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• W2045973674 abstract "Hyaluronan synthases (HASs) are glycosyltransferases that catalyze polymerization of hyaluronan found in vertebrates and certain microbes. HASs transfer two distinct monosaccharides in different linkages and, in certain cases, participate in polymer transfer out of the cell. In contrast, the vast majority of glycosyltransferases form only one sugar linkage. Although our understanding of HAS biochemistry is still incomplete, very good progress has been made since the first genetic identification of a HAS in 1993. New enzymes have been discovered, and some molecular details have emerged. Important findings are the lipid dependence of Class I HASs, the function of HASs as protein monomers, and the elucidation of mechanisms of synthesis by Class II HAS. We propose three classes of HASs based on differences in protein sequences, predicted membrane topologies, potential architectures, mechanisms, and direction of polymerization. Hyaluronan synthases (HASs) are glycosyltransferases that catalyze polymerization of hyaluronan found in vertebrates and certain microbes. HASs transfer two distinct monosaccharides in different linkages and, in certain cases, participate in polymer transfer out of the cell. In contrast, the vast majority of glycosyltransferases form only one sugar linkage. Although our understanding of HAS biochemistry is still incomplete, very good progress has been made since the first genetic identification of a HAS in 1993. New enzymes have been discovered, and some molecular details have emerged. Important findings are the lipid dependence of Class I HASs, the function of HASs as protein monomers, and the elucidation of mechanisms of synthesis by Class II HAS. We propose three classes of HASs based on differences in protein sequences, predicted membrane topologies, potential architectures, mechanisms, and direction of polymerization. Hyaluronan (HA) 2The abbreviations used are:HAhyaluronan (hyaluronic acid)HASHA synthasePmHASPasteurella multocida HASSpHASStreptococcus pyogenes HASMmHASMus musculus HASSeHASStreptococcus equisimilis HASaaamino acid(s)GT2glycosyltransferase 2MDsmembrane domainsXlHASXenopus laevis HASHsHASHomo sapiens HASABCATP-binding cassette. is a linear heteropolysaccharide composed of (→4)-β-d-GlcUA-(1→3)-β-d-GlcNAc(1→) disaccharide repeats and is found in vertebrates and a few select microbes (1Meyer K. Fed. Proc. 1958; 17: 1075-1077PubMed Google Scholar, 2McDonald J.A. Camenisch T.D. Glycoconj. J. 2002; 19: 331-339Crossref PubMed Scopus (77) Google Scholar, 3Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar, 4Toole B.P. J. Clin. Investig. 2000; 106: 335-336Crossref PubMed Scopus (141) Google Scholar, 5Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (453) Google Scholar, 6Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1512) Google Scholar). HA typically resides on the cell surface or in the extracellular space, but is also found inside mammalian cells. Glycosyltransferases that polymerize HA are HA synthases (HASs; EC 2.4.1.212), earlier called HA synthetases (7DeAngelis P.L. CMLS Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (162) Google Scholar, 8Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 9Itano N. Kimata K. IUBMB Life. 2002; 54: 195-199Crossref PubMed Scopus (312) Google Scholar). All HASs use UDP-sugars in the presence of Mg2+ or Mn2+ (Equation 1, overall reaction to form HA). nUDP-GlcUA+nUDP-GlcNAc→2nUDP+(-4GlcUA-β1,3-GlcNAcβ1-)n(Eq. 1) hyaluronan (hyaluronic acid) HA synthase Pasteurella multocida HAS Streptococcus pyogenes HAS Mus musculus HAS Streptococcus equisimilis HAS amino acid(s) glycosyltransferase 2 membrane domains Xenopus laevis HAS Homo sapiens HAS ATP-binding cassette. Typically, n > 103–4; thus, HASs make HA chains of ∼1–10 MDa. The intrinsic fidelity of the two glycosyltransferases of HAS generates the repeating disaccharide structure. Unlike virtually all other vertebrate glycoconjugates, HA is made as a free glycan, not attached to protein or lipid. HASs do not need a primer for HA synthesis; they all initiate HA biosynthesis de novo with only UDP-GlcNAc, UDP-GlcUA, and Mg2+. The UDP-sugars are α-linked and HASs synthesize β-linkages; thus, these are inverting (not retaining) enzymes. UDP-sugars are polymerized from cytoplasmic pools by HAS, and the growing HA chain is extruded out of the cell. The deduced sequences of the genes encoding the HAS proteins were identified using molecular biological techniques (e.g. transposon mutagenesis and expression cloning) (10DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 11Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12DeAngelis P.L. Jing W. Graves M.V. Burbank D.E. Van Etten J.L. Science. 