Chapter 16 Hyaluronan

Essentials of Glycobiology, 3rd edition Chapter 16 Hyaluronan

Authors: Vincent Hascall and Jeffrey D. Esko

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Animal cells and some bacteria produce hyaluronan, a high molecular weight, non- sulfated glycosaminoglycan synthesized at the cell surface and extruded into the extracellular environment. Its biological activity depends on size and binding to a number of hyaluronan-binding proteins. This chapter describes the structure and metabolism of hyaluronan, its chemical and biophysical attributes, and its highly diverse and versatile biological functions.

HISTORICAL AND EVOLUTIONARY PERSPECTIVES

Sulfated glycosaminoglycans were first isolated in the late 1800s, and the isolation of hyaluronic acid (now called hyaluronan) followed in the early 1930s. In their classic paper, Karl Meyer and John Palmer named the “polysaccharide acid of high molecular weight” that they purified from bovine vitreous humor as “hyaluronic acid” (from hyaloid, meaning vitreous), and they showed that it contained “uronic acid (and) an amino sugar.” It took almost 20 years to determine the actual structure of the repeating disaccharide motif of hyaluronan (Figure 16.1). In contrast to the other classes of glycosaminoglycans, hyaluronan is not further modified by sulfation or by epimerization of the glucuronic acid moiety to iduronic acid. Thus, the chemical structure shown in Figure 16.1 is faithfully reproduced by any cell that synthesizes hyaluronan, including animal cells and bacteria.

At first glance, the simplicity of hyaluronan might suggest that it arose early in evolution relative to other glycosaminoglycans. However, this is not the case, because Drosophila melanogaster and Caenorhabditis elegans do not contain the necessary synthases for its assembly. Instead, it appears that hyaluronan arose during the evolution of the notocord shortly before or concurrent with the advent of cartilage and appendicular skeletons. Virtually all cells from vertebrate species can produce hyaluronan, and its expression correlates with tissue expansion and cell motility. As discussed below, hyaluronan has essential roles in development, tissue architecture, cell proliferation, signaling reactions across the plasma membrane, inflammation and microbial virulence.

STRUCTURE AND BIOPHYSICAL PROPERTIES

Hyaluronan has an indefinite and very high degree of polymerization, typically in the range of 104 disaccharides, with an end-to-end length of approximately 10 μm (~1 nm/disaccharide). Thus, a single molecule of hyaluronan could stretch about halfway around the circumference of a typical mammalian cell. The carboxyl groups on the glucuronic acid residues (pKa 4–5) are negatively charged at physiological pH and ionic strength, making hyaluronan polyanionic. The anionic nature of hyaluronan together with spatial restrictions around the glycosidic bonds confer a relatively stiff, random coil structure to individual hyaluronan molecules in most biological settings. Hyaluronan chains occupy a large hydrodynamic volume such that in a solution containing 3–5 mg/ml hyaluronan, individual molecules occupy essentially all of the solvent. This arrangement creates a size-selective barrier in which small molecules can diffuse freely, whereas larger molecules are partially or completely excluded. Additionally, this solution would exhibit high viscosity with viscoelastic properties, conditions found in the vitreous humor of the human eye and in synovial fluid of joints. Hyaluronan in synovial fluids of articular joints is essential for distributing load during joint motion and for protecting the cartilaginous surfaces. Thus, in both eye and joint tissues, the physical properties of hyaluronan relate directly to tissue function.

BIOSYNTHESIS

Hyaluronan biosynthesis is catalyzed by hyaluronan synthases (HAS) (Figure 16.2). The first bona fide HAS gene (spHas) was cloned from Streptococcus, and the protein expressed in Escherichia coli was shown to synthesize high-molecular-weight hyaluronan from the UDP-sugar substrates. The gene shows homology with a Xenopus gene, DG42 (now known as xlHAS1; Chapter 27). The homology was instrumental in the subsequent identification of the three members of the mammalian Has gene family, Has1–3. These genes code for homologous proteins predicted to contain five to six membrane-spanning segments and a central cytoplasmic domain.

As described in Chapter 17, cells synthesize sulfated glycosaminoglycans (heparan sulfate, chondroitin sulfate, and keratan sulfate) on core proteins of proteoglycans as they transit through the Golgi, and elongation of the chains occurs at their nonreducing ends. In contrast, hyaluronan synthesis normally occurs at the inner surface of the plasma membrane in eukaryotic cells and at the cytoplasmic membrane of bacteria that produce hyaluronan capsules. The synthases use the cytosolic substrates UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc) and extrude the growing polymer through the membrane to form extracellular matrices (Figure 16.2). According to the model, the reducing end of the growing chain would have a UDP moiety that is displaced when the next nucleotide sugar is added.

In mammalian cells, division under conditions of hyperglycemia (2-3 times normal glucose level) results in hyaluronan synthesis in the endoplasmic reticulum (ER), Golgi and transport vesicles. Under these conditions, the elongating hyaluronan chains are inserted inappropriately into these compartments, inducing abnormalities in cellular functions, e.g. kidney nephropathy and proteinurea. The activity of the HAS enzymes also can be regulated by phosphorylation and addition of O-GlcNAc (Chapter 19).

Hyaluronan biosynthesis in bacteria involves the expression of multiple enzymes, usually as an operon. For example, in Streptococcus, hasC encodes an enzyme that makes UDP-Glc from UTP and glucose-1-P; hasB encodes the dehydrogenase that converts UDP-Glc to UDP-GlcA; hasD generates UDP-GlcNAc from glucosamine-1-P, acetyl CoA, and UTP; and hasA (spHas) encodes the hyaluronan synthase. The Streptococcus hasA gene encodes a bifunctional protein that contains both transferase activities. Thus, spHas assembles the polysaccharide from the reducing end. The synthase spans the membrane multiple times, presumably forming a pore for hyaluronan extrusion during capsule formation. In contrast, Pasteurella synthesizes hyaluronan by an enzyme that is unrelated to hasA and the mammalian Has gene family. In this case, the enzyme has two separable domains with independent glycosyltransferase activities—one for UDP-GlcNAc and the other for UDP-GlcA, and the elongation is on the non-reducing end.

THE HYALURONIDASES AND HYALURONAN TURNOVER

Animal cells express a set of catabolic enzymes that degrade hyaluronan. The human hyaluronidase gene (HYAL) family is complex, with two sets of three contiguous genes located on two chromosomes, a pattern that suggests two ancient gene duplications followed by a block duplication. In humans, the cluster on chromosome 3p21.3 (HYAL1, 2, and 3) appears to have major roles in somatic tissues. HYAL4 in the cluster on chromosome 7q31.3 codes for a protein that appears to have chondroitinase, but not hyaluronidase, activity; PHYAL1 is a pseudogene; and SPAM1 (sperm adhesion molecule 1, PH-20) is restricted to testes. The role of SPAM1 in fertilization is discussed below.

The turnover of hyaluronan in most tissues is rapid (e.g., a half-life of approximately 1 day in epidermal tissues), but its residence time in some tissues can be quite long and dependent on location (e.g., in cartilage). It has been estimated that an adult human contains approximately 15 g of hyaluronan and that about one-third turns over daily. Turnover appears to occur by receptor-mediated endocytosis and lysosomal degradation either locally or after transport by lymph to lymph nodes or by blood to liver. The endothelial cells of the lymph node and liver sinusoids remove hyaluronan via specific receptors such as LYVE-1 (a homolog of CD44) and HARE (hyaluronan receptor for endocytosis). HARE appears to be the major clearance receptor for hyaluronan delivered systemically by lymph and blood. The current understanding of this catabolic process is that hyaluronidases at the cell surface and in the lysosome cooperate to degrade the chains. Large hyaluronan molecules in the extracellular space interact with cell-surface receptors that internalize fragments produced by a membrane-associated, GPI-anchored hyaluronidase, most likely HYAL2. The fragments are transported into a unique vesicular endosomal compartment and eventually enter a pathway to lysosomes for complete degradation to monosaccharides, probably involving HYAL1 and the two exoglycosidases β-glucuronidase and β-N-acetylglucosaminidase. The importance of this process is demonstrated by the fact that Hyal2-null mice are embryonic lethal and by the identification of a lysosomal storage disorder in a person with a mutation in HYAL1.

Hyaluronan fragments have been suggested to act as an endogenous signal of injury, for example from infection by Group A Streptococcus. The signaling activity of HA fragments is mediated through binding of cell surface receptors, such as CD44, which in turn modulates response through toll-like receptors. Signaling through these and other receptors are affected by the size of the hyaluronan fragments, but the mechanism underlying the dependence of activity on the size of the fragments remains an area of active research.

HYALURONAN FUNCTION IN THE EXTRACELLULAR MATRIX

Hyaluronan has multiple roles in early development, tissue organization, and cell proliferation. The Has2-null mouse exhibits an embryonic lethal phenotype at the time of heart formation, whereas Has1-, Has3-null and Has1/3 compound mutant mice show no obvious developmental phenotype. Interestingly, explanted cells from the Has2-null embryonic heart do not synthesize hyaluronan or undergo epithelial- mesenchymal transformation and migration unless small amounts of hyaluronan are added to the culture medium. This finding indicates that the production of hyaluronan at key points may be essential for many tissue morphogenetic transformations—in this case, formation of the tricuspid and mitral valves.

Many of the activities of hyaluronan depend on binding proteins present on the cell surface and/or secreted into the extracellular matrix. A class of proteins that bind selectively to hyaluronan was first discovered in cartilage. This class is now referred to as the link module family of hyaladherins (Figure 16.3). Proteoglycans were efficiently extracted from this tissue with denaturing solvents and were shown to reaggregate when restored to renaturing conditions. An essential protein, referred to as the link protein (HAPLN-1), was shown to be necessary for stabilizing the proteoglycan aggregates, and subsequently, the structure of the aggregate was defined (Figure 16.4). The link protein contains two homologous repeats of a sequence motif, now named the link module. Proteins that have a link module, including link proteins (HAPLN-1 through 4 in humans), several proteoglycans, and other extracellular matrix proteins, can interact specifically with hyaluronan. The major cartilage proteoglycan, now named aggrecan, also contains a globular domain, the G1 domain, with two homologous link modules that interact with hyaluronan. An additional domain in the link protein (HAPLN-1) cooperatively interacts with a homologous domain in G1, which locks the proteoglycan on the hyaluronan chain. In the absence of the HAPLN-1, aggrecan fails to anchor to hyaluronan. Mice deficient in HAPLN-1 show defects in cartilage development and delayed bone formation (short limbs and craniofacial anomalies). Most mutant mice die shortly after birth as a result of respiratory failure, and the few survivors develop progressive skeletal deformities.

Interestingly, there are four proteoglycan genes with homologous G1 domains that interact with hyaluronan (versican, neurocan, brevican, and aggrecan) (Figure 16.3). Versican is a major component of many soft tissues and is especially important in vascular biology. Neurocan and brevican are expressed predominantly in brain tissue. Versican and aggrecan are anchored to hyaluronan in tissues by similar link protein-dependent mechanisms, and it is likely that neurocan and brevican are organized similarly. Thus, hyaluronan acts as a scaffold on which to build proteoglycan aggregate structures adapted to diverse tissue functions.

An impressive example of the requirement for a hyaluronan-based matrix occurs during the process of cumulus oophorus expansion in the mammalian preovulatory follicle. At the beginning of this process, the oocyte is surrounded by about 1000 cumulus cells tightly compacted and in gap-junction contact with the oocyte. In response to hormonal stimuli, the cumulus cells up-regulate HAS2 and a link module family hyaladherin encoded by tumor necrosis factor-stimulated gene 6 (TSG-6). The expression of these proteins initiates production of hyaluronan and its organization into an expanding matrix around the cumulus cells. Concurrently, the follicle becomes permeable to serum, which introduces an unusual molecule called inter-α-trypsin inhibitor (ITI), composed of the trypsin inhibitor bikunin and two heavy chains all covalently bound to a chondroitin sulfate chain. In a complex process, TSG-6 catalyzes the transfer of heavy chains that are covalently linked to chondroitin sulfate (via an ester bond between the C-terminal aspartate residue of the heavy chains and the C-6 of N-acetylgalactosamine in chondroitin-4-sulfate) onto the newly synthesized hyaluronan by transesterification to C6 of an N- acetylglucosamine residue. In the absence of either TSG-6 or ITI, the matrix does not form, and the phenotype of mice null for either of these molecules is female infertility. At the time of ovulation, hyaluronan synthesis ceases, and ovulation of the expanded cumulus cell–oocyte complex occurs. Prior to fertilization, individual sperm undergo capacitation enabling them to penetrate and fertilize an ovum. During this process, SPAM1/PH20, a GPI-anchored hyaluronidase, redistributes and accumulates in the sperm head. SPAM1 binds hyaluronan in the cumulus, causing an increase in Ca++ flux and sperm motility. It also helps dissolve the cumulus matrix as the sperm moves through the hyaluronan vestment. A soluble form of SPAM1 is secreted during the acrosome reaction. The release of acrosomal hyaluronidase and proteases renders the sperm capable of fusing with the egg and eventually destroys the entire matrix to allow the fertilized oocyte to implant and develop.

HYALURONAN-BINDING PROTEINS WITH LINK MODULES

There are several hyaluronan-binding proteins with homologous link modules (Figure 16.3). The four homologous link proteins belong to a subfamily called the “hyaluronan and proteoglycan link proteins” (HAPLN); these are expressed in many tissues. Four cell-surface receptors have extracellular domains with one link module: CD44, LYVE-1 (lymphatic vessel endothelial hyaluronan receptor), HARE/STABILIN-2 (hepatic hyaluronan clearance receptor), and STABILIN-1, which are expressed on discontinuous endothelial cells and some activated macrophages. Other hyaluronan-binding proteins are secreted and include the chondroitin sulfate proteoglycans that comprise the aggrecan superfamily (aggrecan, versican, brevican, and neurocan) and TNFα-stimulated gene 6 (TSG-6), which has one link module.

