Identification and biochemical characterization of C.elegans ER GT.

BUZZI, L ; MARINO, C; HERNANDEZ E; PARODI A; CASTRO O

Puerto Madryn

Congreso; XLVI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

    Identification and biochemical characterization of C.elegans  ER  GT.   Buzzi, LI,   Marino Bujsle, C.  Hernandez, Evelyn, Parodi, A. and Castro OA.   Quality control mechanisms ensure that newly synthesized glycoproteins reach their properly folded conformation. The UDP-Glc glycoprotein glucosyltransferase (GT) which functions as a conformational sensor is the key element in this mechanism. The human genome encodes two GT homologues, HUGT1 and HUGT2, but only HUGT1p is active. The C.elegans genome encodes two GTs F48E3.3 (CeGT 3.3) and F26H9.8 (CeGT9.8). We determined that C.elegans has a ten fold lower GT activity than rat liver and it shares common features with the GT of other organisms. Bioinformatics analysis showed that CeGT3.3p is around 34% identical to HUGT1p and HUGT2p while CeGT9.8p is 25% identical to them. Fold recognition methods showed that in the N-terminal region there are three consecutive thioredoxin like domains (with an alfa subdomain insertion) of the DsbA-like type in the first 800 residues. The C-terminal region contains a glyco-transf-8 domain responsible for the catalytic activity. We expressed CeGT3.3p and CeGT9.8p in alg6-gpt1- S.pombe cells and only the GeGT3.3p was active. Addition of a C-terminal HA epitope to both proteins abolished CeGT3.3 activity and CeGT9.8 became fully proteolysed. We expressed both proteins in S. cerevisiae but both lacked GT activity when labeled with HA epitope These results suggest that CeGT3.3 could be the ER GT while CeGT9.8p may play another biological role in C.elegans.     La Contiene tres dominios consecutivos de este plegamiento dsba like. Estos dominios tienen alrededor de 170 pares de bases   Tanto en humanos en cele y resto la arquitectura del dominio global es similar. Se pueden predecir tres dominios consecutivos  trx like del tipo DsbA like  que posee una insercion…  Esta estructura se encontró en  dsba disulidi bimd formation facilitation-   Esta estructura se encontró en otra proteínas ej de tándem de trx like se encuentran en  calsecuestrinas   Fig   Se muestra la estructura de un dominio txlike pdb code 3gyk  chain A in cluidibg el dominio de trxlike domain clorodeado depor elementos de estructura secuencia y la inserción alfa coloreada en azul