1997; 278: 1800-1803Crossref PubMed Scopus (111) Google Scholar, 13DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 14Meyer M.F. Kreil G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4543-4547Crossref PubMed Scopus (88) Google Scholar, 15Itano N. Kimata K. Biochem. Biophys. Res. Commun. 1996; 222: 816-820Crossref PubMed Scopus (106) Google Scholar, 16Itano N. Kimata K. J. Biol. Chem. 1996; 271: 9875-9878Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 17Watanabe K. Yamaguchi Y. J. Biol. Chem. 1996; 271: 22945-22948Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Shyjan A.M. Heldin P. Butcher E.C. Yoshino T. Briskin M.J. J. Biol. Chem. 1996; 271: 23395-23399Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 19Spicer A.P. Augustine M.L. McDonald J.A. J. Biol. Chem. 1996; 271: 23400-23406Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 20Spicer A.P. Olson J.S. McDonald J.A. J. Biol. Chem. 1997; 272: 8957-8961Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 21DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Classical biochemical purification methods failed with these membrane-associated enzymes. In 1993, identification of the streptococcal hasA gene and the biochemical demonstration that a single protein species is required to synthesize HA (13DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 22Dougherty B.A. van de Rijn I. J. Biol. Chem. 1994; 269: 169-175Abstract Full Text PDF PubMed Google Scholar) led to the identification of HAS genes in other species, including vertebrates. Only PmHAS is so different that its discovery was not aided by similarity comparisons with the prototypical Streptococcus HAS. The GenBank™ Data Bank lists >20 bacterial, viral, and vertebrate HAS genes or cDNAs. The nomenclature for HASs employs the first letters of the genus and species (e.g. Streptococcus pyogenes HAS = SpHAS). The vertebrate HAS isozymes are named in order of discovery (e.g. murine HAS isozymes = MmHAS1, -2, and -3). Functional testing of recombinant proteins revealed HASs to be the first glycosyltransferases in which a single protein catalyzes the transfer of two different monosaccharides. Studies with purified recombinant SpHAS, SeHAS (23Tlapak-Simmons V.L. Baron C.A. Weigel P.H. Biochemistry. 2004; 43: 9234-9242Crossref PubMed Scopus (46) Google Scholar, 24DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar, 25Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), MmHAS1 (26Yoshida M. Itano N. Yamada Y. Kimata K. J. Biol. Chem. 2000; 275: 497-506Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and PmHAS (27Jing W. DeAngelis P.L. J. Biol. Chem. 2004; 279: 42345-42349Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) confirmed that no other proteins are needed to initiate or catalyze HA synthesis. This finding of dual-action, or bifunctional, enzymes is in contrast to the usual “one enzyme, one sugar linkage” dogma of glycobiology. It was also unexpected that such small proteins (417–588 aa and one of 972 aa) would perform multiple functions, including (a) binding of two distinct UDP-sugars, (b) binding of two distinct HA acceptor or donor species, (c) transfer of two different sugars in two different linkages, (d) repetitive sugar polymerization, and (e) HA transfer across the membrane. Many bacteria and Archaea produce polysaccharides, but only certain animal and human pathogens produce extracellular HA capsules (28DeAngelis P.L. Glycobiology. 