The three-dimensional structure of the link module fold in TSG-6 has been determined by nuclear magnetic resonance and defines a consensus fold of the two α-helices and two triple-stranded antiparallel β-sheets (Figure 16.5). The fold consists of about 100 amino acids and contains four cysteines disulfide-bonded in the pattern Cys1-Cys4 and Cys2-Cys3. This fold has only been found in vertebrates, consistent with the fact that hyaluronan is a relatively recent evolutionary invention. The link module fold is related to that found in the C-type lectins, but it lacks the Ca++ binding motif (Chapter 34). In the case of TSG-6, the interaction of hyaluronan with the protein involves 1) ionic interactions between positively charged amino acid residues and the carboxyl groups of the uronic acids, and 2) hydrophobic interactions between the acetamido side chains of two N- acetylglucosamine residues and hydrophobic pockets on either side of adjacent tyrosines (Figure 16.5). Many of these features are conserved in other members of the hyaluronan-binding proteins. Subgroups, however, differ in the preferred size and length of hyaluronan for binding (e.g., hexasaccharides to decasaccharides).

Some hyaluronan-binding proteins do not contain a link module (RHAMM, ITI, SPACR, SPACRCAN, CD38, CDC37, HABP1/P-32, and IHABP4), and most of these are unrelated to one another by primary sequence. Some of these proteins contain clusters of basic amino acids, referred to as BX7B motifs (where B is either lysine or arginine and X can be any amino acid other than acidic residues), but the actual hyaluronan docking site of the chain with this motif has not been established. Thus, the presence of the BX7B motif should not be taken as proof that the protein interacts with hyaluronan.

HYALURONAN AND CELL SIGNALING

Hyaluronan expression has long been implicated in enhanced cell adhesion and locomotion because it is expressed abundantly during morphogenesis and in both physiological and pathological invasive processes. A search for cell-surface receptors revealed two major hyaluronan-binding proteins, CD44 and RHAMM (receptor for hyaluronan-mediated motility). CD44 is a transmembrane receptor expressed by many cell types, and it varies markedly in glycosylation, oligomerization, and protein sequence because of differential mRNA splicing. CD44H (the isoform expressed by hematopoietic cells) binds to hyaluronan, and the interaction can mediate leukocyte rolling and extravasation in some tissues. Changes in CD44 expression, notably expression of CD44 variants, are associated with a wide variety of tumors and the metastatic spread of cancer. Many cells also express the receptor RHAMM, which is involved in cell motility and cell transformation. The RHAMM pathway is thought to induce focal adhesions to signal the cytoskeletal changes required for elevated cell motility seen in tumor progression, invasion, and metastasis. Like CD44, RHAMM splice variants exist, some of which may be intracellular.

CD44 contains a cytoplasmic domain, a transmembrane segment, and an ectodomain with a single link module that can bind hyaluronan. When hyaluronan binds to CD44, the cytoplasmic tail can interact with regulatory and adaptor molecules, such as SRC kinases, RHO (ras homolog) GTPases, VAV2 (a human proto- oncogene), GAB1 (a GRB2-associated binding protein), and ankyrin and ezrin (which regulate cytoskeletal assembly/disassembly and cell migration). Hyaluronan binding to RHAMM also transduces signals that influence growth and motility, for example, by activating SRC, FAK (focal adhesion kinase), ERK (extracellular mitogen-regulated protein kinase), and PKC (protein tyrosine kinase C) (Chapter 40).

Interaction of hyaluronan with CD44 can also regulate ERBB-family (epithelial growth factor receptor) signaling, thereby activating the PI3K (phophoinositide-3- kinase)–PKB/AKT (protein kinase B) signaling pathway and phosphorylation of FAK and BAD (BCL2-antagonist of cell death), which promote cell survival. RHAMM can interact with and activate ERK1, which can also phosphorylate BAD. Thus, both CD44 and RHAMM interactions with hyaluronan can influence cell survival. These pathways are relevant to tumor cell survival and invasion; their inhibition by hyaluronan oligomers and soluble hyaluronan-binding proteins suggests novel therapeutic approaches for treating cancer (Chapter 47).

HYALURONAN CAPSULES IN BACTERIA

Some pathogenic bacteria (e.g., certain strains of Streptococcus and Pasteurella) produce hyaluronan and deposit it as an extracellular capsule (Figure 16.6; also see Chapter 21). Capsular hyaluronan, like other capsular polysaccharides, increases virulence by helping to shield the microbe from host defenses. For example, the capsule blocks phagocytosis and protects against complement-mediated killing. Because bacterial hyaluronan is identical in structure to host hyaluronan, the capsule can also prevent the formation of protective antibodies. Thus, the formation of hyaluronan capsules by bacteria is a form of molecular mimicry. The capsule also can aid in bacterial adhesion to host tissue, facilitating colonization (Chapter 37). Finally, the production of hyaluronan by invading bacteria can also induce a number of signaling events through hyaluronan-binding proteins that modulate the host physiology, i.e.cytokine production (Chapter 42).

In addition to bacteria, an algal virus (Chlorella) encodes a hyaluronan synthase. The functional significance of viral hyaluronan production is unknown, but could be related to prevention of secondary viral infection, increase in host capacity to produce virus, or viral burst size. The origin of viral HAS is unknown, but based on sequence homology it most likely arose from a vertebrate.

HYALURONAN AS A THERAPEUTIC AGENT

Hyaluronan has been used therapeutically for a number of years. In some countries, patients with osteoarthritis are successfully treated by direct injection of high- molecular-weight hyaluronan into the synovial space of an affected joint. The mechanism of action is complex and probably involves both the viscoelastic properties of the polymer as well as effects on the growth of synovial cells in the joint capsule. Hyaluronan suppresses cartilage degeneration, acts as a lubricant (thereby protecting the surface of articular cartilage), and reduces pain perception.

The application of hyaluronan in ophthalmology is widespread. During surgery for lens replacement due to cataracts, a high potential for injury of fragile intraocular tissues exists, especially for the endothelial layer of the cornea. High-molecular- weight hyaluronan is injected to maintain operative space and structure and to protect the endothelial layer from physical damage. Hyaluronan also has been approved for cosmetic use (e.g., by subdermal injection to fill wrinkles or pockets under the skin).

Low-molecular-weight hyaluronan oligosaccharides (~103–104 D) also have potent biological activities by altering selective signaling pathways. In cancer cells, hyaluronan oligosaccharides induce apoptosis and inhibit tumor growth in vivo. Thus, short hyaluronan chains may prove useful for preventing cancer metastasis by boosting certain immune responses or altering new blood vessel growth. Recently, recombinant forms of the Pasteurella synthase (pmHas) have been engineered to produce hyaluronan oligosaccharides of defined size. This strategy has great promise for exploring the relationship of hyaluronan size to function, which may in turn yield new therapeutic agents with selective activities. ACKNOWLEDGEMENTS

The authors appreciate helpful comments and suggestions from Nicholas Keul, Sumit Rai, Kristin Stanford and Kekoa Taparra.

FURTHER READING

Hascall VC, Wang A, Tammi M, Oikari S, Tammi R, Passi A, Vigetti D, Hanson RW, Hart GW. 2014. The dynamic metabolism of hyaluronan regulates the cytosolic concentration of UDP-GlcNAc. Matrix Biol 35: 14-17. Jiang D, Liang J, Noble PW. 2007. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol 23: 435-461. Liang J, Jiang D, Noble PW. 2016. Hyaluronan as a therapeutic target in human diseases. Adv Drug Deliv Rev 97: 186-203. Maytin EV. 2016. Hyaluronan: More Than Just A Wrinkle Filler. Glycobiology. In press. Petrey AC, de la Motte CA. 2014. Hyaluronan, a Crucial Regulator of Inflammation. Frontiers in immunology 5: 101. Smith PD, Coulson-Thomas VJ, Foscarin S, Kwok JC, Fawcett JW. 2015. "GAG-ing with the neuron": The role of glycosaminoglycan patterning in the central nervous system. Exp Neurol 274: 100-114. Wang A, de la Motte C, Lauer M, Hascall V. 2011. Hyaluronan matrices in pathobiological processes. The FEBS journal 278: 1412-1418. Weigel PH. 2015. Hyaluronan Synthase: The mechanism of initiation at the reducing end and a pendulum model for polysaccharide translocation to the cell exterior. Int J Cell Biol 2015: 367579. Weigel PH, Deangelis PL. 2007. Hyaluronan synthases: A decade-plus of novel glycosyltransferases. J Biol Chem 282: 36777-36781. Zhuo L, Kimata K. 2008. Structure and function of inter-alpha-trypsin inhibitor heavy chains. Connect Tissue Res 49: 311-320.

Figure Legends

FIGURE 16.1. Hyaluronan consists of repeating disaccharides composed of N- acetylglucosamine (GlcNAc) and glucuronic acid (GlcA). It is the largest polysaccharide found in vertebrates, and it forms hydrated matrices. (Electron micrograph provided by Drs. Richard Mayne and Randolph Brewton, University of Alabama at Birmingham.)

FIGURE 16.2. Hyaluronan biosynthesis by hyaluronan synthase (Has) occurs by addition of UDP-sugars (UDP-N-acetyl-glucosamine and UDP-glucuronic acid) to the reducing end of the polymer with release of the anchoring UDP. M++ refers to a metal ion cofactor.

FIGURE 16.3. Modular organization of the link module superfamily of hyaluronan- binding proteins. These proteins contain one or two link modules that bind to hyaluronan. Like many extracellular matrix proteins, the link module superfamily members contain various subdomains, depicted by the following annotated symbols: (IG) immunoglobulin-like domain; (EGF) epidermal growth factor–like domain; (CLECT) C-type lectin domain; (CCP) complement control protein module; (Link) hyaluronan-binding module; (CUB) domain found in some complement proteins, peptidases, and bone morphogenetic protein; (FAS) domain found in fasciclin I family of proteins. (See the SMART database at EMBL for additional information on these domains: http://smart.embl-heidelberg.de/.) (Redrawn, with permission, from Blundell C.D. 2004. In Chemistry and biology of hyaluronan [ed. H.G. Garg and C.A. Hales], pp.189–204, © Elsevier.)

FIGURE 16.4. The large cartilage CS proteoglycan (aggrecan) forms an aggregate with hyaluronan and link protein. Rick: we need to combine the information in these two renderings into one. I like the one on the right which was originally Figure 16.1 in the 2nd edition. But the problem is that Hascall indicates that the model is old fashioned; it lacks the G2 and G3 globular domains on aggrecan and the globular domain on Link protein as shown in the left drawing. These globular domains are the folds shown in the next figure (16.5) and somehow should be added to the drawing on the right. Any questions, let me know. -Jeff

FIGURE 16.5. Structure of the link module. (a) TSG-6 contains a prototypical link module defined by two α helices (α1 and α2) and two triple-stranded antiparallel β sheets (β1,2,6 and β3–5). (Redrawn, with permission, from Blundell C.D. et al. 2003. J. Biol. Chem. 278: 49261–49270, ©American Society for Biochemistry and Molecular Biology.) (b) The hyaluronan-bound conformation of the protein, with a view showing key amino acids. (Redrawn, with permission, from Blundell C.D. et al. 2004. In Chemistry and biology of hyaluronan [eds. H.G. Garg and C.A. Hales], pp. 189–204, ©Elsevier.) (c) A model of the TSG-6 link module/hyaluronan complex. Binding of the hyaluronan to the protein is mediated through ionic interactions between positively charged amino acid residues (green) and the carboxylate groups of the uronic acids, and by a combination of aromatic ring stacking and H-bond interactions between the polysaccharide and aromatic amino acids (red). In addition, hydrophobic pockets (on either side of Tyr59) can accommodate the methyl groups of two N-acetylglucosamine side chains, which is likely to be a major determinant in specificity of link modules for hyaluronan. (Redrawn, with permission, from Blundell C.D. et al. 2005. J. Biol. Chem. 280: 18189–18201, ©American Society for Biochemistry and Molecular Biology.)

FIGURE 16.6. Hyaluronan capsule. Cross-sectioned Streptococcus zooepidemicus cells surrounded by a hyaluronan capsule and observed at differing magnifications. (a) 1 μm. (Reprinted, with permission, from Chong B.F., Blank L.M., McLaughlin R., and Nielsen L.K. 2005. Appl. Microbiol. Biotechnol. 66: 341–351, ©Springer Verlag.) (b) 0.1 μm. (Reprinted from Goh L.-T. 1998. “Effect of culture conditions on rates of intrinsic hyaluronan production by Streptococcus equi subsp. zooepidemicus.” Ph.D. thesis. University of Queensland, Australia.)

Chapter 17 Proteoglycans and Sulfated Glycosaminoglycans

Essentials of Glycobiology, 3rd edition Title: Proteoglycans and Sulfated Glycosaminoglycans

Authors: Ulf Lindahl, John Couchman, Koji Kimata, and Jeffrey D. Esko

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This chapter focuses on the structure, biosynthesis, and general biology of proteoglycans. Topics include a description of the major families of proteoglycans, their characteristic polysaccharide chains (glycosaminoglycans), biosynthetic pathways, and general concepts about proteoglycan function. Proteoglycans, like other glycoconjugates, have many essential roles in biology.

HISTORICAL PERSPECTIVE

The study of proteoglycans dates back to the beginning of the 20th century with investigations of “chondromucoid” from cartilage and anticoagulant preparations from liver (heparin). From 1930 to 1960, great strides were made in analyzing the chemistry of the polysaccharides of these preparations (also known as “mucopolysaccharides”), yielding the structure of hyaluronan (Chapter 16), dermatan sulfate, keratan sulfate, different isomeric forms of chondroitin sulfate, heparin, and heparan sulfate. Together, these polysaccharides came to be known as glycosaminoglycans (sometimes abbreviated as GAGs) to indicate the presence of amino sugars and other sugars in a polymeric form. Subsequent studies provided insights into the linkage of the chains to core proteins. These structural studies paved the way for biosynthetic studies.

