Topological Organization of the Hyaluronan Synthase fromStreptococcus pyogenes

Abstract

Since we first reported (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184) the cloning of the hyaluronan (HA) synthase from Streptococcus pyogenes (spHAS), numerous membrane-bound HA synthases have been discovered in both prokaryotes and eukaryotes. The HASs are unique among enzymes studied to date because they mediate 6 -7 discrete functions in order to assemble a polysaccharide containing hetero-disaccharide units and simultaneously effect translocation of the growing HA chain through the plasma membrane. To understand how the relatively small spHAS performs these various functions, we investigated the topological organization of the protein utilizing fusion analysis with two reporter enzymes, alkaline phosphatase and ␤-galactosidase, as well as several other approaches. From these studies, we conclude that the NH 2 terminus and the COOH terminus, as well as the major portion of a large central domain are localized intracellularly. The first two predicted membrane domains were confirmed to be transmembrane domains and give rise to a very small extracellular loop that is inaccessible to proteases. Several regions of the large internal central domain appear to be associated with, but do not traverse, the membrane. Following the central domain, there are two additional transmembrane domains connected by a second small extracellular loop that also is inaccessible to proteases. The COOH-terminal ϳ25% of spHAS also contains a membrane domain that does not traverse the membrane and may contain extensive re-entrant loops or amphipathic helices. Numerous membrane associations of this latter COOH-terminal region and the central domain may be required to create a pore-like structure through which a growing HA chain can be extruded to the cell exterior. Based on the high degree of similarity among Class I HAS family members, these enzymes may have a similar topological organization for their spHASrelated domains.

Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria

“…Schematic organization and alignment of class I HA synthase proteins. A: The streptococcal, vertebrate, and viral HASs are integral membrane proteins that have a similar predicted overall organization (the streptococcal enzyme is shown here because topological organization data has been obtained (Heldermon et al, 2001). A central conserved region of the enzymes is predicted to contain a catalytic domain that resides in the cytosol (Cyto); this region is used in the alignment in panel B.…”

Functional Characteristics and Catalytic Mechanisms of the Bacterial Hyaluronan Synthases

“…The location of the active sites of spHAS was examined experimentally and confirmed to be intracellular (29). This finding Yes for HA translocation makes sense, since the nucleotide-sugar substrates are produced in the cytoplasm.…”

“…The upper portion of each cartoon represents the outside of the cell membrane (indicated by the parallel lines) and the lower portion is in the cytoplasm. Only the topology of the spHAS has been determined experimentally (29). The topological organization of the seHAS and suHAS are assumed to be the same because their amino acid sequences are >70% identical to spHAS.…”

An Engineered Hyaluronan Synthase

“…reported that the major catalytic regions of eukaryotic HAS seem to exist in the cytoplasmic region by topology research and Kyte-Doolittle hydropathy analysis (5,15,30), although the essential structure of the catalytic domain is still putative and has not been identified previously. In this study, we designed three constructions: (i) a peptide composed of the two common catalytic domains of HAS in the cytoplasmic region and the sequence including a part of the transmembrane region (rhHAS2-(122-414)), (ii) a peptide containing only two catalytic sites (rhHAS2-(122-355)), and (iii) a small peptide lacking one of the catalytic sites (rhHAS2-(122-347)) as illustrated in Fig.…”