2002; 12: 9R-16RCrossref PubMed Google Scholar). HA is found in some Gram-positive streptococci (e.g. S. pyogenes, S. equisimilis, Streptococcus uberis, and Streptococcus zooepidemicus) and in Gram-negative Type A P. multocida. Mutant microbes without HA capsules are less virulent than parental strains because of increased susceptibility to host defenses, e.g. complement and phagocytosis (29Wessels M.R. Bronze M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12238-12242Crossref PubMed Scopus (114) Google Scholar, 30Chung J.Y. Wilkie I. Boyce J.D. Townsend K.M. Frost A.J. Ghoddusi M. Adler B. Infect. Immun. 2001; 69: 2487-2492Crossref PubMed Scopus (28) Google Scholar). The streptococcal HASs are ∼70% identical to each other and ∼25% identical to the vertebrate HASs. PmHAS is totally different from all other HASs. HA is not found in all animals and arose ∼500 million years ago in some chordates (31DeAngelis P.L. Anat. Rec. 2002; 268: 317-326Crossref PubMed Scopus (88) Google Scholar). Thus far, primitive extant animals, including hydra, sponges, insects (e.g. Drosophila), nematodes (e.g. Caenorhabditis), echinoderms, and tunicates (Urochordata, a branch of Chordata), do not contain HA or HAS genes (31DeAngelis P.L. Anat. Rec. 2002; 268: 317-326Crossref PubMed Scopus (88) Google Scholar, 32Toyoda H. Kinoshita-Toyoda A. Selleck S.B. J. Biol. Chem. 2000; 275: 2269-2275Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). In contrast, candidate genes encoding enzymes that make chondroitin/chondroitin sulfate and heparan sulfate, structurally related glycosaminoglycans, are in all animal genomes down to primordial hydra. This distribution is not simply because HAS cannot function in cells of lower organisms because MmHAS2 expression in fly results in high HA production in vivo (33Takeo S. Fujise M. Akiyama T. Habuchi H. Itano N. Matsuo T. Aigaki T. Kimata K. Nakato H. J. Biol. Chem. 2004; 279: 18920-18925Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Multiple HAS isozymes encoded by three to four genes are present in chordates (34Spicer A.P. McDonald J.A. J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). No known animal possesses only one HAS gene that might suggest an early ancestor/founder, but there is evidence for two rounds of gene duplication (34Spicer A.P. McDonald J.A. J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Xenopus has three active HAS genes and one pseudogene, whereas mice and humans lack a nonfunctional gene. The mammalian HAS1, -2, and -3 isozymes are ∼55–70% identical and >90% identical within an isozyme family. Knock-out mouse developmental studies show that HAS2 is essential (35Camenisch T.D. Spicer A.P. Brehm-Gibson T. Biesterfeldt J. Augustine M.L. Calabro Jr., A. Kubalak S. Klewer S.E. McDonald J.A. J. Clin. Investig. 2000; 106: 349-360Crossref PubMed Scopus (719) Google Scholar), but HAS1 and HAS3 have lesser roles. A phycodnavirus (Chlorella virus) that infects freshwater green algae also possesses a HAS, CvHAS (12DeAngelis P.L. Jing W. Graves M.V. Burbank D.E. Van Etten J.L. Science. 1997; 278: 1800-1803Crossref PubMed Scopus (111) Google Scholar), with a sequence more similar to vertebrate than streptococcal HASs, but the evolutionary pathway of this gene is unclear. Early in viral infection, algal cells are coated with HA fibers (36Graves M.V. Burbank D.E. Roth R. Heuser J. DeAngelis P.L. Van Etten J.L. Virology. 1999; 257: 15-23Crossref PubMed Scopus (64) Google Scholar), but their function is unknown. This is the first and only report of HA naturally occurring in plants and the first report of a viral carbohydrate-producing enzyme. The various types of HASs have biochemical and protein sequence differences (Table 1); apparently, multiple classes of HASs evolved independently to perform the same overall synthesis reaction by two distinct modes. New sugars may be added to the reducing (Equation 2, reducing end elongation reaction) 3These glycosyl-UDPs are shown with the reducing end on the right and highlighted differentially (italics or boldface) to illustrate the fate of the various UDP groups in this reaction. or nonreducing (Equation 3, nonreducing end elongation reaction) terminus of the HA polymer (supplemental Fig. 1). HA-GlcNAc-UDP+GlcUA-UDP+GlcNAc-UDP→UDP+UDP+HA-GlcNAc(β1,4)GlcUA(β1,3)GlcNAc-UDP(Eq. 2) UDP-GlcUA+UDP-GlcNAc+HA→UDP+UDP+GlcUA(β1,3)GlcNAc(β1,4)-HA(Eq. 3) HASs were previously