From the Department of Biochemistry and Molecular
Biology, University of Oklahoma Health Sciences Center, Oklahoma City,
Oklahoma 73190, ¶ Department of Biomedical Engineering, The
Cleveland Clinic Foundation, Cleveland, Ohio 44195, and
Department of Anatomy, University of Kuopio,
70211 Kuopio, Finland
In 1934, Meyer and Palmer isolated a novel, high
Mr glycosaminoglycan from the vitreous of the
eye (1). They showed that this substance contained a hexuronic acid, an
amino sugar, and no sulfoesters and proposed the name hyaluronic acid
(hyaluronan, HA),1 from the Greek
hyaloid (vitreous) and uronic acid. It took 20 years before
Weissmann and Meyer (2) finally established the precise structure of
the repeating disaccharide unit of hyaluronic acid
(GlcA It is now clear that a single protein utilizes both sugar
substrates to synthesize HA (10). The abbreviation HAS, for the HA synthase, has gained widespread support for
designating this class of enzymes and should now be accepted as
standard nomenclature. Markovitz et al. (11) 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
(12) 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 (13) also showed that HAS
activity cofractionated with plasma membrane markers in mouse
oligodendroglioma cells. HAS assembles high Mr HA 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 (14) 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 (15). However, Prehm (14) 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 (16) or the
bacterial, K5 (17), or K4 (18) 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 (11-13, 19-21). Although the streptococcal (19, 20) and murine oligodendroglioma enzymes (21) 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 (19) 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 (22) 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 (20) used digitonin to solubilize HAS from
streptococcal membranes in an active form. van de Rijn and Drake (23)
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 (24, 25), 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. (24, 25) 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 (26), 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 (10). The
S. 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 (26).
HasC is a UDP-glucose pyrophosphorylase, which is required to convert
glucose 1-phosphate and UTP to UDP-glucose (27). Co-transfection of
both hasA and hasB genes into either acapsular
Streptococcus strains or Enteroccus faecalis
conferred them with the ability to synthesize HA and form a capsule
(24, 25). 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 (24), 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 (25) was 5-10% identical to a
family of yeast chitin synthases and 30% identical to the
Xenopus laevis protein DG42 (developmentally expressed during gastrulation (28)), whose function was
unknown at the time. DeAngelis and Weigel (10) expressed the active recombinant spHAS in Escherichia coli and showed that this
single purified gene product synthesizes high Mr
HA 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 (11). 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 (29-34), and a third gene has now been found (35). Fig.
2 compares the predicted amino acid sequences of spHAS,
human HAS1 and HAS2, mouse HAS1, HAS2, and HAS3, and frog HAS.
Further, preliminary studies2 have also
identified the authentic HAS gene from Group C Streptococcus
equisimilis (seHAS); the seHAS protein has a high level
of identity to the spHAS enzyme. Membranes prepared from E. coli expressing recombinant seHAS synthesize HA when both
substrates are provided. These results confirm that the earlier report
of Lansing et al. (22) claiming to have cloned the Group C
HAS was wrong. Unfortunately, several studies have employed antibody to
this uncharacterized 52-kDa streptococcal protein to investigate what
was believed to be eukaryotic HAS (36-41). In view of subsequent
developments, it must be considered that these reports reached
incorrect conclusions and should be reexamined, since they were not in
fact studying HAS.
Itano and Kimata (29) used expression cloning in a mutant mouse mammary
carcinoma cell line, unable to synthesize HA, to clone the first
putative mammalian HAS cDNA (mmHAS1). Subclones defective in HA
synthesis fell into three separate classes that were complementary for
HA synthesis in somatic cell fusion experiments, suggesting that at
least three proteins are required. Two of these classes maintained some
HA synthetic activity, whereas one showed none. The latter cell line
was used in transient transfection experiments with cDNA prepared
from the parental cells to identify a single protein that restored HA
synthetic activity. Sequence analyses revealed a deduced primary
structure for a protein of ~65 kDa with a predicted membrane topology
similar to that of spHAS (25). mmHAS1 is 30% identical to spHAS and
55% identical to DG42. The same month this report appeared, three
other groups submitted papers describing cDNAs encoding what was
initially thought to be the same mouse and human enzyme. However,
through an extraordinary circumstance, each of the four laboratories
had discovered a separate HAS isozyme in both species. Using a similar functional cloning approach to that of Itano and Kimata (29), Shyjan
et al. (30) identified the human homolog of HAS1. A
mesenteric lymph node cDNA library was used to transfect murine
mucosal T lymphocytes that were then screened for their ability to
adhere in a rosette assay. Adhesion of one transfectant was inhibited by antisera to CD44, a known cell surface HA-binding protein, and was
abrogated directly by pretreatment with hyaluronidase. Thus, rosetting
by this transfectant required synthesis of HA. Cloning and sequencing
of the responsible cDNA identified hsHAS1. Itano and Kimata (31)
also reported a human HAS1 cDNA isolated from a fetal brain
library. The hsHAS1 cDNAs reported by the two groups, however,
differ in length; they encode a 578 (30) or a 543 (31) amino acid
protein. HAS activity has only been demonstrated for the longer
form.
