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• W2015196611 abstract "Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has long been known to play structural roles in vertebrates. Recently, it has become increasingly obvious that this linear polysaccharide has many more uses than simply scaffolding or space filler. HA has been found to be involved in development, cell signaling, cell motility, and metastasis. These roles are often dictated by the length of the HA polymer, which can vary from a few to about 10,000 sugar residues in length. Three distinct isoforms of HA synthase exist in mammals. It has been shown previously by others that each isoform produces HA that differs in size distribution, but the regulatory mechanism is not yet known. Mutations have been described that alter the size distribution of the HA produced by the streptococcal HA synthases. We show that by mutating one particular amino acid residue of a vertebrate HA synthase, depending on the introduced side chain, the size of HA produced can be either reduced or increased. We postulate that several cysteine residues and a serine residue may be involved in binding directly or indirectly to the nascent HA chain. These data support the theory that the relative strength of the interaction between the catalyst and the polymer may be a major factor in HA size control. Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has long been known to play structural roles in vertebrates. Recently, it has become increasingly obvious that this linear polysaccharide has many more uses than simply scaffolding or space filler. HA has been found to be involved in development, cell signaling, cell motility, and metastasis. These roles are often dictated by the length of the HA polymer, which can vary from a few to about 10,000 sugar residues in length. Three distinct isoforms of HA synthase exist in mammals. It has been shown previously by others that each isoform produces HA that differs in size distribution, but the regulatory mechanism is not yet known. Mutations have been described that alter the size distribution of the HA produced by the streptococcal HA synthases. We show that by mutating one particular amino acid residue of a vertebrate HA synthase, depending on the introduced side chain, the size of HA produced can be either reduced or increased. We postulate that several cysteine residues and a serine residue may be involved in binding directly or indirectly to the nascent HA chain. These data support the theory that the relative strength of the interaction between the catalyst and the polymer may be a major factor in HA size control. HA 1The abbreviations used are: HA, hyaluronan, hyaluronate, or hyaluronic acid; HAS, HA synthase; GlcUA, glucuronic acid; MALLS, multi-angle laser light scattering; MANT, N-methylanthraniloyl.1The abbreviations used are: HA, hyaluronan, hyaluronate, or hyaluronic acid; HAS, HA synthase; GlcUA, glucuronic acid; MALLS, multi-angle laser light scattering; MANT, N-methylanthraniloyl. is a glycosaminoglycan composed of repeats of the alternating disaccharide (→4)-β-d-GlcUA(1→3)-β-d-GlcNAc(1→). The enzymes that catalyze the formation of HA, the HA synthases, are dual action glycosyltransferases that catalyze the transfer of both GlcUA and GlcNAc (1Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 2Spicer A.P. McDonald J.A. J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Birds and mammals, including humans, each contain three HAS isoforms called HAS1, 2, and 3 (1Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 2Spicer A.P. McDonald J.A. J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). These enzymes share considerable homology with each other as well as with HASs from group A and C Streptococcus bacteria and a virus (3DeAngelis P.L. Cell Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (160) Google Scholar). The membrane-associated enzymes utilize UDP-linked sugar precursors in the cytosol and extrude the growing HA chain