divided into two groups (7DeAngelis P.L. CMLS Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (162) Google Scholar): Class II containing PmHAS and Class I containing all other HASs. Further analysis is required to determine catalytic mechanisms, but note that the two UDP-sugars are acceptors and the HA-UDP chain is the donor during synthesis at the reducing end, whereas the converse occurs for nonreducing end growth (8Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar).TABLE 1Proposed HAS classification systemClass IRNClass IIMembersSpHAS, SeHAS, SuHAS (MmHAS, HsHAS)XlHAS1 (CvHAS)PmHASNo. of GT2 modulesOneOneTwoPredicted topologyIntegral membraneIntegral membranePeripheral (or soluble)HA chain growthReducingNonreducingNonreducingIntrinsic polymerizationProcessiveProcessiveNonprocessiveNo. of UDP-sugar sites??TwoNo. of HA polymer sites??Two (probably) Open table in a new tab The Class I streptococcal, vertebrate, and viral HASs (Fig. 1) (7DeAngelis P.L. CMLS Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (162) Google Scholar) share aa sequence similarities, especially in their central regions. These enzymes have a single glycosyltransferase 2 (GT2) family (see www.cazy.org/) module with similarity to chitin synthases. Class I HASs are integral membrane proteins with four to six predicted trans-MDs and one to two membrane-associated MDs (8Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). The overall topology for SpHAS was determined by a reporter protein fusion approach (37Heldermon C. DeAngelis P.L. Weigel P.H. J. Biol. Chem. 2001; 276: 2037-2046Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The active sites are intracellular (37Heldermon C. DeAngelis P.L. Weigel P.H. J. Biol. Chem. 2001; 276: 2037-2046Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), as are the UDP-sugars. All Class I HAS topologies are predicted to be comparable because the first two-thirds of these proteins are similar to SpHAS. Class II PmHAS (Fig. 1) (7DeAngelis P.L. CMLS Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (162) Google Scholar) has two GT2 family modules. PmHAS is a peripheral membrane protein with neither intrinsic affinity for bilayers nor lipid requirement for activity. Truncation analysis suggests that a C-terminal region (aa 703–972) docks peripherally with integral membrane transport proteins common to many encapsulated Gram-negative bacteria (38Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar). Specific phospholipids (e.g. cardiolipin) are required for activity of membrane-bound or purified SeHAS or SpHAS (23Tlapak-Simmons V.L. Baron C.A. Weigel P.H. Biochemistry. 2004; 43: 9234-9242Crossref PubMed Scopus (46) Google Scholar, 25Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 39Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar, 40Weigel P.H. Kyossev Z. Torres L.C. J. Biol. Chem. 2006; 281: 36542-36551Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and MmHAS2. The functional catalytic unit of SeHAS or SpHAS, as assessed by radiation inactivation analysis, is a HAS monomer with ∼16 cardiolipins (41Tlapak-Simmons V.L. Kempner E.S. Baggenstoss B.A. Weigel P.H. J. Biol. Chem. 1998; 273: 26100-26109Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), as illustrated in supplemental Fig. 2. XlHAS1 is also active as a monomer with an additional mass of ∼23 kDa that was not identified (42Pummill P.E. Kempner E.S. DeAngelis P.L. J. Biol. Chem. 2001; 276: 39832-39835Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Kimata et al. 4K. Kimata and co-workers, personal communication. found that dodecyl maltoside-solubilized recombinant insect cell-derived MmHAS2 requires subsequent addition of one particular phospholipid for activity. Thus, it seems likely that all Class I HAS family members are lipid-dependent, and the historic difficulties in purifying active detergent-solubilized Class I HASs can be attributed to the loss of tightly bound phospholipid (40Weigel P.H. Kyossev Z. Torres L.C. J. Biol. Chem. 2006; 281: 36542-36551Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). New sugars can be added to a linear saccharide chain at either the nonreducing (Equation 3) or reducing (Equation 2) terminus during