ENHANCED FOLDING AND PROCESSING OF A DISULFIDE MUTANT OF THE HUMAN ASIALOGLYCOPROTEIN RECEPTOR H2B SUBUNIT

Unfolded forms of the H2b subunit of the human asialoglycoprotein rece ptor, a galactose-specific C-type lectin, are degraded in the endoplas mic reticulum (ER), whereas folded forms of the protein can mature to the cell surface (Wikstrom, L., and Lodish, H. F. (1993) J. Biol. Chem . 268, 14412-14416). There are eight cysteines in the exoplasmic domai n of the protein, forming four disulfide bonds in the folded protein, We have constructed double cysteine to alanine mutants for each of the four disulfide bonds and examined the folding and metabolic fate of e ach of the mutants in transfected 3T3 fibroblasts, We find that mutati on of the two cysteines nearest to the transmembrane region (C1) does not prevent proper folding of the protein, whereas mutations of the ot her three disulfides prevent proper folding of the protein and all of the mutant proteins are degraded in the ER, A normal (similar to 20%) fraction of the C1 mutant protein exits the endoplasmic reticulum and is processed in the Gels complex, and it does so at a faster rate comp ared to the wild-type, Furthermore, the folded form of this mutant pro tein is more resistant to unfolding by dithiothreitol than the wild-ty pe. The CI mutant protein is expressed on the cell surface and can for m a functional receptor with the H1 subunit with similar binding affin ities for natural ligands as that of the wild-type receptor, The same fraction of newly made mutant and wild-type proteins (similar to 80%) remain in the ER, but the mutant protein is degraded more quickly, Thu s, the presence of the C1 disulfide bond in the wild-type receptor bot h reduces the rate of protein folding and exit to the Golgi and slows the rate of ER degradation of the portion (similar to 80%) of the rece ptor that never folds properly.