Exploiting O-GlcNAc transferase promiscuity to dissect site-specific O-GlcNAcylation.

Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.

The Bacteroidetes Q-rule and glutaminyl cyclase activity increase the stability of extracytoplasmic proteins.

IMPORTANCE: Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase have N-terminal glutamine converted to pyroglutamate. The reaction is catalyzed by the periplasmic enzyme glutaminyl cyclase (QC), which is essential for the growth of Porphyromonas gingivalis and other periodontopathogens. Apparently, pyroglutamyl formation stabilizes extracytoplasmic proteins and/or protects them from proteolytic degradation in the periplasm. Given the role of P. gingivalis as the keystone pathogen in periodontitis, P. gingivalis QC is a promising target for the development of drugs to treat and/or prevent this highly prevalent chronic inflammatory disease leading to tooth loss and associated with severe systemic diseases.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to -25°C. P355's stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (k cat ) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

A top-down approach to uncover the hidden ligandome of low-density lipoprotein receptor-related protein 1 in cartilage.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a cell-surface receptor ubiquitously expressed in various tissues. It plays tissue-specific roles by mediating endocytosis of a diverse range of extracellular molecules. Dysregulation of LRP1 is involved in multiple conditions including osteoarthritis (OA) but little information is available about the specific profile of direct binding partners of LRP1 (ligandome) for each tissue, which would lead to a better understanding of its role in disease states. Here, we investigated adult articular cartilage where impaired LRP1-mediated endocytosis leads to tissue destruction. We used a top-down approach involving proteomic analysis of the LRP1 interactome in human chondrocytes, direct binding assays using purified LRP1 and ligand candidates, and validation in LRP1-deficient fibroblasts and human chondrocytes, as well as a novel Lrp1 conditional knockout (KO) mouse model. We found that inhibition of LRP1 and ligand interaction results in cell death, alteration of the entire secretome and transcriptional modulations in human chondrocytes. We identified a chondrocyte-specific LRP1 ligandome consisting of more than 50 novel ligand candidates. Surprisingly, 23 previously reported LRP1 ligands were not regulated by LRP1-mediated endocytosis in human chondrocytes. We confirmed direct LRP1 binding of HGFAC, HMGB1, HMGB2, CEMIP, SLIT2, ADAMTS1, TSG6, IGFBP7, SPARC and LIF, correlation between their affinity for LRP1 and the rate of endocytosis, and some of their intracellular localization. Moreover, a conditional LRP1 KO mouse model demonstrated a critical role of LRP1 in regulating the high-affinity ligands in cartilage in vivo. This systematic approach revealed the specificity and the extent of the chondrocyte LRP1 ligandome and identified potential novel therapeutic targets for OA.

A Protein Corona Modulates Interactions of α-Synuclein with Nanoparticles and Alters the Rates of the Microscopic Steps of Amyloid Formation.

Nanoparticles (NPs) can modulate protein aggregation and fibril formation in the context of amyloid diseases. Understanding the mechanism of this action remains a critical next step in developing nanomedicines for the treatment or prevention of Parkinson's disease. α-Synuclein (α-Syn) can undergo interactions of different strength with nanoparticles, and these interactions can be prevented by the presence of a protein corona (PC) acquired during the exposure of NPs to serum proteins. Here, we develop a method to attach the PC irreversibly to the NPs, which enables us to study in detail the interaction of α-Syn and polyethylenimine-coated carboxyl-modified polystyrene NPs (PsNPs-PEI) and the role of the dynamics of the interactions. Analysis of the kinetics of fibril formation reveals that the NPs surface promotes the primary nucleation step of amyloid fibril formation without significantly affecting the elongation and fragmentation steps or the final equilibrium. Furthermore, the results show that even though α-Syn can access the surface of NPs that are precoated with a PC, due to the dynamic nature of the PC proteins, the PC nevertheless reduces the acceleratoring effect of the NPs. This effect is likely to be caused by reducing the overall amount of weakly interacting α-Syn molecules on the NP surface and the access of further α-Syn required for fibril elongation. Our experimental approach provides microscopic insight into how serum proteins can modulate the complex interplay between NPs and amyloid proteins.

Identification of polyphenol oxidases in potato tuber (Solanum tuberosum) and purification and characterization of the major polyphenol oxidases.