The 1970s marked a turning point in the field, when improved isolation and chromatographic procedures were developed for the purification and analysis of tissue proteoglycans and glycosaminoglycans. Density-gradient ultracentrifugation allowed separation of the large aggregating proteoglycans from cartilage, revealing a complex of proteoglycan, hyaluronan, and link protein. Also during this period, it was realized that the production of proteoglycans was a general property of animal cells and that proteoglycans and glycosaminoglycans were present on the cell surface, inside the cell, and in the extracellular matrix (ECM). This observation led to a rapid expansion of the field and the eventual appreciation of proteoglycan function in cell adhesion, signaling, and other biological activities (see Chapter 38). Today, studies with somatic cell mutants (Chapter 49) as well as experiments using gene knockout and silencing techniques in a variety of model organisms, such as nematode worms (Caenorhabditis elegans), fruit flies (Drosophila melanogaster), African clawed frogs (Xenopus laevis), zebrafish (Danio rerio), and mice (Mus musculus), are aimed at extending our understanding of the role of proteoglycans in development and physiology (Chapters 25-27) and human diseases (Chapters 41- 47). The application of a variety of newly developed analytical tools, including mass spectrometry (Chapter 50) and glycan arrays (Chapter 48), are leading to a better understanding of proteoglycan structure and function.

PROTEOGLYCAN AND GLYCOSAMINOGLYCAN COMPOSITION

Proteoglycans consist of a core protein and one or more covalently attached glycosaminoglycan chains (Figure 17.1). Glycosaminoglycans are linear polysaccharides, whose disaccharide building blocks consist of an amino sugar (glucosamine that is N-acetylated or N-sulfated or N-acetylgalactosamine) and a uronic acid (glucuronic acid or iduronic acid) or galactose. Figure 17.2 depicts short segments of glycosaminoglycans and their characteristic features. Hyaluronan does not occur covalently linked to proteoglycans, but instead interacts noncovalently with some proteoglycans via hyaluronan-binding motifs (Chapter 16). Generally, invertebrates produce the same types of glycosaminoglycans as vertebrates, except that hyaluronan is not present and the chondroitin chains tend to be nonsulfated. Most proteoglycans also contain N- and O-glycans typically found on glycoproteins (see Chapters 9 and 10). The glycosaminoglycan chains are much larger than these other types of glycans (e.g., a 20 kD glycosaminoglycan chain contains ~80 sugar residues, whereas a typical biantennary N-glycan contains 10–12 residues). Both the composition of the glycosaminoglycan chain, the structure of the protein core, and the distribution of the proteoglycan determine the biological activities associated with proteoglycans.

PROTEOGLYCANS ARE DIVERSE IN STRUCTURE AND FUNCTION

Virtually all mammalian cells produce proteoglycans and secrete them into the ECM, insert them into the plasma membrane, or store them in secretory granules. The ECM, an essential component of all multicellular animals, determines the physical characteristics of tissues and many of the biological properties of the cells embedded in it. The major components of the ECM are fibrillar proteins that provide tensile strength and elasticity (e.g., various collagens and elastins), adhesive glycoproteins (e.g., fibronectin, laminins, and tenascins), and proteoglycans that interact with other ECM components to promote ECM assembly, govern its physical properties and serve as a reservoir of biologically active small proteins such as growth factors. A single cell type can express multiple proteoglycans. Vascular endothelial cells, for example, synthesize several different cell surface proteoglycans, secretory granule proteoglycans, as well as several ECM proteoglycans.

Compared to the hundreds, perhaps thousands, of glycoproteins that carry N- and O- linked glycans, relatively few proteins have been identified that carry glycosaminoglycans (~17 with heparan sulfate, approximately ~20 with chondroitin/dermatan sulfate, and ~8 with keratan sulfate). Nevertheless, tremendous structural variation of proteoglycans exists due to a number of factors. First, many proteoglycans can be substituted with one or two types of glycosaminoglycan chains, for example glypicans contain heparan sulfate whereas syndecan-1 contains both heparan sulfate and chondroitin sulfate chains. Some proteoglycans contain only one glycosaminoglycan chain (e.g., decorin), whereas others have more than 100 chains (e.g., aggrecan). Another source of variability lies in the stoichiometry of glycosaminoglycan chain substitution. For example, syndecan-1 has five attachment sites for glycosaminoglycans, but not all of the sites are used equally. Other proteoglycans can be “part time,” that is, they may exist with or without a glycosaminoglycan chain or with only a truncated oligosaccharide. A given proteoglycan present in different cell types often exhibits differences in the number of glycosaminoglycan chains, their lengths, and the arrangement of sulfated residues along the chains. Thus, a preparation of any one proteoglycan (defined by its core protein) represents a diverse population of molecules, each potentially representing a unique structural entity. These characteristics, typical of all proteoglycans, create enormous diversity and potential biological variation in activity.

Mammalian proteoglycans – form and function

The major classes of proteoglycans can be classified by their distribution, homologies, and function. Table 17.1 provides an overview of many of the known proteoglycans.

The aggrecan family of ECM proteoglycans (also known as lecticans) consists of aggrecan, versican, brevican, and neurocan. In all four members, the protein moiety contains an amino-terminal domain capable of binding hyaluronan, a central region that contains covalently bound chondroitin sulfate chains, and a carboxy-terminal domain containing a C-type lectin domain (Chapter 34). Aggrecan is the best-studied member of this family, because it represents the major proteoglycan in cartilage where it forms a stable matrix capable of withstanding compressive forces by water desorption and resorption. Versican, which is produced predominantly by connective tissue cells, undergoes alternative splicing events that generate a family of proteins. Neurocan is expressed in the late embryonic central nervous system (CNS) and can inhibit neurite outgrowth. Brevican is expressed in the terminally differentiated CNS, particularly in perineuronal nets.

The small leucine-rich proteoglycans (SLRPs) contain leucine-rich repeats flanked by cysteines in their central domain. At least nine members of this family are known and some carry chondroitin sulfate, dermatan sulfate, or keratan sulfate chains. These proteoglycans help to stabilize and organize collagen fibers, but have other roles in innate immunity and regulation of growth factor signaling.

The interstitial proteoglycans and aggrecan family of proteoglycans appear to be unique to vertebrates. C. elegans and D. melanogaster express other proteoglycans, suggesting that the core proteins have undergone enormous diversification during evolution, presumably to accommodate different needs of the organism. In contrast, the biosynthetic machinery has been evolutionarily conserved, demonstrating conservation of function for the glycosaminoglycan chains.

Basement membranes are highly specialized thin layers of the ECM that lie flush against epithelial cells and surround muscle and fat cells. Major components are laminins, nidogens, and collagens, as well as three unrelated basement membrane proteoglycans, one of which is a collagen (perlecan, agrin, and Type XVIII collagen). These proteoglycans interact with other basement membrane components and cell surface adhesion receptors, but may also be important reservoirs of heparan sulfate-binding growth factors.

The membrane-bound proteoglycans are diverse. The syndecan family consists of four members, each with a short hydrophobic domain that spans the membrane, linking the larger extracellular domain containing the glycosaminoglycan attachment sites to a smaller intracellular cytoplasmic domain. The syndecans are expressed in a tissue-specific manner and facilitate cellular interactions with a wide range of extracellular ligands, such as growth factors and matrix molecules. Because of their membrane-spanning properties, the syndecans can transmit signals from the extracellular environment to the intracellular cytoskeleton via their cytoplasmic tails. Syndecans are very sensitive to proteolytic cleavage by matrix metalloproteases, resulting in shedding of the ectodomains bearing the glycosaminoglycan chains that retain potent biological activity (Chapter 38). C. elegans and D. melanogaster express only one syndecan (Chapter 26).

Glypicans carry only heparan sulfate chains, which can bind a wide array of factors essential for development and morphogenesis. Six glypican family members exist in mammals, only two are expressed in D. melanogaster and C. elegans. Each member of the glypican family of cell-surface proteoglycans has a glycosylphosphatidylinositol anchor attached at the carboxyl terminus, which embeds them in the outer leaflet of the plasma membrane (Chapter 12). The amino- terminal portion of the protein has multiple cysteine residues and a globular shape that distinguishes the glypicans from the syndecan ectodomains, which tend to be extended structures (Figure 17.1).

A number of other membrane proteoglycans are expressed on the surface of many different cell types including the widespread CD44, NG2 (also known as CSPG4), phosphacan (PTPζ), thrombomodulin and invariant chain of the MHC class II system. Serglycin is the major cytoplasmic secretory granule proteoglycan that is present in endothelial, endocrine, and hematopoietic cells. Depending on the species, it has a variable number of glycosaminoglycan attachment sites that can carry chondroitin sulfate or heparin chains. Heparin is a highly sulfated form of heparan sulfate (discussed below).

To a large extent, the biological functions of proteoglycans depend on the interaction of the glycosaminoglycan chains with different protein ligands. Table 17.2 lists examples of proteins known to interact with glycosaminoglycans. Proteins that bind to the sulfated glycosaminoglycan chains appear to have evolved by convergent evolution (i.e., they do not contain a specific fold present in all glycosaminoglycan binding proteins, in contrast to other groups of glycan binding proteins). These interactions have profound physiological effects and are discussed further in Chapter 38.

LINKAGES OF GLYCOSAMINOGLYCANS TO PROTEINS

Different subtypes of sulfated glycosaminoglycans are attached to their core proteins by unique linkages. There are two types of keratan sulfate, distinguished by the nature of their linkage to protein (Figure 17.3). KS I, originally described in cornea, is found on an N-glycan linked to protein through an asparagine residue (Chapter 9). KS II (skeletal keratan sulfate) is found on an O-glycan core 2 structure and is thus linked through N-acetylgalactosamine (GalNAc) to serine or threonine (Chapter 10). The structural features in control of keratan sulfate substitution remain unclear. Notably, in humans and bovine, the large chondroitin sulfate proteoglycan found in cartilage (aggrecan) contains a segment of 4–23 hexapeptide repeats (E-E/L-P-F-P-S) where the keratan sulfate chains are located, whereas aggrecan in rats and other rodents lacks this motif and does not contain keratan sulfate .

Two classes of glycosaminoglycan chains, chondroitin sulfate/dermatan sulfate and heparan sulfate/heparin, are linked to serine residues in core proteins by way of xylose (Figure 17.4). Xylosyltransferase initiates the process using UDP-xylose as donor. Two isoforms of the enzyme are known in vertebrates (Xylt1 and Xylt2), but only one isozyme exists in C. elegans and D. melanogaster. A glycine residue invariably lies to the carboxy-terminal side of the serine attachment site, but a perfect consensus sequence for xylosylation does not exist. At least two acidic amino acid residues are usually present, and they can be located on one or both sides of the serine, usually within a few residues. Several proteoglycans contain clustered glycosaminoglycan attachment sites, raising the possibility that xylosyltransferase could act in a processive manner. Xylosylation is an incomplete process in some proteoglycans, which may explain why proteoglycans with multiple potential attachment sites contain different numbers of chains in different cells.

After xylose addition, a linkage tetrasaccharide assembles by the transfer of two galactose residues catalyzed by unique members of the β4 galactosyl-, β3 galactosyl- and β3 glucuronosyltransferase families of enzymes (Figure 17.4). This intermediate can undergo phosphorylation at the C-2 position of xylose and in the case of chondroitin sulfate, sulfation of the galactose residues. In general, phosphorylation and sulfation occur substoichiometrically, but phosphorylation may be transient. Phosphorylation occurs early in the assembly process and creates the preferred substrate for B4GALT7; a phosphatase removes the phosphate at a later stage of biosynthesis. Galactose sulfation is found only in chondroitin sulfate, but its function remains unclear.

The linkage tetrasaccharide lies at a bifurcation in the biosynthetic pathway. Two types of reactions occur: addition of β4-linked N-acetylgalactosamine (GalNAc), which initiates chondroitin sulfate assembly, or addition of α4-linked N- acetylglucosamine (GlcNAc), which initiates heparan sulfate assembly (Figure 17.4). Genetic evidence from studies of C. elegans suggests that GalNAc addition during chondroitin assembly is mediated by the same enzyme that is involved in chain polymerization (SQV5), but biochemical evidence suggests that more than one enzyme may exist in vertebrates. In heparin/heparan sulfate formation, the addition of the first GlcNAc residue is catalyzed by an enzyme called EXTL3, which differs from the transferases involved in heparan polymerization (called EXT1 and EXT2). These enzymes are important control points because they ultimately regulate the type of glycosaminoglycan chain that will assemble. Control of the addition of β4GalNAc or α4GlcNAc appears to be manifested at the level of enzyme recognition of the polypeptide substrate.

GLYCOSAMINOGLYCAN BIOSYNTHESIS

Keratan sulfate

Keratan sulfate chains contain a mixture of nonsulfated (Galβ4GlcNAcβ3), monosulfated (Galβ4GlcNAc6Sβ3), and disulfated (Gal6Sβ4GlcNAc6Sβ3) disaccharide units (Figure 17.2).The biosynthesis of the poly-N-acetyllactosamine backbone is described in Chapter 10. At least two classes of sulfotransferases, one or more GlcNAc 6-O-sulfotransferases (e.g., Chst6), and one Gal 6-O-sulfotransferase (Chst1) catalyze the sulfation reactions. These enzymes, like other sulfotransferases, use activated sulfate (PAPS [3′-phosphoadenyl-5′-phosphosulfate]) as a high-energy donor (Chapter 5). GlcNAc 6-O-sulfation occurs on the nonreducing terminal GlcNAc residue, as a prerequisite to further chain elongation, whereas sulfation of galactose residues takes place on nonreducing terminal and internal galactose residues, with a preference for galactose units adjacent to a sulfated GlcNAc. Sulfation of a nonreducing terminal galactose residue blocks further elongation of the chain, providing a potential mechanism for controlling chain length. The poly-N- acetyllactosamine chains of KS I are generally longer than those of KS II, and may contain up to 50 disaccharide units (20–25 kD). The relationship of enzymes involved in KS I and KS II sulfation is unclear. The chains can be fucosylated and sialylated as well (Chapter 14).