through the plasma membrane (1Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 4Prehm P. Biochem. J. 1984; 220: 597-600Crossref PubMed Scopus (291) Google Scholar). The roles that HA plays in the vitreous humor of the eye (5Meyer K. Palmer J. J. Biol. Chem. 1934; 107: 629-634Abstract Full Text PDF Google Scholar) and in the formation of cartilage (6Hardingham T.E. Muir H. Biochim. Biophys. Acta. 1972; 279: 401-405Crossref PubMed Scopus (415) Google Scholar) have been known for many years. Considering the high concentration of HA (>100 mg/liter) in these areas (7Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2041) Google Scholar, 8Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1418) Google Scholar), it is not surprising that structural functions were the first biological uses discovered. Only recently have a wider variety of roles for HA been discovered. In vertebrates, HA is also involved in development, cell migration, and signaling (9Tammi M.I. Day A.J. Turley E.A. J. Biol. Chem. 2002; 277: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Many hyaluronan-binding proteins (10Day A.J. Prestwich G.D. J. Biol. Chem. 2002; 277: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar) are implicated strongly in signal transduction processes (11Turley E.A. Noble P.W. Bourguignon L.Y. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar); thus, HA is much more than just a space-filling molecule. The sizes of the HA molecules involved appear to dictate their biological activities (12Camenisch T.D. McDonald J.A. Am. J. Respir. Cell Mol. Biol. 2000; 23: 431-433Crossref PubMed Scopus (81) Google Scholar, 13Toole B.P. Wight T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). For example, HA with an average size of >106 Da appears to be involved in maintaining structure and viscosity (14Laurent T.C. Laurent U.B. Fraser J.R. Immunol. Cell Biol. 1996; 74: A1-A7Crossref PubMed Scopus (389) Google Scholar), mediating cell-matrix adhesion (15Zimmerman E. Geiger B. Addadi L. Biophys. J. 2002; 82: 1848-1857Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and reducing contact inhibition and promoting migration and metastasis (16Ichikawa T. Itano N. Sawai T. Kimata K. Koganehira Y. Saida T. Taniguchi S. J. Invest. Dermatol. 1999; 113: 935-939Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17Itano N. Sawai T. Miyaishi O. Kimata K. Cancer Res. 1999; 59: 2499-2504PubMed Google Scholar, 18Kosaki R. Watanabe K. Yamaguchi Y. Cancer Res. 1999; 59: 1141-1145PubMed Google Scholar, 19Liu N. Lapcevich R.K. Underhill C.B. Han Z. Gao F. Swartz G. Plum S.M. Zhang L. Gree S.J. Cancer Res. 2001; 61: 1022-1028PubMed Google Scholar, 20Itano N. Atsumi F. Sawai T. Yamada Y. Miyaishi O. Senga T. Hamaguchi M. Kimata K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3609-3614Crossref PubMed Scopus (261) Google Scholar). However, HA with an average size of <106 Da has been implicated in angiogenesis (21West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (956) Google Scholar, 22West D.C. Kumar S. Exp. Cell Res. 1989; 183: 179-196Crossref PubMed Scopus (308) Google Scholar, 23Sattar A. Rooney P. Kumar S. Pye D. West D.C. Scott I. Ledger P. J. Invest. Dermatol. 1994; 103: 576-579Crossref PubMed Scopus (155) Google Scholar, 24Lees V.C. Fan T.P. West D.C. Lab. Invest. 1995; 73: 259-266PubMed Google Scholar, 25Deed R. Rooney P. Kumar P. Norton J.D. Smith J. Freemont A.J. Kumar S. Int. J. Cancer. 1997; 71: 251-256Crossref PubMed Scopus (227) Google Scholar, 26Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003PubMed Google Scholar, 27Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 28Slevin M. Kumar S. Gaffney J. J. Biol. Chem. 2002; PubMed Google Scholar), cellular proliferation and migration (22West D.C. Kumar S. Exp. Cell Res. 1989; 183: 179-196Crossref PubMed Scopus (308) Google Scholar, 23Sattar A. Rooney P. Kumar S. Pye D. West D.C. Scott I. Ledger P. J. Invest. Dermatol. 