Gene expression, purification and characterization of recombinant human neutrophil collagenase.

Human neutrophil collagenase (HNC) is a member of a family of matrix metalloproteinases (MMP). HNC is capable of cleaving all three alpha-chains of types I, II and III collagens. In rheumatoid and osteo-arthritis, MMP members have been implicated in the pathology associated with these diseases due to the accelerated breakdown of the extracellular matrix of articular cartilage. A cDNA coding for the HNC catalytic domain (lacking both the propeptide and C-terminal fragments) was sub-cloned into the pETlla prokaryotic expression vector. The cloned fragment encodes a protein that extends from amino acids (aa) Met100 through Gly262 of the full-length proenzyme, which as a result, would not require proteolytic or chemical activation. The HNC construct was expressed in Escherichia coli and recombinant mature, truncated neutrophil collagenase (re-mNC-t) was produced at high levels (approx. 30% of total bacterial protein). The re-mNC-t protein was extracted from inclusion bodies by solubilization in 6 M urea, followed by ion-exchange chromatography. The protein was refolded to an active conformation in the presence of Ca2+ and Zn2+. A final purification step on size-exclusion chromatography yielded 30 mg per liter of active re-mNC-t with minor autodegradative products. Alternatively, hydroxamate affinity chromatography was used to obtain pure, non-degraded re-mNC-t (20-25 mg per liter). The catalytic activity of re-mNC-t was abolished by known MMP inhibitors and the Ki measurement against actinonin was similar to that of HNC prepared from human blood.

Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli.

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

Pseudoadrenal mass: unusual presentation of bronchogenic cyst.

Isolated abdominal bronchogenic cysts are extremely rare. We report the fourth such case in an asymptomatic 4-year-old girl who initially presented for evaluation of urinary tract infection and new-onset urinary incontinence. Ultrasound, computed tomography, and magnetic resonance imaging findings were consistent with an adrenal mass. At exploration, the patient was found to have a mass in the area of the gastroesophageal junction and a normal left adrenal gland. The final pathology confirmed the diagnosis of a bronchogenic cyst. Abdominal bronchogenic cysts, although rare, should be considered in the diagnosis of a retroperitoneal mass.

Evidence for the inorganic nature of the cyanolyzable sulfur of molybdenum hydroxylases.

Activation of the desulfo forms of milk xanthine oxidase, chicken liver xanthine dehydrogenase, and aldehyde oxidase with S2- is greatly facilitated in the presence of reducing agents. Upon anaerobic incubation with 1 mM S2- and 1 mM dithionite, desulfo xanthine oxidase and chicken liver xanthine dehydrogenase prepared by cyanide treatment of active enzymes, are activated to the specific activity predicted by their molybdenum content. Routine preparations containing desulfo molecules are also similarly activated to the extent predicted. Cyanide-inactivated chicken liver xanthine dehydrogenase was reconstituted with 35S2- in the presence of dithionite. 85% of enzyme-bound radioactivity was shown to be in the form of cyanolyzable sulfur, by comparison of enzyme activity, bound radioactivity, and 35SCN- yields from exposure of labeled enzyme to cyanide. This radiolabeled enzyme allowed the determination of the following. 1) The cyanolyzable sulfur is largely removed from the polypeptide by incubation at 37 degrees C for one hour in 1% sodium dodecyl sulfate, pH 7, or for 15 min in 6 M guanidinium chloride, pH 6.2. 2) The cyanolyzable sulfur is "acid labile." [35S]Methylene blue is formed in the theoretical quantity from oxidized or substrate-reduced enzyme under the standard conditions for labile sulfur analysis by the methylene blue method. These data strongly support the conclusion that the cyanolyzable sulfur is a terminal sulfur ligand of the Mo atom, and is not part of an organic moiety.

Clostridial pyruvate oxidoreductase and the pyruvate-oxidizing enzyme specific to nitrogen fixation in Klebsiella pneumoniae are similar enzymes.

The chemical characterization, EPR properties, and mechanism of pyruvate:flavodoxin (ferredoxin) oxidoreductase from Klebsiella pneumoniae and Clostridium thermoaceticum have been investigated. A simple, specific, and sensitive assay and an efficient purification (based on the high affinity of these enzymes for a dye attached to agarose) are reported. The observed iron content of 8 atoms/subunit is twice that reported by others, whereas the contents of lipoate and flavin are less than 0.1 mol/subunit, in agreement with previous reports. Spectroscopic evidence suggests that the iron is present in Fe4S4(2+,1+) clusters. Reduction of the enzyme requires the presence of CoA as well as 1.1 pyruvate/subunit, which is very nearly the theoretical amount required the reduce two Fe4S(2+,1+) clusters. In the absence of CoA, stoichiometric amounts of pyruvate are decarboxylated, but the Fe/S centers are not reduced. We conclude that the K. pneumoniae and C. thermoaceticum enzymes are adapted to rapid reduction of low potential 1-e- carriers, similar to the pyruvate oxidoreductase of Halobacterium (Kerscher, L., and Oesterhelt, D. (1977) FEBS Lett. 83, 197-201), but different in that an Fe/S center-radical pair is used in the latter enzyme in place of the pair of Fe4S4 centers we find. The K. pneumoniae and C. thermoaceticum oxidoreductases appear to be mechanistically closely related to the Clostridium acidiurici enzyme (Uyeda, K., and Rabinowitz, J. C. (1971) J. Biol. Chem. 246, 3111-3119), differing as a class from the lipoate-containing, pyridine nucleotide-reducing enzyme present in aerobes (Reed, L. J. (1974) Accts. Chem. Res. 2, 740-746). The function of the Klebsiella enzyme is to supply electrons to nitrogenase. This is accomplished in vitro with purified components via a nif-specific flavodoxin or other low potential 1-e- carriers such as viologen dyes or ferredoxins. The in vivo molar ratio of nitrogenase to the physiological reduction system, estimated from activity measurements of individual components in crude extracts, was 0.4:0.03:2:1 pyruvate oxidoreductase:flavodoxin:nitrogenase component II:nitrogenase component I.

