Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

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 inflammation-associated 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.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

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.

Crystallization and preliminary X-ray diffraction data of the complex of recombinant tick anticoagulant peptide (rTAP) and bovine factor Xa.

The complex of bovine factor Xa and recombinant tick anticoagulant peptide (rTAP) was crystallized in two different crystal forms using polyethylene glycol as a precipitant. Form I belongs to space group P42212 with unit-cell dimensions a = b = 133.1, c = 68.8 A. It contains one complex per asymmetric unit and diffracts to 3.0 A resolution. Form II belongs to P41212 (or P43212) with dimensions a = b = 126.5, c = 146.7 A; it contains two complexes per asymmetric unit and diffracts to 2.5 A. The crystals of both forms consist of factor Xa (MW = 45.3 kDa) and rTAP (MW = 6.7 kDa).

Unexpected binding mode of tick anticoagulant peptide complexed to bovine factor Xa.

The structure of recombinant tick anticoagulant peptide (rTAP) complexed to bovine factor Xa at 3.0 A resolution reveals the structural basis for the specificity and the high affinity of rTAP. Three N-terminal residues, Tyr501, Asn502 and Arg503, play a critical role in the complex formation as suggested by earlier mutagenic studies and the ornithodorin-thrombin complex. Unexpectedly, the side-chain of Tyr501 is located in the S1 pocket, although factor Xa favors arginine as a P1 residue. Arg503 is located at the aryl binding pocket and forms a salt-bridge with Glu97 of factor Xa. The autolysis loop, which is disordered in the uninhibited factor Xa structure, is involved in the formation of the complex as a part of the secondary binding site. The C-terminal helix of rTAP interacts with factor Xa as a secondary binding determinant. The N-terminal residues of rTAP reorganize during the formation of the factor Xa-rTAP complex from the conformation found in the solution into an extended conformation. The presence of the secondary binding site confirms the proposed two-step kinetic mechanism based on the results of a mutagenesis study.

Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris.

A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leech Hirudo medicinalis, was cloned and expressed in the methylotrophic yeast Pichia pastoris. The HIR gene was inserted into the P. pastoris pPic9K expression vector such that the gene's expression is under alcohol oxidase (AOX1) promoter control and the HIR coding sequence is fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor signal sequence. A Tn903Kan(r) determinant and His4+ gene are also present on pPic9K, affording a method for selecting chromosomal integrants of the HIR gene. Following electroporation of the DNA into the P. pastoris strain GS115 (his-4), His+ transformants were recovered and plated on medium containing increasing concentrations of the aminoglycoside antibiotic G418. The resulting His+ G418-resistant transformants were grown in shake flasks and screened for those that secreted recombinant hirudin (rHIR) to the growth medium. Clones exhibiting rHIR production and secretion were retained for fermentation studies where optimization of growth conditions was found to dramatically increase rHIR expression. One clone that was retained for further characterization secreted rHIR at a level of 1.5 g/liter. Using a straightforward two-step chromatography procedure, the rHIR was purified to > 97% with a recovery yield of 63%. The purified rHIR had the predicted N-terminal amino acid sequence and exhibited the same thrombin inhibition kinetics as a variety of HIR isoforms produced in other heterologous systems. Based on these data, P. pastoris offers an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.

Production and purification of human fibroblast collagenase (MMP-1) expressed in the methylotrophic yeast Pichia pastoris.

The cDNA that encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was expressed in the methylotrophic yeast Pichia pastoris. The proMMP-1 encoding DNA was fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal in the P. pastoris pPIC9 expression plasmid, transformed into strain GS115 (His-), and His+ Muts (slow methanol utilization) transformants were selected. Full-length proenzyme and processed forms of the protein could be detected in yeast culture supernatants following shake flask and 10-liter fermentations. The protein was purified to greater than 95% homogeneity. The recombinant proMMP-1 was comparable to the native fibroblast material based on (i) migration of the full-length molecule as a 52-kDa protein on reducing SDS-PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuric acetate to yield processed protein species, (iv) degradation of gelatin as monitored by zymogram gels, and (v) enzymatic activity. These data suggest that the P. pastoris expression system offers a convenient and efficient means to produce and purify MMP-1.