1994; 103: 576-579Crossref PubMed Scopus (155) Google Scholar, 26Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003PubMed Google Scholar, 28Slevin M. Kumar S. Gaffney J. J. Biol. Chem. 2002; PubMed Google Scholar, 29Suzuki M. Kobayashi H. Kanayama N. Nishida T. Takigawa M. Terao T. Biochim. Biophys. Acta. 2002; 1591: 37-44Crossref PubMed Scopus (20) Google Scholar, 30Kobayashi H. Suzuki M. Kanayama N. Nishida T. Takigawa M. Terao T. Int. J. Cancer. 2002; 102: 379-389Crossref PubMed Scopus (46) Google Scholar, 31Nasreen N. Mohammed K.A. Hardwick J. Van Horn R.D. Sanders K. Kathuria H. Loghmani F. Antony V.B. Oncol. Res. 2002; 13: 71-78PubMed Google Scholar, 32Fujita Y. Kitagawa M. Nakamura S. Azuma K. Ishii G. Higashi M. Kishi H. Hiwasa T. Koda K. Nakajima N. Harigaya K. FEBS Lett. 2002; 528: 101Crossref PubMed Scopus (97) Google Scholar), and inflammation (33Noble P.W. Lake F.R. Henson P.M. Riches D.W. J. Clin. Invest. 1993; 91: 2368-2377Crossref PubMed Scopus (305) Google Scholar, 34McKee C.M. Penno M.B. Cowman M. Burdick M.D. Strieter R.M. Bao C. Noble P.W. J. Clin. Invest. 1996; 98: 2403-2413Crossref PubMed Scopus (682) Google Scholar, 35Noble P.W. McKee C.M. Cowman M. Shin H.S. J. Exp. Med. 1996; 183: 2373-2378Crossref PubMed Scopus (276) Google Scholar, 36Hodge-Dufour J. Noble P.W. Horton M.R. Bao C. Wysoka M. Burdick M.D. Strieter R.M. Trinchieri G. Pure E. J. Immunol. 1997; 159: 2492-2500PubMed Google Scholar, 37McKee C.M. Lowenstein C.J. Horton M.R. Wu J. Bao C. Chin B.Y. Choi A.M. Noble P.W. J. Biol. Chem. 1997; 272: 8013-8018Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 38Horton M.R. Burdick M.D. Strieter R.M. Bao C. Noble P.W. J. Immunol. 1998; 160: 3023-3030PubMed Google Scholar, 39Horton M.R. McKee C.M. Bao C. Liao F. Farber J.M. Hodge-DuFour J. Pure E. Oliver B.L. Wright T.M. Noble P.W. J. Biol. Chem. 1998; 273: 35088-35094Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 40Oertli B. Beck-Schimmer B. Fan X. Wuthrich R.P. J. Immunol. 1998; 161: 3431-3437PubMed Google Scholar, 41Rockey D.C. Chung J.J. McKee C.M. Noble P.W. Hepatology. 1998; 27: 86-92Crossref PubMed Scopus (92) Google Scholar, 42Horton M.R. Shapiro S. Bao C. Lowenstein C.J. Noble P.W. J. Immunol. 1999; 162: 4171-4176PubMed Google Scholar, 43Fitzgerald K.A. Bowie A.G. Skeffington B.S. O'Neill L.A. J. Immunol. 2000; 164: 2053-2063Crossref PubMed Scopus (124) Google Scholar, 44Ohkawara Y. Tamura G. Iwasaki T. Tanaka A. Kikuchi T. Shirato K. Am. J. Respir. Cell Mol. Biol. 2000; 23: 444-451Crossref PubMed Scopus (109) Google Scholar, 45Termeer C.C. Hennies J. Voith U. Ahrens T. Weiss J.M. Prehm P. Simon J.C. J. Immunol. 2000; 165: 1863-1870Crossref PubMed Scopus (325) Google Scholar, 46Horton M.R. Boodoo S. Powell J.D. J. Biol. Chem. 2002; 277: 43757-43762Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 47Termeer C. Benedix F. Sleeman J. Fieber C. Voith U. Ahrens T. Miyake K. Freudenberg M. Galanos C. Simon J.C. J. Exp. Med. 2002; 195: 99-111Crossref PubMed Scopus (1145) Google Scholar). The various vertebrate HAS isozymes produce HA of different sizes (48Itano 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 (693) Google Scholar). Therefore, it is likely that cells can manipulate the properties of HA produced by controlling the expression of the different isozymes depending on the particular developmental stage, tissue type, and external stimuli (2Spicer A.P. McDonald J.A. J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 49Jacobson A. Brinck J. Briskin M.J. Spicer A.P. Heldin P. Biochem. J. 2000; 348: 29-35Crossref PubMed Scopus (184) Google Scholar, 50Rosa F. Sargent T.D. Rebbert M.L. Michaels G.S. Jamrich M. Grunz H. Jonas E. Winkles J.A. Dawid I.B. Dev. Biol. 1988; 129: 114-123Crossref PubMed Scopus (85) Google Scholar, 51Koprunner M. Mullegger J. Lepperdinger G. Mech. Dev. 2000; 90: 275-278Crossref PubMed Scopus (38) Google Scholar, 52Sayo T. Sugiyama Y. Takahashi Y. Ozawa N. Sakai S. Ishikawa O. Tamura M. Inoue S. J. Invest. Dermatol. 