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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.

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Essentials of Glycobiology. 2nd edition.

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Chapter 15Hyaluronan

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This chapter describes the structure and metabolism of the nonsulfated glycosamino-glycan hyaluronan and how its chemical attributes contribute to its highly diverse and versatile biological functions. Chapter 16 describes sulfated glycosaminoglycans.

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 hyaluronic acid (from hyaloid [meaning vitreous] and uronic acid) and they showed that it contained “uronic acid (and) an amino sugar.” It took almost 20 years to determine the actual structure of the disaccharide that forms the repeating disaccharide motif of hyaluronan (Figure 15.1). 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 15.1 is faithfully reproduced by any cell that synthesizes hyaluronan, including animal cells and bacteria.

FIGURE 15.1. Hyaluronan consists of repeating disaccharides composed of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA).

FIGURE 15.1

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 (more...)

At first glance, the simplicity of hyaluronan might suggest that it arose early in evolution relative to other glycosaminoglycans (see Chapter 16). 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. The simplicity of hyaluronan’s structure may be the key to its success, because numerous hyaluronan-binding proteins have evolved (often referred to as hyaladherins) and several enzymes responsible for its synthesis and degradation are present. 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 organization, cell proliferation, signaling reactions across the plasma membrane, and microbial virulence.

STRUCTURE AND BIOPHYSICAL PROPERTIES

As described in the next section, hyaluronan is the only glycosaminoglycan synthesized in the cytoplasm at the plasma membrane, with the growing polymer being extruded into the extracellular environment. This allows hyaluronan to have an indefinite and very large degree of polymerization, typically in the range of 104 disaccharides (~3.7 × 106 D as the sodium salt) and 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 predominantly 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 individual molecules of high molecular weight in a 3–5 mg/ml physiological solution 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. Such a solution would have a swelling pressure and exhibit high viscosity with viscoelastic properties, conditions found in the vitreous humor of the human eye and in 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 synthesis is catalyzed by hyaluronan synthases (HAS), each of which contains dual catalytic activities required for the transfer of N-acetylglucosamine and glucuronic acid units from the corresponding nucleotide sugars (Figure 15.2). 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; see Chapter 25). 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.

FIGURE 15.2. Hyaluronan biosynthesis by hyaluronan synthase (Has) occurs by addition of sugars (N-acetyl-glucosamine and glucuronic acid) to the reducing end of the polymer.

FIGURE 15.2

Hyaluronan biosynthesis by hyaluronan synthase (Has) occurs by addition of sugars (N-acetyl-glucosamine and glucuronic acid) to the reducing end of the polymer. M++ refers to a metal ion cofactor. Symbol Key: Image symbol_key_small.jpg

As described in Chapter 16, 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 substrates UDP-GlcA and UDP-GlcNAc and extrude the growing polymer through the membrane (Figure 15.2). According to the model, the reducing end of the growing chain would have a UDP moiety that is displaced when the next sugar is added.

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 and uses a nucleotide sugar as the acceptor. 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 from the nonreducing end 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.

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 molecule1, 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 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 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- and Has3-null mice as well as Has1/3 compound mutant mice show no obvious phenotype. Interestingly, explanted cells from the Has2-null embryonic heart do not synthesize hyaluronan or undergo endothelial-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 15.3). 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, was shown to be necessary for stabilizing the proteoglycan aggregates, and subsequently, the structure of the aggregate was defined (see Chapter 16, Figure 16.1). The link protein contains two repeats of a motif, the link module, that interact specifically with hyaluronan and form the backbone on which the proteoglycan aggregate assembles. The 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 cooperatively interacts with a homologous domain in G1, which locks the proteoglycan on the hyaluronan chain. Absence of the link molecule fails to anchor the proteoglycan, and null mice deficient in link protein 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.

FIGURE 15.3. Modular organization of the link module superfamily of hyaluronan-binding proteins.

FIGURE 15.3

Modular organization of the link module superfamily of hyaluronan-binding proteins. These proteins contain one or more link modules that bind to hyaluronan. Like many extracellular matrix proteins, the link module superfamily members contain various subdomains, (more...)

Interestingly, there are four proteoglycan genes with homologous G1 domains interspersed in the genome (versican, neurocan, brevican, and aggrecan), and each contains a contiguous homologous link molecule (Figure 15.3). 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 mechanisms, and it is likely that neurocan and brevican are organized similarly. Thus, hyaluronan acts as a scaffold on which to build proteoglycan 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 (see Chapter 12). In a complex process, TSG-6 catalyzes the transfer of heavy chains from chondroitin sulfate onto the newly synthesized hyaluronan. 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: THE LINK PROTEIN MOTIF

Many of the hyaluronan-binding proteins described above have in common a protein motif called the link module, first described in cartilage link protein (Figure 15.4). The link proteins belong to a subfamily called the hyaluronan and proteoglycan link proteins (HAPLN) and are expressed in many tissues. Four cell-surface receptors have extracellular domains with link module motifs: CD44, LYVE-1 (lymphatic vessel endothelial hyaluronan receptor), HARE/STABILIN-2 (hepatic hyaluronan clearance receptor), and STA-BILIN-1, which are expressed on discontinuous endothelial cells and some activated macrophages. The other members of the superfamily are secreted and include the chondroitin-sulfate-containing proteoglycans that comprise the aggrecan superfamily (aggrecan, versican, brevican, and neurocan) and TSG-6.

