We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn 341 -Phe 342 and Glu 373 -Ala 374 . While several matrix metalloproteinases have been shown to cleave at Asn 341 -Phe 342 , an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu 373 -Ala 374 and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammationassociated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu 373 -Ala 374 site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.Aggrecan is the major proteoglycan of cartilage and is responsible for its compressibility and stiffness. Aggrecan contains two N-terminal globular domains, G 1 and G 2 , separated by a proteolyticaly sensitive interglobular domain, followed by a glycosaminoglycan attachment region and a C-terminal globular domain (G 3 ). The G 1 domain of aggrecan interacts with hyaluronic acid and link protein to form large aggregates containing multiple aggrecan monomers that are trapped within the cartilage matrix. Cleavage of aggrecan has been shown to occur at Asn 341 -Phe 342 and Glu 373 -Ala 374 within the interglobular domain, with the cleaved aggrecan being free to exit the matrix since it lacks the G 1 domain, which is responsible for formation of the high molecular weight complexes. Results from several studies suggest that cleavage at the Glu 373 -Ala 374 site is responsible for the increased aggrecan degradation observed in inflammatory joint disease. Products resulting from cleavage at the Glu 373 -Ala 374 site have been shown to accumulate in cartilage explants and chondrocyte cultures treated with interleukin-1 and retinoic acid (1-5) and in the synovial fluid of patients with osteoarthritis and inflammatory joint disease (6, 7). While several characterized matrix metalloproteases 1 have been shown to cleave at the Asn 341 -Phe 342 site (8 -14), they are not responsible for the observed cleavage at Glu 373 -Ala 374 . A novel proteolytic activity, termed "aggrecanase," has been hypothesized to be respo...

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000-to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0-or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme•sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 × 10 5 M −1 s −1 ) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.The reduction of oxygen to hydrogen peroxide by free reduced flavin is thermodynamically favorable but kinetically slow because the 2-electron reduction of triplet oxygen by a diamagnetic organic molecule is spin-forbidden. In fact, the 2-electron reduction of oxygen by reduced flavin proceeds via an initial 1-electron transfer step that generates a flavin radical/ superoxide anion radical pair in a spin-allowed but energetically unfavorable, rate-determining reaction. The term oxygen activation is used in reference to the accelerated rates of oxygen reduction observed with flavoprotein oxidases and other enzymes that reduce molecular oxygen (1,2). A series of elegant studies by Klinman and Roth identified His516 as the site of oxygen activation in the flavoenzyme glucose oxidase and showed that the reaction required the protonated form of this residue (2-4). Surprisingly little is, however, currently known about the detailed mechanism of oxygen activation by other flavoprotein oxidases, especially regarding the specific role of active site residues in these reactions.

The minimal size of the recombination site required for efficient FLP recombinase-catalyzed recombination in vitro is no more than 28 base pairs, which includes parts of two 13-base-pair inverted repeats and all of an 8-base-pair spacer. The FLP recombinase cleaves the DNA at the boundaries of the spacer, becomes covalently linked to the spacer DNA via a 3' phosphate, and leaves a free 5' hydroxyl at the other end of the 8-base-pair spacer. The efficiency of recombination is reduced if the size of the spacer in a recombinant site is increased or decreased by 1 base pair, while the spacer in the second site is unaltered. Recombination between two sites with identical 1-base-pair additions or deletions in the spacer, however, is relatively unaffected. This result suggests that pairing of sequences in the spacer region is important in FLP-promoted recombination events. The sequence asymmetry utilized by the recombinase to determine the orientation of the site is located uniquely within the spacer region. spacer (9). The third repeat was shown to be unnecessary for cleavage (9). This work extends these results. The boundaries of the minimal site required for recombination in vitro are defined, and sequences that determine functional site asymmetry are localized to the spacer region. Modest flexibility in the size of the spacer region is demonstrated. In addition, the location of the cleavage site is confirmed. Some of these results have been described in a preliminary report (10).The 2-,gm plasmid is a 6318-base-pair (bp) circular DNA molecule common to many yeast strains (1). The plasmid encodes a site-specific recombination system that inverts unique sequences separating a pair of 599-bp repeats. The availability of the plasmid sequence (2) (6)(7)(8).Much of our recent effort has been directed at characterizing the recombination site for this reaction in vitro. The most prominent structural features of this site are shown in Fig. 1. A series of three 13-bp repeats is evident. The second and third are inverted relative to one another and separated by an 8-bp spacer. The first copy of this repeat is immediately adjacent to and in the same orientation as the second, as shown in Fig. 1. The third repeat contains a 1-bp mismatch relative to repeats 1 and 2 (t in sequence, Fig. 1). An Xba I restriction site accounts for 6 of the 8 bp in the spacer. Destruction of this restriction site by using methods that add or delete 4 bp has been shown to abolish this recombination event in vivo in yeast (3) and E. coli (5) and in vitro (7,8).It has been demonstrated that FLP protein binds to all 3 of the 13-bp repeats and cleaves the site at the boundaries of the MATERIALS AND METHODSBacterial Strains, Enzymes, and Reagents. Plasmids were maintained in the E. coli strain HB101 (ref. 11, p. 504). Bacteriophage M13 mp8 and its derivatives were maintained in the strain JM101 (12). FLP protein was partially purified as described (8). FLP protein fractions used were free of detectable nuclease. The large fragment of T4 DNA ligas...

A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a pre-organized binding site for superoxide anion, an obligatory intermediate in the 2-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. Firstly, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX•chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analog (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX•chloride complex and a ternary MSOX•chloride•MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule, and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1 and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride: (i) acts as an oxygen surrogate; (ii) is an effective probe of polar oxygen activation sites; (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.