Based on the molecular identification of spHAS as an authentic HA
synthase and regions of near identity among DG42, spHAS, and NodC (a
Most recently Spicer et al. (35) used a PCR approach to
identify a third HAS gene in mammals. The mmHAS3 protein is 554 amino
acids long (Fig. 2) and 57, 71, 56, and 28% identical, respectively, to mmHAS1, mmHAS2, DG42, and spHAS. Spicer et al. (42) have also localized the three human and mouse genes to three different chromosomes (HAS1 to hsChr 19/mmChr 17; HAS2 to hsChr 8/mmChr 15; HAS3
to hsChr 16/mmChr 8). Localization of the three HAS genes on different
chromosomes and the appearance of HA throughout the vertebrate class
suggest that this gene family is ancient and that isozymes appeared by
duplication early in the evolution of vertebrates. The high identity
(~30%) between the bacterial and eukaryotic HASs also indicates a
common ancestoral gene. Perhaps primitive bacteria usurped the HAS gene
from an early vertebrate ancestor before the eukaryotic gene products
became larger and more complex. Alternatively, the bacteria could have
obtained a larger vertebrate HAS gene and deleted regulatory sequences nonessential for enzyme activity.
The discovery of X. laevis DG42 by Dawid and co-workers (28)
played a significant role in these recent developments, even though
this protein was not known to be an HA synthase. Nonetheless, that DG42
and spHAS were 30% identical was critical for designing oligonucleotides that allowed identification of mammalian HAS2 (32,
34). Ironically, definitive evidence that DG42 is a bona fide HA synthase was reported only after the discoveries of the mammalian isozymes, when DeAngelis and Achyuthan (43) expressed the
recombinant protein in yeast (an organism that cannot synthesize HA)
and showed that it synthesizes HA when isolated membranes are provided
with the two substrates. Meyer and Kreil (44) also showed that lysates
from cells transfected with cDNA for DG42 synthesize elevated
levels of HA. Now that its function is known, DG42 can, therefore, be
designated xlHAS (Fig. 2).
Structural Features of the HAS Proteins Fig. 3 depicts the common predicted structural
features shared by all the HAS proteins, including a large central
domain and clusters of 2-3 transmembrane or membrane-associated
domains at both the amino and carboxyl ends of the protein. The central
domain, which comprises up to ~88% of the predicted intracellular
HAS protein sequences, probably contains the catalytic regions of the
enzyme (10, 25). This predicted central domain is 264 amino acids long
in spHAS (63% of the total protein) and 307-328 residues long in the
eukaryotic HAS members (54-56% of the total protein). The exact
number and orientation of membrane domains and the topological
organization of extracellular and intracellular loops have not yet been
experimentally determined for any HAS. spHAS is the only HAS family
member to date that has been purified and partially characterized (10).
Initial studies using spHAS/alkaline phosphatase fusion proteins
indicate that the N terminus, C terminus, and the large central domain
of spHAS are, in fact, inside the cell (45). spHAS has 6 cysteines,
whereas HAS1, HAS2, and HAS3 have 13, 14, and 14 Cys residues,
respectively. Two of the 6 Cys residues in spHAS are conserved and
identical in HAS1 and HAS2. Only one conserved Cys residue is found at
the same position (Cys-225 in spHAS) in the HAS family members shown in
Fig. 2. This may be an essential Cys whose modification by sulfhydryl
poisons partially inhibits enzyme activity.2 The possible
presence of disulfide bonds or the identification of critical Cys
residues needed for any of the multiple HAS functions noted below has
not yet been elucidated for any members of the HAS family.
In addition to the proposed unique mode of synthesis at the plasma
membrane, the HAS enzyme family is highly unusual in the large number
of functions required for the overall polymerization of HA. At least
six discrete activities could be present within the HAS enzyme: binding
sites for each of the two different sugar nucleotide precursors
(UDP-GlcNAc and UDP-GlcA), two different glycosyltransferase
activities, one or more binding sites that anchor the growing HA
polymer to the enzyme (perhaps related to a
B-X7-B motif (46)), and a ratchet-like transfer
reaction that moves the growing polymer one sugar at a time. This later
activity is likely coincident with the stepwise advance of the polymer through the membrane. All of these functions, and perhaps others as yet
unknown, are present in a relatively small protein ranging in size from
419 (spHAS) to 588 (xlHAS) amino acids.
Although all the available evidence supports the conclusion that only
the spHAS protein is required for HA biosynthesis in bacteria or
in vitro, it is possible that the larger eukaryotic HAS
family members are part of multicomponent complexes (47). Since the
eukaryotic HAS proteins are ~40% larger than spHAS, their additional
protein domains could be involved in more elaborate functions such as
intracellular trafficking and localization, regulation of enzyme
activity, and mediating interactions with other cellular
components.