The relationship of Mo, molybdopterin, and the cyanolyzable sulfur in the Mo cofactor.

Reconstitution of the apoprotein of the molybdoenzyme nitrate reductase in extracts of the Neurospora crassa mutant nit-1 with molybdenum cofactor released by denaturation of purified molybdoenzymes is efficient in the absence of exogenous MoO2-4 under defined conditions. Evidence is presented that this molybdate-independent reconstitution is due to transfer of intact Mo cofactor, a complex of Mo and molybdopterin (MPT), the organic constituent of the cofactor. This complex can be separated from denatured protein by gel filtration, and from excess MoO2-4 by reverse-phase HPLC. Sulfite oxidase, native xanthine dehydrogenase, and cyanolyzed xanthine dehydrogenase are equipotent Mo cofactor donors. Other well-studied inactive forms of xanthine dehydrogenase are also shown to be good cofactor sources. Using xanthine dehydrogenase specifically radiolabeled in the cyanolyzable sulfur, it is shown that this terminal ligand of Mo is rapidly removed from Mo cofactor under the conditions used for reconstitution.

Drosophila melanogaster ma-l mutants are defective in the sulfuration of desulfo Mo hydroxylases.

Xanthine dehydrogenase was purified more than 1500-fold from crude extracts of wild type Drosophila melanogaster. Like the bovine milk and chicken liver enzymes, the purified Drosophila enzyme was inactivated by cyanide, and the cyanide-inactivated desulfo enzyme was reactivated by anaerobic incubation with 1 mM sulfide and 1 mM dithionite. Application of the resulfuration procedure to crude extracts of Drosophila ma-l flies which slow pleiotropic deficiencies of xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase led to the emergence of xanthine dehydrogenase and aldehyde oxidase activities. Representatives of all the five known complementation groups of ma-l mutants were amenable to activation; 59-95% of wild type xanthine dehydrogenase activity and 1-7% of wild type aldehyde oxidase activity were reconstituted. Evidence for the identity of in vitro reconstituted xanthine dehydrogenase from ma-l mutants with wild type enzyme is presented. Since the inactive xanthine dehydrogenase and aldehyde oxidase proteins present in ma-l mutants are identical with the catalytically inactive desulfo forms obtained by cyanide treatment of active enzymes, these data constitute evidence for genetic control of the incorporation of the cyanolyzable sulfur of Mo hydroxylases.

Comparison of native matrix metalloproteinases and their recombinant catalytic domains using a novel radiometric assay.

A novel radiometric assay was developed for human fibroblast collagenase (matrix metalloproteinase-1, MMP-1), stromelysin (MMP-3), and their recombinant catalytic domains. Using this assay we were able to compare the native MMPs with the respective catalytic domains in terms of inhibitor affinities and peptide hydrolysis. The assay works on the same principle as an assay developed for carboxypeptidase (Rossier et al., Anal. Biochem. 1989, 178, 27-31) and is based on a synthetic peptide substrate, [1-benzoyl-14C]benzoyl-Pro-Leu-Ala-Leu-Trp- NH(CH2)4N(CH3)2(bnzPLALW-NX). The generation of product is measured by selective solvent extraction of radioactive product directly into scintillation cocktail; the entire assay, including the radioactivity measurement, is completed in a single 1-ml tube (96-well format) without removal or transfer of phases. Results of steady-state measurements demonstrated that peptide hydrolysis follows Michaelis-Menten kinetics with the fibroblast MMPs and their C-terminal deleted forms. The kinetic constants for hydrolysis of bnz-PLALW-NX, and for inhibition by actinonin, a natural peptide-hydroxamate, are essentially the same for the native and the C-terminally deleted MMPs.

Structure of human neutrophil collagenase reveals large S1' specificity pocket.

The crystal structure of the catalytic domain of human neutrophil collagenase complexed with a peptide transition state analogue has been determined to a resolution of 2.1 A. The structure of the neutrophil enzyme, when compared with the three dimensional structure of the corresponding human fibroblast collagenase, shows differences in the first, S1', of the three enzyme specificity subsites on the carboxy-terminal side of the substrate scissile bond. The S1' pocket in the neutrophil collagenase is significantly larger than the equivalent site in the fibroblast enzyme, suggesting that the former enzyme has a broader range of possible substrates. Such differences also suggest approaches for the design of selective matrix metalloproteinase inhibitors.