Potato is widely consumed across the globe. Understanding and inhibiting the oxidation caused by polyphenol oxidase (PPO) could improve shelf life and increase the nutritional and economic value of potato proteins. This study aimed to identify and quantify all expressed PPOs in potato tuber (Solanum tuberosum) and to purify and characterize the major PPOs responsible for oxidase activity. Four different PPOs were expressed in potato tuber, with 2 PPOs constituting the majority. These 2 PPOs were copurified and characterized. Both potato juice and the purified PPOs had an optimum pH of 5 and were stable over a broad pH range (6-11). The optimum temperature and stability varied, with potato juice having an optimum at 30 °C and showing a gradual decline in oxidase activity with increasing incubation temperature, while the purified PPOs had an optimum temperature and were stable up to 40 °C. Reducing agents effectively inhibited oxidase activity, while chelators did not.

An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution.

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a 'fuzzy complex' that follows a 'dynamic zipper' mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.

PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins.

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two ß-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the "attachment complex" composed of PorU, PorZ, and the integral OM ß-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface.IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

Transport of a Peptide from Bovine αs1-Casein across Models of the Intestinal and Blood-Brain Barriers.

The effect of food components on brain growth and development has attracted increasing attention. Milk has been shown to contain peptides that deliver important signals to the brains of neonates and infants. In order to reach the brain, milk peptides have to resist proteolytic degradation in the gastrointestinal tract, cross the gastrointestinal barrier and later cross the highly selective blood-brain barrier (BBB). To investigate this, we purified and characterized endogenous peptides from bovine milk and investigated their apical to basal transport by using human intestinal Caco-2 cells and primary porcine brain endothelial cell monolayer models. Among 192 characterized milk peptides, only the αS1-casein peptide 185PIGSENSEKTTMPLW199, and especially fragments of this peptide processed during the transport, could cross both the intestinal barrier and the BBB cell monolayer models. This peptide was also shown to resist simulated gastrointestinal digestion. This study demonstrates that a milk derived peptide can cross the major biological barriers in vitro and potentially reach the brain, where it may deliver physiological signals.

Mapping and identification of soft corona proteins at nanoparticles and their impact on cellular association.

The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identify a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins are revealed as modulators of nanoparticle-cell association mainly through their dynamic nature. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.

Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

FAM20C-Mediated Phosphorylation of MEPE and Its Acidic Serine- and Aspartate-Rich Motif.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed in bone and teeth where it has multiple functions. The C-terminus of MEPE contains a mineral-binding, acidic serine- and aspartate-rich motif (ASARM) that is also present in other noncollagenous proteins of mineralized tissues. MEPE-derived ASARM peptides function in phosphate homeostasis and direct inhibition of bone mineralization in a phosphorylation-dependent manner. MEPE is phosphorylated by family with sequence similarity 20, member C (FAM20C), which is the main kinase phosphorylating secreted phosphoprotein. Although the functional importance of protein phosphorylation status in mineralization processes has now been well-established for secreted bone and tooth proteins (particularly for osteopontin), the phosphorylation pattern of MEPE has not been previously determined. Here we provide evidence for a very high phosphorylation level of this protein, reporting on the localization of 31 phosphoresidues in human MEPE after coexpression with FAM20C in HEK293T cells. This includes the finding that all serine residues located in the canonical target sequence of FAM20C (Ser-x-Glu) were phosphorylated, thus establishing the major target sites for this kinase. We also show that MEPE has numerous other phosphorylation sites, these not being positioned in the canonical phosphorylation sequence. Of note, and underscoring a possible important function in mineralization biology, all nine serine residues in the ASARM were phosphorylated, even though only two of these were positioned in the Ser-x-Glu sequence. The presence of many phosphorylated amino acids in MEPE, and particularly their high density in the ASARM motif, provides an important basis for the understanding of structural and functional interdependencies in mineralization and phosphate homeostasis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Tracing the In Vivo Fate of Nanoparticles with a "Non-Self" Biological Identity.

Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, "non-self" incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis.

Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a 'pedal bin' mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.

Apolipoprotein E Triggers Complement Activation in Joint Synovial Fluid of Rheumatoid Arthritis Patients by Binding C1q.