biosynthesis depending on the nature of the enzyme (supplemental Fig. 1). SpHAS was first reported to add sugars to the nonreducing end (43Stoolmiller A.C. Dorfman A. J. Biol. Chem. 1969; 244: 236-246Abstract Full Text PDF PubMed Google Scholar), but later studies using crude membranes (44Prehm P. Biochem. J. 1983; 211: 191-198Crossref PubMed Scopus (120) Google Scholar, 45Bodevin-Authelet S. Kusche-Gullberg M. Pummill P.E. DeAngelis P.L. Lindahl U. J. Biol. Chem. 2005; 280: 8813-8818Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) or purified enzymes (46Tlapak-Simmons V.L. Baron C.A. Gotschall R. Haque D. Canfield W.M. Weigel P.H. J. Biol. Chem. 2005; 280: 13012-13018Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) demonstrated that SeHAS and SpHAS elongate HA at the reducing end. In each cycle of sugar addition at the reducing end, the released UDP is derived from the previously added monosaccharide (i.e. HA-UDP). The hyaluronyl-UDP is activated by its attachment to UDP and is the donor in each transfer reaction, analogous to protein and fatty acid synthesis (47Robbins P.W. Bray D. Dankert B.M. Wright A. Science. 1967; 158: 1536-1542Crossref PubMed Scopus (105) Google Scholar, 48Weigel P.H. IUBMB Life. 2002; 54: 201-211Crossref PubMed Scopus (71) Google Scholar). The direction of synthesis for eukaryotic HASs is less clear; either some reports are in error, or both types of elongation occur, although the HAS sequences are all similar. Recombinant amphibian XlHAS1 adds sugars at the nonreducing end (45Bodevin-Authelet S. Kusche-Gullberg M. Pummill P.E. DeAngelis P.L. Lindahl U. J. Biol. Chem. 2005; 280: 8813-8818Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), but native mouse (49Prehm P. Biochem. J. 2006; 398: 469-473Crossref PubMed Scopus (30) Google Scholar) and human (50Asplund T. Brinck J. Suzuki M. Briskin M.J. Heldin P. Biochim. Biophys. Acta. 1998; 1380: 377-388Crossref PubMed Scopus (45) Google Scholar) HASs extend HA at the reducing end. Nonreducing end addition was reported for recombinant truncated Escherichia coli-derived HsHAS2 (51Hoshi H. Nakagawa H. Nishiguchi S. Iwata K. Niikura K. Monde K. Nishimura S. J. Biol. Chem. 2004; 279: 2341-2349Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), although only very small oligosaccharides were made. Recombinant E. coli-derived PmHAS elongates exogenous HA oligosaccharide acceptors (e.g. tetrasaccharides) in vitro by stepwise addition of individual monosaccharides to the nonreducing terminus (supplemental Fig. 1) (52DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These acceptors are not absolutely required for HA formation by PmHAS, but stimulate polymerization by bypassing the relatively slow initiation step. A biotechnological spin-off (Hyalose, LLC) capitalizing on this kinetic feature is the production of size-defined HA polymers; in vitro reactions employing stoichiometrically controlled purified PmHAS, acceptors, and UDP-sugars are synchronized to yield essentially monodisperse HA products in the range of ∼20 to ∼2000 kDa (27Jing W. DeAngelis P.L. J. Biol. Chem. 2004; 279: 42345-42349Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In light of new data, the original system of two HAS classes (7DeAngelis P.L. CMLS Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (162) Google Scholar) needs to be revised (Table 1). We propose to maintain all integral membrane HAS proteins with a single GT2 module in Class I, but further subdivide these into Class I-R (reducing end polymerization; e.g. Streptococcus, mouse, and human HASs) and Class I-N (nonreducing end polymerization; e.g. XlHAS1). Another reason to keep the original Class I is that these HASs appear to both catalyze sugar transfer and facilitate HA transfer across the membrane. We anticipate that initial assignments of some members to the Class I-N or Class I-R group will require more experimental confirmation and may change with future studies. The very distinct PmHAS remains the only known Class II HAS. No three-dimensional structure is available for any HAS, but models for HAS architecture, function, and mechanisms of catalysis have been discussed (supplemental Fig. 1 and 2) (7DeAngelis P.L. CMLS Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (162) Google Scholar, 8Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 9Itano N. Kimata K. IUBMB Life. 2002; 54: 195-199Crossref PubMed Scopus (312) Google Scholar, 28DeAngelis P.L. Glycobiology. 