2002; 118: 43-48Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The basis for size control of HA product has recently begun to be investigated (53Heldermon C. Kumari K. Tlapak-Simmons V.L. Weigel P.H. Weigel P.H. New Frontiers in Medical Sciences: Redefining Hyaluronan. Elsevier Science, Padua, Italy1999: 41-50Google Scholar). It is likely that the differences in the HAS sequences of the different isoforms are responsible at least in part for the variation in HA size distribution. One or more residues that are conserved between species but vary between isoforms may be responsible for the differences in HA size distribution. This hypothesis is based on the fact that when the different recombinant enzymes are expressed in the same system, the HA product size differs. In most experiments, a recombinant vector was used to express a HAS gene either in a foreign host, yeast, or in a HA-deficient mammalian cell line. In a direct comparison utilizing membrane preparations from yeast, spHAS (from Streptococcus pyogenes) made larger HA polymers than xlHAS1 (from Xenopus laevis, originally called DG42) in vitro (54Pummill P.E. Achyuthan A.M. DeAngelis P.L. J. Biol. Chem. 1998; 273: 4976-4981Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Murine HAS1, 2, and 3 were also directly compared in vitro and in vivo (48Itano 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 (693) Google Scholar). First, membrane preparations derived from mammalian cells transfected with the various murine HAS isozymes demonstrated that the relative product size was HAS2 > HAS1 > HAS3. Second, HA in the culture medium from mammalian cells transfected with HAS1, 2, or 3 also showed differences; HAS1 and 3 appeared to produce HA with more equivalent size distributions in vivo but still smaller than HAS2. Another group showed that the size distribution of HA products formed by xlHAS1 and xlHAS2 differed substantially when examined in vitro (51Koprunner M. Mullegger J. Lepperdinger G. Mech. Dev. 2000; 90: 275-278Crossref PubMed Scopus (38) Google Scholar). In addition to regulation via the innate kinetics or enzymological properties of the HASs, control of the precursor levels has been hypothesized as being important for size control. Certain treatments (e.g. proinflammatory cytokines) can cause cells to increase the expression of UDP-Glc dehydrogenase (55Spicer A.P. Kaback L.A. Smith T.J. Seldin M.F. J. Biol. Chem. 1998; 273: 25117-25124Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The actual levels of UDP-GlcUA were not measured, but it was postulated that HA production could be regulated by such a mechanism. The predominant HA size control mechanism, whether intrinsic factors (e.g. enzymological properties of the synthase), extrinsic factors (e.g. UDP-sugar substrate concentration or substrate/enzyme ratio), or a combination of both, has not been reported. Recently, it was shown that mutation of certain single cysteine residues in spHAS and seHAS (from Streptococcus equisimilis) cause the enzyme preparations to either produce HA of smaller or slightly larger average size distribution than that of wild type enzyme in vitro (53Heldermon C. Kumari K. Tlapak-Simmons V.L. Weigel P.H. Weigel P.H. New Frontiers in Medical Sciences: Redefining Hyaluronan. Elsevier Science, Padua, Italy1999: 41-50Google Scholar, 56Kumari 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). We have used site-directed mutagenesis to examine the difference in the size distribution of HA produced by various mutants of a vertebrate enzyme, xlHAS1. We have not only discovered a variety of single mutations that cause either a smaller or a larger HA product but also one particular amino acid that can be mutated to give rise to either a smaller or a larger HA product depending on the substituting residue. We have also examined these mutants to assess whether there is a relationship between enzyme kinetics and chain length. Production of Recombinant xlHAS1 Wild Type, Cysteine Mutants, and Ser77 Mutant Enzymes—All of the reagents were from Sigma or Fisher