expression of glycosylation-dependent fashion; however, the non-glycosylated calnexin with either MHC class I heavy chains, heavy-chain- protein will only co-immunoprecipitate if aggregated [23]. β#microglobulin dimers [11] or the T-cell receptor ε-subunit [12] Finally, two recent studies have shown that binding of ribo- in Drosophila melanogaster cells. The above studies have provided nuclease to calnexin and calreticulin is independent of pro- correlative evidence that calnexin and calreticulin are involved in tein conformation and solely dependent upon whether the subunit assembly of oligomeric proteins. Direct evidence for protein bears monoglucosylated oligosaccharides [24,25]. These such a function for calnexin and calreticulin has been provided studies have cast doubt on the functional relevance of non- by the observations that both lectins increase the efficiency of glycosylated and deglycosylated proteins forming stable inter- folding and assembly and suppress degradation of HA syn- actions with calnexin and calreticulin and suggest that an thesized in microsomes [13]. Similarly, calnexin has been shown aggregation phenomenon is responsible. This has favoured the to have similar effects on MHC class I expressed in D. melano- idea that calnexin and calreticulin function exclusively as lectins. gaster cells [14]. To extend these studies with a different model protein, we have Calnexin [7,15] and calreticulin [16,17] have been shown to used acquisition of enzymic activity, the definitive measure of associate specifically with proteins bearing monoglucosylated protein folding, to investigate the specificities and roles of oligosaccharides. Parodi and co-workers [18] have demonstrated interactions of calnexin and calreticulin with glycoproteins. Using Abbreviations used: BiP, Ig heavy-chain-binding protein; CHO, Chinese hamster ovary; dNM, deoxynojirimycin; ER, endoplasmic reticulum; HA, influenza haemagglutinin; IAA, iodoacetic acid; NLT, benzoyl-asparaginyl-leucinyl-threoninyl-N-methylamide; SP-cells, semi-permeabilized cells; tPa, tissue-type plasminogen activator. 1 To whom correspondence should be addressed. 114 S. Allen and N. J. Bulleid co-immunoprecipitation of tissue-type plasminogen activator stock in DMSO) then incubated on ice for 5 min. To terminate (tPA) with antisera raised to calnexin and calreticulin and analysis permeabilization, KHM (8 ml) was added, and the cells were of the precipitates by SDS}PAGE, it was demonstrated that tPA pelleted then incubated in 50 mM Hepes, pH 7±2, containing interacts with both lectins during its synthesis in a manner 90 mM potassium acetate (10 ml), on ice for 10 min. The cells dependent upon glucose trimming of its oligosaccharides. To were pelleted and resuspended in KHM (100 µl). Endogenous investigate whether the binding of calnexin and calreticulin was mRNA was removed by adding calcium chloride to a con- required for the correct folding of tPA, we studied the kinetics of centration of 1 mM and staphylococcal nuclease to 10 µg}ml and folding of tPA synthesized in the presence or absence of incubating at room temperature for 12 min. The reaction was deoxynojirimycin (dNM) in semi-permeabilized cells (SP-cells). terminated by the addition of EGTA to a concentration of No significant difference in the specific activity of tPA synthesized 4 mM. The cells were pelleted and resuspended in KHM (100 µl). in the presence and the absence of dNM was observed. This demonstrated that the oligosaccharide-dependent interactions of In vitro transcription and translation of tPA calnexin and calreticulin with tPA were not required for the folding of this protein. When calnexin and calreticulin immuno- Synthesis of wild-type tPA mRNA with T7 RNA polymerase has precipitates were analysed for tPA activity, they were found to been described previously [30]. The mRNA transcript encoding $& be enzymically active. This indicated that calnexin and cal- wild-type tPA was translated and the S-labelled products were reticulin could form stable oligosaccharide-dependent inter- translocated using the following translation reaction mixtures. actions with native tPA during biosynthesis in the endoplasmic When microsomes were used, each translation reaction consisted reticulum. of 17±5 µl of Flexi Lysate, 0±5 µl of KCl (1 M), 0±5 µl of amino acids (2 mM) lacking methionine, 1 µl of nuclease-treated dog $& EXPERIMENTAL pancreas microsomes, 1 µl of EASYTAG2 [ S]methionine (10 µCi}µl), 3±5 µl of RNase-free water and 1 µl of tPA mRNA Materials (1 mg}ml). When SP-cells were used, each translation reaction The plasmid pKC3T containing cDNA coding for wild-type tPA consisted of 17±5 µl of Flexi Lysate, 0±5 µl of KCl (1 M), 0±5 µlof amino acids (2 mM) lacking methionine, 4 µl of nuclease-treated was a gift from Dr. Mary-Jane Gething (University of Mel- $& bourne, Melbourne, Australia). The CEM and CEM-NK2 cells SP-cells, 1 µl of EASYTAG2 [ S]methionine (10 µCi}µl), 0±5 µl were gifts from Dr. Peter Cresswell (University of Yale, New of RNase-free water and 1 µl of tPA mRNA (1 mg}ml). Where Haven, CT, U.S.A.). Benzoyl-asparaginyl-leucinyl-threoninyl- indicated, NLT was added to a final concentration of 2 mM to N-methylamide (NLT) was a gift from Dr. Stephen High inhibit core N-linked glycosylation. Where indicated, dNM was (University of Manchester, Manchester, U.K.). Rabbit reticulo- added to a final concentration of 1 mM to inhibit glucose cyte lysate (Flexi Lysate2), amino acid mixture lacking meth- trimming by glucosidase I and glucosidase II. The translation ionine, RNasin and T7 RNA polymerase were purchased from mixtures were incubated at 30 mC for 60 min unless otherwise $& Promega (Madison, WI, U.S.A.). EASYTAG2 [ S]methionine indicated. When the NLT and dNM were included, the trans- was purchased from New England Nuclear (Dreiech, Germany). lation mixtures were pre-incubated at 30 mC for 10 min before the dNM was purchased from Oxford Glycosystems (Oxford, U.K.). addition of tPA mRNA. All translations were terminated by Protein A–Sepharose 4B was purchased from Zymed (San alkylation, by adding iodoacetic acid to a final concentration of Francisco, CA, U.S.A.). Tissue-culture reagents and mouse anti- 20 mM followed by incubation on ice for 10 min; this also (human tPA) monoclonal antibody (clone L172D) was purchased ensured that any remaining free thiols were blocked before from Gibco Life Technologies (Glasgow, Scotland, U.K.). Goat further processing as described below. anti-(human tPA) polyclonal antibody was purchased from Alpha Laboratories (Eastleigh, Hants., U.K.).