Deep,Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.Lysine acetylation (Kac)¹ is a well conserved, reversible post-translational modification (PTM) involved in multiple cellular processes (1). Acetylation is regulated by two classes of enzymes: lysine acetyltransferases (KATs) and histone deacetylases (HDACs) (2–4). This modification was originally identified as a nuclear event on histone proteins and has been long appreciated for its role in epigenetic and DNA-dependent processes. With the help of a growing number of large-scale acetylation studies, it has become evident that lysine acetylation is ubiquitous, also occurring on cytoplasmic and mitochondrial proteins and has a role in signaling, metabolism, and immunity (1, 4–6). Therefore, the examination of lysine acetylation on nonhistone proteins has gained a prominent role in PTM analysis.To date, the identification of large numbers of acetylation sites has been challenging because of the substoichiometric nature of this modification (7, 8). Additionally, global acetylation is generally less abundant than phosphorylation and ubiquitylation (1). The introduction of antibodies specific for lysine acetylation has significantly improved the ability to enrich and identify thousands of sites (9–14). A landmark study by Choudhary et al. used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome (10). A more recent study by Lundby et al. investigated the function and distribution of acetylation sites in 16 different rat tissues, and identified, in aggregate, 15,474 acetylation sites from 4541 proteins (12).Although anti-acetyl lysine antibodies have been a breakthrough for globally mapping acetylation sites (9–12), it remains a challenge to identify large numbers of lysine acetylation sites from a single sample, as is now routinely possible for phosphorylation and ubiquitylation (13, 15–18). To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for (1) alternative anti-acetyl lysine antibodies with higher specificity, (2) optimized antibody usage parameters, and (3) robust proteomic workflows that permit low to moderate protein input. In this study, we describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples.     

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter: ROLE OF LYSINE 63-LINKED UBIQUITIN CHAINS*

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys⁶³ linked chain(s), and Jen1-Lys³³⁸ is one of the target residues. Ubiquitin-Lys⁶³-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys⁶³-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.Ubiquitylation is one of the most prevalent protein post-translational modifications in eukaryotes. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes, including DNA repair, signaling, and intracellular trafficking. Ubiquitylation serves as a key signal mediating the internalization of plasma membrane receptors and transporters, followed by their intracellular transport and subsequent recycling or lysosomal/vacuolar degradation (1, 2). In Saccharomyces cerevisiae, transporters usually display both constitutive and accelerated endocytosis regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions. Ubiquitylation of these cell surface proteins acts as a signal triggering their internalization (1). A single essential E3⁴ ubiquitin ligase, Rsp5, has been implicated in the internalization of most, if not all, endocytosed proteins (3). Rsp5 is the unique member in S. cerevisiae of the HECT (homologous to E6AP COOH terminus)-ubiquitin ligases of the Nedd4/Rsp5 family (4). In a few cases, Rsp5-dependent cell surface ubiquitylation was shown to involve PY-containing adapters that bind to Rsp5 (5–7). Rsp5-mediated ubiquitylation is also required for sorting into multivesicular bodies (MVBs) of endosomal membrane proteins that come from either the plasma membrane (through endocytosis) or the Golgi (through vacuolar protein sorting (VPS) pathway) (8). Although much progress has been made in elucidating the mechanistic basis of various steps in protein trafficking, the precise requirement for a specific type and length of Ub chains at various stages of the endocytic pathway remains to be addressed.The ubiquitin profile needed for proper internalization has been established for some yeast membrane proteins (1). The α-factor receptor Ste2 was described as undergoing monoubiquitylation on several lysines (multimonoubiquitylation). The a-factor receptor, Ste3p; the general transporter of amino acids, Gap1; the zinc transporter, Ztr1; and the uracil transporter, Fur4, have been shown to be modified by short chains of two to three ubiquitins, each attached to one, two, or more target lysine residues (oligo-ubiquitylation). Among them, Fur4 and Gap1 were the only transporters demonstrated to undergo plasma membrane oligo-ubiquitylation with ubiquitin residues linked via ubiquitin-Lys⁶³ (9, 10). In addition, the two siderophore transporters Arn1 and Sit1 were also shown to undergo Lys⁶³-linked cell surface ubiquitylation (11, 12). Whether these four transporters are representative of a larger class of plasma membrane substrates remains to be determined. Little is known about the type of ubiquitylation involved and/or required for sorting to MVBs. Some MVB cargoes appear to