MEMBRANE TOPOLOGY ANALYSIS OF CYCLIC GLUCAN SYNTHASE, A VIRULENCE DETERMINANT OF BRUCELLA ABORTUS

CIOCCHINI AE, ROSET MS, IÑÓN DE IANNINO N, UGALDE RA.

Iguazú, Misiones, Argentina.

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB 2004).; 2004

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Brucella abortus cyclic glucan synthase (Ba-Cgs) is an integral inner membrane protein of 316 kDa (2,831 residues) responsible for the synthesis of cyclic b-1,2-glucan by a novel mechanism in which the enzyme itself acts as protein intermediate. Ba-Cgs uses UDP-glucose as donor-sugar and has the three enzymatic activities required for the synthesis of the cyclic polysaccharide, ie: initiation, elongation and cyclization. Cyclic glucan is required in Brucella abortus for effective host interaction and full expression of virulence. To gain further insight into the structure and mechanism of action of Ba-Cgs we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the cgs gene and the genes encoding alkaline phosphatase (phoA) and b-galactosidase (lacZ) and site-directed chemical labeling of lysine residues. We found that Ba-Cgs is a polytopic membrane protein with the amino and carboxyl terminus located in the cytoplasm and containing six transmembrane segments; I (residues 419-441),II (residues 452-474), III (residues 819-841), IV (residues 847-869), V (residues 939-961) and VI (residues 968-990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.