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Glucosidase II is an ER resident glycoprotein involved in the processing of N-linked glycans and probably a component of the ER quality control of glycoproteins. For cloning of glucosidase II cDNA, degenerate oligonucleotides based on amino acid sequences derived from proteolytic fragments of purified pig liver glucosidase II were used. An unamplified cDNA library from pig liver was screened with a 760 bp glucosidase II specific cDNA fragment obtained by RT-PCR. A 3.9 kb glucosidase II cDNA with an open reading frame of about 2.9 kb was obtained. The glucosidase II sequence did not contain known ER retention signals nor hydrophobic regions which could represent a transmembrane domain; however, it contained a single N-glycosylation site close to the amino terminus. All studied pig and rat tissues exhibited an mRNA of approximately 4.4 kb with varying tissue expression levels. The authenticity of the identified cDNA with that coding for glucosidase II was proven by overexpression in CHO cells. Mouse lymphoma PHAR 2.7 cells, deficient in glucosidase II activity, were shown to be devoid of transcripts.

Mutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t 1/2 of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t 1/2 ‫؍‬ 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t 1/2 ‫؍‬ 1.8 hours) but in a proteasome-and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants. The endoplasmic reticulum (ER) represents a site of quality control of glycoprotein folding. 1,2 Misfolded glycoproteins are recognized and retained by the concerted action of chaperones, lectins, and modifying enzymes such as UDP-glucose:glycoprotein glucosyltransferase and glucosidase II. 3 Glycoproteins failing to achieve their correct conformation might become retrotranslocated to the cytosol 4 and degraded by the ubiquitin-proteasome pathway, a process referred to as ER-associated protein degradation. [5][6][7] Quality control of protein folding is of importance in congenital diseases caused by point mutations that result in the synthesis of misfolded glycoproteins. 8 Mutations in the water channel aquaporin-2 (AQP2) can cause nephrogenic diabetes insipidus (NDI), in which patients are unable to concentrate urine in response to the anti-diuretic hormone arginine-vasopressin. 9 -11 If not corrected, this defect results in a deregulated whole-body water homeostasis that is accompanied by various symptoms of dehydration. 12 AQP2 belongs to the large family of AQPs, 13,14 and is a 29-kd polytope membrane protein that contains a single N-glycosylation site and two phosphorylation sites, and is present in the principal cells of renal collecting ducts. 15,16 In states of hypernatremia or hypovolemia, translocation of phosphorylated AQP2 homotetramers from vesicles to the apical plasma membrane of the principal cells is triggered by a signal transduction cascade induced by arginine-vasopressin. 16 -18 Alike to normal human kidney, 19 wild-type (wt) AQP2, when expressed in Xenopus ooc...

Chicken mitochondrial and Escherichia coli aspartate aminotransferases K258H, in which the active site lysine residue has been exchanged for a histidine residue, retain partial catalytic competence [Ziak et al. (1993) Eur. J. Biochem. 211, 475-484]. Mutant PLP and PMP holoenzymes and the complexes of the latter (E. coli enzyme) with sulfate and 2-oxoglutarate, as well as complexes of the mitochondrial apoenzyme with N-(5'-phosphopyridoxyl)-L-aspartate or N-(5'-phosphopyridoxyl)-L-glutamate, were crystallized and analyzed by means of X-ray crystallography in order to examine how the side chain of histidine 258 can substitute as a general acid/base catalyst of the aldimine-ketimine tautomerization in enzymic transamination. The structures have been solved and refined at resolutions between 2.1 and 2.8 A. Both the closed and the open conformations, identical to those of the wild-type enzyme, were observed, indicating that the mutant enzymes of both species exhibit the same conformational flexibility as the wild-type enzymes, although in AspAT K258H the equilibrium is somewhat shifted toward the open conformation. The replacement of the active site K258 by a histidine residue resulted only in local structural adaptations necessary to accommodate the imidazole ring. The catalytic competence of the mutant enzyme, which in the forward half-reaction is 0.1% of that of the wild-type enzyme, suggests that the imidazole group is involved in the aldimine-ketimine tautomerization. However, the imidazole ring of H258 is too far away from C alpha and C4' of the coenzyme-substrate adduct for direct proton transfer, suggesting that the 1,3-prototropic shift is mediated by a water molecule. Although there is enough space for a water molecule in this area, it has not been detected. Dynamic fluctuations of the protein matrix might transiently open a channel, giving a water molecule fleeting access to the active site.

In tissues of higher organisms homopoly-