Hyaluronan Synthases
In 1934, Meyer and Palmer isolated a novel, highMr glycosaminoglycan from the vitreous of the eye (1Meyer K. Palmer J.W. J. Biol. Chem. 1934; 107: 629-634Abstract Full Text PDF Google Scholar). They showed that this substance contained a hexuronic acid, an amino sugar, and no sulfoesters and proposed the name hyaluronic acid (hyaluronan, HA), 1The abbreviations used are:HAhyaluronanHAShyaluronan synthaseRTreverse transcriptionPCRpolymerase chain reactionMDmembrane domain. from the Greekhyaloid (vitreous) and uronic acid. It took 20 years before Weissmann and Meyer (2Weissmann B. Meyer K. J. Am. Chem. Soc. 1954; 27: 1753-1757Crossref Scopus (219) Google Scholar) finally established the precise structure of the repeating disaccharide unit of hyaluronic acid (GlcAβ(1→3)GlcNAcβ(1→4)). The number of repeating disaccharides in an HA molecule can exceed 30,000, a Mr>107. MedLine surveys for reports describing the structure, synthesis, degradation, and biology of HA reveal a steadily increasing interest in this biopolymer during the four decades following the determination of its structure: 790 papers published from 1966 to 1975; 2200 from 1976 to 1985; over 3300 from 1986 to 1996. During this time, HA has been identified in virtually every tissue in vertebrates and has achieved widespread use in various clinical applications, most notably and appropriately as an intra-articular matrix supplement (3Balazs E.A. J. Rheumatol. 1993; 39: 3-9Google Scholar) and in eye surgery. This period has also seen a transition from the original perception that HA is primarily a passive structural component in the matrix of a few connective tissues and in the capsule of certain strains of bacteria to a recognition that this ubiquitous macromolecule is dynamically involved in many biological processes: from modulating cell migration and differentiation during embryogenesis (4Toole B.P. Curr. Opin. Cell Biol. 1990; 2: 839-844Crossref PubMed Scopus (393) Google Scholar) to regulation of extracellular matrix organization and metabolism (5Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar) to important roles in the complex processes of metastasis, wound healing, and inflammation (6Hall C.L. Turley E.A. J. Neuro-Oncology. 1995; 26: 221-229Crossref PubMed Scopus (78) Google Scholar, 7Lesley J. Kincade H.R. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar). Further, it is becoming clear that HA is highly metabolically active and that cells focus much attention on the processes of its synthesis and catabolism. For example, the half-life of HA in tissues ranges from 1 to 3 weeks in cartilage (8Morales T.I. Hascall V.C. J. Biol. Chem. 1988; 263: 3632-3638Abstract Full Text PDF PubMed Google Scholar) to <1 day in epidermis (9Tammi R. Säämänen A.-M. Maibach H.I. Tammi M. J. Invest. Dermatol. 1991; 97: 126-130Abstract Full Text PDF PubMed Google Scholar). In this report, we describe recent advances that provide exciting new insights into the biosynthetic side of these metabolic processes. hyaluronan hyaluronan synthase reverse transcription polymerase chain reaction membrane domain. It is now clear that a single protein utilizes both sugar substrates to synthesize HA (10DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar). The abbreviation HAS, for theHAsynthase, has gained widespread support for designating this class of enzymes and should now be accepted as standard nomenclature. Markovitz et al. (11Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Abstract Full Text PDF PubMed Google Scholar) successfully characterized the HAS activity from Streptococcus pyogenes and discovered the enzyme's membrane localization and its requirements for sugar nucleotide precursors and Mg2+. Prehm (12Prehm P. Biochem. J. 1984; 220: 597-600Crossref PubMed Scopus (293) Google Scholar) found that elongating HA, made by B6 cells, was digested by hyaluronidase added to the medium and proposed that HAS resides at the plasma membrane. Philipson and Schwartz (13Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Abstract Full Text PDF PubMed Google Scholar) also showed that HAS activity cofractionated with plasma membrane markers in mouse oligodendroglioma cells. HAS assembles high MrHA that is simultaneously extruded through the membrane into the extracellular space (or to make the cell capsule in the case of bacteria) as glycosaminoglycan synthesis proceeds. This mode of biosynthesis is unique among macromolecules since nucleic acids, proteins, and lipids are synthesized in the nucleus, endoplasmic reticulum/Golgi, cytoplasm, or mitochondria. The extrusion of the growing chain into the extracellular space would also allow unconstrained polymer growth, thereby achieving the exceptionally large size of HA, whereas confinement of synthesis within a Golgi or post-Golgi compartment could limit the overall amount or length of the polymers formed. High concentrations of HA within a confined lumen could also create a high viscosity environment that might be deleterious for other organelle functions. In 1983, Prehm (14Prehm P. Biochem. J. 1983; 211: 191-198Crossref PubMed Scopus (120) Google Scholar) proposed a novel mechanism for HA synthesis