RESUMEN
Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of calnexin and calreticulin were largely interchangeable. We also determined that a soluble form of calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin bound to a similar set of glycoproteins as calnexin. Therefore, the different topological environments of calnexin and calreticulin are important in determining their distinct substrate specificities.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Retículo Endoplásmico/química , Ribonucleoproteínas/fisiología , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Calnexina , Calreticulina , Línea Celular , Membrana Celular/química , Citoplasma/química , Drosophila melanogaster , Ratones , Mutagénesis , Pliegue de Proteína , Estructura Terciaria de Proteína , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Especificidad por Sustrato , Factores de Tiempo , Transfección , Microglobulina beta-2/metabolismoRESUMEN
Calreticulin (CRT) is thought to be a molecular chaperone that interacts with glycoproteins exclusively through a lectin site specific for monoglucosylated oligosaccharides. However, this chaperone function has never been directly demonstrated nor is it clear how lectin-oligosaccharide interactions facilitate glycoprotein folding. Using purified components, we show that CRT suppresses the aggregation not only of a glycoprotein bearing monoglucosylated oligosaccharides but also that of non-glycosylated proteins. Furthermore, CRT forms stable complexes with unfolded, non-glycosylated substrates but does not associate with native proteins. ATP and Zn(2+) enhance CRT's ability to suppress aggregation of non- glycoproteins, whereas engagement of its lectin site with purified oligosaccharide attenuates this function. CRT also confers protection against thermal inactivation and maintains substrates in a folding-competent state. We conclude that in addition to being a lectin CRT possesses a polypeptide binding capacity capable of discriminating between protein conformational states and that it functions in vitro as a classical molecular chaperone.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Chaperonas Moleculares/fisiología , Ribonucleoproteínas/fisiología , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Calreticulina , Cloruros/metabolismo , Citrato (si)-Sintasa/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Escherichia coli/metabolismo , Glicosilación , Inmunoglobulinas/metabolismo , Lectinas/metabolismo , Malato Deshidrogenasa/metabolismo , Microscopía Electrónica , Oligosacáridos/metabolismo , Unión Proteica , Pliegue de Proteína , Conejos , Dispersión de Radiación , Temperatura , Factores de Tiempo , Compuestos de Zinc/metabolismoRESUMEN
Although calnexin is thought to function as a molecular chaperone for glycoproteins, a prevalent view is that it cannot distinguish between protein conformational states, binding solely through its lectin site to monoglucosylated oligosaccharides. Using purified components in vitro, calnexin effectively prevented the aggregation not only of glycoproteins bearing monoglucosylated oligosaccharides but also proteins lacking N-glycans, an effect enhanced by ATP. It also suppressed the thermal denaturation of nonglycosylated proteins and enhanced their refolding in conjunction with other cellular components. Calnexin formed stable complexes with unfolded conformers of these proteins but not with the native molecules. Therefore, in addition to being a lectin, calnexin functions as a bona fide molecular chaperone capable of interacting with polypeptide segments of folding glycoproteins.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Chaperonas Moleculares/metabolismo , Lectinas de Plantas , Proteínas de Soja , Adenosina Trifosfato/farmacología , Animales , Proteínas de Unión al Calcio/efectos de los fármacos , Calnexina , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , Perros , Glucosa , Glicosilación , Calor , Lectinas/química , Lectinas/metabolismo , Modelos Químicos , Chaperonas Moleculares/efectos de los fármacos , Oligosacáridos/farmacología , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin. We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222 as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antiportadores/metabolismo , Antígenos H-2/metabolismo , Inmunoglobulinas , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Sitios de Unión/genética , Transporte Biológico Activo , Línea Celular , Membrana Celular/inmunología , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Antígenos H-2/genética , Antígeno de Histocompatibilidad H-2D , Humanos , Cinética , Sustancias Macromoleculares , Proteínas de Transporte de Membrana , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Calnexin, a membrane protein of the endoplasmic reticulum, is generally thought to function as a molecular chaperone, based on indirect or correlative evidence. To examine calnexin's functions more directly, we reconstituted the assembly of class I histocompatibility molecules in the absence or presence of calnexin in Drosophila melanogaster cells. Calnexin enhanced the assembly of class I heavy chains with beta 2-microglobulin as much as 5-fold. The improved assembly appeared largely due to more efficient folding of heavy chains, as evidenced by increased reactivity with a conformation-sensitive monoclonal antibody and by a reduction in the level of aggregates. Similar findings were obtained in mouse or human cells when the interaction of calnexin with class I heavy chains was prevented by treatment with the oligosaccharide processing inhibitor castanospermine. The ability of calnexin to facilitate castanospermine. The ability of calnexin to facilitate heavy chain folding and to prevent the formation of aggregates provides compelling evidence that calnexin functions as a bona fide molecular chaperone.