2002; 12: 9R-16RCrossref PubMed Google Scholar, 48Weigel P.H. IUBMB Life. 2002; 54: 201-211Crossref PubMed Scopus (71) Google Scholar, 53Williams K.J. Halkes K.M. Kamerling J.P. DeAngelis P.L. J. Biol. Chem. 2006; 281: 5391-5397Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Two types of putative functional domains, termed Domains A and B, were proposed to be present in many β-glycosyltransferases that use nucleoside diphosphosugars as donors (54Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar). Processive enzymes, which add many sugars without releasing their polymer chain, possess both Domains A and B, whereas enzymes that add a single sugar usually have only Domain A. Class I-R and Class I-N HASs do not release their growing chains during synthesis and possess both putative Domains A and B. Class II PmHAS possesses two tandem copies of Domain A and no Domain B (38Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar). Studies of SeHAS have provided insight into the molecular organization of Class I HASs (55Kumari K. Baggenstoss B.A. Parker A.L. Weigel P.H. J. Biol. Chem. 2006; 281: 11755-11760Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 56Kumari K. Weigel P.H. Glycobiology. 2005; 15: 529-539Crossref PubMed Scopus (11) Google Scholar). Four cysteine residues, conserved in the Class I family, are clustered very close together at the inner surface of the membrane and are close to or within UDP-sugar-binding sites (56Kumari K. Weigel P.H. Glycobiology. 2005; 15: 529-539Crossref PubMed Scopus (11) Google Scholar). These results indicate that HAS active site(s) are at the membrane-protein junction and support the “Pore Hypothesis” for HA synthesis (25Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). supplemental Fig. 2 shows the first experimentally based model for the organization and nearest neighbors of the six MDs of SeHAS (55Kumari K. Baggenstoss B.A. Parker A.L. Weigel P.H. J. Biol. Chem. 2006; 281: 11755-11760Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) that create this putative pore. Kinetic analyses of Class II PmHAS and its mutants show that two relatively independent active sites with separate UDP-sugar-binding sites (one each in Domains A1 and A2) exist in one protein (38Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar, 57Jing W. DeAngelis P.L. Glycobiology. 2003; 13: 661-671Crossref PubMed Scopus (65) Google Scholar). A pair of PmHAS variants, one disrupted at Domain A1 and the other at Domain A2, acted together to polymerize HA at rates similar to the wild-type enzyme (38Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar). HA synthesis is thus nonprocessive because HA chains must be released by one mutant PmHAS enzyme to be acted on by the other mutant enzyme. Cross-competition between GlcUA- and GlcNAc-terminated oligosaccharides for PmHAS-mediated elongation was not observed (53Williams K.J. Halkes K.M. Kamerling J.P. DeAngelis P.L. J. Biol. Chem. 2006; 281: 5391-5397Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), indicating at least two acceptor-binding sites. Studies of optimal HA oligosaccharide acceptor length suggest that ∼3 or 4 monosaccharide units of the nascent HA chain are bound by the enzyme (53Williams K.J. Halkes K.M. Kamerling J.P. DeAngelis P.L. J. Biol. Chem. 2006; 281: 5391-5397Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The number and the nature of UDP-sugar- and HA-binding site(s) for Class I HASs are still as unclear as in 1959, when the first schematic models were presented (58Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Abstract Full Text PDF PubMed Google Scholar). Mutations at conserved aa thought to be in UDP-sugar-binding or catalytic sites often inactivate HA polymerization completely; thus, key aa for various HAS functions have been difficult to map. However, mutations in MmHAS1 motifs that are conserved in other Domain A/B glycosyltransferases resulted in formation of short chitin