unless noted otherwise. The construction and the use of the xlHAS1 expression plasmid for studies in yeast were previously described (54Pummill P.E. Achyuthan A.M. DeAngelis P.L. J. Biol. Chem. 1998; 273: 4976-4981Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 57DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Basically, the xlHAS1 polypeptide was cloned into the pYES2 vector (Invitrogen) under control of the GAL1 promoter to form pYES/DG+. Site-directed mutagenesis was performed on pYES/DG+ using a QuikChange™ kit (Stratagene). Based on the preliminary results from photoaffinity labeling experiments probing the active site of xlHAS1, 2P. E. Pummill, R. R. Drake, and P. L. DeAngelis, unpublished observation. the Ser77 and Tyr107 codons were altered using pairs of synthetic oligonucleotides containing partially degenerate codons. For Ser77, the codon NNY (where N = any base and Y is C or T) was used to obtain a variety of mutants. For Tyr107, the codon TYT was used to obtain the Y107F mutant (Y107S was also generated but not investigated). Plasmids derived from independent transformants were sequenced to determine the identity of the substitution at residues 77 or 107 as well as to verify the entire open reading frame. The following Ser77 mutants were generated: alanine (S77A), aspartate (S77D), phenylalanine (S77F), isoleucine (S77I), threonine (S77T), valine (S77V), and tyrosine (S77Y/R271G; a secondary mutation was found upon sequencing). The cysteine mutants (C117S, C210S, C239S, C298S, C304S, C307S, and C337S) were obtained in a manner similar to that previously described (58Pummill P.E. DeAngelis P.L. J. Biol. Chem. 2002; 277: 21610-21616Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The plasmids were transformed into Saccharomyces cerevisiae BJ5461 yeast (a pleiotrophic protease-deficient strain; Yeast Genetic Stock Center, Berkeley, CA) by the lithium acetate/poly(ethylene glycol) method (59Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1667) Google Scholar). Yeast with recombinant plasmids were routinely grown to a suitable biomass (A600 reached 0.3) in uracil-deficient synthetic medium with 0.1% glucose and 5% glycerol. Upon induction with galactose (final concentration, 1%), xlHAS1 wild type or mutant enzyme accumulated in the plasma membrane fraction. The crude membranes were prepared by disruption with silica/zirconia beads (0.5 mm) in a MiniBead-Beater-8 (Biospec) and harvested by ultracentrifugation. The membrane pellet was suspended in 50 mm Tris, pH 7.5, 0.1 mm EDTA, 1 μm E-64, 1 mm benzamidine, 0.2 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride, and 5 μg/ml pepstatin. Protein was quantitated by the Coomassie dye binding assay (Pierce) using a bovine serum albumin standard (60Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Polysaccharide Synthase Assays and Analyses—The incorporation of sugars into high molecular mass HA polysaccharide was monitored using UDP-[14C]GlcUA (∼290 mCi/mmol; PerkinElmer Life Sciences) and/or UDP-[3H]GlcNAc (29.2 Ci/mmol; PerkinElmer Life Sciences) precursors as described previously (54Pummill P.E. Achyuthan A.M. DeAngelis P.L. J. Biol. Chem. 1998; 273: 4976-4981Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 57DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Briefly, the crude membranes were incubated at 30 °C in Tris buffer, pH 7.5, with MgCl2 and the UDP-sugar precursors. Unincorporated, labeled UDP-sugars were separated from the HA product using paper chromatography. HA at the origin of the paper strip was detected by liquid scintillation counting. The assays were set so that <5% of the radiolabeled substrate was consumed and the enzyme concentration was in the linear range. All of the HAS assays throughout this work were performed in duplicate, and the values were averaged. The apparent Km values for the substrates were obtained by holding one radiolabeled UDP-sugar at a constant and saturating concentration while titrating the other