that was distinctly different from that for other glycosaminoglycans, such as chondroitin sulfate and heparan sulfate. These latter glycosaminoglycans are elongated on core proteins by transfer of an appropriate sugar from a sugar nucleotide onto the nonreducing terminus of a growing chain (15Hascall V.C. Heinegard D.K. Wight T.N. Hay E.D. Cell Biology of Extracellular Matrix. Plenum Publishing Corp., New York1991: 149-175Crossref Google Scholar). However, Prehm (14Prehm P. Biochem. J. 1983; 211: 191-198Crossref PubMed Scopus (120) Google Scholar) proposed that HA synthesis occurs at the reducing terminus of a growing HA chain by a two-site mechanism (Fig. 1). In this mechanism, the reducing end sugar of the growing HA chain (either in the GlcNAc or GlcA site) would remain covalently bound to a terminal UDP, and the next sugar to be added from the second site would be transferred as the UDP-sugar onto the reducing end sugar with displacement of its terminal UDP. The HA chain would then be in the second site. This unusual mode of synthesis does not occur with the eukaryotic heparan sulfate synthase (16Bame K.J. Cheifetz S. Massague J. Lindahl U. Esko J.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2267-2271Crossref PubMed Scopus (236) Google Scholar) or the bacterial, K5 (17Lidholt K. Fjelstad M. Jann K. Lindahl U. Carbohydr. Res. 1994; 255: 87-101Crossref PubMed Scopus (20) Google Scholar), or K4 (18Lidholt K. Fjelstad M. J. Biol. Chem. 1997; 272: 2682-2687Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) polysaccharide synthases, each of which utilizes the same nucleotide sugar substrates, and it remains to be verified using purified recombinant HAS. Several studies attempted to solubilize, identify, and purify HAS from strains of Streptococci that make a capsular coat of HA as well as from eukaryotic cells (11Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Abstract Full Text PDF PubMed Google Scholar, 12Prehm P. Biochem. J. 1984; 220: 597-600Crossref PubMed Scopus (293) Google Scholar, 13Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Abstract Full Text PDF PubMed Google Scholar, 19Prehm P. Mausolf A. Biochem. J. 1986; 235: 887-889Crossref PubMed Scopus (33) Google Scholar, 20Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar, 21Ng K.F. Schwartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Abstract Full Text PDF PubMed Google Scholar). Although the streptococcal (19Prehm P. Mausolf A. Biochem. J. 1986; 235: 887-889Crossref PubMed Scopus (33) Google Scholar, 20Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar) and murine oligodendroglioma enzymes (21Ng K.F. Schwartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Abstract Full Text PDF PubMed Google Scholar) were successfully detergent-solubilized and studied, efforts to purify an active HAS for further study or molecular cloning remained unsuccessful for decades. Prehm and Mausolf (19Prehm P. Mausolf A. Biochem. J. 1986; 235: 887-889Crossref PubMed Scopus (33) Google Scholar) used periodate-oxidized UDP-GlcA or UDP-GlcNAc to affinity label a protein of ∼52 kDa in streptococcal membranes that co-purified with HA. This led to a report (22Lansing M. Lellig S. Mausolf A. Martini I. Crescenzi F. Oregon M. Prehm P. Biochem. J. 1993; 289: 179-184Crossref PubMed Scopus (40) Google Scholar) claiming that the Group C streptococcal HAS had been cloned, which was unfortunately erroneous. This study failed to demonstrate expression of an active synthase and may have actually cloned a peptide transporter. Triscott and van de Rijn (20Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar) used digitonin to solubilize HAS from streptococcal membranes in an active form. van de Rijn and Drake (23van de Rijn I. Drake R.R. J. Biol. Chem. 1992; 267: 24302-24306Abstract Full Text PDF PubMed Google Scholar) selectively radiolabeled three streptococcal membrane proteins of 42, 33, and 27 kDa with 5-azido-UDP-GlcA and suggested that the 33-kDa protein was HAS. As shown later (24DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Abstract Full Text PDF PubMed Google Scholar, 25DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar), however, HAS actually turned out to be the 42-kDa protein. Despite these efforts, progress in understanding the regulation and mechanisms of HA synthesis was essentially stalled, since there were no molecular probes for HAS mRNA or HAS protein. A major breakthrough occurred in 1993 when DeAngelis et al. (24DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Abstract Full Text PDF PubMed Google Scholar, 25DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar) reported the molecular cloning and characterization of the Group A streptococcal gene encoding the protein HasA, known to be in an operon required for bacterial HA synthesis (26Dougherty B.A. van de Rijn I. J. Biol. Chem. 1993; 268: 7118-7124Abstract Full Text PDF PubMed Google Scholar), although the function of this protein, which we now propose to designate as spHAS (the S. pyogenes HAS), was unknown. spHAS was subsequently proven to be responsible for HA elongation (see below) and was the first glycosaminoglycan synthase identified and cloned