oligosaccharides instead of HA (26Yoshida M. Itano N. Yamada Y. Kimata K. J. Biol. Chem. 2000; 275: 497-506Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Class II PmHAS in vivo is probably docked with the capsular polysaccharide transport machinery, which helps retain the HA chain in the microenvironment of the two active sites and mediates HA export. Transporters typically use ∼12 trans-MDs to create a pore for substrate transfer (e.g. lactose permease (59Kaback H.R. C. R. Biol. 2005; 328: 557-567Crossref PubMed Scopus (78) Google Scholar)). Class I HAS proteins have six to eight MDs, but these enzymes might use multiple interactions with the phospholipids essential for activity to create a larger pore-like passage through which HA could move (supplemental Fig. 2) (25Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 41Tlapak-Simmons V.L. Kempner E.S. Baggenstoss B.A. Weigel P.H. J. Biol. Chem. 1998; 273: 26100-26109Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The paradox is as follows: how can HAS bind and retain HA tightly enough to be processive, yet the HA chain move easily within the enzyme after every one to two sugar additions? Forces generated by Brownian motion might separate HA from HAS. One answer is that if HA is within an intraprotein pore, HAS can more readily release and rebind the HA without complete dissociation. An alternative explanation is that HA is transported by an ABC system (60Ouskova G. Spellerberg B. Prehm P. Glycobiology. 2004; 14: 931-938Crossref PubMed Scopus (41) Google Scholar, 61Prehm P. Schumacher U. Biochem. Pharmacol. 2004; 68: 1401-1410Crossref PubMed Scopus (60) Google Scholar), which can export many different bacterial polysaccharides (62Silver R.P. Prior K. Nsahlai C. Wright L.F. Res. Microbiol. 2001; 152: 357-364Crossref PubMed Scopus (43) Google Scholar). In this model, HAS synthesizes intracellular HA, which is recognized and transported by a membrane-bound ABC transport system. An ABC transport system seems unlikely to be involved in HA externalization for the Class I HASs because this model requires that Enterococcus faecalis, Bacillus subtilis, and Drosophila express ABC transport systems that lack specificity and can transport a foreign polysaccharide, HA (13DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 33Takeo S. Fujise M. Akiyama T. Habuchi H. Itano N. Matsuo T. Aigaki T. Kimata K. Nakato H. J. Biol. Chem. 2004; 279: 18920-18925Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 63Widner B. Behr R. Von Dollen S. Tang M. Heu T. Sloma A. Sternberg D. DeAngelis P.L. Weigel P.H. Brown S. Appl. Environ. Microbiol. 2005; 71: 3747-3752Crossref PubMed Scopus (227) Google Scholar). Perhaps HA can be brought to the cell surface by a combination of both HAS pore and ABC transporter mechanisms. HA size (i.e. mass) is now recognized to be an important factor governing the ability of HA to alter cell behavior (4Toole B.P. J. Clin. Investig. 2000; 106: 335-336Crossref PubMed Scopus (141) Google Scholar, 5Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (453) Google Scholar). In particular, very small HA oligosaccharides (e.g. ∼10–20 sugars) have potent effects in many in vitro or ex vivo systems, including angiogenesis models. It is generally assumed that small HA oligosaccharides would be derived from large extracellular matrix HA, which is degraded inside cells via exo-β-glycosidases and hyaluronidases (64Stern R. Glycobiology. 2003; 13: 105R-115RCrossref PubMed Scopus (290) Google Scholar) during local or systemic turnover (6Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1512) Google Scholar). However, it is also possible that HASs might be regulated to synthesize very small HA, thus producing small HA oligosaccharides directly. Tissue HA levels vary during embryonic and oocyte development and wound healing (3Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar, 6Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1512) Google Scholar). The levels of the three HAS mRNAs or isozymes also vary from amphibians to mammals, but data required for a cohesive view of HAS regulation are still being generated. Many connections have been reported between the proliferation, survival, and metastasis of cancer cells and the relative expression of HAS isoforms or HA levels (65Adamia S. Maxwell C.A. Pilarski L.M. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005; 5: 3-14Crossref PubMed Scopus (138) Google Scholar). The relative specific activities of the three HsHAS isozymes and the size distributions of their in vitro HA products are different (66Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar). Changing a single aa in XlHAS1 alters the HA size distribution in vitro to either a bigger or smaller size depending on the substitution (67Pummill P.E. DeAngelis P.L. J. Biol. Chem. 2003; 278: 19808-19814Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and many mutations of SpHAS or SeHAS result in smaller HA (55Kumari K. Baggenstoss B.A. Parker A.L. Weigel P.H. J. Biol. Chem. 2006; 281: 11755-11760Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 68Kumari K. Tlapak-Simmons V.L. Baggenstoss B.A. Weigel P.H. J. Biol. Chem. 2002; 277: 13943-13951Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In all cases, the biochemical explanation for how HASs control HA size is unknown. One hypothesis is that the more time a chain is held and acted on by HAS, the larger the HA; conversely, if the chains are readily released by HAS, then the HA will be smaller. Another possibility is that HA is released when HA retention forces within a HA·HAS complex are exceeded by the HA release forces (e.g. movement or collisions of the growing HA chain) (48Weigel P.H. IUBMB Life. 2002; 54: 201-211Crossref PubMed Scopus (71) Google Scholar). Increasing the ionic strength of an in vitro XlHAS1 reaction yields longer HA (67Pummill P.E. DeAngelis P.L. J. Biol. Chem. 2003; 278: 19808-19814Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), suggesting that hydrophobic forces (e.g. between hydrophobic aa side chains and the hydrophobic faces of sugars in HA) are involved in nascent HA retention. The demonstration for HsHAS3 that HASs can be phosphorylated (69Goentzel B.J. Weigel P.H. Steinberg R.A. Biochem. J. 2006; 396: 347-354Crossref PubMed Scopus (42) Google Scholar) and the finding that mammalian HAS trafficking and catalytic activity are dynamic and controlled (70Rilla K. Siiskonen H. Spicer A.P. Hyttinen J.M. Tammi M.I. Tammi R.H. J. Biol. Chem. 2005; 280: 31890-31897Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) also add new levels of complexity for how HA production and HAS location are regulated. Several areas stand out to us as focal points for future progress. Biochemistry—The identity, number, and nature of substrate-binding sites for any Class I HAS are unknown. Whether the initiating or terminating sugars in released HA chains are unique is unclear. Knowing these details could aid in the understanding of HAS regulation and development of useful drug targets. Cell Biology—The field needs better HAS isozyme-specific antibodies that allow immunodetection of native nondenatured HAS1, -2, or -3 so that the cellular localization and distribution of each isoform can be elucidated in different cell types or tissues. Understanding HA homeostasis requires knowing how both the HAS and hyaluronidase genes and proteins are regulated. HA Biology—Regulation of HA quantity and size has potentially great significance for altering cell behavior in health and disease (e.g. inflammation and cancer). Determining whether small HA oligomers are present in vivo outside cells and have effects similar to those found in vitro and in model systems is a priority. Are there also other hitherto unidentified regulatory proteins that alter HAS activity or localization? We look forward to the next decade and many more answers to these and other questions. We thank members of our laboratories, past and present, and the many participants in the Society for Glycobiology, International Society of Hyaluronan Sciences, and Proteoglycan Gordon Conference meetings for stimulating conversations and feedback. Download .zip (.04 MB) Help with zip files" @default.
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• W2045973674 title "Hyaluronan Synthases: A Decade-plus of Novel Glycosyltransferases" @default.
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