UDP-sugar. Analysis of HA Product Size Distribution—The reactions were analyzed by either high performance gel filtration chromatography or agarose gel electrophoresis. For gel filtration analyses, the membranes (40–1400 μg of total protein) were incubated with 0.3–0.6 mm UDP-[14C]GlcUA (0.1–0.2 μCi) and 1.2–2.4 mm unlabeled UDP-GlcNAc in 50 mm Tris, pH 7.5, and 20 mm MgCl2 at 30 °C for various times. Control experiments validated that the HA product size was unaffected by the variations in UDP-sugar and enzyme concentrations. Membranes and the other reaction components were prewarmed separately to 30 °C prior to mixing to start the HAS reaction. At the end of the reaction time, the samples were either stopped by the addition of SDS to 2% (final concentration, w/v) or by addition of SDS to 0.5% followed by Pronase® treatment (final concentration, 1 mg/ml; overnight at 37 °C; Roche Applied Sciences). After Pronase digestion, the unincorporated precursors and small molecules were removed by ultrafiltration with a Microcon® 3 unit (three buffer changes; 3000 Da molecular mass cutoff; Amicon). The sizes of HA polymers were analyzed by gel filtration chromatography on a Phenomenex PolySep-GFC-P 5000 or 6000 column (300 × 7.8 mm) eluted with 0.2 m sodium nitrate at 0.6 ml/min on a Waters 600E system. All of the samples were clarified by centrifugation at 16,000 × g for 5 min prior to injection. Radioactive components were detected with a LB508 Radioflow Detector (EG & G Berthold) and Zinsser mixture (1.8 ml/min). The columns were standardized with various size dextrans (580, 145, 50, and 20 kDa) or, more appropriately, MANT-labeled HA with average molecular masses of 1300, 600, and 80 kDa (determined by MALLS). Each radiolabeled sample was spiked with an internal fluorescent dextran standard; elution times were reproducible to within 0.1 min. Because of discrepancies in the elution times of dextrans compared with HA, HA standards were made by substoichiometric labeling (∼1 MANT/50 monosaccharides) of hydroxyl groups of the streptococcal HA polysaccharide (1300 kDa by MALLS) with N-methylisatoic anhydride (61DeAngelis P.L. Anal. Biochem. 2000; 284: 167-169Crossref PubMed Scopus (12) Google Scholar). The 600-kDa standard was obtained by subfractionation of bulk HA using preparative high pressure liquid chromatography. Extended ultrasonication (1% acetone in water, 2-min intervals for 30 min total on ice) of the bulk HA with a Heat Systems Ultrasonic W-380 sonicator with a MicrotipTM (power setting 4) was used to produce the 80-kDa standard. The size determination of HA standards was performed by MALLS. The HA polymers (100 μg) were loaded on two tandem Toso Biosep TSK-GEL columns (6000PWXL followed by 4000PWXL; each 7.8 mm × 30 cm) and eluted in 50 mm sodium phosphate, 150 mm NaCl, pH 7, at 0.5 ml/min. The eluant flowed through an Optilab DSP interferometric refractometer and then a Dawn DSF laser photometer (632.8 nm; Wyatt Technology, Santa Barbara, CA) in the multi-angle mode. The software package of the manufacturer was used to determine the absolute average molecular mass using a dn/dC coefficient of 0.153. Some of the reactions treated with Pronase/Microcon were run on a 1.35% agarose gel in 1× TAE (40 mm Tris acetate, 2 mm EDTA, pH 8.5) at 30 V for several hours. The gels were dried, and the labeled HA was visualized using a PhosphorImager™ and ImageQuant© software from Molecular Dynamics. The identity of the radiolabeled material as authentic HA was assessed by Streptomyces HA lyase digestion. Prior to gel drying, the 1-kb DNA ladder standard (Stratagene) was visualized by photography of ethidium bromide fluorescence upon exposure to UV light. HA sizes were estimated by comparison with the DNA standard according to a previous study (62Lee H.G. Cowman M.K. Anal. Biochem. 