Identification and concerted function of two receptor binding surfaces on basic fibroblast growth factor required for mitogenesis.

Members of the fibroblast growth factor (FGF) family promote angiogenesis and wound repair, modulate early developmental events and survival of neurons, and have been associated with the pathogenesis of various diseases. FGFs interact with specific FGF receptors (FGFRs) and heparan sulfate proteoglycans on cell surfaces to mediate mitogenesis. Using protein structure-based site-directed mutagenesis of basic FGF (bFGF), we have identified two FGFR binding sites on bFGF which act in concert to initiate signal transduction. Both FGFR binding surfaces are distinct from the heparan sulfate proteoglycan binding domain. The primary, higher affinity, binding interaction comprises a cluster of solvent exposed hydrophobic amino acids (Tyr-24, Tyr-103, Leu-140, and Met-142), and two polar residues (Arg-44 and Asn-101). The hydrophobic contacts dominate the primary binding interaction and provide approximately 75% of the binding affinity. The secondary FGFR binding site on bFGF has an approximately 250-fold lower affinity and is composed of amino acids Lys-110, Tyr-111, and Trp-114 in a surface-exposed type I beta-turn (formerly known as the putative receptor binding loop). Binding of FGFR to both bFGF surfaces in a stoichiometry of 2FGFR:1bFGF is required for growth factor mediated cell proliferation. This represents a mechanism for the fibroblast growth factor/receptor family in which FGF facilitates FGFR dimerization and subsequent signal transduction events as a monomeric ligand.

Multiple sites of the propeptide region of human stromelysin-1 are required for maintaining a latent form of the enzyme.

Latency of matrix metalloproteinase 3 (MMP-3) is regulated by the interaction of a free cysteine residue (Cys-75) in the conserved amino acid sequence Pro-Arg-Cys-Gly-Val-Pro-Asp located in the COOH-terminal portion of the propeptide with a chelated zinc atom in the active site of the catalytic domain. Proteolytic activation of full-length human pro-MMP-3 involves the removal of approximately 35 amino acids from the NH2-terminal portion of the propeptide, forming a 53-kDa unstable intermediate that undergoes intermolecular autocatalysis to form the 45-kDa mature active enzyme. In this study, we have evaluated the contribution of the NH2-terminal 35 amino acids to the maintenance of latency. Full-length human pro-MMP-3 was expressed in Escherichia coli and refolded to form latent pro-MMP-3 capable of activation by chymotrypsin or aminophenylmercuric acetate. Renaturation of pro-MMP-3 expressed in bacteria with 20 or more amino acids removed from the NH2-terminal region of the propeptide yielded only an active enzyme. COS-7 cells transiently transfected with pro-MMP-3 expression vectors containing the single amino acid substitutions Y20A, L21A, and C75S also secreted active forms of the enzyme. These data suggest that simultaneous interactions of the NH2- and COOH-terminal regions of the propeptide are required for maintenance of the latent form of the enzyme.

Production of human matrix metalloproteinase 3 (stromelysin) in Escherichia coli.

Full-length human matrix metalloproteinase 3 (prostomelysin or proMMP-3) was produced in Escherichia coli as an intracellular insoluble aggregate that could be solubilized and refolded to yield an activatable proenzyme. The refolded protein was purified to > 95% homogeneity. The recombinant proMMP-3 (re-proMMP-3) could be activated by agents known to stimulate self-catalyzed cleavage of native fibroblast proMMP-3. The N-terminal amino-acid sequence of the re-proMMP-3 and its activation products indicated that they were the same as those obtained with the natural material.

Intracranial hypertension and cerebrospinal fluid production in dogs: effects of furosemide.

