Identification of a membrane-localized cysteine cluster near the substrate-binding sites of the Streptococcus equisimilis hyaluronan synthase

Hyaluronan synthase (HAS) is a membrane-bound enzyme that catalyzes the synthesis of hyaluronan (HA) in both eukaryotes (Itano, et al. 2002) and prokaryotes (Weigel, 2002). HA is a linear glycosaminoglycan composed of the repeating hetero-disaccharide unit: GlcUAs(1,3)GlcNAcs (1,4). HA is a component of extracellular matrices in all vertebrates, and is present in large amounts for special functions in cartilage, synovial fluid, dermis and the vitreous humor of eye (Abatangelo and Weigel, 2000). This glycosaminoglycan plays critical roles during fertilization, embryogenesis, development and differentiation (Laurent and Fraser, 1992; Toole, 1997; Knudson and Knudson, 1993, Fenderson et al., 1993). In Group A and Group C streptococcal strains, HA forms a capsule that helps these cells evade the host immune system during infection (Kass and Seastone, 1944; Wessels et al. 1994). Progress in understanding the molecular basis for HA biosynthesis accelerated greatly after 1993, when the Streptococcus pyogenes HAS (spHAS) gene was first cloned (DeAngelis et al., 1993). Other members of the HAS family were subsequently identified from mammalian, avian and amphibian species (DeAngelis et al., 1997, 1998; Fulop et al. 1997; Itano et al., 1996; Shyjan et al. 1996; Spicer et al., 1996, Spicer and McDonald, 1998; Watanabe et al. 1996), and also from Group C Streptococcus equisimilis (seHAS) and Streptococcus uberis (Kumari and Weigel, 1997; Ward et al., 2001). The HAS proteins are organized into two groups. The Class I enzymes contain all but one reported HAS; the single Class II enzyme (from Pasteurella multocida) differs from all other HASs in its structure and mechanism of HA biosynthesis (DeAngelis, 1999). The Class I HASs from prokaryotes and vertebrates are ~30% identical, and share a common membrane topology (Weigel et al., 1997; Heldermon et al., 2001). Although Class I HASs function as glycosyltransferases, they are very different from the vast majority of glycosyltransferases in that their UDP-sugar substrates are acceptors rather than donors (Robbins et al., 1967), because sugar addition occurs at the reducing end of growing HA chains (Prehm, 1983; Asplund et al., 1998; Tlapak-Simmons and Weigel, 2002). The growing HA chain remains bound to the enzyme at the cell membrane, while it is extruded into the extracellular space, and then ultimately shed into the extracellular space or assembled into a pericellular coat around eukaryotic cells or a capsule around bacterial cells. Therefore, despite their relatively small sizes (e.g. seHAS is ~49 kDa), HASs perform multiple discrete functions in order to synthesize HA (Tlapak-Simmons et al., 1999b; Weigel 2002). Radiation inactivation studies demonstrated that the streptococcal (Tlapak-Simmons et al., 1998) and amphibian (Pummill et al., 2001) Class I HASs function as protein monomers, but require phospholipids for activity, probably in both cases. Cardiolipin, phosphatidylserine or phosphatidic acid activate purified spHAS and seHAS, whereas other lipids do not activate these enzymes (Tlapak-Simmons et al., 1999a). The activating lipids for the eukaryotic HASs have not yet been identified. The recombinant streptococcal enzymes have been purified, characterized kinetically, and studied more extensively than other Class I HASs (Tlapak-Simmons et al., 1999a; Tlapak-Simmons et al., 1999b; Tlapak-Simmons et al., 2004). Yoshida et al. 2000 purified and kinetically characterized recombinant mouse HAS1, but no other eukaryotic Class I enzyme has been purified. All the vertebrate and prokaryote recombinant Class I HASs synthesize high molecular mass HA, although the three mammalian isozymes produce HA of different size distributions (Itano et al., 1999; Brinck and Heldin, 1999; Koprunner et al., 2000). Generally, cells expressing HAS1 or HAS3 synthesize and secrete HA of molecular mass 2 MDa. We are using seHAS as a model enzyme to study structure-function relationships within the Class I HAS family. The eukaryotic HASs contain about 14 Cys residues, whereas seHAS has only four, and these latter Cys residues are generally conserved among the vertebrate HASs. Since investigators have known for many decades (Markovitz et al., 1959) that HASs are sensitive to oxidation, we recently investigated whether one or more of the conserved Cys residues is vital for seHAS function. Surprisingly, although Cys modification inhibits HAS activity, the Cys-null mutants of seHAS and spHAS are active (Heldermon et al., 2001; Kumari et al., 2002). Also, these enzymes do not contain disulfide bonds. The present study examined the structural and functional roles of the four cysteines in seHAS, using combinations of site directed mutagenesis, chemical modification and substrate protection studies. The results were previously reported in preliminary form (Kumari et al., 1999).