Chondroitin sulfate

Vertebrate chondroitin sulfate consists of repeating sulfate-substituted GalNAcβ4GlcAβ3 disaccharide units polymerized into long chains (Figure 17.2). In contrast, invertebrates such as C. elegans and D. melanogaster make either nonsulfated or low sulfated chains, respectively. The assembly process for the backbone appears to be highly conserved, based on the presence of homologous genes for all of the reactions (Chapters 25 and 26). As described above, the assembly process is initiated by the transfer of GalNAcβ3 to the linkage tetrasaccharide (Figure 17.4). In both vertebrates and invertebrates, the polymerization step is catalyzed by one or more bifunctional enzymes (chondroitin synthases) that have both β3 glucuronosyltransferase and β4 N-acetylgalactosaminyltransferase activities. Vertebrates also express homologs that can transfer individual sugars to the chain. Chondroitin polymerization also requires the action of the chondroitin polymerizing factor (Chpf), a protein that lacks independent activity but collaborates with the polymerases to enhance the formation of polymers. Sulfation of chondroitin in vertebrates is a complex process, with multiple sulfotransferases involved in 4-O-sulfation and 6-O-sulfation of GalNAc residues (Figure 17.5). Additional enzymes exist for epimerization of glucuronic acid (GlcA) to iduronic acid (IdoA) in dermatan sulfate (Dse1-2), sulfation at the C-2 position of the uronic acids, and other patterns of sulfation found in unusual species of chondroitin (Table 17.2). The location of sulfate groups is easily assessed using bacterial chondroitinases (ABC, B, and ACII) that cleave the chains into disaccharides. Many chains are hybrid structures containing more than one type of chondroitin disaccharide unit. For example, dermatan sulfate is defined as having one or more IdoA-containing disaccharide units (chondroitin sulfate B) as well as GlcA- containing disaccharides (chondroitin sulfate A and C). Animal cells also degrade chondroitin sulfate in lysosomes using a series of exolytic activities (Chapter 44).

Heparan sulfate

Heparan sulfate assembles as a copolymer of GlcNAcα4GlcAβ4 (Figure 17.5), which then undergoes extensive modification reactions, catalyzed by at least four families of sulfotransferases and one epimerase. N-acetylglucosamine N-deacetylase/N- sulfotransferases (Ndst1-4) act on a subset of GlcNAc residues to generate N- sulfated glucosamine (GlcNSO3) units, many of which occur in clusters along the chain. Generally, the enzyme deacetylates GlcNAc and rapidly adds sulfate to the free amino group to form GlcNSO3, but a small number of glucosamine residues with unsubstituted amino groups may arise from incomplete N-sulfation. An epimerase (Glce), different from the one involved in dermatan sulfate synthesis, then acts on some GlcA residues, followed by 2-O-sulfation of some of the IdoA units (catalyzed by Hs2st). Some glucuronic units also undergo 2-O-sulfation by the same enzyme. The addition of 2-O-sulfate groups to GlcA or IdoA blocks the epimerization reaction. Next, 6-O-sulfotransferases (Hs6st1-3) add sulfate groups to selected glucosamine residues. Finally, certain subsequences of sulfated sugar residues and uronic acid epimers provide targets for 3-O-sulfotransferases (Hs3st1-6).

In contrast to chondroitin chains, which tend to have long tracts of fully modified disaccharides, the modification reactions in heparan sulfate biosynthesis occur in clusters along the chain, with regions devoid of sulfate separating the modified tracts. In general, the reactions proceed in the order indicated, but they often fail to go to completion, resulting in tremendous chemical heterogeneity. The disaccharide composition of the chains can be readily assessed using bacterial heparin lyases or chemical degradation methods (which are more useful for differentiating GlcA/IdoA) but direct sequencing of the chains has proven difficult because of their heterogeneity. New mass spectrometry methods are making significant inroads into sequencing of glycosaminoglycans (Chapter 50).

Regulation of glycosaminoglycan assembly

The specific arrangement of sulfated residues and uronic acid epimers in heparin/ heparan sulfate and dermatan sulfate gives rise to binding sequences for ligands. The three examples shown in Figure 17.5 demonstrate minimal sequences that can interact with fibroblast growth factors (FGFs), antithrombin and heparin cofactor II. More modified sequences can interact as well, and the binding of most FGFs is actually more sensitive to overall sulfation than to the specific position of the sulfate groups. Binding of glycosaminoglycans to proteins is described in greater detail in Chapter 38. A major question remains regarding how the enzymes and biosynthetic pathways are regulated to achieve tissue-specific expression of ligand-binding sequences.

During the last decade, most if not all of the enzymes involved in glycosaminoglycan synthesis have been purified and molecularly cloned from mammals and model organisms. Several important features have emerged from these studies, which may shed light on how different protein binding sequences arise.

• Several of the enzymes appear to have dual catalytic activities. Thus, a single protein bearing two catalytic domains catalyzes N-deacetylation of GlcNAc residues and subsequent N-sulfation (Ndsts) in heparan sulfate formation. The same is true of the copolymerases, which transfer GlcNAc and GlcA (heparan sulfate) and GalNAc and GlcA (chondroitin sulfate) from the corresponding UDP- sugars to the growing polymer. In contrast, the epimerases and O-sulfotransferase activities appear to be unique properties of independent enzymes. • In several cases, multiple isozymes exist that can catalyze either a single or a pair of reactions. Thus, four N-deacetylase/N-sulfotransferases, three 6-O- sulfotransferases, and seven 3-O-sulfotransferases have been identified in heparan sulfate biosynthesis. Their tissue distribution varies and differences exist in substrate preference, which may cause differences in the pattern of sulfation. However, some overlap in expression and in substrate utilization occurs as well. Multiple isozymes of 4-O- and 6-O-sulfotransferases also can participate in chondroitin sulfate formation. • The polymerization and polymer modification reactions probably colocalize in the same stacks of the Golgi complex. Thus, the enzymes may form supramolecular complexes that coordinate these reactions. The composition of these complexes may play a part in regulating the fine structure of the chains. • In general, the composition of heparan sulfate, and likely chondroitin and dermatan sulfate, on a given proteoglycan varies more between cell types than that of heparan sulfate on different core proteins expressed in the same cell. This observation suggests that each cell type may express a unique array of enzymes and potential regulatory factors. The mechanisms behind the generation of apparently cell-specific glycosaminoglycan chains through regulated, yet partly stochastic modification reactions remain poorly understood. • Recombinant enzymes and new synthetic schemes are increasingly used to generate defined glycosaminoglycan oligosaccharides, which can be used to probe ligand-binding affinities and specificities.

HEPARIN VERSUS HEPARAN SULFATE

Considerable confusion exists regarding the definition of heparin and heparan sulfate . Heparin is produced by a limited number of cells, notably connective tissue- type mast cells and bipotential glial progenitor cells, whereas heparan sulfate is made by virtually all types of cells. During biosynthesis, heparin undergoes more extensive sulfation and uronic acid epimerization, such that more than 80% of the GlcNAc residues are N-deacetylated and N-sulfated and more than 70% of the glucuronic acids undergo epimerization to IdoA. Heparin derived from porcine and bovine entrails is prepared commercially by selective precipitation and is sold by pharmaceutical companies as an anticoagulant due to its capacity to bind to antithrombin. The active sequence is a pentasaccharide shown in Figure 17.5, which is now sold as a purely synthetic anticoagulant (Arixtra). Low-molecular-weight heparins are derived from commercial unfractionated heparin by chemical or enzymatic cleavage, depending on the brand. Selectively desulfated forms of heparin are also available commercially, some of which lack anticoagulant activity, but still retain other potentially useful properties (e.g., inhibition of inflammation and cell proliferation, and antimetastatic activity). Heparan sulfate also can contain anticoagulant activity, but typical preparations from cells or tissues are much less active than heparin. Care should be taken in extrapolating data obtained with heparin (e.g. binding to heparin-Sepharose) versus binding to heparan sulfate and heparan sulfate proteoglycans; binding to heparin can occur due to the high charge content of the polysaccharide, whereas the same factor might bind to heparan sulfate with lower affinity or not at all. On the other hand, specific protein-binding motifs expressed in subspecies of heparan sulfate may occur also in heparin, although concealed by additional, redundant, sulfate residues. PROTEOGLYCAN PROCESSING AND TURNOVER

Cells secrete matrix proteoglycans directly into the extracellular environment (e.g., members of the aggrecan family, the basement membrane proteoglycans, SLRPs, and serglycin). However, others are shed from the cell surface through proteolytic cleavage of the core protein through matrix metalloproteases (e.g., the syndecans). Cells also internalize a large fraction of cell-surface heparan sulfate proteoglycans by endocytosis. These internalized proteoglycans first encounter proteases and heparanase, an endo-ß-glucuronidase, and the core protein and the heparan sulfate chains are cleaved endolytically. The resulting smaller heparan sulfate fragments eventually appear in the lysosome and undergo complete degradation by way of a series of exoglycosidases and sulfatases (Chapter 44). Chondroitin sulfate and dermatan sulfate proteoglycans follow a similar endocytic route. One of human hyaluronidases (Hyal-4) has been found to be involved in endolytic degradation of chondroitin sulfate.

Cells secrete heparanase as well. Extracellular heparanase can cleave heparan sulfate chains at restricted sites, resulting in release of growth factors or chemokines immobilized on heparan sulfate proteoglycans at cell surfaces or in the ECM. In particular, invading cells secrete heparanase. Thus, heparanase may act with matrix metalloproteases to remodel the ECM.

A family of plasma membrane endosulfatases (Sulfs) can remove sulfate groups from internal 6-O-sulfated glucosamine residues. This post-assembly processing of the chains at the cell surface results in altered response of cells to growth factors and morphogens. The mammalian genome contains other sulfatases of unknown function, raising the possibility that other post-assembly processing reactions of glycosaminoglycans may occur.

Acknowlegements

The authors appreciate helpful comments and suggestions from Andreas Geissner, Marissa Martinez and Yuko Naito-Matsui.

FURTHER READING

Filmus J, Capurro M. 2014. The role of glypicans in Hedgehog signaling. Matrix Biol 35: 248-252. Friand V, David G, Zimmermann P. 2015. Syntenin and syndecan in the biogenesis of exosomes. Biol Cell 107: 331-341. Gallagher J. 2015. Fell-Muir Lecture: Heparan sulphate and the art of cell regulation: a polymer chain conducts the protein orchestra. Int J Exp Pathol 96: 203-231. Hultgardh-Nilsson A, Boren J, Chakravarti S. 2015. The small leucine-rich repeat proteoglycans in tissue repair and atherosclerosis. J Intern Med 278: 447- 461. Mizumoto S, Yamada S, Sugahara K. 2015. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins. Curr Opin Struct Biol 34: 35-42. Neill T, Schaefer L, Iozzo RV. 2015. Decoding the matrix: instructive roles of proteoglycan receptors. Biochemistry 54: 4583-4598. Thacker BE, Xu D, Lawrence R, Esko JD. 2013. Heparan sulfate 3-O-sulfation: A rare modification in search of a function. Matrix Biol 35: 60-72. Vlodavsky I, Iozzo RV, Sanderson RD. 2013. Heparanase: multiple functions in inflammation, diabetes and atherosclerosis. Matrix Biol 32: 220-222. Wight TN, Kinsella MG, Evanko SP, Potter-Perigo S, Merrilees MJ. 2014. Versican and the regulation of cell phenotype in disease. Biochim Biophys Acta 1840: 2441-2451. Xu D, Esko JD. 2014. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83: 129-157. Yurchenco PD. 2011. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb Perspect Biol 3.

Figure Legends

FIGURE 17.1. Proteoglycans consist of a protein core (brown) and one or more covalently attached glycosaminoglycan chains ([blue] heparan sulfate ; [yellow] chondroitin sulfate /dermatan sulfate ). Membrane proteoglycans either span the plasma membrane (type I membrane proteins) or are linked by a GPI anchor. ECM proteoglycans are usually secreted, but some proteoglycans can be proteolytically cleaved and shed from the cell surface (not shown).

FIGURE 17.2. Glycosaminoglycans consist of repeating disaccharide units composed of an N-acetylated or N-sulfated hexosamine and either a uronic acid (GlcA or IdoA) or galactose. Hyaluronan lacks sulfate groups, but the rest of the glycosaminoglycans contain sulfates at various positions. Dermatan sulfate is distinguished from chondroitin sulfate by the presence of IdoA. Heparan sulfate is the only glycosaminoglycan that contains an N-sulfated hexosamine. Keratan sulfates lack uronic acids and instead consist of sulfated galactose and GlcNAc residues. Reducing termini are to the right in all sequences

FIGURE 17.3. Keratan sulfates contain a sulfated poly-N-acetyllactosamine chain linked to either asparagine or serine/threonine residues. Chst1 and Chst5 add sulfate groups at the indicated positions. The actual order of the various sulfated and nonsulfated disaccharides occurs somewhat randomly along the chain.

FIGURE 17.4. The biosynthesis of chondroitin sulfate (left chain) and heparan sulfate (right chain) is initiated by the formation of a linkage region tetrasaccharide. Addition of the first hexosamine commits the intermediate to either chondroitin sulfate (GalNAc) or heparan sulfate (GlcNAc).

FIGURE 17.5. Biosynthesis of chondroitin sulfate/dermatan sulfate involves the polymerization of GalNAc and GlcA units and a series of modification reactions including O-sulfation and epimerization of GlcA to IdoA. Heparan sulfate biosynthesis involves copolymerization of GlcNAc and GlcA residues. Chain polymerization and modification by sulfation and uronic acid epimerization are thought to occur simultaneously.

