Calnexin (CNX) is a membrane protein of the ER that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin (CRT), a soluble analog of CNX, is thought ...to possess similar functions but these have never been directly demonstrated. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that CRT 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 CNX and CRT were largely interchangeable. We also determined that a soluble form of CNX (residues 1–387) can functionally replace its membrane-bound counterpart. However, the different topological environments of CNX and CRT are important in determining their distinct substrate specificities. CNX and CRT bind to glycoprotein substrates in part through a lectin site specific for monoglucosylated (Glc1Man7–9GlcNAc 2) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that CNX and CRT can also bind to polypeptide segments of both glycosylated and non-glycosylated proteins. We examined whether polypeptide-based interactions occur between CNX and its substrates in cultured cells. We demonstrate that CNX is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between CNX and diverse proteins in such a system and that the strength of the latter interaction varies substantially between protein substrates. ERp57 is a thiol oxidoreductase that binds to CNX and CRT and, at least in vitro, can promote disulfide bond formation in glycoproteins that interact with CNX or CRT. As yet, there is no direct information concerning how ERp57 functions in class I biogenesis. By using a heterologous Drosophila expression system, we showed that ERp57 does not affect the formation of the disulfide bond in cells expressing free H chain. Furthermore, the rate of class I molecules folding and assembly with β2m remains unchanged in the presence of ERp57. These are important findings that challenge the current view of ERp57 function in class I biogenesis.
The presence of ventricular myosin light chains in the atria of children with congenital heart disease was demonstrated by two-dimensional polyacrylamide gel electrophoresis, peptide mapping, and ...Western blot analysis. Ventricular myosin light chains were present in 27% of biopsies from 91 children with different forms of congenital heart disease. Perimembranous ventricular septal defects and tetralogy of Fallot were associated with the presence of ventricular myosin light chains in 50% of patients. The presence of ventricular myosin light chains in these atria did not correlate with pressure or volume overload. Analysis of myosin heavy chain isotype in the same biopsies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and Western blot analysis indicated that there was no detectable expression of ventricular myosin heavy chain (beta-subunit), suggesting that the genes for the myosin heavy chains and light chains are not expressed coordinately.
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