PubMed · 8753398
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S Matsushima, T Tanaka. 1995. [Calcium-binding proteins].. https://pubmed.ncbi.nlm.nih.gov/8753398/
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The roles of the endoplasmic-reticulum lectins calnexin and calreticulin in the folding of tissue-type plasminogen activator (tPA) have been investigated using an in vitro translation system that reconstitutes these processes as they would occur in the intact cell. Using co-immunoprecipitation of newly synthesized tPA with antibodies to calnexin and calreticulin, it was demonstrated that the interaction of tPA with both lectins was dependent upon tPA glycosylation and glucosidase trimming. When tPA was synthesized in the presence of semi-permeabilized cells under conditions preventing complex formation with calnexin and calreticulin, the translation product had a specific plasminogenolytic activity identical with that when synthesized under conditions permitting interactions with both lectins. Furthermore, complexes of tPA bound to calnexin and calreticulin were shown to be enzymically active. These results demonstrate that calnexin and calreticulin can form a stable interaction with correctly folded tPA; however, such interactions are not required for the synthesis of enzymically active tPA.
Perturbation of the normal functioning of the endoplasmic reticulum (ER) increases the expression of lumenal proteins, such as grp78, and calreticulin. These proteins are retained within the compartment by a salvage mechanism involving the recognition of a C-terminal tetra-peptide sequence by the KDEL receptor. We have investigated whether disrupting normal ER function concomitantly increases the expression of the mRNAs encoding the two mammalian isoforms of the receptor, erd2.1 and erd2.2. Inhibition of N-linked glycosylation of proteins by tunicamycin had no effect upon the levels of the single mRNA species recognized by the erd2.1 probe, or the multiple transcripts detected with the erd2.2 cDNA probe. ER Ca2+ store depletion by thapsigargin did not increase erd2.1 mRNA, but actually caused a decrease in erd2.2 mRNA. Both thapsigargin, and tunicamycin, increased calreticulin secretion from the cells, although this might be due to more than simply saturation of KDEL receptor binding.
Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum. Because they have an affinity for monoglucosylated N-linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding. To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells. Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution. In this system, addition of a glucose donor, UDP-glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP-glucose enhanced the amount of folded transferrin and reduced the disulfide-linked aggregates. Analysis of transferrin folding in briefly heat-treated microsomes revealed that UDP-glucose was also effective in elimination of heat-induced misfolding. Incubation of the microsomes with an alpha-glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex. Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.