undergo monoubiquitylation (8), whereas Sna3, an MVB cargo of unknown function, undergoes Lys⁶³-linked ubiquitylation (13). Lys⁶³-linked ubiquitin chains were also recently reported to be required, directly or indirectly, for MVB sorting of the siderophore transporter, Sit1, when trafficking through the VPS pathway in the absence of its external substrate (11). In agreement with the possibility that additional membrane-bound proteins might undergo Lys⁶³-linked ubiquitylation, a proteomic study aiming to uncover ubiquitylated yeast proteins showed that Lys⁶³-ubiquitin chains are far more abundant than previously thought (14).The transport of monocarboxylates, such as lactate and pyruvate, as well as ketone bodies across the plasma membrane is essential for the metabolism of cells of various organisms. A family of monocarboxylate transporters has been reported that includes mainly mammalian members (15). In S. cerevisiae, two monocarboxylate-proton symporters have been described, Jen1 and Ady2 (16, 17). These transporters exhibit differences in their mechanisms of regulation and specificity. Jen1 is a lactate-pyruvate-acetate-propionate transporter induced in lactic or pyruvic acid-grown cells (18). Ady2, which accepts acetate, propionate, or formate, is present in cells grown in non-fermentable carbon sources (19). Jen1 has unique regulatory characteristics and has been extensively studied. It was the first secondary porter of S. cerevisiae characterized by heterologous expression in Pichia pastoris at both the cell and the membrane vesicle levels (20). The addition of glucose to lactic acid-grown cells very rapidly triggers loss of Jen1 activity and repression of JEN1 gene expression (21, 22). Newly synthesized Jen1-GFP fusion protein is sorted to the plasma membrane in an active and stable form, and loss of Jen1-GFP activity upon glucose addition is the result of its endocytosis followed by vacuolar degradation (23). Data from large scale analyses based on mass spectrometry approaches led to the detection of two sites of ubiquitylation for Jen1, one located in the N terminus of the protein and the second in the central loop (14), and several sites of phosphorylation in the N terminus, central loop, and C terminus of the protein (14, 24). In the present study, we aimed at further characterizing the internalization step of endocytosis of the transporter Jen1 and the potential role of the phosphorylation and ubiquitylation events required for its correct endocytic trafficking.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.The commercialization of antibodies that recognize lysine residues modified with a di-glycine remnant (K-ε-GG)¹ has significantly transformed the detection of endogenous protein ubiquitination sites by mass spectrometry (1–5). Prior to the development of these highly specific reagents, proteomics experiments were limited to identification of up to only several hundred ubiquitination sites, which severely limited the scope of global ubiquitination studies (6). Recent proteomic studies employing anti-K-ε-GG antibodies have enhanced our understanding of ubiquitin biology through the identification of thousands of ubiquitination sites and the analysis of the change in relative abundance of these sites after chemical or biological perturbation (1–3, 5, 7). Use of stable isotope labeling by amino acids in cell culture (SILAC) for quantification has enabled researchers to better understand the extent of ubiquitin regulation upon proteasome inhibition and precisely identify those protein classes, such as newly synthesized proteins or chromatin-related proteins, that see overt changes in their ubiquitination levels upon drug treatment (2, 3, 5). Emanuel et al. (1) have combined genetic and proteomics assays implementing the anti-K-ε-GG antibody to identify hundreds of known and putative Cullin-RING ligase substrates, which has clearly demonstrated the extensive role of Cullin-RING ligase ubiquitination on cellular protein regulation.Despite the successes recently achieved with the use of the anti-K-ε-GG antibody, increased sample input (up to ∼35 mg) and/or the completion of numerous experimental replicates have been necessary to achieve large numbers of K-ε-GG sites (>5,000) in a single SILAC-based experiment (1–3, 5). For example, it has been recently shown that detection of more than 20,000 unique ubiquitination sites is possible from the analysis of five different murine tissues (8). However, as the authors indicate, only a few thousands sites are detected in any single analysis of an individual tissue sample (8). It is recognized that there is need for further improvements in global ubiquitin technology to increase the depth-of-coverage attainable in quantitative proteomic experiments using moderate amounts of protein input (9). Through systematic study and optimization of key pre-analytical variables in the preparation and use of the anti-K-ε-GG antibody as well as the proteomic workflow, we have now achieved, for the first time, routine quantification of ∼20,000 nonredundant K-ε-GG sites in a single SILAC triple encoded experiment starting with 5 mg of protein per SILAC channel. This represents a 10-fold improvement over our previously published method (3).     