Table 17.1 See separate file.

TABLE 17.2 Types of chondroitin sulfates Chondroitin sulfate type Disaccharide repeat Source A GlcAβ1–3GalNAc4S cartilage and other tissues B IdoAα1–3GalNAc4S skin; tendon C GlcAβ1–3GalNAc6S cartilage and other tissues D GlcA2Sβ1–3GalNAc6S shark cartilage; brain E GlcAβ1–3GalNAc4,6diS squid; secretory granules This list is not meant to be exhaustive because many types of chondroitins exist with unusual modifications. For example, dermatan sulfate disaccharide can also contain sulfate at the C-2 position of IdoA and sulfate at C-6 instead of C-4, and 2-O-sulfated and 3-O-sulfated GlcA has been described in some cartilage chondroitin sulfate.

TABLE 17.3 Examples of proteins that bind to sulfated glycosaminoglycans Growth Cell/matrix Coagulation/fibrinolysis Lipolysis Inflammation factors and interactions morphogens Laminin Antithrombin Lipoprotein Cytokines (IL- FGFs and lipase 2, IL-7 IL-8) FGF receptors Fibronectin Heparin cofactor II Hepatic Chemokines HGF; scatter lipase (e.g., MIP-1β, factor SDF-1, etc.) Vitronectin Tissue factor pathway ApoE TNF-α VEGF inhibitor Thrombospondin Thrombin ApoB L and P TGF-β selectins Tenascin Protein C inhibitor, ApoA-V Extracellular BMPS Superoxide dismutase Various TPA and PAI-1 Microbial Hedgehogs, collagens adhesins Hedgehog interaction proteins Amyloid proteins Wnts

Review Article Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior Hindawi Publishing Corporation International Journal of Cell Biology Volume 2015, Article ID 367579, 15 pages http://dx.doi.org/10.1155/2015/367579

Review Article Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior

Paul H. Weigel

Department of Biochemistry & Molecular Biology, Te Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190,USA

Correspondence should be addressed to Paul H. Weigel; [email protected]

Received 2October 2014; Accepted 14 January 2015

Academic Editor: Howard Beverley Osborne

Copyright © 2015 Paul H. Weigel. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for asynthasepore-mediatedpolysaccharidetranslocationprocessanddescribeapossiblemechanism(thePendulumModel)and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. Te synthesis of chitin-UDP oligomers by HAS confrms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. Tese new fndings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

1. Introduction and Overview of demonstration that only the HAS protein was required to HA Biosynthesis synthesize HA [5] then led to the identifcation of hasA genes in S. equisimilis [6]andS. uberis [7]andvertebrate Cell-free biosynthesis of HA was demonstrated in 1959 using homologues of these HAS genes in many species [8–10].Te Streptococcus membranes [1]. Te enzyme responsible, HA frst active HAS was purifed when the recombinant enzymes synthase (HAS), is a membrane protein that requires only from Group A (SpHAS) and Group C (SeHAS) Streptococcus Mg and two sugar-UDP substrates (GlcUA-UDP and were overexpressed in E. coli SURE cells [11]. GlcNAc-UDP)+2 to polymerize HA chains. (To be consistent Mammalian genomes have three diferent HAS genes in using the standard convention of showing the reducing (HAS1, HAS2, and HAS3) that are expressed at specifc times end of any glycan or saccharide to the right, we do not and specifc tissues during development, aging, wound heal- use the normal convention for nucleotide-sugars (e.g., UDP- ing, and under normal or pathologic conditions or in diseases GlcNAc); instead HA-UDP, GlcNAc-UDP, and GlcUA-UDP such as cancer [12, 13]. HA, which is found in only some are abbreviated to show their reducing ends to the right.) No prokaryotes but is a general ubiquitous extracellular matrix one was able to identify any streptococcal or eukaryotic HA component in vertebrates [14, 15], is a linear heteropolysac- synthase gene until 1993 when the hasA gene was identifed charide composed of the repeating disaccharide: (-3)- -D- and cloned, and the S. pyogenes HAS protein was expressed N-acetylglucosamine- (1,4)-D-glucuronic acid- (1-). Tis [2–4]. Identifcation of the hasA gene and the biochemical unsulfated glycosaminoglycan is a major component! in ! ! 2 International Journal of Cell Biology cartilage and dermis, and in synovial and vitreous fuids. HA HA-GlcUA-UDP chain HA-GlcNAc-UDP chain plays an important role during fertilization, embryogenesis, 7 7 development, and diferentiation [16, 17]andisalsoinvolved Outside in many diverse cellular functions and behaviors, such as Membrane cell migration, phagocytosis, and proteoglycan assembly [18]. 3 4 3 4 Additionally, HA plays important roles during wound healing and is used as a drug delivery vehicle, a cosmetic ingredient, 5 6 5 6 UDP and an analgesic device [19–21]. 1 2 1 UDP- 2

UDP- All known HASs, except one, are related structurally, -UDP -UDP show high sequence identity or similarity, share similar multi- membrane-domain organizations (with 6–8membrane domains), predicted topologies, and processive mechanisms, = GlcNAc and constitute a large family, the Class I HASs [10, 22].Te = GlcUA only known Class II HAS, from Pasteurella multocida,is Figure 1: Schematic model of HAS showing the functions needed diferent from Class I HASs in membrane attachment (having for HA chain growth at the reducing end and transfer to the cell asinglemembranedomain),geneandproteinsequence, surface. HAS uses multiple discrete functions (numbers 1–7) to domain organization, and having a distributive (nonproces- assemble each HA disaccharide (red squares are GlcNAc and green sive) mechanism. Tis review focuses on the characteristics circles are GlcUA). Te same HAS protein is indicated in two of Class I streptococcal and mammalian HASs. diferent situations, at sequential times, as it alternately adds HA- Streptococcal and mammalian HASs in membranes [23– GlcUA-UDP to a new GlcNAc-UDP, using functions 1,3, and5 26]oraspurifedenzymes[27] elongate HA at the reducing (lef), and then adds HA-GlcNAc-UDP to a new GlcUA-UDP, using end and do not require an exogenous primer to begin HA functions 2,4, and 6(right). In this example (variant 1; Table 1) the sugar-UDPs are sequentially added in a continuous alternating synthesis. HAS initiates biosynthesis using just the two sugar- manner and each cohort of needed functions cycles between being UDP substrates, although we now know that the enzyme active (larger black numbers) and inactive (smaller gray numbers) makes a self-primer using only GlcNAc-UDP (described within the active site domains (gray ovals). Te functions required below). HAS is an unusual enzyme in that it uses four sub- to add GlcNAc-UDP to HA-GlcUA-UDP are (lef):1, GlcNAc- strates (i.e., two sugar-nucleotides and two types of HA-UDP UDP acceptor binding; 3, HA-GlcUA-UDP donor binding; 5, HA- chains, with either GlcUA or GlcNAc at the reducing end) and GlcUA-UDP: GlcNAc-UDP, 1,3(HA-GlcUA-) transferase; and 7, two glycosyltransferase activities within the same protein. HA translocation through the membrane. Te functions required DNA and RNA polymerases utilize template molecules to to add GlcUA-UDP to HA-GlcNAc-UDP! are (right): 2, GlcUA- direct synthesis of products with only one type of bond UDP acceptor binding; 4, HA-GlcNAc-UDP donor binding; 6, HA- between monomers. Heteropolysaccharide synthases, such GlcNAc-UDP: GlcUA-UDP, 1,4(HA-GlcNAc-) transferase; and 7, HA translocation. as HAS (which makes a [GlcNAc( 1,4)GlcUA( 1,3)]n-UDP polymer), create de novo two diferent glycoside linkages in an ! alternating manner. Te HA product! afer each sugar! addition then becomes a substrate for the next sugar addition. In the presence of exogenous precursors, membrane-bound HASs Table 1:Tree variations of the Pendulum hypothesis. use at least seven binding or catalytic functions (Figure 1)to synthesize disaccharide units at the reducing end of a growing Variant Disaccharideassembly Glycosyl-UDPsites HA-UDP chain. Class I HAS enzymes are processive; they do 1Sequential Four independent sites not rebind and extend HA chains once they are released. 2Simultaneous Tree independent sites SpHAS, the only Class I HAS whose topology has been 3Alternating Two or three dependent sites determined experimentally [28], has the N- and C-terminus Te mechanism for adding sugars to the reducing end of HA could entail and majority of the SpHAS protein inside the cell (Figure 2). polymerization of a disaccharide unit by either a sequential (i.e.,onesugar StrepHASs have six membrane domains (MDs), four of which at a time) or a concerted (i.e., simultaneous) mechanism. For the sequential pass through the membrane giving two small loops of the assembly of a disaccharide unit (variant 1), the enzyme would need two protein exposed to the extracellular side. Te other two MDs, glycosyl-UDP binding sites for addition of each sugar, one for a HA-UDP and one for a sugar-UDP. Since there are two types of HA-UDP species, one within the large central catalytic domain and one in the enzyme would need four glycosyl-UDP binding sites to assemble each the C-terminal one-third of the protein, interact with the disaccharide unit. For disaccharide assembly at the reducing end in a membrane as amphipathic helices or reentrant loops but do concerted way (variant 2), HAS would require three glycosyl-UDP binding not appear to span the membrane. Te presence of two MDs sites, one each for GlcNAc-UDP,GlcUA-UDP,and a specifc HA-UDP donor chain (i.e., HA-GlcUA-UDP or HA-GlcNAc-UDP), depending on which of in HAS that are amphipathic and do not cross the membrane the two possible HA disaccharide units was assembled. Another variation is is intriguing because these might be particularly well suited that HAS contains only one donor and one acceptor glycosyl-UDP binding for the formation of an intraprotein pore. Vertebrate HASs site, whose specifcities alternate as the two sugars are assembled one at a contain an additional C-terminal region of 130–160 aa with time (variant 3). If there is a single donor binding site, its specifcity would alternately recognize HA-GlcUA-UDP and HA-GlcNAc-UDP. Tere could two trans-MDs. Te amino 75% of the larger eukaryotic be two separate sugar-UDP sites, but if there is a single acceptor binding site, HAS family members is homologous to SpHAS∼ with the same its specifcity would also alternate in a reciprocal fashion with the HA-UDP predicted domain organization,∼ so the overall topological site to bind GlcUA-UDP or GlcNAc-UDP. International Journal of Cell Biology 3

12 4 5 6 V10 Y55 A215 Y233 F319 3 C367 C226 S379 K398 DD-260 DSD-161 N EE-171 UDP-sugar C262 binding region C DAD-153 C281 ED-77

