Passa al contenuto
Merck

Identification of a novel protein binding motif within the T-synthase for the molecular chaperone Cosmc.

The Journal of biological chemistry (2014-03-13)
Rajindra P Aryal, Tongzhong Ju, Richard D Cummings
ABSTRACT

Prior studies suggested that the core 1 β3-galactosyltransferase (T-synthase) is a specific client of the endoplasmic reticulum chaperone Cosmc, whose function is required for T-synthase folding, activity, and consequent synthesis of normal O-glycans in all vertebrate cells. To explore whether the T-synthase encodes a specific recognition motif for Cosmc, we used deletion mutagenesis to identify a cryptic linear and relatively hydrophobic peptide in the N-terminal stem region of the T-synthase that is essential for binding to Cosmc (Cosmc binding region within T-synthase, or CBRT). Using this sequence information, we synthesized a peptide containing CBRT and found that it directly interacts with Cosmc and also inhibits Cosmc-assisted in vitro refolding of denatured T-synthase. Moreover, engineered T-synthase carrying mutations within CBRT exhibited diminished binding to Cosmc that resulted in the formation of inactive T-synthase. To confirm the general recognition of CBRT by Cosmc, we performed a domain swap experiment in which we inserted the stem region of the T-synthase into the human β4GalT1 and found that the CBRT element can confer Cosmc binding onto the β4GalT1 chimera. Thus, CBRT is a unique recognition motif for Cosmc to promote its regulation and formation of active T-synthase and represents the first sequence-specific chaperone recognition system in the ER/Golgi required for normal protein O-glycosylation.

MATERIALI
N° Catalogo
Marchio
Descrizione del prodotto

Sigma-Aldrich
Phosphorylase b from rabbit muscle, lyophilized powder, ≥20 units/mg protein, 2× crystallization
Sigma-Aldrich
Phosphorylase b from rabbit muscle, For use as a marker in SDS-PAGE