The Active Streptococcal Hyaluronan Synthases (HASs) Contain a Single HAS Monomer and Multiple Cardiolipin Molecules

Abstract

The functional sizes of the two streptococcal hyaluronan synthases (HASs) were determined by radiation inactivation analysis of isolated membranes. The native enzymes in membranes from Group A Streptococcus pyogenes HAS and Group C Streptococcus equisimilis HAS were compared with the recombinant proteins expressed in Escherichia coli membranes. Based on their amino acid sequences, the masses of these four proteins as monomers are ϳ48 kDa. In all cases, loss of enzyme activity was a simple single exponential function with increasing radiation dose. The functional sizes calculated from these data were identical for the four HASs at ϳ64 kDa. In contrast, the sizes of the proteins estimated by the loss of antibody reactivity on Western blots were essentially identical at 41 kDa for the four HAS species, consistently lower than the functional size by ϳ23 kDa. Matrix-assisted laser desorption time of flight mass spectrometry analysis of purified S. pyogenes HAS-H 6 and S. equisimilis HAS-H 6 gave masses that differed by <0.07% from the predicted monomer sizes, which confirms that neither protein is posttranslationally modified or covalently attached to another protein. Ongoing studies indicate that the purified HAS enzymes require cardiolipin (CL) for maximal activity and stability. When irradiated membranes were detergent solubilized and the extracts were incubated with exogenous CL, the residual level of HAS activity increased. Consequently, the calculated functional size decreased by ϳ23 kDa to the expected size of the HAS monomer. The ϳ23-kDa larger size of the functional HAS enzyme, compared with the HAS monomer, is due, therefore, to CL molecules. We propose that the active streptococcal HA synthases are monomers in complex with ϳ16 CL molecules.

Functional Molecular Mass of a Vertebrate Hyaluronan Synthase as Determined by Radiation Inactivation Analysis

“…Radiation inactivation studies of spHAS and seHAS, the homologous enzymes from Group A and C Streptococcus, respectively, have indicated that ϳ16 molecules of lipid and a monomer of protein comprise the functional HA polymerization unit (21). In the past, it was speculated that other molecules or a complex in the mammalian cell is involved or required for HAS activity (11,22,23).…”

An Engineered Hyaluronan Synthase

“…This may be caused by the limit of the spatial size of the carbohydrate-binding cavity or pocket of the rhHAS2 peptides. Although the essentials in the mechanism of HA synthesis by native human HASs expressed in mammalian cells have not been discussed thoroughly (19 -21), rhHAS2 peptides expressed in E. coli in this study will become convenient tools for further investigation of the rela- tionship between HAS functions and cardiolipin or ectoprotein kinase (27,(31)(32)(33) and their use as a novel class of engineered glycosyltransferases for medicinal purposes.…”

“…Preparation of Membrane Fraction-Membranes from E. coli were obtained by modifications of a protoplast method as reported previously (27,28). The fusion plasmids for the rhHAS2 fusion constructs were grown in JM109 cells.…”

Functional Characteristics and Catalytic Mechanisms of the Bacterial Hyaluronan Synthases

“…To determine if the active HAS is a monomer or a larger oligomer, we used radiation inactivation analysis (26). This approach uses very high-energy radiation to inactivate the enzyme.…”