Yutaka Fujii

Membrane Type 1 Matrix Metalloproteinase Digests Interstitial Collagens and Other Extracellular Matrix Macromolecules

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (ΔMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for α1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for α2(I) chains. ΔMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of ΔMT1 and MMP-1 indicate that ΔMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of ΔMT1 is 8-fold higher than that of MMP-1. ΔMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as α1-proteinase inhibitor and α2-macroglobulin. The activity of ΔMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.

Cloning and Crystal Structure of Hematopoietic Prostaglandin D Synthase

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

CDNA CLONING, EXPRESSION, AND MUTAGENESIS STUDY OF LIVER-TYPE PROSTAGLANDIN F SYNTHASE

Prostaglandin (PG) F synthase catalyzes the reduction of PGD2 to 9α,11β-PGF2 and that of PGH2 to PGF2α on the same molecule. PGF synthase has at least two isoforms, the lung-type enzyme (K m value of 120 μm for PGD2 (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O. (1985) J. Biol. Chem. 260, 7035–7041) and the liver-type one (K m value of 10 μm for PGD2 (Chen, L. -Y., Watanabe, K., and Hayaishi, O. (1992)Arch. Biochem. Biophys. 296, 17–26)). The liver-type enzyme was presently found to consist of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with aM r of 36,742. Sequence analysis indicated that the bovine liver PGF synthase had 87, 79, 77, and 76% identity with the bovine lung PGF synthase and human liver dihydrodiol dehydrogenase (DD) isozymes DD1, DD2, and DD4, respectively. Moreover, the amino acid sequence of the liver-type PGF synthase was identical with that of bovine liver DD3. The liver-type PGF synthase was expressed in COS-7 cells, and its recombinant enzyme had almost the same properties as the native enzyme. Furthermore, to investigate the nature of catalysis and/or substrate binding of PGF synthase, we constructed and characterized various mutant enzymes as follows: R27E, R91Q, H170C, R223L, K225S, S301R, and N306Y. Although the reductase activities toward PGH2 and phenanthrenequinone (PQ) of almost all mutants were not inactivated, the K m values of R27E, R91Q, H170C, R223L, and N306Y for PGD2 were increased from 15 to 110, 145, 75, 180, and 100 μm, respectively, indicating that Arg27, Arg91, His170, Arg223, and Asn306 are essential to give a low K m value for PGD2 of the liver-type PGF synthase and that these amino acid residues serve in the binding of PGD2. Moreover, the R223L mutant among these seven mutants especially has a profound effect on k cat for PGD2 reduction. TheK m values of R223L, K225S, and S301R for PQ were about 2–10-fold lower than the wild-type value, indicating that the amino acid residues at 223, 225 and 301 serve in the binding of PQ to the enzyme. On the other hand, the K m value of H170C for PGH2 was 8-fold lower than that of the wild type, indicating that the amino acid residue at 170 is related to the binding of PGH2 to the enzyme and that Cys170 confer high affinity for PGH2. Additionally, the 5-fold increase in k cat/K m value of the N306Y mutant for PGH2 compared with the wild-type value suggests that the amino acid at 306 plays an important role in catalytic efficiency for PGH2.

Membrane-type 1 MMP (MMP-14) cleaves at three sites in the aggrecan interglobular domain

An aggrecan G1‐G2 substrate was used to determine sites within the interglobular domain that were susceptible to cleavage by MT1‐MMP. Degradation products were identified by Western blotting with neo‐epitope antibodies specific for MMP‐derived N‐ and C‐terminal sequences. The results showed that MT1‐MMP cleaved at the N341‐F342 and D441‐L442 bonds, as shown for other MMPs, and also at a site 13 amino acids C‐terminal to the N341‐F342 site. The G2 product of this additional cleavage was identified by sequence analysis and revealed an N‐terminus commencing T355VxxPDVELPLP. The data are consistent with MT1‐MMP cleavage at three sites in the aggrecan interglobular domain; one at N342‐F342, a second at D441‐L442 and a third at Q354‐T355.

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4-imidazo-lidine+ ++)-2,5-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.

