High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity.

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.

Crystal structure of glycosylasparaginase from Flavobacterium meningosepticum.

The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 angstroms resolution. This enzyme is a glycoamidase that cleaves the link between the asparagine and the N-acetylglucosamine of N-linked oligosaccharides and plays a major role in the degradation of glycoproteins. The three-dimensional structure of the bacterial enzyme is very similar to that of the human enzyme, although it lacks the four disulfide bridges found in the human enzyme. The main difference is the absence of a small random coil domain at the end of the alpha-chain that forms part of the substrate binding cleft and that has a role in the stabilization of the tetramer of the human enzyme. The bacterial glycosylasparaginase is observed as an (alphabeta)2-tetramer in the crystal, despite being a dimer in solution. The study of the structure of the bacterial enzyme allows further evaluation of the effects of disease-causing mutations in the human enzyme and confirms the suitability of the bacterial enzyme as a model for functional analysis.

Molecular cloning and sequence analysis of flavastacin: an O-glycosylated prokaryotic zinc metalloendopeptidase.

A new zinc metalloendopeptidase that cleaves peptides on the amino-terminal side of aspartic acid was isolated from the cultural filtrate of Flavobacterium meningosepticum. The gene for this new enzyme was cloned into pBluescript, and the complete nucleotide sequence was determined. Over 40% of the deduced amino acid sequence was verified independently by direct protein microsequencing. The most important structural features of this new enzyme include (i) the presence of an unusual O-linked oligosaccharide of unknown function located at a unique consensus site near the C-terminus and (ii) a characteristic extended zinc-binding site and corresponding Met-turn that places this metalloendopeptidase in the astacin family. This is the first example of a prokaryotic enzyme related to the eukaryotic astacin group; it is being designated hereafter as flavastacin.

Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum.

Flavobacterium meningosepticum, Elder strain (ATCC 33958), secretes into the medium a neutral zinc endoprotease as a major component of the extracellular proteins. The enzyme was purified to homogeneity in a simple two-step procedure involving ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular weight of this metalloprotease was determined to be about 27,000 (P27) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P27 was comparable to thermolysin in the relative rates of elastin-orcein, azocasein, and azoalbumin hydrolysis. P27 and thermolysin hydrolyzed equally well 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the same primary sites that are susceptible to cleavage by vertebrate collagenases, Gly-Ile, and Gly-Leu. P27 was also capable of partially hydrolyzing Type I acid-soluble calf skin collagen and slowly hydrolyzing N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala, a bacterial collagenase substrate not cleaved by thermolysin. P27 was further differentiated from thermolysin from the inability of the former to hydrolyze N-[3-(2-furyl)acryloyl]-Gly-Leu-NH2. In addition, a vertebrate elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide was hydrolyzed by P27 but not by thermolysin. P27 is a newly described and unique enzyme from the standpoint of substrate specificity and from the fact that it is resistant to inhibition by phosphoramidon, an inhibitor of a number of zinc endopeptidases, including thermolysin.

Carboxypeptidase H. A regulatory peptide-processing enzyme produced by human hepatoma Hep G2 cells.

Human hepatoma (Hep G2) cells have been shown to secrete nanogram quantities of carboxypeptidase N (Grimwood, B. G., Plummer, T. H., Jr., and Tarentino, A. (1988) J. Biol. Chem. 263, 14397-14401). A second carboxypeptidase with an acidic pH optimum (pH 5.5) is also secreted at levels 2-3-fold greater than carboxypeptidase N. This enzyme was partially purified from the conditioned medium and compared with pure bovine pituitary carboxypeptidase H. The two enzymes behaved in a similar fashion in DE52 ion-exchange chromatography and on gel filtration, with the Hep G2 enzyme being slightly larger than the bovine pituitary enzyme (52-54 versus 50-52 kDa). Both enzymes hydrolyzed COOH-terminal basic amino acids from typical synthetic substrates as well as from natural leuenkephalin peptides and were identical based on pH activity profiles, inhibition by EDTA or guanidinoethyl mercaptosuccinic acid, and stimulation by Co2+ ions. Inhibition of enzyme secretion from Hep G2 cells by tunicamycin indicated that the Hep G2 enzyme was glycosylated. This finding was confirmed by a parallel deglycosylation of the Hep G2 and bovine pituitary carboxypeptidase H enzymes with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Immunoblots using mouse antiserum to bovine pituitary carboxypeptidase H revealed that the Hep G2 enzyme was immunocross-reactive with the bovine enzyme but was slightly larger in size (54 versus 52 kDa). Continuous [35S]methionine labeling and purification to near homogeneity using an affinity matrix corroborated the observations that the secreted Hep G2 carboxypeptidase H was slightly larger than bovine pituitary carboxypeptidase H. The Hep G2-secreted enzyme in pulse-chase experiments was initially detected intracellularly after a 15-min pulse as a single protein of about 54 kDa and was present in the 30-min chase medium with no evidence for pre- or postsecretion proteolytic processing. The human adrenergic cell line IMR-32 continuously labeled with [35S]methionine also secreted carboxypeptidase H of the same size as the Hep G2 enzyme.