1994; 219: 278-287Crossref PubMed Scopus (257) Google Scholar). Immunochemical Detection of Polypeptides—The xlHAS1 and mutant proteins were quantitated by Western blot analysis for assessment of the relative specific HAS activity. After SDS-PAGE separation, the proteins in the gel were transferred to nitrocellulose by semi-dry transfer. The blot was blocked with bovine serum albumin and incubated with the primary reagent composed of serum (1:1,000) from rabbits immunized with a fusion protein containing 1–166 residues of xlHAS1 (gift of I. Dawid; 50). Protein A-alkaline phosphatase detection with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium was used to visualize the immunoreactive bands. We have reported previously that xlHAS1 synthesizes HA at a rate of ∼80 monosaccharides/s and produces chains of between 3 × 106 and >2 × 107 Da (54Pummill P.E. Achyuthan A.M. DeAngelis P.L. J. Biol. Chem. 1998; 273: 4976-4981Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). These sizes were based on results obtained on a Sephacryl S-500HR gel filtration column using dextrans (α1→6 glucans) as standards. In this study, we have determined that these dextran polymers elute after HA polymers of similar size (dextrans are probably more compact than HA polymer of the same molecular mass) on the Phenomenex PolySep columns. Based on the MANT-labeled authentic HA standards, we have revised the HAS-catalyzed elongation rate for xlHAS1 to ∼3 monosaccharides/s in vitro and the final product chain length to between 5 × 104 and 9 × 105 Da in vitro. This rate is similar to that of mmHAS3 (from mouse) expressed in fibroblasts (48Itano 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 (693) Google Scholar), and the size range is similar to or slightly larger than that obtained for xlHAS1 expressed in COS-1 cells (2Spicer A.P. McDonald J.A. J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 51Koprunner M. Mullegger J. Lepperdinger G. Mech. Dev. 2000; 90: 275-278Crossref PubMed Scopus (38) Google Scholar), as determined by agarose gel analysis. We discovered that certain xlHAS1 mutants with substitutions for the serine at position 77 synthesize HA products with different size distribution (Table I). Of the Ser77 mutants created, S77F and S77I created a larger HA product and S77T created a smaller HA product. On the other hand, S77A, S77D, S77V, S77Y/R271G, and Y107F all made HA with an average size similar to that of wild type xlHAS1 (data not shown). As can be seen in Fig. 1, the average size of HA produced by S77T starts to fall behind that of wild type after ∼5 min. The average size of HA produced by S77T is smaller than that of wild type even after incubation for 60 min. S77F and S77I make HA with a larger average product size than wild type for all time points after 5 min.Table IEnzyme expression, HAS activity, and substrate affinityxlHAS1 enzymeProtein expressionHAS activityKmSize of HA compared with WTUDP-GlcNAcUDP-GlcUAWT++++400 ± 100; 260a0.6 mM UDP-GlcUA.190 ± 40S77A++++130a0.6 mM UDP-GlcUA.90equivalentS77D++NDbND, not determined.NDequivalentS77F++++160; 70a0.6 mM UDP-GlcUA.90 ± 50largerS77I+++90a0.6 mM UDP-GlcUA.50largerS77T++++460; 250a0.6 mM UDP-GlcUA.210 ± 60smallerS77V++++160a0.6 mM UDP-GlcUA.NDequivalentS77Y/R271G+++220; 150a0.6 mM UDP-GlcUA.90 ± 40equivalentY107F++++NDNDequivalentC117S++++340120largerC239S++++320110largerC337S++++400 ± 100700 ± 170smallera 0.6 mM UDP-GlcUA.b ND, not determined. Open table in a new tab We also tested several cysteine to serine mutants from our previous study (58Pummill P.E. DeAngelis P.L. J. Biol. Chem. 2002; 277: 21610-21616Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) for changes in the size of the HA product. As shown in Fig. 2, C117S and C239S consistently made larger HA than that of wild type, whereas C337S made HA that was consistently smaller. No difference was observed with any of the other cysteine to serine mutants (data not shown). The results obtained from the gel filtration experiments shown in Figs. 1 and 2 were confirmed by electrophoretic analysis of the HA produced by wild type and mutant enzymes. The average HA product size was larger than that" @default.