Expression of bovine lung prostaglandin F synthase in Escherichia coli

The full-length bovine lung prostaglandin(PG) F synthase cDNA was constructed from partial cDNA clones and ligated into bacterial expression vector pUC8 to develop expression plasmid pUCPF1. This plasmid permitted the synthesis of bovine lung PGF synthase in Escherichia coli. The recombinant bacteria overproduced a 36-KDa protein that was recognized by anti-PGF synthase antibody, and the expressed protein was purified to apparent homogeneity. The expressed protein reduced not only carbonyl compounds including PGD2 and phenanthrenequinone but also PGH2; and the Km values for phenanthrenequinone, PGD2, and PGH2 of the expressed protein were 0.1, 100, and 8 microM, respectively, which are the same as those of the bovine lung PGF synthase. The protein produced PGF2 alpha from PGH2, and 9 alpha, 11 beta-PGF2 from PGD2 at different active sites. Moreover, the structure of the purified protein from Escherichia coli was essentially identical to that of the native enzyme in terms of C-terminal sequence, sulfhydryl groups, and CD spectra except that the nine amino acids provided by the lac Z gene of the vector were fused to the N-terminus. These results indicate that the expressed protein is essentially identical to bovine lung PGF synthase. We confirmed that PGF synthase is a dual function enzyme catalyzing the reduction of PGH2 and PGD2 on a single enzyme and that it has one binding site for NADPH.

Enzymatic formation of prostaglandin F2α in human brain

Prostaglandin (PG)E2 9-ketoreductase, which catalyzes the conversion of PGE2 to PGF2α, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE2, and turnover number were 34,000, 8.2, 6.5–7.5, 1.0 mM, and 7.6 min−1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA2, PGE1, and 13,14-dihydro-15-keto PGF2α, but not that of PGD2, 11β-PGE2, PGH2, PGJ2, or Δ12-PGJ2. The reaction product formed from PGE2 was identified as PGF2α, by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE2 was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE2 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF2α in human brain.

CCN1 (Cyr61) Is Overexpressed in Human Osteoarthritic Cartilage and Inhibits ADAMTS-4 (Aggrecanase 1) Activity

ADAMTS‐4, also called aggrecanase 1, is considered to play a key role in aggrecan degradation in human osteoarthritic (OA) cartilage, but information about regulators of ADAMTS‐4 aggrecanase activity remains limited. We undertook this study to search for molecules that modulate ADAMTS‐4 activity.

Effect of fructose 1,6-bisphosphate on the activity of liver pyruvate kinase aftter limited proteolysis with cathepsin B

Treatment of rat liver-type pyruvate kinase with rabbit liver cathepsin B at pH 7.0 caused loss of activity in the standard assay with 0.6 mM of phosphoenolpyruvate. The modified enzyme exhibited about 10% of the original activity when assayed with 2.0 mM of the substrate. No detectable change in the subunit molecular weight of the enzyme occurred during inactivation. On addition of 4 microM fructose 1,6-bisphosphate the activity of the treated enzyme was restored to that of the original enzyme. Limited proteolysis of the enzyme by cathepsin B appears to enhance the requirement for the positive effector, fructose 1,6-bisphosphate.

Stable preparation of aldose reductase isoenzymes from human placenta.

An efficient, large-scale purification has been achieved for two aldose reductase isoenzymes from human placenta in stable form. The procedure included ammonium sulfate fractionation (45-75%), followed by chromatographies on Matrex Red A, DE-52 cellulose, and Matrex Orange A. The preparations were stable for at least 3 months at 3 degrees C. IC50 values toward sorbinil were similar to those reported for crude or partially purified enzymes, indicating that they retained native structures during the purification steps. The molecular weights of purified GAR1 and GAR2, named according to their order of elution with a salt gradient from a Matrex Red A column, were 36,600 and 40,300, respectively. Kinetic studies indicate that GAR1 belongs to an aldose reductase (a low-Km form) and GAR2 to an aldehyde reductase (a high-Km form). GAR2, an aldehyde reductase, was also active in the reduction of D-glucose, with an apparent Km comparable to that of GAR1 but with a Vmax only 14% that of GAR1.