A method using chronically prepared, anesthetized dogs was devised for studying the effects of treatments of intracranial hypertension induced by applying a reversible extradural mass lesion while simultaneously measuring production of cerebrospinal fluid. This was measured with a ventricular-cisternal perfusion technique in which the rate of cisternal outflow could be controlled by a pump and matched to the inflow, allowing intracranial pressure to fluctuate despite simultaneous measurement of cerebrospinal fluid formation. Elevations of intracranial pressure to the range 20 to 35 Torr were induced and maintained during perfusion, but elevations above 35 Torr would not permit continued perfusion. At normal intracranial pressure, 10 Torr or less, rates of cerebrospinal fluid formation were the same whether the outflow controlling pump or free outflow was used. Formation of cerebrospinal fluid decreased progressively as intracranial pressure increased above 20 Torr. It also decreased with time after the start of perfusion during the course of 5 h, but returned to the initial range during the control phase of subsequent experiments in the same animal. Furosemide, 3 mg kg-1, i.v., had no significant effect on rate of formation but did induce a small decrease in ICP in time-controlled experiments in which i.v. fluid replacement limited net fluid losses to 20 ml kg-1 with no change in mean arterial or central venous pressures.

Synergistic effect of methotrexate and radiation on gliosarcoma in tissue culture and in rats.

In vitro and in vivo studies were performed on rat gliosarcoma cells (RT-9) to investigate the synergistic effect of methotrexate and radiation against malignant brain tumors. For in vitro study, RT-9 cells were cultured on microtiter plates and treated with methotrexate, radiation, or both. For in vivo study, the cells were implanted into the cerebrums of CD-Fischer rats, and intraperitoneal methotrexate, whole brain radiation, or both were given. Although synergistic suppression of cell growth with combined therapy was observed in the in vitro study, synergism was less clearly demonstrated in tumor-bearing rats because of systemic toxicity of the therapy.

Cloning and characterization of the xyl genes from Escherichia coli.

Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10 -15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.

Genetic characterization of the glutamate dehydrogenase gene (gdhA) of Salmonella typhimurium.

Salmonella typhimurium mutants, either devoid or glutamate dehydrogenase activity or having a thermolabile glutamate dehydrogenase protein, were used to identify the structural gene (gdhA) for this enzyme. Transductions showed that the mutations producing these phenotypes were linked to both the pncA and nit genes, placing the gdhA locus between 23 and 30 U on the S. typhimurium chromosome. Additional transductions with several Tn10 insertions established the gene order as pncA-gdhA-nit. Since few genetic markers exist in this region of the chromosome, Hfr strains were constructed to orient the pncA-gdhA-nit cluster with outside genes. Conjugation experiments provided evidence for the gene order pyrD-pncA-gdhA-nit-trp. To further characterize gdhA, we used Mu cts d1 (Apr lac) insertions in this gene to select numerous strains containing deletions with various endpoints. Transductions of these deletions with strains containing different gdh mutations and with a mutant having a thermolabile glutamate dehydrogenase protein permitted us to construct a deletion map of the gdhA region.

Bacteriophage P1 as a vehicle for Mu mutagenesis of Salmonella typhimurium.

We developed a procedure using bacteriophage P1 as a vector for transferring Mu phage deoxyribonucleic acid into Salmonella typhimurium. Mu phage transferred in this manner yielded lysogenic auxotrophs, and we demonstrated that specific deletions and lac gene fusions can be selected.

Regulation of nitrogen utilization of hisT mutants of Salmonella typhimurium.

Mutations in the hisT gene of Salmonella typhimurium alter pseudouridine synthetase I, the enzyme that modifies two uridines in the anticodon loop of numerous transfer ribonucleic acid species. We have examined two strains carrying different hisT mutations for their ability to grow on a variety of nitrogen sources. The hisT mutants grew more rapidly than did hisT+ strains with either arginine or proline as the nitrogen source and glucose as the carbon source. The hisT mutations were transduced into new strains to show that these growth properties were due to the hisT mutations. The hisT mutations did not influence the growth of mutants having altered glutamine synthetase regulation. Assays of the three primary ammonia-assimilatory enzymes, glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, showed that glutamate synthase activities were lower in hisT mutants than in isogenic hisT+ controls; however, the glutamate dehydrogenase activity was about threefold higher in the hisT strains grown in glucose-arginine medium. The results suggest that the controls for enzyme synthesis for nitrogen utilization respond either directly or indirectly to transfer ribonucleic acid species affected by the hisT mutation.

Rapid methods for generalized transduction of Salmonella typhimurium mutants.

A procedure has been developed that allows the propagation of generalized transducing phage directly on cells growing on solid media. After the donor cells are killed with chloroform, the phage can be transferred directly to recipient cells and transductants can be selected.