Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase fromStreptococcus agalactiaeNEM316

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surfaceassociated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P2 1 and P2 1 2 1 2 1 , respectively. The structure was solved by molecular replacement and structure refinement is now in progress.

Structure ofStreptococcus agalactiaeglyceraldehyde-3-phosphate dehydrogenase holoenzyme reveals a novel surface

“…Hanging drops were prepared by mixing 2 ml protein plus cofactor mixture, 0.5 ml water and 0.5 ml reservoir solution containing seed suspension in a cover slip, which was then sealed over a 500 ml reservoir and incubated at 295 K. The reservoir solution for growing the crystal used for structure determination consisted of 28% PEG 4000, 0.1 M MES buffer pH 6.5. Recently, crystallization of S. agalactiae in an isomorphous crystal form has been reported (Nagarajan & Ponnuraj, 2014). These crystals were also grown from PEG 4000 and PEG 3350 at 287 K, but the crystallization buffer was composed of 0.1 M Tris-HCl pH 8.0-9.0.…”

Partial catalytic Cys oxidation of human GAPDH to Cys-sulfonic acid.

“…GAPDH is mainly a cytoplasmic enzyme, but it has been reported to moonlight as a cell surface and/or extracellular enzyme in healthy human cells 49,50 , in human cells subjected to stresses such as micronutrient starvation, hypoxia, infection, and cancer [50][51][52][53][54] , and to act as a extracellular effector in fungi and bacteria [55][56][57][58][59][60][61][62] . For recent reviews on GAPDH inhibitors and their multiple roles in several pharmacological applications see 63,64. In our study, HsGAPDH was purified from the supernatant of HEK293F cells treated with the mannosidase inhibitors kifunensine and kept for 4 days at 37°C after transfection with a DNA plasmid for recombinant expression of an endoplasmic reticulum glycoprotein.…”