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• W2015196611 title "Alteration of Polysaccharide Size Distribution of a Vertebrate Hyaluronan Synthase by Mutation" @default.
• W2015196611 cites W1502415301 @default.
• W2015196611 cites W1569446107 @default.
• W2015196611 cites W1599301214 @default.
• W2015196611 cites W1964320911 @default.
• W2015196611 cites W1964730873 @default.
• W2015196611 cites W1969196669 @default.
• W2015196611 cites W1970976508 @default.
• W2015196611 cites W1977689971 @default.
• W2015196611 cites W1983020647 @default.
• W2015196611 cites W1987632841 @default.
• W2015196611 cites W1989846203 @default.
• W2015196611 cites W2001729687 @default.
• W2015196611 cites W2002552794 @default.
• W2015196611 cites W2003199291 @default.
• W2015196611 cites W2004038487 @default.
• W2015196611 cites W2007035508 @default.
• W2015196611 cites W2008678914 @default.
• W2015196611 cites W2009275174 @default.
• W2015196611 cites W2010427736 @default.
• W2015196611 cites W2011488277 @default.
• W2015196611 cites W2024054831 @default.
• W2015196611 cites W2026718707 @default.
• W2015196611 cites W2033306446 @default.
• W2015196611 cites W2034767282 @default.
• W2015196611 cites W2034807753 @default.
• W2015196611 cites W2035536358 @default.
• W2015196611 cites W2042705358 @default.
• W2015196611 cites W2047025323 @default.
• W2015196611 cites W2047439787 @default.
• W2015196611 cites W2053802748 @default.
• W2015196611 cites W2058245773 @default.
• W2015196611 cites W2061095488 @default.
• W2015196611 cites W2062003642 @default.
• W2015196611 cites W2064349021 @default.
• W2015196611 cites W2065736289 @default.
• W2015196611 cites W2069649792 @default.
• W2015196611 cites W2075922357 @default.
• W2015196611 cites W2087427950 @default.
• W2015196611 cites W2088128469 @default.
• W2015196611 cites W2088687466 @default.
• W2015196611 cites W2089764248 @default.
• W2015196611 cites W2101837780 @default.
• W2015196611 cites W2126659405 @default.
• W2015196611 cites W2134819882 @default.
• W2015196611 cites W2138083101 @default.
• W2015196611 cites W2139966914 @default.
• W2015196611 cites W2146505397 @default.
• W2015196611 cites W2151615110 @default.
• W2015196611 cites W2166818857 @default.
• W2015196611 cites W2167704983 @default.
• W2015196611 cites W2172200283 @default.
• W2015196611 cites W2323623488 @default.
• W2015196611 cites W2339327983 @default.
• W2015196611 cites W2346166755 @default.
• W2015196611 cites W348026187 @default.
• W2015196611 cites W4243231451 @default.
• W2015196611 cites W4244406676 @default.
• W2015196611 cites W4293247451 @default.
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