DAE-79

DD-103

ED-116

Figure 2: Membrane organization of HAS domains and conserved potential glycosyl-UDP binding regions. Te experimentally determined topology of SpHAS [28] is modifed to incorporate the discovery [46]thatallfourCysresiduesofSeHAS(whitecircles)areatthemembrane- protein interface and are located in or very near to the sugar-UDP binding sites. Tese four Cys residues are positionally conserved in the Class I HAS family. Te SeHAS numbering shows the amino acids at the cytoplasmic junctions of the six MDs (white numbers1 –6). Te parallel lines (gray) between C262and C281 indicate the close proximity ( 5A) of these residues; they are not disulfde bonded. Eight “DXD”- or “XDD”-equivalent motifs in SeHAS, potential glycosyl-UDP binding sites (rectangle boxes), are either conserved just among the streptococcal enzymes (light gray) or also among the eukaryotic HASs (white); a∼ few exceptions are discussed in the text. In some motifs, a streptococcal acidic residue is shaded white to indicate its conservation in the HAS family. organization of all the Class I HASs is predicted to be similar 3. Mammalian HAS Activity Is [10]. Regulated by Precursor Availability, Posttranslational Modifications and 2. HAS Activity Is Regulated by Protein-Protein Interactions Its Lipid Environment In streptococcal [37], B. subtilis [38], or mammalian [39]cells Radiation inactivation studies [29] showed that the “active expressing HAS and making HA, the consumption of the two unit” mass of SeHAS or SpHAS is 23kDa more than a precursor sugar-UDPs is extraordinarily high compared to monomer, but smaller than a HAS dimer. Te additional cells not making HA. To enable HA synthesis cells must have 23kDa was identifed as phospholipid∼ (CL). Te active strep- greater expression levels of the biosynthetic enzymes and tococcal enzymes are HAS protein monomers in complex greater fux rates in the precursor metabolic pathways; this with 14–18 molecules of CL or other phospholipids. Similarly, ofen results in higher steady-state precursor concentrations, active XlHAS1is a monomer of 69 kDa with an additional but a more important factor is that the rate of precursor 20 kDa of unknown components [30], probably phospho- synthesis supports the high rate of HAS precursor use. HAS lipid although this was not con∼frmed. Kinetic characteri- regulation in mammalian cells is more complicated than in ∼zation of purifed StrepHASs [31]andhumanHAS2[32] bacteria and several groups have identifed a range of diferent confrmed that activity of these enzymes is regulated by, or mechanisms, including transcriptional and posttranslational dependent on, lipids. Purifed StrepHASs have low activity control [40, 41]. Mammalian HAS2has been studied the most without lipid and are activated 10-fold by exogenous CL and is regulated posttranslationally by phosphorylation [42], [11, 33], with high specifcity for particular fatty acyl chains O-GlcNAcylation, and GlcNAc-UDP levels [43, 44], and by [34]. SeHAS is highly activated∼ by oleoyl (C18:1) CL, but ubiquitination and dimerization [45]. It is not known if any almost completely inactive with myristyl (C14:0) CL.T e of these regulatory mechanisms alters HAS monosaccharide activity of purifed human HAS2in reconstituted liposomes assembly activity or HA translocation activity, but since these is greatly infuenced by cholesterol and the available lipids two functions are coupled, altering one activity is expected to [32]andmanipulatingplasmamembranecholesterolcontent alter both. in diferent cell types causes them to make less HA [35, 36]. Tus, Class I HASs are either lipid-dependent or regulated by 4. HA Synthases Elongate HA at their lipid and cholesterol microenvironment. Strong positive the Reducing End modulation by cholesterol might also serve to minimize intracellular HA synthesis, which could be detrimental to Stoolmiller and Dorfman [47] reported that the SpHAS adds many cellular pathways and functions if in excess. new sugars to the nonreducing end, but other studies with 4 International Journal of Cell Biology membranes from streptococci [24] or eukaryotic cells [23, 26] chain across the cell membrane into the extracellular space show that HA synthesis occurs at the reducing end. Purifed [11]. An alternative proposal was that an ABC transport SeHAS and SpHAS [27]orSpHASincrudemembranes[25] system is required for the appearance of extracellular HA [50, also add sugar-UDP units at the reducing end. Te mech- 51], as for many bacterial polysaccharides [52]; HAS would anism for polysaccharide biosynthesis is diferent if chain synthesize intracellular HA that, while still being assembled, growth is from the reducing or nonreducing end. When a would be exported by a nearby membrane-bound ABC sugar is added from a sugar-nucleotide (making it the donor) transport system. to the nonreducing end of a polysaccharide (the acceptor), It seemed unlikely that an ABC transport system was the nucleotide (e.g., UDP) is released. However, for reducing involved in HA translocation for several reasons: (i) It is unex- end elongation, the growing polymer chain is always attached pected that ABC polysaccharide transporters in E. faecalis, B. to UDP. Reaction (1) shows the reaction for HA disaccharide subtilis, and fruit fies would have such low specifcity for their assembly (D = disaccharide units). During HA synthesis, the normal substrate that they would efectively transport HA. UDP released at each transfer step comes from the HA-UDP (ii) Since multiple MDs are not needed for just HA synthesis intermediate formed by addition of the previous sugar. In (e.g., the Class II P. mu lto c i d a HAS contains one membrane each cycle of monosaccharide addition, the released UDP is anchor and, unlike Class I HASs, can be expressed as an active derived from the last monosaccharide added: soluble truncated protein [53]), the topological organization ofHASenzymes,containing6–8membrane domains, is more HAD -UDP GlcUA- consistent with a translocation function [54]. (iii) HAS activ- ity is lipid-dependent or modulated by its lipid environment, ( ) UDP + HAD -GlcUA-%&' (1) consistent with an inherently intimate organization within GlcNAc-UDP the membrane bilayer, as expected for an HA translocation !→ +( ) %&' function, but not the independent ABC transport model. (iv) ↓HAD -GlcUA-GlcNAc-UDP Te sugar-UDP binding sites of Strep and mammalian HASs are at the inner membrane surface [46], which better fts a Te donor HA-UDP%&'+ transfers( ) a hyaluronyl- (HA-) chain to model in which the HA-UDP chain is extended near or within the new sugar-UDP (acceptor) without cleavage of the latter the membrane and translocated through the enzyme to the high-energy linkage to UDP; the UDP released is from the exterior (Figure 2). (v) Class I HASs are processive enzymes, HA-UDP donor. Tissituationisanalogoustothatforprotein meaning they do not release their HA-UDP chains during and fatty acid synthesis [48]. synthesis; dissociation of HA from HAS does not occur [5, Te IUBMB nomenclature for HAS glycosyltransferase 55].Tis characteristic strongly supports a Pore model. HA- activities (EC 2.4.1.212) is diferent compared to that for typ- UDP that is bound by weak HA-HAS interactions would ical glycosyltransferases (Figure 1). An enzyme that utilizes be released, moved, and elongated continuously within the GlcNAc-UDP to add to the nonreducing end of GlcUA would enzyme, while still being retained by the topological con- create a GlcNAc( 1,4)GlcUA linkage, whereas the HAS straint of being within a pore. In the ABC model, HA-HAS transferase activity adding GlcNAc-UDP at the reducing end interactions are reversible, as for the nonprocessive PmHAS, creates the GlcUA(! 1,3)GlcNAc linkage. Systematic naming which dissociates from and then rebinds HA afer every sugar of a transferase activity specifes the donor: acceptor, group addition [53]. transferred.Tus, addition! of a GlcUA residue to a GlcNAc Te strong biochemical logic supporting a Pore Translo- at the reducing end of the growing HA chain is catalyzed by cation Model was confrmed by multiple studies showing an activity that adds a hyaluronyl chain from HA-GlcNAc- that an ABC transporter Model for HA translocation is not UDP to GlcUA-UDP. Tis is a HA-GlcNAc( )UDP: correct. Tomas and Brown [56]foundthatABCtransporters GlcUA( )UDP, (1,4) hyaluronyltransferase. Similarly, are not involved in HA translocation by breast cancer cells an activity adding GlcNAc-UDP to a HA-GlcUA-UDP)1→ chain and Medina et al. [57]showedthatpurifedSeHASmediates is a HA-GlcUA()1→ !)UDP: GlcNAc( )UDP, (1,3) hya- luminal dye efux when added to liposomes, demonstrating luronyltransferase. the presence of an intraprotein pore. Hubbard et al. [58] )1→ )1→ ! found that SeHAS, incorporated into liposomes, delivers HA 5. HA Translocation to the Cell Exterior Is directly to the internal lumen, demonstrating that HAS pos- Mediated by the HAS Protein Itself sesses the predicted HA translocation function. Misra et al. [59]showedthatABCtransporterMDR1 Te active sites of HAS and the sugar-UDP substrates are expression is regulated by changes in the pericellular HA coat. inside cells [28], so how do the large HA products (e.g., Coordinately regulated expression of ABC transporters and 40,000 sugars long; 8MDa) reach the surface or extra- HAS provides an alternative interpretation of studies impli- cellular space? Only the exogenous HAS protein (gene) and cating a role for transporters in HA transfer. Two independent >sufcient GlcNAc-UDP> and GlcUA-UDP are required for HA cellular protective mechanisms (provided by pericellular HA biosynthesis and secretion by heterologous cells that cannot coats and ABC multidrug transporters) may have coevolved normally make HA, including E. faecalis [3], B. subtilis [38] in vertebrates to be coordinately regulated in a complex man- and D. melanogaster [49]. Based on these fndings and the ner in response to environmental cues; this could explain why lipid dependence and the topology of HAS, we proposed that HAS and extracellular HA levels are lower in cells treated with HAS must have the ability to translocate the growing HA inhibitors of multidrug transporter function [50]. Another International Journal of Cell Biology 5 explanation for inhibition of HA translocation by ABC (GlcNAc)n-UDP transporter inhibitors is that these inhibitors alter uridine uptake or salvage pathways and change uridine nucleotide 320 pools, which inhibits HAS (e.g., controls were not preformed 1 to verify that substrate sugar-UDP levels did not decrease or 3 240 that the potent HAS inhibitor UDP did not increase). 2 Finally, the fnding that a bacterial cellulose synthase creates an intraprotein pore, in which the product cellulose 4 is synthesized and translocated [60], confrms the principle 160 we proposed in 1999 [11]thatglycosyltransferasessuchasHA

and cellulose synthases can mediate both polysaccharide (units) Mass/charge synthesis and translocation. 80

1 2 3 4 6. HAS Synthesizes Chitin and 0 Chitosyl-UDP Oligosaccharides 0 120 Glycosidase treatment (min) SeHAS synthesizes chitin oligomers, (GlcNAc- 1,4) [61], n Figure 3: Glycosidase treatment converts larger GlcNAc -UDP as reported for XlHAS1[62]andMmHAS1[63]. More oligomers to GlcNAc-UDP. SeHAS membranes were incubated importantly, however, and consistent with reducing end sugar ! ! for 30min with UDP-GlcNAc alone, Folch extracted,( fractionated) addition, we found that SeHAS also makes novel chitin over a size exclusion column, and samples were either untreated oligomers attached to -GlcNAc( )UDP at the reducing (0min) or treated (120 min) with jack bean hexosaminidase.T e end [61]. SeHAS incubated with only GlcNAc-UDP makes a samples were then analyzed by MALDI-TOF MS to identify and series of (GlcNAc- 1,4)n-GlcNAc()1→ )UDP oligomers (for quantify m/z signals of candidate oligomeric chitin-UDP fragments –15) corresponding to (GlcNAc) -UDP through corresponding to –4 (boldface white or black numbers). (GlcNAc) -UDP products.! SeHAS)1→ membranes incubated Te presence of chitin linkages was confrmed by the ability of +without=2 substrate or with only GlcUA-UDP2 show no signals glycosidase treatment+=1 to shifspecies with 3or4 sugars to products in this region.7 Product identifcations were confrmed by with 1(GlcNAc-UDP) or 2sugars. Additional MS/MS analysis of the MS-MS fragmentation and digestion with jack bean - - starting sample ions (not shown) revealed smaller members of the acetylglucosaminidase (e.g., all species ultimately yielded expected oligomer series, including GlcNAc-UDP. GlcNAc-UDP). For example, tri- and tetraoligomers! were, confrmed to contain 1,4-linked GlcNAc residues attached Oligo-chitin-HA-UDP chain to GlcNAc-UDP because treatment with jack bean hex- osaminidase converted! almost all the initial oligomers to Out GlcNAc-UDP or (GlcNAc) -UDP (Figure 3).Tus, HAS syn- Membrane thesizes (GlcNAc- 1,4) -GlcNAc( )UDP oligomers. – -UDP Tese unusual sugar-nucleotide2 species, activated by - -UDP -UDP

attachment to UDP,! are unstable1 7 and) readily1→ cleaved to yield UDP-

UDP- chitin oligomers, explaining the ability of Class I HASs) to UDP- make chitin. Although HAS does not require an exogenous primer to Figure 4: HAS initiation of HA synthesis using a self-made oligo- make HA, these novel self-made GlcNAc -UDP products chitosyl-UDP primer results in HA chains with a chitin oligosaccha- could potentially serve as endogenous primers that enable ride cap at the nonreducing end. HAS makes chitin oligomers when HAS to initiate HA chain assembly.( In this proposed)! scenario incubated with GlcNAc-UDP (red squares) alone. We found that the (Figure 4), the nonreducing end of all HA chains retains enzyme frst makes chitin oligomers linked to UDP at the reducing this initial chitin oligomer primer, and all HA molecules end [61], consistent with the mechanism of addition at the reducing have a novel non-HA structure (a chitin oligosaccharide end, and that these molecules could then serve as self-primers for HA disaccharide synthesis (GlcUA; green circles). cap) at their nonreducing end. Ongoing studies support this hypothesis [64], including HAS-dependent m/z signals indi- cating hybrid chitin-HA species such as GlcNAc (GlcUA- GlcNAc) in ovine testicular hyaluronidase-digested sam- under conditions (e.g., exposure to a single sugar-UDP) that ples (Figure 5(b)). Empty vector membranes without6 HAS may not normally be encountered in cells, it remains to be (Figure 52(a))orSeHASmembranesincubatedwithoutsub- demonstrated if these interesting products are also made in strate (not shown) show no signals in this region. Te vivo.StudiesareinprogresstodetermineifHAmolecules hybrid chitin-HA digestion fragments can be afnity puri- made by streptococcal and mammalian Class I HASs contain fed, fractionated by TLC or PAGE, and shown to contain a nonreducing end chitin oligosaccharide cap. Te presence multiple nonreducing GlcNAc residues that are releasable of a chitin cap would have important physiologic impli- by treatment with jack bean hexosaminidase, as in Figure 3. cations for the polarity of HA chains and the potential Since we studied chitin-UDP oligomer products in vitro only, ability of chitin-like binding proteins in the biomatrix or 6 International Journal of Cell Biology

6000 SeHAS membranes HA synthesis requires making the frst disaccharide using No UDP-GlcNAc 5000 a non-HA substrate, so an acceptor site for the GlcUA to No UDP-GlcA be added and a unique transferase activity (used only once 4000 for each HA molecule synthesized) are needed (Box 1(B), 3000 functions 6and 7). Subsequent steady-state HA disaccharide 2000 synthesis requires seven functions, using two binding sites noted in (A) and (B) (functions 2and6 ), two additional Signal intensity (a.u.) intensity Signal 1000 hyaluronyl-UDP species binding sites, and two additional 0 corresponding transferase activities (Box 1(C), functions 8– 1850 1900 1950 2000 2050 2100 2150 11). Tefnal function is the translocation activity of HAS, m/z value which acts in a continuous manner during HA chain elonga- (a) tion but is listed separately to emphasize its novel and separate nature, as a “spatial” rather than chemical catalytic process 6000 GlcNAc6-HA4 2017.9 SeHAS membranes (Box 1(D), function 12).Tus, an astounding 12 discrete Predicted mass +5mM UDP-GlcNAc 5000 [M + Na]+2017.7 functions are attributable to Class I HASs in order for these +5mM UDP-GlcA 2047.9 4000 enzymes to initiate, assemble, and transfer the oligo-chitosyl- HA-UDP polysaccharide to the cell exterior; it is not known if 3000 2081.0 released HA chains are still attached to UDP at their reducing 2000 ends or if chains are released because this group has been lost 2114.0 and elongation has therefore stopped, resulting in HA release. Signal intensity (a.u.) intensity Signal 1000 0 1850 1900 1950 2000 2050 2100 2150 8. A Pendulum Model for m/z value Polysaccharide Translocation