We identified apolipoprotein E (ApoE) as one of the proteins that are found in complex with complement component C4d in pooled synovial fluid of rheumatoid arthritis (RA) patients. Immobilized human ApoE activated both the classical and the alternative complement pathways. In contrast, ApoE in solution demonstrated an isoform-dependent inhibition of hemolysis and complement deposition at the level of sC5b-9. Using electron microscopy imaging, we confirmed that ApoE interacts differently with C1q depending on its context; surface-bound ApoE predominantly bound C1q globular heads, whereas ApoE in a solution favored the hinge/stalk region of C1q. As a model for the lipidated state of ApoE in lipoprotein particles, we incorporated ApoE into phosphatidylcholine/phosphatidylethanolamine liposomes and found that the presence of ApoE on liposomes increased deposition of C1q and C4b from serum when analyzed using flow cytometry. In addition, posttranslational modifications associated with RA, such as citrullination and oxidation, reduced C4b deposition, whereas carbamylation enhanced C4b deposition on immobilized ApoE. Posttranslational modification of ApoE did not alter C1q interaction but affected binding of complement inhibitors factor H and C4b-binding protein. This suggests that changed ability of C4b to deposit on modified ApoE may play an important role. Our data show that posttranslational modifications of ApoE alter its interactions with complement. Moreover, ApoE may play different roles in the body depending on its solubility, and in diseased states such as RA, deposited ApoE may induce local complement activation rather than exert its typical role of inhibition.

FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvß3 integrin.

Osteopontin (OPN) is a ubiquitously expressed, multifunctional, and highly phosphorylated protein. OPN contains two neighboring integrin-binding motifs, RGD and SVVYGLR, which mediate interaction with cells. Phosphorylation and proteolytic processing affect the integrin-binding activities of OPN. Here we report that the kinase, FAM20C, phosphorylates Ser146 in the 143 RGDSVVYGLR152 motif of OPN and that Ser146 is phosphorylated in vivo in human and bovine milk. Ser146 is located right next to the RGD motif and close by the regulatory thrombin and plasmin cleavage sites in the OPN sequence. Phosphorylation of Ser146 could potentially affect the proteolytic processing and the integrin-binding activities of OPN. We show that phosphorylation of Ser146 does not affect the susceptibility of OPN for thrombin or plasmin cleavage. However, phosphorylation of Ser146 significantly reduces the RGD-mediated interaction with the αv ß3 integrin in MDA-MB-435 and Moαv cells. This suggests a new mechanism by which specific phosphorylation of OPN can regulate interaction with the αv ß3 integrin and thereby affect OPN-cell interaction.

The interactome of stabilized α-synuclein oligomers and neuronal proteins.

While it is generally accepted that α-synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA-stabilized human αSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs-binding partners. We identify in total 29 proteins associated with DHA-αSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium-potassium pump (Na+ /K+ ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic αSOs.

Matrix-degrading protease ADAMTS-5 cleaves inter-α-inhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients.

Destruction of the cartilage matrix in joints is an important feature of arthritis. Proteolytic degradation of cartilage glycoproteins can contribute to the loss of matrix integrity. Human inter-α-inhibitor (IαI), which stabilizes the extracellular matrix, is composed of the light-chain serine proteinase inhibitor bikunin and two homologous heavy chains (HC1 and HC2) covalently linked through chondroitin 4-sulfate. Inflammation promotes the transfer of HCs from chondroitin 4-sulfate to hyaluronan by tumor necrosis factor-stimulated gene-6 protein (TSG-6). This reaction generates a covalent complex between the heavy chains and hyaluronan that can promote leukocyte invasion. This study demonstrates that both IαI and the HC-hyaluronan complex are substrates for the extracellular matrix proteases ADAMTS-5 and matrix metalloprotease (MMP) -3, -7, and -13. The major cleavage sites for all four proteases are found in the C terminus of HC2. ADAMTS-5 and MMP-7 displayed the highest activity toward HC2. ADAMTS-5 degradation products were identified in mass spectrometric analysis of 29 of 33 arthropathic patients, indicating that ADAMTS-5 cleavage occurs in synovial fluid in arthritis. After cleavage, free HC2, together with TSG-6, is able to catalyze the transfer of heavy chains to hyaluronan. The release of extracellular matrix bound HC2 is likely to increase the mobility of the HC2/TSG-6 catalytic unit and consequently increase the rate of the HC transfer reaction. Ultimately, ADAMTS-5 cleavage of HC2 could alter the physiological and mechanical properties of the extracellular matrix and contribute to the progression of arthritis.