FIGURE 15.4. Structure of the link module.

FIGURE 15.4

Structure of the link module. (a) TSG-6 contains a prototypical link fold defined by two α helices (α1 and α2) and two triple-stranded antiparallel β sheets (β1,2,6 and β3–5). (Redrawn, with permission, (more...)

The three-dimensional structure of the module 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. The link module consists of about 100 amino acids and contains four cysteines disulfide-bonded in the pattern Cys1-Cys4 and Cys2-Cys3. This fold has so far 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 (see Chapter 31). 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 15.4). Many of these features are conserved in other members of the superfamily. Different subgroups of the link module superfamily differ in size and length of hyaluronan recognized (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 docking site of the chain with this motif has not been established. Thus, the presence of this 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 are associated with a wide variety of tumors and the metastatic spread of cancer, although as with other tumor-associated factors, a strict correlation does not exist. 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 interacts 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) (see Chapter 37).

Interaction of hyaluronan with CD44 can also regulate ERBB-family (epithelial growth factor receptor) signaling, 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 (see Chapter 44).

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 15.5; also see Chapter 20). 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. 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 (see Chapter 34). 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, for example, cytokine production (see Chapter 39).

FIGURE 15.5. Hyaluronan capsule.

FIGURE 15.5

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. (more...)

In addition to bacteria, an algal virus (Chlorella) also 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 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. It can also suppress prostaglandin E2 and IL-1 production, which in turn can affect proliferation of synovial cells.

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 has also 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. 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.

FURTHER READING

  1. Day AJ, Prestwich GD. Hyaluronan-binding proteins: Tying up the giant. J Biol Chem. 2002;277:4585–4588. [PubMed: 11717315]
  2. DeAngelis PL. Microbial glycosaminoglycan glycosyltransferases. Glycobiology. 2002;12:9R–16R. [PubMed: 11825882]
  3. Itano N, Kimata K. Mammalian hyaluronan synthases. IUBMB Life. 2002;54:195–199. [PubMed: 12512858]
  4. Tammi MI, Day AJ, Turley EA. Hyaluronan and homeostasis: A balancing act. J Biol Chem. 2002;277:4581–4584. [PubMed: 11717316]
  5. Turley EA, Noble PW, Bourguignon LYW. Signaling properties of hyaluronan receptors. J Biol Chem. 2002;277:4589–4592. [PubMed: 11717317]
  6. Weigel PH. Functional characteristics and catalytic mechanisms of the bacterial hyaluronan synthases. IUBMB Life. 2002;54:201–211. [PubMed: 12512859]
  7. Hascall VC, Majors AK, De La Motte CA, Evanko SP, Wang A, Drazba JA, Strong SA, Wight TN. Intracellular hyaluronan: A new frontier for inflammation? Biochim. Biophys. Acta. 2004;1673:3–12. [PubMed: 15238245]
  8. Toole BP. Hyaluronan: From extracellular glue to pericellular cue. Nat. Rev. Cancer. 2004;4:528–539. [PubMed: 15229478]
  9. Zhuo L, Hascall VC, Kimata K. Inter-α-trypsin inhibitor, a covalent protein-glycosaminoglycan-protein complex. J Biol Chem. 2004;279:38079–38082. [PubMed: 15151994]
  10. Day AJ, de la Motte CA. Hyaluronan cross-linking: A protective mechanism in inflammation. Trends Immunol. 2005;26:637–643. [PubMed: 16214414]
  11. Taylor KR, Gallo RL. Glycosaminoglycans and their proteoglycans: Host-associated molecular patterns for initiation and modulation of inflammation. FASEB J. 2006;20:9–22. [PubMed: 16394262]
  12. Volpi N. Therapeutic applications of glycosaminoglycans. Curr Med Chem. 2006;13:1799–1810. [PubMed: 16787222]
  13. Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol. 2007;23:435–461. [PubMed: 17506690]
  14. Weigel PH, DeAngelis PL. Hyaluronan synthases: A decade-plus of novel glycosyltransferases. J Biol Chem. 2007;282:36777–36781. [PubMed: 17981795]
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Copyright © 2009, The Consortium of Glycobiology Editors, La Jolla, California.
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