(b) Weproposedanovelmechanismin2004[37]bywhichasin- Figure 5: Mass spectral evidence for chitin-HA in HA made by gle membrane-bound HAS lipid complex could simultane- SeHAS. Empty vector (a) or SeHAS (b) membranes were preincu- ously extend a polymer chain at its reducing end and extrude bated for 30min with UDP-GlcNAc, UDP-GlcUA was added, and the growing chain through the⋅ membrane (Figures 6 and 7), incubation proceeded for several minutes. Te membranes were in a process not requiring other proteins or ATP. Te model heated to release HA and subjected to two cycles of Folch extraction. also applies to other membrane polysaccharide synthases that Extracted HA products were speed vacuum concentrated, digested use two transferase sites to make hetero- or homopolysaccha- with ovine testicular hyaluronidase, and the resulting oligomers were subjected to afnity selection over carbograph. Eluted material rides (e.g., cellulose). Te model involves continuous “swing- was surveyed by MALDI-TOF MS to identify “hybrid” fragments ing” movement by enzyme domains (pendulum-like) and has corresponding to a chitin oligomer cap linked to HA disaccharides, variations (three of which are noted in Table 1), depending such as the GlcNAc -(GlcUA-GlcNAc) species shown. on whether the catalytic mechanism utilizes independent glycosyl-UDP binding sites (e.g., variants 1and 2) or one 6 2 site with alternating specifcity (e.g., variant 3). Disaccharide on cell surfaces to orient, align, and organize individual HA assembly in variant 1or 2is sequential or simultaneous, polymers into bundles; for example, cables or fbers [65]. respectively, whereas assembly would necessarily be one sugar at a time in a variant 3mechanism. Key features of the Pendulum Model are presented below to describe Pendulum 7. Class I HAS Performs Multiple Model variant 1, but similar central points and considerations Binding and Catalytic Function in apply to variant 2. Order to Synthesize HA (i) HAS Has Two Functional Domains Tat Act as “Arms.” Each We noted above the seven functions that all Class I HAS arm contains an active site for one of the glycosyltransferase enzymes possess in order to catalyze the steady-state assem- functions, a binding site for one of the acceptor sugar-UDPs, bly of GlcNAC( 1,3)GlcUA( 1,4) disaccharides during HA and a binding site for one of the donor HA-UDP species synthesis. Te recent discovery that HASs are also able to (Figure 6(a)).Te right arm (pink) contains the GlcNAc- make oligo-chitosyl-UDP! species! and that these can serve UDP acceptor binding site, the HA-GlcNAc-UDP donor as self-primers for subsequent HA assembly means that this binding site, and the (1,4) hyaluronyltransferase activity that enzyme family also possesses more binding site and catalysis makes the GlcNAc- (1,4)-GlcUA linkage. Some residues functions than previously recognized. Tese functions are participating in interactions (e.g., binding) needed for each summarized in Box 1,usingthestandardtransferasenomen- function might be in either! arm or in other HAS domains not clature, and listed in order of use in the initiation and assem- shown in Figures 6 and 7. bly of HA. Te synthesis of chitin-UDP species requires three binding sites with diferent structural specifcities and two (ii) Only One Arm Is Active at a Time and Teir Activities Are transferase activities (Box 1(A), functions 1–5). Initiation of Reciprocal. When one arm is active as a transferase, the other International Journal of Cell Biology 7

Functions and activities of Class I HA synthases needed for HA synthesis.

(A) Synthesis of GlcNAc n-UDP Self Primer (1) Donor site for: GlcNAc( 1 )UDP (2) Acceptor site( for:) GlcNAc( 1 )UDP (3) Transferase activity: GlcNAc()1→ )UDP: GlcNAc( 1 )UDP, GlcNAc( 1,4) (4) Donor site for: [GlcNAc() 1→,4)] ( 1 )UDP (5) Transferase activity: [GlcNAc() 1→,4)] ( 1 )UDP: GlcNAc() → 1 )UDP, [GlcNAc(! 1,4)] ! ! ) → GlcNAc( 1 )UDP +GlcNAc( 1 )UDP ! UDP +GlcNAc( 1,4)GlcNAc( 1 )UDP ! ! ) → ) → ! GlcNAc( 1 )UDP ) → ) → ...... (3) → ! ) → GlcNAc( 1,4)GlcNAc( 1,4)GlcNAc( 1 (5))UDP + UDP [GlcNAc( 1 )UDP]↙ ) → [GlcNAc( 1,4)]! GlcNAc((5) !1 )UDP + ) UDP→ ↙+ ) → (B) Synthesis of frst HA disaccharide !+2 (6) Acceptor site for: GlcUA( 1 ! )UDP ) → + (7) Transferase: [GlcNAc( 1,4)] GlcNAc( 1 )UDP: GlcUA( 1 )UDP, [GlcNAc( 1,4)] ) → GlcNAc( 1,4) GlcNAc( 1 )UDP +GlcUA(! 1 )UDP ! ! [GlcNAc() →1,4)] GlcNAc((7) 1),4)GlcUA(→ 1 )UDP! + UDP ! [ ! ] ) → ) → ...... → (C)Steady-statesynthesisofHA ! (8) Donor site for: Hyaluronyl-GlcUA( 1,3)GlcNAc(! 1 )UDP ! ) → (9) Donor site for: Hyaluronyl-GlcNAc( 1,4)GlcUA( 1 )UDP (10) Transferase: Hyaluronyl-GlcNAc(! 1 )UDP: UDP() → 1 )GlcUA, hyaluronyl-GlcNAc( 1,4) (11) Transferase: Hyaluronyl-GlcUA( 1! )UDP: UDP-GlcNAc,) → hyaluronyl-GlcUA( 1,3) ) → 10 & 11 ) → ! (HA)D-UDP +UDP-GlcUA+UDP-GlcNAc HA D+1-UDP + UDP +UDP ) → ( ) ( ) ! (D) Steady-state extrusion of HA-UDP across the membrane through a HAS pore (12) Hyaluronyl-UDP translocation activity ...... →( )

Box 1: Summary of HAS functions required for HA biosynthesis. At least twelve discrete binding and catalytic function are required for Class I HA synthases to create a (GlcNAc) -UDP self-primer (A, functions 1–5), to initiate HA disaccharide synthesis (B, functions 6and 7), and to then assemble HA disaccharide units in a continuous manner (C, functions 8–11), while the growing HA-UDP chain is also continuously translocated through the HAS! lipid complex pore to the cell surface or exterior (D, function 12). Two functions for steady-state HA disaccharide synthesis (C) are also used to make (GlcNAc) -UDP (#2) or the frst HA disaccharide (#6). ⋅ ! serves as an acceptor binding site. Each arm can “swing” the binding and catalytic sites (Figure 6(c)). Even if binding to one of three functionally diferent positions (Figure 6(b)) of the alternate acceptor and donor could occur, the active that correspond to conformations in which it is active as sites would not be aligned for functional sugar transfer adonorbindingsiteandtransferase,inactive,andactive (Figure 6(c), lef). as an acceptor binding site. For example, the right (pink) arm can be active as a donor binding site for HA-GlcNAc- (iii) HA-UDP Translocation Is Coupled to Sugar Addition.As UDP and the ( 1,4) transferase (Figure 6(b),lef),inactive each arm moves from one side to the other through a sugar (Figure 6(b), center), or active as an acceptor binding site for addition cycle, the HA chain is extruded through the enzyme GlcNAc-UDP (Figure! 6(b), right). In the same relative posi- and lipid bilayer (each row in Figure 7)onesugaratatime tions, the lefarm is similarly active as an acceptor binding (or two at a time if concerted disaccharide synthesis occurs). site for GlcUA-UDP (Figure 6(b),lef),inactive(Figure 6(b), With each new sugar-UDP addition, the bound HA-UDP center), or active as the ( 1,3) transferase and donor binding chain is passed from one HAS arm to the other (e.g., like a site for HA-GlcUA-UDP (Figure 6(b), right). Te relationship person pulling up a rope, hand-over-hand) and as the arms of the arms in a neutral! inactive conformation (Figure 6(b), swing, the nonreducing end of bound HA-UDP is simulta- center) creates misalignment of glycosyl-UDP acceptor and neously moved away from the intracellular active sites. Te donor sites that is not suitable for glycosyl binding or transfer. synchronized arm movement provides the force needed to In contrast, when the arms have moved to either the lefor move the bound HA-chain through the protein pore and cell the right position, the alignment of glycosyl-UDP donor and membrane to the cell exterior. Te bound HA-chain does not acceptor are favorable for the respective transferase activity to dissociate from HAS during continuous elongation because function. In either of the two functional positions, in which it is always bound to one arm or the other as it cycles between transferase activities add sugar-UDP to the growing HA-UDP the two arms and it is always topologically constrained by chain, there is only one appropriate active arrangement of all being within the HAS-lipid pore. 8 International Journal of Cell Biology

Outside HAS HAS HAS HAS

HA-GlcUA transferase polysaccharide Membrane HA-G HA-GlcUA transferas pore Inside (

,4) 1, l cUA cUA

transferase transferase HA-GlcUA-UDP HA-GlcNAc-UDP

Donor binding transferase Donor binding region region -GlcNAc transferase UDP-

UDP- e

1, 3) UDP- UDP- transferase HA-GlcNAc

( HA

HA-GlcNAc transferase HA-GlcNAc UDP- UDP- In position to transfer In position to transfer In neutral position: HA-GlcUA to GlcUA-UDP GlcNAc-UDP HA-GlcNAc to Acceptor binding site Acceptor binding site GlcUA-UDP no HA-chain transfer GlcNAc-UDP (a) (b)

HAS HAS

HA-GlcUA transferase HA-GlcUA HA-GlcUA transferase HA-GlcUA

UDP- UDP- HA-GlcNAcUDP- transferase HA-GlcNAcUDP- transferase Donor HA-UDP and Donor HA-UDP and sugar-UDP acceptor sugar-UDP acceptor are not aligned-no transfer aligned-transfer occurs (c)

Figure 6:Te Pendulum Model for HAS Translocation of HA. (a) Organization of hyaluronyl transferase domains and glycosyl-UDP binding sites within HAS. Te scheme shows the cell membrane (gray) and three overall domains of the HAS lipid complex: a pore region (purple) containing HAS MDs through which a growing HA chain is passed to the exterior, and two catalytic domains that behave as swinging arms (blue and pink). Each arm contains one of the two functional hyaluronyl transferase activities and binding⋅ sites needed to add either an HA-GlcUA-UDP donor chain to GlcNAc-UDP [lefarm (blue); the (1,3)-hyaluronyl transferase] or an HA-GlcNAc-UDP donor chain to GlcUA-UDP [right arm (pink); the (1,4)-hyaluronyl transferase]. Tefgure also illustrates that an individual sugar-UDP binding site is part of the HA-UDP binding site on each arm. (b) Te interactions between! the glycosyl-UDP binding sites and hyaluronyl transferase domains change as the domain arms move. T! e three positions, from lefto right, indicate three conformations in which HAS is either able to create the GlcNAc (1,4)GlcUA bond, unable to perform either transferase function, or able to create the GlcUA (1,3)GlcNAc bond. Te lefand right positions also illustrate that when the enzyme is in position to catalyze one of the transferase reactions, the growing HA chain is bound primarily to! one arm, whereas the sugar-UDP substrate is bound to the other arm. In the central panel, a! neutral or inactive position, the individual sugar binding sites in the HA-binding region on each arm are “misaligned” so that they are unable to bind HA at the same time. (c) HAS hyaluronyl transferase activities require correct alignment between the glycosyl-UDP binding sites on opposite domain arms. Transferase function depends on how the two arms are aligned with respect to the ability to bind substrates or to perform catalysis. Te relative positioning of HA-UDP and sugar-UDP binding sites are shown with the complementary glycosyl-UDP substrates bound incorrectly and not aligned for creating a glycoside bond (lef) or with the right substrates bound and aligned correctly for successful -GlcNAc- (1,4)-GlcUA-bond formation (right). !

(iv) Other Variants of the Pendulum Model.Tegeneral is intrinsically more complicated and may thus be less likely, scheme for other variants of the Pendulum Model is slightly especially given the large number of potential glycosyl- diferent compared to that for variant 1(Table 1). In variant UDP binding sites in the Class I HAS family (Figure 2 and 2thethreeglycosyl-UDPbindingsitesmightbeinone Section 10 below). arm, but the transferase sites and the HA-binding sites for Akeymechanisticfeaturetoconsiderforconcertedaddi- moving the chain could be on the other arm or both arms. tion of a disaccharide unit to HA-UDP by adding both UDP- Te glycosyl-UDP binding sites could be on two arms in sugars simultaneously is that HAS would use only one of variant 3, and simultaneous disaccharide assembly could the two possible HA-UDP donors, either HA-GlcUA-UDP or occur in swinging from one position to the other. Release of HA-GlcNAc-UDP and thus make only one of the two possible UDP products could occur as the arms swing to the other HA disaccharide units (i.e., HA-GlcUA( 1,3)GlcNAc-UDP position, transfer (and translocate) the HA-UDP chain, and versus HA-GlcNAc( 1,4)GlcUA-UDP, resp.). Although no “reload” for another round of disaccharide assembly. An data currently support one mechanism! over another, the alternating specifcity mechanism (variant 3) involving fewer presence of a chitin! cap at the nonreducing end defnes the sites, whose glycosyl-UDP binding specifcity cycles between frst HA disaccharide made (Box 1)asGlcNAc( 1,4)GlcUA two species (e.g., HA-GlcNAc-UDP and HA-GlcUA-UDP), and makes it likely that subsequent coordinated disaccharide ! International Journal of Cell Biology 9

HAS HAS HAS

UDP

- UDP- UDP-

UDP- -UDP

UDP UDP

HAS HAS HAS

UDP

UDP- -UDP UDP-

UDP-

UDP UDP-

HAS HAS HAS

UDP

P-

D U

UDP-

UDP-

UDP- UDP

Figure 7:Te Pendulum Model: Arm movement and HA transfer between arms drives HA chain translocation through the HAS lipid complex to the cell exterior. Te changing alignment of the HA-binding regions on the two arms in the two extreme positions (lefand right) creates the ability of the enzyme to move the HA chain from one arm to the other (shown in each row). When the arms swing from⋅ one extreme position to the other, the HA chain is transferred from the frst arm to the other arm as the HA-binding site alignments move out of (neutral position) and then back into register. A “time-lapse” of HAS action is illustrated in the nine panels as the enzyme adds three new sugars to an HA-UDP chain of seven sugars. Te enzyme goes through three stages of arm movement (in each row) to add each new sugar. Afer assembly of each disaccharide, the enzyme arms are in the same starting position (e.g., the lefpanels in top and bottom rows). Te sugars “crossing” the membrane are shown outside of the enzyme in the top row to help orient the reader, and then within the intraprotein pore in the middle and bottom rows. An animation of this process showing chain translocation through assembly of an HA 10-mer is at http://www.glycoforum.gr.jp/science/hyaluronan/HA06a/Pendulum Hypothesis Anima.fles/slide0001.htm.