Biochemical Characterization and Function of Complexes Formed by Hyaluronan and the Heavy Chains of Inter-��-inhibitor (HC��HA) Purified from Extracts of Human Amniotic Membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic Extract A (70.1 ± 6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (IαI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein (TSG-6). This HC·HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced mac ro phage apo pto sis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.Hyaluronan (HA)⁴ is widely distributed in extracellular matrices, tissues, body fluids, and even in intracellular compartments (reviewed in Refs. 1 and 2). The molecular weight of HA ranges from 200 to 10,000 kDa depending on the source (3), but can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions (1). Investigations over the last 15 years have suggested that low M_r HA can induce the gene expression of proinflammatory mediators and proangiogenesis, whereas high molecular weight (HMW) HA inhibits these processes (4–7).Several proteins have been shown to bind to HA (8) such as aggrecan (9), cartilage link protein (10), versican (11), CD44 (12, 13), inter-α-inhibitor (IαI) (14, 15), and tumor necrosis factor-α stimulated gene-6 protein (TSG-6) (16, 17). IαI consists of two heavy chains (HCs) (HC1 and HC2), both of which are linked through ester bonds to a chondroitin sulfate chain that is attached to the light chain, i.e. bikunin. Among all HA-binding proteins, only the HCs of IαI have been clearly demonstrated to be covalently coupled to HA (14, 18). However, TSG-6 has also been reported to form stable, possibly covalent, complexes with HA, either alone (19, 20) or when associated with HC (21).The formation of covalent bonds between HCs and HA is mediated by TSG-6 (22–24) where its expression is often induced by inflammatory mediators such as tumor necrosis factor-α and interleukin-1 (25, 26). TSG-6 is also expressed in inflammatory-like processes, such as ovulation (21, 27, 28) and cervical ripening (29). TSG-6 interacts with both HA (17) and IαI (21, 24, 30–33), and is essential for covalently transferring HCs on to HA (22–24). The TSG-6-mediated formation of the HC·HA complex has been demonstrated to play a crucial role in female fertility in mice. The HC·HA complex is an integral part of an expanded extracellular “cumulus” matrix around the oocyte, which plays a critical role in successful ovulation and fertilization in vivo (22, 34). HC·HA complexes have also been found at sites of inflammation (35–38) where its pro- or anti-inflammatory role remain arguable (39, 40).Immunostaining reveals abundant HA in the avascular stromal matrix of the AM (41, 42).⁵ In ophthalmology, cryopreserved AM has been widely used as a surgical graft for ocular surface reconstruction and exerts clinically observable actions to promote epithelial wound healing and to suppress inflammation, scarring, and angiogenesis (for reviews see Refs. 43–45). However, it is not clear whether HA in AM forms HC·HA complex, and if so whether such an HC·HA complex exerts any of the above therapeutic actions. To address these questions, we extracted AM with buffers of increasing salt concentration. Because HMW HA was found to form the HC·HA complex and was mainly extractable by isotonic solutions, we further purified it from the isotonic AM extract and reconstituted it in vitro from three defined components, i.e. HMW HA, serum IαI, and recombinant TSG-6. Our results showed that the HC·HA complex is an active component in AM responsible for the suppression of TGF-β1 promoter activity, linkable to the scarring process noted before by AM (46–48) and by the AM soluble extract (49), as well as for the promotion of macrophage death, linkable to the inflammatory process noted by AM (50) and the AM soluble extract (51).