Mutation of Cys672 allows recombinant expression of activatible macrophage-stimulating protein.

We readily produced recombinant pro-macrophage stimulating protein in a mammalian expression system, but it was only weakly active after proteolytic activation. Active macrophage stimulating protein is a disulfide-bonded heterodimer, but in our hands, the subunits of recombinant macrophage stimulating protein were mostly not disulfide bonded. Molecular modeling of the serine proteinase domain of macrophage stimulating protein based on homology to human trypsin suggested that macrophage stimulating protein, but not plasminogen or hepatocyte growth factor, has a Cys residue (672) in close proximity to the Cys residue (578) that forms the intersubunit disulfide link with the other subunit. We hypothesized that Cys672 might interfere with intersubunit disulfide formation by forming an intrasubunit disulfide with Cys578 and therefore mutated Cys672 to Ala. After kallikrein activation, the subunits of Cys672 --> Ala macrophage stimulating protein were fully disulfide linked, and the mutant macrophage stimulating protein had 10-20-fold higher specific activity than the wild type recombinant macrophage stimulating protein.

Chicken macrophage stimulating protein is a ligand of the receptor protein-tyrosine kinase Sea.

Affinity chromatography, employing the extracellular domain of the Sea receptor, was used to enrich Sea-binding proteins from chicken serum. One isolated protein bound both a Sea-immunoglobulin fusion protein and an antisera raised against murine macrophage stimulating protein. Amino-terminal sequencing of the dual-reactive protein yielded sequences which were identical to the predicted alpha and beta subunits of chicken macrophage stimulating protein. The partially purified chicken macrophage stimulating protein caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol. Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophage-stimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.

1.56 A structure of mature truncated human fibroblast collagenase.

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 A resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded beta-sheet and three alpha-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase.

Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest.

The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.

Identification of incompletely processed potential carboxypeptidase E substrates from CpEfat/CpEfat mice.

In an attempt to identify peptides that may be involved in the obese phenotype observed in CpEfat/CpEfat mice (deficient in Carboxypeptidase E, CpE) samples from fourteen neuroendocrine tissues in wild-type and CpEfat/CpEfat mice were obtained. Peptides were purified from these tissues and potential CpE substrate peptides were enriched using an anhydrotrypsin column that captures peptides with basic C-termini. Bound peptides were subjected to tryptic digestion and followed by liquid chromatography-mass spectrometry analysis. The relative levels of CpEfat/CpEfat versus wild-type peptides were determined by comparison of the ion intensities. Peptide ions elevated in the CpEfat/CpEfat samples were identified by targeted liquid chromatography-tandem mass spectrometry. From those ions, 27 peptides derived from known neuropeptides (including CpE substrates) were identified, together with another 25 peptides from proteins not known to be components of the neuropeptide processing pathway. The known CpE substrates identified included the recently discovered proSAAS, granin-like neuroendocrine peptide precursor that inhibits prohormone processing. The approach demonstrated the feasibility of using an affinity-based method for identifying differences in specific classes of peptides between normal and mutant mice.

Tolerance of normal aorta to oversized dual balloon valvuloplasty. Observations in a swine model: technical note.

The damage threshold during aortic valvuloplasty was determined in 12 normal swine subjected to inflation of oversized dual balloons. Catheters with combined balloon diameters of 1.2-2.0 times the aortic annulus were selected. Following completion of the procedure, the animals were sacrificed and examined for pathology. With combined balloon diameters less than or equal to 1.7 times the aortic annulus, there was no gross or microscopic damage. In animals with combined diameters equal to 2 times the aortic annulus, there was rupture of the aorta. This data provides further basis for the selection of catheter sizes in dual balloon aortic valvuloplasty.

Assignments and structure determination of the catalytic domain of human fibroblast collagenase using 3D double and triple resonance NMR spectroscopy.

We report here the backbone 1HN, 15N, 13C alpha, 13CO, and 1H alpha NMR assignments for the catalytic domain of human fibroblast collagenase (HFC). Three independent assignment pathways (matching 1H, 13C alpha, and 13CO resonances) were used to establish sequential connections. The connections using 13C alpha resonances were obtained from HNCOCA and HNCA experiments; 13CO connections were obtained from HNCO and HNCACO experiments. The sequential proton assignment pathway was established from a 3D (1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and 15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement with recent X-ray structures of human fibroblast collagenase and human neutrophil collagenase as well as similar to recent NMR structures of a highly homologous protein, stromelysin. All three helices were located; a five-stranded beta-sheet (four parallel strands, one antiparallel strand) was also determined. beta-Sheet regions were identified by cross-strand d alpha N and d NN connections and by strong intraresidue d alpha N correlations, and were corroborated by observing slow amide proton exchange. Chemical shift changes in a selectively 15N-labeled sample suggest that substantial structural changes occur in the active site cleft on the binding of an inhibitor.