assembly would utilize the three bound substrates indicated 9. The Bioenergetics of HA Translocation in reaction (2),sothatdonorHA-GlcUA-UDPwillbe covalently linked to GlcNAc-UDP, as it in turn is linked to Any mechanism of HA translocation by HAS must account GlcUA-UDP in sequential coupled reactions that would be for several potential bioenergetic obstacles: (i) the energy essentially simultaneous: barrier associated with transfer of a hydrophilic HA chain across a hydrophobic membrane lipid bilayer, (ii) the energy required to move an HA molecule might become progres- HA-GlcUA-UDP GlcNAc-UDP GlcUA-UDP sively greater as it elongates to larger mass and hydrodynamic (2) volume [66], and (iii) the energy to cause conformational HA-GlcUA-GlcNAc-GlcUA-UDP+ + 2UDP changes and movement of domains within the enzyme that

.→ + 10 International Journal of Cell Biology could be physically responsible for rapid chain movement increased at the cell surface, the network of interact- across the membrane without releasing the chain. For per- ing chains would diminish the efects of Brownian- spective, an 8MDa HA chain proportionally enlarged to generated forces acting to release the HA, thus human scale would be equivalent to a 1mm wide thread resulting in a stabilized gel-like cell surface coat still 30 m long. anchored to multiple HAS molecules. In the extreme of this scenario, HA synthesis would slow or stop as >(i) How Does HAS Overcome the Hydrophobic Membrane forces generated by the HA coat network increased, Barrier? Membrane transporters, such as lac permease with fnally providing too much resistance for HAS to 12 MDs [67], mediate sugar transfer through an intraprotein translocate HA. pore created by interactions among many MDs. HASs appear to compensate for not having enough MDs to form such an (iii) What Are the Energy Sources for HA Translocation? At intraprotein pore by their interactions with phospholipids least three possible energy sources could contribute to an [11, 32]. HAS lipid interactions could allow the enzyme to HA translocation mechanism (Figure 8), as estimated below: create a larger pore-like passage within the enzyme, through glycosyl-UDP hydrolysis [4–8kJ/mol], H-bond formation which the growing⋅ HA chain could pass [11, 57, 68]. HAS lipid [12–24 kJ/mol], and ion-pair reactions and the potential complexes would present exterior hydrophobic interactions energy of electrochemical gradients [4–8kJ/mol]. If these with fatty acyl groups in the bilayer, while engaging HA on⋅ the energy sources contribute to the HA translocation mecha- interior pore surface with hydrophilic interactions; thus, an nism, a conservative estimate of the total free energy change HA chain moving through a HAS lipid pore could efectively for HA translocation is 30kJ/mol or 7kcal/mol (1kcal = bypass the hydrophobic membrane barrier. 4.18 kJ) of HA disaccharide units moved across the mem- ⋅ brane. Tis is equivalent to 1ATP and would be a favorable (ii) How Could HAS Move HA Chains with Masses Tat Get ∼ ∼ bioenergetic situation, explaining how an ATP-independent Progressively Greater? Intuitively, one might expect that the translocation process is feasible. energy needed to overcome the inertia of, and move, a long HA-UDP chain (e.g., 4MDa) would be much greater than the (a) UDP-Sugar Hydrolysis.Te free energy for Glc-UDP energy needed to move a short HA oligosaccharyl-UDP chain hydrolysis is 7.3–7.6kcal/mol and the diference (e.g., 4kDa). However, two factors may make this apparent between Glc-UDP hydrolysis and glucoside bond for- “molecular weight-lifing” less difcult than it appears. mation is 0.6–1.0kcal/mol [72]. Values for hydroly- sis of GlcNAc-UDP and GlcUA-UDP are not avail- (a) HA Segmentation.Tesegmented,semi-independent able but are∼ presumably somewhat greater, due to movement of discrete portions (roughly 100 sugars destabilizing repulsive and steric factors. Free energy long) of a large HA chain [69–71]couldcreatean diferences between sugar-UDP hydrolysis and glyco- upper limit for the “apparent” mass of HA that side bond formation for GlcNAc and GlcUA are likely HAS would need to “move” to translocate a discrete similar to Glc, 0.6–1.0kcal/mol. Tus, the available section. If segments of 100 sugars behave semi- “excess” free energy to perform additional work independently for brief periods (e.g., msec), during beyond creating∼ two glycoside bonds could be 1- 2kcal/mol ( 4–8kJ/mol) of HA disaccharide units which translocation is favored,∼ then the apparent mass of HA against which HAS needs to generate a (Figure 8,#1). ∼ translocation force would only be 20 kDa. As a chain (b) Electrochemical∼ Gradients or External Ion Reactions was initiated and elongated, HAS might initially May Provide Additional Energy. HA translocation to translocate it rapidly and then∼ progressively more the cell exterior must necessarily be associated with slowly until the segmental length was approached, oneor moreelectrochemicalgradientsthat couldpro- and a steady-state translocation rate was then vide additional energy for the translocation process. reached. Te observed kinetic profle for how HA HAS might recognize only one of 4possible UDP- mass increases during synthesis begins very rapidly GlcUA species as the correct substrate during cataly- and then becomes slower [55]. sis, depending on the state of the carboxyl≥ group. Te carboxyl group could be dissociated and free (i.e., an (b) Increased Release Forces.Tenetforcesgeneratedby anion) or neutral and bound to either H ,Na ,orK BrownianmotionmaytendtopulltheHAchainaway (Mg is also a possibility). In most of these+ + cases,+ a from the enzyme and thus facilitate the translocation favorable+2 energetic situation would occur when newly action of HAS. A possible chain release mechanism, added GlcUA is transferred to the exterior: proto- in fact, could be the balance between the Brownian nated carboxyl groups would readily dissociate releas- and other forces acting to release the HA and the HA- ing H ,negativelychargedcarboxylgroupswould binding forces mediated by sites within the enzyme bind to+ extracellular cations (e.g., Na ), and bound acting to retain the HA [66]. In contrast, cells with K ions would exchange with Na ions,+ which are a pericellular HA coat might efectively stabilize and in+ excess. Such ion-pair reactions would+ yield energy stop HA synthesis, because the presence of nearby (that is difcult to estimate) and tend to increase HAS-HA complexes could augment and increase the (pull) the equilibrium for translocation, in a manner forces favoring HA retention. As HA chain density similar to removal of a reaction product. International Journal of Cell Biology 11

2. X + release intrachain H-bonds, by creating up to two H-bonds + X + + X between adjacent sugars (similar to H-bonds between X X amino acids to form protein -helices). Additional X energy could be captured for chain translocation Outside X 3. H-bond formation if HAS couples the formation) of several new H- X Membrane bonds with HA chain movement and the rotation of alternate newly added sugars occurring within or just X external to the HAS pore (Figure 8,#3). Assuming an average value of 6kJ/mol ( 1.5kcal/mol) for intra-HA H-bonds, then translocation could yield up

UDP- to 12–24 kJ/mol (3–6kcal/mol)∼ associated∼ with the formation of 2–4H-bonds per mol HA disaccharides. -UDP X -UDP

1. Glycosyl-UDP hydrolysis 10. Class I HASs Contain Multiple Potential UDP Glycosyl-UDP Binding Sites Figure 8:Tree energy sources may drive the HAS-mediated translocation of HA-UDP. Te diagram illustrates three sources of HASs contain only one conserved “DXD” motif (DSD energy (numbers 1–3) that could contribute to an overall favorable in SeHAS; Figure 2), typically found in the active sites161 of free energy change to drive translocation (dashed black arrow), many glycosyltransferases and whose carboxyl groups are equivalent to 1ATP per disaccharide, as discussed in the text. 1. coordinated with Mg and the phosphoryl groups of a Two HA-UDP bonds are hydrolyzed to make two glycoside bonds nucleotide-sugar [76]. However, the same “DXD” function per disaccharide. 2. Energy can be captured by extracellular release +2 ∼ can be mediated by essentially any cluster in which two of of a cation (X ; bound to the intracellular GlcUA-UDP substrate three contiguous residues are Asp or Glu [77]. StrepHASs and incorporated+ into HA by HAS) as the GlcUA is released from HAS and any associated restraints on the –CO Xgroupbythe also contain a second DAD motif, which is conserved translocation process. Subsequent reactions in the extracellular in the HAS family, except for153 chicken HAS2and chlorella environment (e.g., ion-pair association, dissociation,2 or exchange) HAS (Asp is conserved in all HASs). A DAE motif in and the coupling of a released ion, such as K ,toacellularelec- SeHAS and153 SpHAS, but not in SuHAS, is present79 in 10 of 13 trochemical gradient (potential) would provide+ favorable energetics. eukaryotic HASs. Related “XDD” motifs are also present in 3. Up to four H-bonds (blue lines between sugars at far right) all HASs at multiple positions: for example, SeHAS GDD ; could be formed as each new GlcNAc-GlcUA disaccharide (red ED (EN in Chlorella HAS); ED (conserved positionally squares and green circles, resp.) is released to the exterior, free of 260 constraints imposed by being bound to HAS. For example, two H- as EE,77 EXE, DE, or EXD). Five of116 the seven “XDD” motifs bonds between the released disaccharide GlcNAc and GlcUA and conserved in the StrepHASs are conserved positionally (i.e., two H-bonds between the GlcUA in the disaccharide released during within 6residues)inallHASs,withtheexceptionofXlHAS1 the previous synthetic cycle and the new disaccharide GlcNAc. Te (in which 3of the 5motifs are conserved, but fve others are gray circular arrow indicates a glycosidic bond that rotates (e.g., so also present).≤ Tus, all Class I HASs except XlHAS1contain that the N-acetyl and carboxyl group of adjacent sugars are on the at least six conserved DXD or XDD motifs and, therefore, same side of the chain) allowing the formation of new H-bonds. all HASs potentially have enough glycosyl-UDP binding sites to assemble a disaccharide unit in either a coordinated or simultaneous manner (Table 1). Te situation with bound K might be particularly favorable because HA translocation+ would also then 11. HAS as a Glycosyltransferase be coupled to the cellular K ion gradient (high intra- Family Member and Consequent cellular and low outside),+ providing an additional energy source for overall HA synthesis and translo- Structural Predictions cation [73]. Given the intracellular abundance of K , HAS is a member of the GT2family [78–80]intheCAZy ion composition and pH, the K -GlcUA-UDP species + database (http://www.cazy.org/)andcatalyzesaninverted is likely the most abundant form of GlcUA-UDP and, + mechanism (i.e., creating a -linked glycoside from the - thus, likely to be the normal HAS substrate. Consis- linked precursor). Tese family members require a divalent tent with this, purifed SeHAS is more active in the metal ion and contain at least one DXD motif and a GT-A fold presence of K than Na ions [33].Te energy avail- ! ) (related to the Rossmann fold), consisting of a seven-stranded able if one K per disaccharide is transferred to the + + -sheet fanked by -helices. Te active site in GT-2proteins extracellular environment is 1-2kcal/mol (4–8kJ/ + is created by the association of the large -sheet with a smaller mol) of HA disaccharides (Figure 8,#2).Tis value, as -sheet. Present in perhaps 5,000 proteins, a basic GT-A noted above, is likely underestimated because it lacks ! ) ∼ fold provides a versatile “platform” for creating a variety of the contributions from associated ion-pair reactions. ! !catalytic situations. > (c) H-Bond formation.Experimentalandmodelingstud- HAS is an exceptional GT2family member due to its mul- ies [69, 71, 74, 75]indicatethatHAformsmultiple tiple MDs, which might create inherent topologic constraints 12 International Journal of Cell Biology in regions of homology to the GT-A fold (making modeling HA-UDP: A hyaluronyl group with -linked UDP at the essentially impossible). Based on extensive sequence sim- reducing end ilarity through a 260 aa region between MD2and MD4 HAS: HA synthase ) (Figure 2), the HAS catalytic domain might adopt an overall MD: Membrane domain fold similar to other∼ GT2family members, including cellulose SeHAS: Streptococcus equisimilis HAS synthase [81]. Similarities between HA and cellulose syn- SpHAS: Streptococcus pyogenes HAS thases include N-terminal nucleotide binding motifs, a puta- StrepHAS: Streptococcal HA synthases tive catalytic base, a QxxRW motif associated with processiv- SuHAS: Streptococcus uberis HAS ity, and that both proteins create an intraprotein pore for their XlHAS: Xenopus laevis. products [58, 60].Te GT2family model predicts that the N- terminal subdomain binds the sugar-UDP donor and the C- Conflict of Interests terminal subdomain binds the acceptor. For enzymes such as HAS that add to the reducing end the normal designation of Te author declares that there is no confict of interests donor and acceptor binding sites may be switched. Tus, in regarding the publication of this paper. HAS an N-terminal subdomain binding site for sugar-UDP could be an acceptor site. Alternatively, if this was conserved Acknowledgments as a donor site, then it would accommodate an HA-UDP substrate. Te ultimate answers to many of the molecular and Research from the author’s laboratory was supported by mechanistic questions posed here will likely only be revealed the National Institutes of Health Grant GM35978 from the when the structure of HAS is ultimately determined at Institute of General Medical Sciences. Te author also thanks sufcient atomic resolution. Dr. Christopher West for very helpful discussions and input.

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