The unexpected finding that there are multiple vertebrate HAS genes
encoding different synthases strongly supports the emerging consensus
that HA is an important regulator of cell behavior and not simply a
structural component in tissues. Thus, in less than 6 months, the field
moved from one known, cloned HAS (spHAS) to recognition of a multigene
family that promises rapid, numerous, and exciting future advances in
our understanding of the synthesis and biology of HA.
(1
3)GlcNAc
(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 (3) 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 (4) to regulation of extracellular matrix organization
and metabolism (5) to important roles in the complex processes of
metastasis, wound healing, and inflammation (6, 7). 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 (8) to <1 day in epidermis (9). In this report, we describe
recent advances that provide exciting new insights into the
biosynthetic side of these metabolic processes.
Fig. 1.
Proposed mechanism of HA synthesis. The
repeating disaccharide (shown in brackets) is synthesized by
extension of the polymer at the reducing end via a two-site mechanism
(14), as described in the text. Definitive evidence for this unusual
mode of saccharide synthesis using purified, recombinant HAS has not yet been obtained.
[View Larger Version of this Image (11K GIF file)]
Fig. 2.
The HAS protein family. Protein
sequences of HAS family members from human (30, 31, 33), mouse (29, 32,
34, 35), bacteria (25), and frog (28) are shown. Asterisks
denote residues conserved in all family members. The shaded
and boldface areas are amino acids identical to the smallest
member of the family shown, spHAS. These presumably include any
specific residues necessary to create an active HAS. Solid
dots above the sequences indicate those amino acids conserved in
all members of the broader -glycosyltransferase family including
NodC, chitin synthases, and cellulose synthase (48). The
diamond denotes the Cys residue (Cys-225 in spHAS) conserved
within the family. The approximate midpoints predicted for MD1-MD7 are
indicated above the sequences. Three or more possible whole or partial
HA-binding domains (B-X7-B, where B is a basic
residue (46)) and many potential phosphorylation sites are present in
all family members (not shown). The carboxyl region of the central
domain, Ala-145-Pro-317, in spHAS is particularly conserved, with few
gaps, in the family. hs, Homo sapiens;
mm, Mus musculis; sp, S. pyogenes; xl, X. laevis.
[View Larger Version of this Image (102K GIF file)]
-GlcNAc transferase nodulation factor in Rhizobium), Spicer et al. (32) used a degenerate RT-PCR approach to
clone a mouse embryo cDNA encoding a second distinct enzyme, which
is designated mmHAS2. Transfection of mmHAS2 cDNA into COS cells directed de novo production of an HA cell coat detected by a
particle exclusion assay, thereby providing strong evidence that the
HAS2 protein can synthesize HA. Using a similar approach, Watanabe and
Yamaguchi (33) screened a human fetal brain cDNA library to
identify hsHAS2. Fulop et al. (34) independently used a
similar strategy to identify mmHAS2 in RNA isolated from ovarian
cumulus cells actively synthesizing HA, a critical process for normal cumulus oophorus expansion in the pre-ovulatory follicle. Cumulus cell-oocyte complexes were isolated from mice immediately after initiating an ovulatory cycle, before HA synthesis begins, and at later
times when HA synthesis is just beginning (3 h) or already apparent (4 h). RT-PCR showed that HAS2 mRNA was absent initially but expressed
at high levels 3-4 h later suggesting that transcription of HAS2
regulates HA synthesis in this process. Both hsHAS2 and mmHAS2 are 552 amino acids in length and are 98% identical. mmHAS1 is 583 amino acids
long and 95% identical to hsHAS1, which is 578 amino acids long.
Fig. 3.
Proposed membrane topology for the HAS
family. Very similar hydropathy plots and primary structure
(28-71% identity) among all the HAS isozymes suggest that they are
similarly organized within the membrane. The scheme depicts the N and C
termini and the large central domain, between MD2 and MD3, inside the
cell. The larger eukaryotic HASs (thick line) have
additional amino acids in all regions (see Fig. 2) compared with the
bacterial HASs (thin line), except for the highly conserved
carboxyl 178 residues of the central domain and MD1-MD5. In
particular, the carboxyl ~25% of the eukaryotic HASs has two
additional predicted membrane domains (MD6 and MD7), missing in the
bacterial proteins. The conserved Cys is indicated by the circled
C. MD5 can be modeled as an amphipathic helix, which would orient
the C terminus of all HAS members inside.
[View Larger Version of this Image (16K GIF file)]
We thank Drs. Andrew Spicer and John McDonald for sharing their recent studies prior to publication. We thank Debbie Blevins for help preparing the manuscript, Coy Heldermon for help with the figures, and Dr. Paul DeAngelis for helpful discussions.