Characterization of the carboxypeptidase N secreted by Hep G2 cells.

Human hepatoma (Hep G2) cells secrete nanogram quantities of carboxypeptidase enzymes which are capable of hydrolyzing COOH-terminal lysine and arginine residues. A carboxypeptidase with a neutral pH optimum (greater than pH 7.0) was partially purified from the conditioned medium and compared with pure plasma carboxypeptidase N. The two enzymes behaved in a similar manner on gel filtration (apparent Mr = 280,000), DE52 ion exchange chromatography, and concanavalin A-affinity chromatography and were indistinguishable enzymatically and immunologically. Immunoblots of the Hep G2 and plasma carboxypeptidase N before and following deglycosylation with peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase F revealed a similar, if not identical, multimeric structure. A second carboxypeptidase with a lower molecular weight and a pH optimum of 5.0 was also detected in the Hep G2 medium.

High performance liquid chromatographic quantitation of carboxypeptidase activity secreted by human hep G2 cells.

A new high performance liquid chromatographic method has been developed for the determination of carboxypeptidase N activity which quantitates the furylacryloyl-alanine released by enzymatic cleavage of furylacryloyl-alanyl-lysine or furylacryloyl-alanyl-arginine. A short isocratic gradient elutes the substrate and product in less than 7 min and multiple analyses are facilitated by an automatic sample injector. The microassay readily detects and quantitates carboxypeptidase N activity secreted into culture medium. It was determined that approximately 1 X 10(6) Hep G2 cells at early confluence secreted 1 ng of carboxypeptidase N in 24 h. The microassay will also detect as little as 51 pg of purified carboxypeptidase N or 8 pg of carboxypeptidase B.

Viral contamination of a subline of Toxoplasma gondii RH.

A subline of Toxoplasma gondii RH was determined to be contaminated with a viral agent, apparently lymphocytic choriomeningitis virus.

Toxofactor associated with Toxoplasma gondii infection is toxic and teratogenic to mice.

A toxin associated with Toxoplasma gondii infection was obtained from the trophozoites and culture medium used to propagate the parasite in cell cultures. The toxin, named Toxofactor (TF), administered parenterally or nonparenterally in adult mice, produces transient symptoms of lethargy, ruffled fur, and body weight loss. Organ changes which accompanied the outward symptoms included hepatosplenomegaly and involuted thymus. TF activity was detected in extracts of the blood, peritoneal fluid, liver, and spleen of infected mice. Severe damage to embryonal and fetal development was induced when TF was administered during pregnancy. Resorption, abortion, and congenital abnormalities were produced, dependent upon the stage of development at the time of exposure. Adult mice which had reacted to and recovered from an initial intraperitoneal injection to TF were protected against a secondary challenge from TF. Fetal development was also protected from damage when TF was used to challenge adults previously exposed to TF. Mouse and rabbit anti-TF sera neutralized TF activity in the adult. In no instance did control mice show any deleterious effect when exposed to soluble cell lysate from the uninfected cell line (BHK-21) used to propagate the organism plus the used medium from these same uninfected cells. TF activity was not attributed to bacterial, myocoplasmal, or viral contamination. TF toxic activity is labile to elevated temperature and high or low pH, which also destroy its protective properties. TF activity was sensitive to trypsin and was obtained in the elution fraction (alpha-methyl-D-mannoside) from affinity chromatography (concanavalin A-Sepharose 4B). Ultrafiltration indicated the molecular weight to be between 50,000 and 100,000. TF, apparently a glycoprotein, was quantitated for activity by a weight loss assay. A unit of activity was defined as the minimum quantity of TF (highest dilution) which produced at least a 10% average body weight loss in adult Nya:NYLAR female mice between days 7 and 12 post-intraperitoneal injection.

Infective Toxoplasma gondii trophozoites attenuated by ultraviolet irradiation.

Both the virulence and infectivity of Toxoplasma gondii trophozoites are affected by increasing fluences of ultraviolet irradiation. All mice received unirradiated virulent trophozoites intraperitoneally died in an average of 5 days. All mice inoculated with trophozoites irradiated with 20 J m-2 died in an average of 8 days. Fluences of 35, 55, and 70 J m-2 resulted in 90, 20 and 0% deaths, respectively. Fluences up to 720 M m-2 did not increase the number of nonviable trophozoites. The minimum fluence which prevented mouse deaths (70 J m-2) also prevented trophozoite proliferation in baby hamster kidney (BHK-21) cell cultures. Minimum fluence-irradiated trophozoites attached to or entered BHK-21 cells or both at a rate similar to that for controls. BHK-21 cells fixed and stained 18 h after inoculation with unirradiated trophozoites contained both single and multiple trophozoites. In contrast, cell cultures inoculated with minimum fluence-irradiated trophozoites contained only single, nonreplicated trophozoites. No proliferation of trophozoites in these cultures was found as long as observed, 14 days after inoculation. Fluences of ultraviolet irradiation can therefore be determined which allow infection of cells but prevent trophozoite proliferation.