and then successfully expressed (10DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar). TheS. pyogenes HA synthesis operon encodes two other proteins. HasB is a UDP-glucose dehydrogenase, which is required to convert UDP-glucose to UDP-GlcA, one of the substrates for HA synthesis (26Dougherty B.A. van de Rijn I. J. Biol. Chem. 1993; 268: 7118-7124Abstract Full Text PDF PubMed Google Scholar). HasC is a UDP-glucose pyrophosphorylase, which is required to convert glucose 1-phosphate and UTP to UDP-glucose (27Crater D.L. Dougherty B.A. van de Rijn I. J. Biol. Chem. 1995; 270: 28676-28680Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Co-transfection of both hasA and hasB genes into either acapsularStreptococcus strains or Enteroccus faecalis conferred them with the ability to synthesize HA and form a capsule (24DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Abstract Full Text PDF PubMed Google Scholar, 25DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar). This provided the first strong evidence that HasA is an HA synthase. The elusive HA synthase gene was finally cloned by a transposon mutagenesis approach (24DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Abstract Full Text PDF PubMed Google Scholar), in which an acapsular mutant Group A strain was created containing a transposon interruption of the HA synthesis operon. Known sequences of the transposon allowed the region of the junction with streptococcal DNA to be identified and then cloned from wild-type cells. The encoded spHAS (25DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar) was 5–10% identical to a family of yeast chitin synthases and 30% identical to theXenopus laevis protein DG42 (developmentally expressed during gastrulation (28Rosa 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 (86) Google Scholar)), whose function was unknown at the time. DeAngelis and Weigel (10DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar) expressed the active recombinant spHAS in Escherichia coli and showed that this single purified gene product synthesizes high MrHA when incubated in vitro with UDP-GlcA and UDP-GlcNAc, thereby showing that both glycosyltransferase activities required for HA synthesis are catalyzed by the same protein, as first proposed in 1959 (11Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Abstract Full Text PDF PubMed Google Scholar). This set the stage for the almost simultaneous identification of eukaryotic HAS cDNAs in 1996 by four laboratories revealing that HAS is a multigene family encoding distinct isozymes. Two genes (HAS1 and HAS2) were quickly discovered in mammals (29Itano N. Kimata K. J. Biol. Chem. 1996; 271: 9875-9878Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 30Shyjan 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, 31Itano N. Kimata K. Biochem. Biophys. Res. Commun. 1996; 222: 816-820Crossref PubMed Scopus (106) Google Scholar, 32Spicer 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, 33Watanabe K. Yamaguchi Y. J. Biol. Chem. 1996; 271: 22945-22948Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 34Fulop C. Salustri A. Hascall V.C. Arch. Biochem. Biophys. 1997; 337: 261-266Crossref PubMed Scopus (136) Google Scholar), and a third gene has now been found (35Spicer 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). Fig.2 compares the predicted amino acid sequences of spHAS, and mouse and and HAS. Further, studies and P. in have also identified the HAS gene from Group C Streptococcus the protein has a high of to the spHAS from E. coli recombinant synthesize HA when both substrates are These that the report of et al. (22Lansing M. Lellig S. Mausolf A. Martini I. Crescenzi F. Oregon M. Prehm P. Biochem. J. 1993; 289: 179-184Crossref PubMed Scopus (40) Google Scholar) claiming to have cloned the Group C HAS was studies have to this streptococcal protein to was to be eukaryotic HAS M. E. Prehm P. Res. 1992; 54: PubMed Scopus Google Scholar, Turley E.A. Prehm P. Biochem. J. 1993; PubMed Scopus Google Scholar, M. C. Prehm P. E. Arch. 1993; PubMed Scopus Google Scholar, Prehm P. J. 1994; PubMed Scopus Google Scholar, Prehm P. J. 1995; PubMed Scopus Google Scholar, A. Prehm P. B. U. 1996; PubMed Scopus (20) Google Scholar). In of it be that these reports and should be since were not in HAS. and Kimata (29Itano N. Kimata K. J. Biol. Chem. 1996; 271: 9875-9878Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) used expression cloning in a mutant mouse cell to synthesize HA, to the first HAS in HA synthesis into three that were for HA synthesis in cell that at three proteins are Two of these HA whereas one showed The latter cell was used in with from the cells to a single protein that HA a structure for a protein of kDa with a predicted membrane to that of spHAS (25DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar). is 30% identical to spHAS and identical to The same this report three other papers describing cDNAs encoding was to be the same mouse and However, through an each of the four laboratories had discovered a HAS in both a cloning approach to that of and Kimata (29Itano N. Kimata K. J. Biol. Chem. 1996; 271: 9875-9878Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), al. 