Monitoring acquisition of glycoprotein tertiary and quaterenary structures in the endoplasmic reticulum

JJ CARAMELO OA CASTRO Y ARMANDO PARODI

Ashburn, Virginia, USA. Septembere 26-29 2006

Otro; Howard Hughes Meeting of International Research Scholar; 2006

Howard Hughes Medical Institute

Monitoring acquisition of glycoprotein  tertiary and quaternary structures in the endoplasmic reticulum ARMANDO J. PARODI, Leloir Institute Foundation,Buenos Aires, ArgentinaRMANDO J. PARODI, Leloir Institute Foundation,Buenos Aires, Argentina J.J. Caramelo, O.A. Castro, A.J. Parodi ?¡ Leloir Institute Foundation, Buenos Aires, Argentina Foundation, Buenos Aires, Argentina ?¡ Leloir Institute Foundation, Buenos Aires, Argentina ?¡ Both the folding status of glycoprotein monomers and the assembly of multimeric glycoprotein complexes are stringently controlled in the endoplasmic reticulum (ER) by a glucosyltransferase (GT) that creates monoglucosylated structures recognized by ER-resident lectins (calnexin and calreticulin). Monoglucosylated glycoproteinlectin interaction prevents exit to the Golgi of structurally faulty macromolecules. Given that it only glucosylates incompletely assembled glycoprotein multimers and glycoprotein monomers that do not display their native structures, GT serves as a folding/assembly sensor. To explore the structural determinants for GT-mediated glucosylation, we used as acceptor substrates a family of chemically glycosylated proteins derived from chymotrypsin inhibitor 2. Structural characterization of species showing higher glucose acceptor capacity indicated that GT recognizes solvent-accessible hydrophobic  patches in molten globule-like conformers mimicking glycoprotein advanced folding stages. Further,to determine if the GT-derived quality control is operative with conformers displaying structures  closely resembling those of native species and that, therefore, no alternative ER retaining mechanisms are required when glycoproteins approach their native folding, we generated and used as acceptor substrates neoglycoproteins bearing conformations mimicking the very last-stage folding structures adopted by glycoproteins entering the ER lumen. Results obtained demonstrate that GT is an exquisite  sensor of glycoprotein conformations, able to recognize folding intermediates only slightly differingfrom the native species. Finally, results obtained using isolated subunits of a homotetrameric glycoprotein (soybean agglutinin) as acceptor substrate showed that GT is also able to recognize hydrophobic interfaces exposed by properly folded subunits not yet forming part of complete multimeric complexes, thus ensuring ER retention of incompletely assembled multimeric species. The same mechanism operates, therefore, to monitor glycoprotein acquisition of both tertiary and  quaternary structures complexes are stringently controlled in the endoplasmic reticulum (ER) by a glucosyltransferase (GT) that creates monoglucosylated structures recognized by ER-resident lectins (calnexin and calreticulin). Monoglucosylated glycoproteinlectin interaction prevents exit to the Golgi of structurally faulty macromolecules. Given that it only glucosylates incompletely assembled glycoprotein multimers and glycoprotein monomers that do not display their native structures, GT serves as a folding/assembly sensor. To explore the structural determinants for GT-mediated glucosylation, we used as acceptor substrates a family of chemically glycosylated proteins derived from chymotrypsin inhibitor 2. Structural characterization of species showing higher glucose acceptor capacity indicated that GT recognizes solvent-accessible hydrophobic  patches in molten globule-like conformers mimicking glycoprotein advanced folding stages. Further,to determine if the GT-derived quality control is operative with conformers displaying structures  closely resembling those of native species and that, therefore, no alternative ER retaining mechanisms are required when glycoproteins approach their native folding, we generated and used as acceptor substrates neoglycoproteins bearing conformations mimicking the very last-stage folding structures adopted by glycoproteins entering the ER lumen. Results obtained demonstrate that GT is an exquisite  sensor of glycoprotein conformations, able to recognize folding intermediates only slightly differingfrom the native species. Finally, results obtained using isolated subunits of a homotetrameric glycoprotein (soybean agglutinin) as acceptor substrate showed that GT is also able to recognize hydrophobic interfaces exposed by properly folded subunits not yet forming part of complete multimeric complexes, thus ensuring ER retention of incompletely assembled multimeric species. The same mechanism operates, therefore, to monitor glycoprotein acquisition of both tertiary and  quaternary structures Both the folding status of glycoprotein monomers and the assembly of multimeric glycoprotein complexes are stringently controlled in the endoplasmic reticulum (ER) by a glucosyltransferase (GT) that creates monoglucosylated structures recognized by ER-resident lectins (calnexin and calreticulin). Monoglucosylated glycoproteinlectin interaction prevents exit to the Golgi of structurally faulty macromolecules. Given that it only glucosylates incompletely assembled glycoprotein multimers and glycoprotein monomers that do not display their native structures, GT serves as a folding/assembly sensor. To explore the structural determinants for GT-mediated glucosylation, we used as acceptor substrates a family of chemically glycosylated proteins derived from chymotrypsin inhibitor 2. Structural characterization of species showing higher glucose acceptor capacity indicated that GT recognizes solvent-accessible hydrophobic  patches in molten globule-like conformers mimicking glycoprotein advanced folding stages. Further,to determine if the GT-derived quality control is operative with conformers displaying structures  closely resembling those of native species and that, therefore, no alternative ER retaining mechanisms are required when glycoproteins approach their native folding, we generated and used as acceptor substrates neoglycoproteins bearing conformations mimicking the very last-stage folding structures adopted by glycoproteins entering the ER lumen. Results obtained demonstrate that GT is an exquisite  sensor of glycoprotein conformations, able to recognize folding intermediates only slightly differingfrom the native species. Finally, results obtained using isolated subunits of a homotetrameric glycoprotein (soybean agglutinin) as acceptor substrate showed that GT is also able to recognize hydrophobic interfaces exposed by properly folded subunits not yet forming part of complete multimeric complexes, thus ensuring ER retention of incompletely assembled multimeric species. The same mechanism operates, therefore, to monitor glycoprotein acquisition of both tertiary and  quaternary structures