THE C. ELEGANS UDP-GLUCOSE:GLYCOPROTEIN GLUCOSYLTRANSFERASE HOMOLOGUES HAVE NOT IDENTICAL FUNCTIONS

BUZZI, LUCILA INÉS; SIMONETTA, SERGIO H; PARODI, ARMANDO; RAYES, DIEGO; ALKEMA, MARK; CASTRO, OLGA A.

Valparaiso y Santiago de Chile

Otro; Small Brains Big Ideas Plus; 2012

Facultad de Medicina, Universidad de Chile

The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the key component of the glycoprotein folding quality control mechanism that takes place in the endoplasmic reticulum (ER). It behaves as a sensor of glycoprotein conformation as it exclusively glucosylates glycoproteins not displaying their native conformations. The addition of this glucose residue enables the interaction of folding intermediates with Calnexin/Calreticulin. An enzymatically active UGGT is encoded by a single gene in S. pombe, D. melanogaster, T. cruzi and plants. There are two homologues coding for UGGT-like proteins in Euteleostomi and in the genus Caenorhabditis. Both UGGT homologues (HUGT1 and HUGT2) are expressed in human cells, the former but not the latter displayed UGGT activity and was upregulated under ER stress conditions. Bioinformatics analysis showed that in C.elegans there are two genes uggt-1 and uggt-2 coding for UGGT homologues. Here we report that C. elegans expresses an active UGGT protein (UGGT-1), while UGGT-2 lacks enzymatic activity. Chimeric proteins combining the N-terminus and C-terminus domain of C. elegans and S. pombe showed that the C- terminal domain of CeUGGT-2 is not able to recognize UDP-Glucose (UDP-Glc). Western Blot analysis showed that all these proteins are expressed in the ER and have the correct molecular weight. uggt-2 is an essential gene and heterozygous uggt-2 +/- worms present a delay in development. uggt-2 -/- eggs are arrested and those that manage to develop to L1 look sluggish and sick and cannot progress to further larval stages. We constructed transgenic worms carrying the Puggt-1::gfp construct and found that UGGT-1 is expressed in cells of the nervous system, but fosmid constructions demonstrated that it is expressed in the intestine, the pharynx and the reproductive system as well. Real time experiments indicate that UGGT-1 is induced under ER stress conditions. Depletion of UGGT-1 by RNA interference resulted in a reduced lifespan and that of UGGT-1 and UGGT-2 in a delay in development.