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) identified the of A was used to murine that were then for ability to in a of one was by to a known cell protein, and was by with by this required synthesis of HA. and of the responsible identified and Kimata N. Kimata K. Biochem. Biophys. Res. Commun. 1996; 222: 816-820Crossref PubMed Scopus (106) Google Scholar) also reported a isolated from a The cDNAs reported by the two however, in a 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) or a N. Kimata K. Biochem. Biophys. Res. Commun. 1996; 222: 816-820Crossref PubMed Scopus (106) Google Scholar) amino acid protein. HAS activity has been for the form. on the molecular identification of spHAS as an HA synthase and of among spHAS, and in et al. 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) used a approach to a mouse encoding a second distinct which is of into cells de of an HA cell coat by a thereby strong evidence that the protein can synthesize HA. a and Yamaguchi K. Yamaguchi Y. J. Biol. Chem. 1996; 271: 22945-22948Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) a to et al. C. Salustri A. Hascall V.C. Arch. Biochem. Biophys. 1997; 337: 261-266Crossref PubMed Scopus (136) Google Scholar) used a to in isolated from cells HA, a for in the were isolated from an before HA synthesis and at later when HA synthesis is or showed that mRNA was expressed at high later that transcription of HA synthesis in this and are amino in length and are is amino and identical to which is amino et al. (35Spicer 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) used a approach to a third HAS gene in The protein is amino (Fig. and and to and et al. A.P. McDonald J.A. 1997; PubMed Google Scholar) have also the three and mouse genes to three different (HAS1 to to to of the three HAS genes on different and the of HA the class that this gene family is and that by in the of The high the bacterial and eukaryotic also a bacteria the HAS gene from an before the eukaryotic gene and the bacteria could have a HAS gene and sequences for The of laevis DG42 by Dawid and (28Rosa 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 (86) Google Scholar) a in these recent this protein was not known to be an HA synthase. that DG42 and spHAS were 30% identical was for that allowed identification of A.P. Augustine M.L. McDonald J.A. J. Biol. Chem. 1996; 271: 23400-23406Abstract Full Text Full Text PDF PubMed Scopus (156) Google C. Salustri A. Hascall V.C. Arch. Biochem. Biophys. 1997; 337: 261-266Crossref PubMed Scopus (136) Google Scholar). evidence that DG42 is a HA synthase was reported the of the when DeAngelis and P.L. A.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus (50) Google Scholar) expressed the recombinant protein in yeast that synthesize and showed that it synthesizes HA when isolated membranes are provided with the two Meyer and Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) also showed that from cells with for DG42 synthesize of HA. that its function is DG42 be (Fig. 3 the predicted structural by the HAS proteins, a large and of or at both the amino and of the protein. The which to of the predicted HAS protein the of the (10DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar, 25DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar). This predicted is amino in spHAS of the and in the eukaryotic HAS of the The number and of membrane and the organization of extracellular and have not been for HAS. spHAS is the HAS family to that has been purified and characterized (10DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar). studies using proteins that the C and the large of spHAS in the cell C. DeAngelis P. Weigel P. Biol. 1996; Scholar). spHAS has whereas and have and Two of the in spHAS are and identical in and one is found at the same in in the HAS family shown in This may be an whose by The of or the identification of for of the HAS has not been for of the HAS In to the proposed unique mode of synthesis at the plasma the HAS family is highly unusual in the large number of required for the overall of HA. activities could be within the HAS for each of the two different sugar nucleotide precursors and two different glycosyltransferase one or that the growing HA polymer to the to a B. R. Turley E.A. J. 1994; PubMed Scopus Google Scholar)), and a transfer reaction that the growing polymer one sugar at a time. This later activity is with the of the polymer through the membrane. of these and as are in a protein in size from to amino Although the evidence the that the spHAS protein is required for HA biosynthesis in bacteria it is that the eukaryotic HAS family are of N. Biochem. J. 1986; PubMed Scopus Google Scholar). the eukaryotic HAS proteins are spHAS, protein could be involved in such as and regulation of and with other The that there are HAS genes encoding different synthases the that HA is an important of cell and not a structural component in in the from one cloned HAS to recognition of a multigene family that and exciting advances in understanding of the synthesis and biology of HA. and McDonald for recent studies to for the for with the and DeAngelis for