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Calnexina , Membrana Celular/efectos de los fármacos , Membrana Celular/inmunología , Células Cultivadas , Drosophila melanogaster , Antígeno HLA-B27/química , Antígeno HLA-B27/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Indolizinas/farmacología , Ratones , Oligosacáridos/metabolismo , Pliegue de Proteína , Transfección , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
The transporter associated with antigen processing (TAP) delivers cytosolic peptides into the endoplasmic reticulum (ER) where they bind to nascent class 1 histocompatibility molecules. Class 1-peptide complexes are then displayed at the cell surface for recognition by cytotoxic T lymphocytes. Immunoprecipitation of either TAP or class 1 molecules revealed an association between the transporter and diverse class 1 products. TAP bound preferentially to heterodimers of the class 1 heavy chain and beta 2-microglobulin, and the complex subsequently dissociated in parallel with transport of class 1 molecules from the ER to the Golgi apparatus. The TAP-class 1 complexes could also be dissociated in vitro by the addition of class 1-binding peptides. The association of class 1 molecules with TAP likely promotes efficient capture of peptides before their exposure to the lumen of the ER.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Presentación de Antígeno , Proteínas Portadoras/metabolismo , Antígenos H-2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Secuencia de Aminoácidos , Animales , Transporte Biológico , Proteínas Portadoras/inmunología , Línea Celular , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Sueros Inmunes , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Microglobulina beta-2/metabolismoRESUMEN
Assembled class I histocompatibility molecules, consisting of heavy chain, beta 2-microglobulin, and peptide ligand, are transported rapidly to the cell surface. In contrast, the intracellular transport of free heavy chains or peptide-deficient heavy chain-beta 2-microglobulin heterodimers is impaired. A 90-kilodalton membrane-bound chaperone of the endoplasmic reticulum (ER), termed calnexin, associates quantitatively with newly synthesized class I heavy chains, but the functions of calnexin in this interaction are unknown. Class I subunits were expressed alone or in combination with calnexin in Drosophila melanogaster cells. Calnexin retarded the intracellular transport of both peptide-deficient heavy chain-beta 2-microglobulin heterodimers and free heavy chains. Calnexin also impeded the rapid intracellular degradation of free heavy chains. The ability of calnexin to protect and retain class I assembly intermediates is likely to contribute to the efficient intracellular formation of class I-peptide complexes.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana/metabolismo , Microglobulina beta-2/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Calnexina , Línea Celular , Drosophila melanogaster , Aparato de Golgi/metabolismo , Datos de Secuencia Molecular , Temperatura , TransfecciónRESUMEN
Previously, we showed that an 88-kD protein (p88) associates rapidly and quantitatively with newly synthesized murine major histocompatibility complex class I molecules within the endoplasmic reticulum (ER). This interaction is transient and dissociation of p88 appears to be rate limiting for transport of class I molecules from the ER to the Golgi apparatus. In this report, we examine the relationship between p88 interaction and assembly of the ternary complex of class I heavy chain beta 2-microglobulin (beta 2m), and peptide ligand. In both murine and human beta 2m-deficient cells, in which little or no transport of class I heavy chains is observed, p88 remained associated with intracellular heavy chains throughout their lifetime. In murine RMA-S cells, which are apparently defective in accumulating peptide ligands for class I within the ER, prolonged association of p88 with "empty" heavy chain-beta 2m heterodimers was also observed. However, p88 dissociated slowly in parallel with the slow rate of ER to Golgi transport of empty class I molecules in these cells. The close correlation between p88 association and impaired class I transport suggests that p88 functions to retain incompletely assembled class I molecules in the ER. We propose that conformational changes in class I heavy chains induced by the binding of both beta 2m and peptide are required for efficient p88 dissociation and subsequent class I transport.