Directed evolution of a fungal peroxidase.

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.

Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 A resolution.

Laccase catalyses the oxidation of a variety of organic substrates coupled to the reduction of oxygen to water. It is widely believed to be the simplest representative of the ubiquitous blue multi-copper oxidase family. Laccase is implicated in a wide spectrum of biological activities and, in particular, plays a key role in morphogenesis, development and lignin metabolism in fungi and plants. The structure of laccase from the fungus Coprinus cinereus has been determined by X-ray crystallography at a resolution of 2.2 A. Laccase is a monomer composed of three cupredoxin-like beta-sandwich domains, similar to that found in ascorbate oxidase. In contrast to ascorbate oxidase, however, the mononuclear type-1 Cu site lacks the axial methionine ligand and so exhibits trigonal planar coordination, consistent with its elevated redox potential. Crucially, the structure is trapped in a Cu depleted form in which the putative type-2 Cu atom is completely absent, but in which the remaining type-1 and type-3 Cu sites display full occupancy. Type-2 Cu depletion has unexpected consequences for the coordination of the remaining type-3 Cu atoms.

Crystallization and preliminary X-ray analysis of the laccase from Coprinus cinereus.

The laccase from the fungus Coprinus cinereus has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small plate-like crystals of an enzymatically deglycosylated form of the enzyme have been grown by the hanging-drop method using polyethylene glycol as precipitant. These crystals diffract to at least 2.2 A. They belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.4, b = 85.7, c = 143.1 A with a single molecule of laccase in the asymmetric unit.

Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine.

Factor VII is a multidomain, vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol. Chem. 264, 20320-20325). In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation. Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis. Using a combined strategy of amino acid sequencing, carbohydrate and amino acid composition analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII. Approximately equal amounts of the three glycan structures were observed in plasma factor VII, whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated. In addition to the O-linked glycan structures observed at serine 52, a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII. Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative. Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60. In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII, we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue. Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay. The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa. In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa. These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor, or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of recombinant human blood coagulation factor VIIa amidolytic and proteolytic activity by zinc ions.

Although it is well established that calcium is an essential cofactor in blood coagulation, recent experimental evidence suggests that zinc may also play an important role in hemostasis. In the present study, we have examined the effect of zinc ions on the amidolytic and proteolytic activity of recombinant factor VIIa in the presence of physiological levels of calcium ions. The amidolytic activity of factor VIIa was inhibited half-maximally by 20 microM zinc. The amidolytic activity of a derivative of factor VIIa lacking the gamma-carboxyglutamic acid domain was also inhibited half-maximally by 20 microM zinc, suggesting that the mechanism of zinc inhibition of factor VIIa amidolytic activity did not involve its gamma-carboxyglutamic acid residues. The amidolytic activity of a complex of recombinant tissue factor and factor VIIa was inhibited half-maximally by 70 microM zinc. In contrast to the results obtained with factor VIIa, the amidolytic activities of other human vitamin K-dependent coagulation proteases including factor Xa, thrombin and activated protein C were not appreciably affected by 50-100 microM zinc. The proteolytic activation of factor X by a complex of factor VIIa and relipidated tissue factor apoprotein was inhibited half-maximally by 40 microM zinc, whereas activation of factor IX in this system was inhibited half-maximally by 70 microM zinc ions. Considerably higher levels of zinc (approximately 100 microM) were required to inhibit half-maximally the rate of factor X activation by a complex of factor VIIa and functional tissue factor on the surface of either a human bladder carcinoma cell line, J82, or stimulated human umbilical vein endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions.

Previous studies have shown that extrinsic pathway inhibitor (EPI) is an effective inhibitor of factor Xa alone or factor VIIa-tissue factor complex in the presence of factor Xa. Since tissue factor exposure is implicated in thrombogenesis, we hypothesized that EPI may be valuable in the treatment of some thromboembolic episodes. Furthermore, EPI may be an important factor in bleeding complications in hemophiliacs. In the present study, human EPI was expressed in baby hamster kidney cells using a mammalian expression vector. Transfected cells expressed 1-2 micrograms/ml of recombinant EPI (rEPI) which was purified to homogeneity by heparin-Sepharose chromatography, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Purified rEPI exhibited a specific activity of 30,000 units/mg and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 42,000. In addition, the NH2-terminal sequence of rEPI was identical to that of HepG2 EPI and HeLa EPI. The ability of rEPI to inhibit factor X activation by a complex of factor VIIa-tissue factor was then examined in the presence and absence of plasma concentrations of human factors VIII and IX. Using relipidated human brain tissue factor apoprotein, rEPI inhibited the factor VIIa-mediated activation of factor X half-maximally at 2.5 and 1 nM in the presence and absence of factors VIII and IX, respectively. Using monolayers of a human bladder carcinoma cell line (J82) as the source of tissue factor, the activation of factor X by cell-bound factor VIIa was inhibited half-maximally by 5 nM rEPI in the presence of factors VIII and IX. The proteolytic activity of J82 cell-bound factor Xa toward prothrombin was inhibited half-maximally at approximately 5 nM rEPI, while the amidolytic activity of factor Xa in solution was inhibited by rEPI with a Ki of 130 pM. Recombinant EPI also inhibited the amidolytic activity of factor VIIa half-maximally at 10 nM rEPI in the presence of relipidated tissue factor apoprotein and calcium. These results indicate that, in the presence of plasma concentrations of factors VIII and IX, at least 10 times the plasma concentration of EPI is required to reduce factor VIIa-dependent factor X activation one order of magnitude in vitro. In the absence of functional factor VIII and IX, rEPI at plasma levels was a potent inhibitor of factor VIIa-mediated factor X activation, and this activity presumably accounts for the inability of hemophiliacs to initiate hemostasis via the extrinsic pathway.

Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor.

Previous studies indicated that factor VIIa, in complex with tissue factor, readily activates either factor X or factor IX in the presence of calcium ions. In order to assess the relative physiological importance of the activation of factor IX versus the activation of factor X by recombinant factor VIIa, we have obtained steady-state kinetic parameters for the factor VIIa catalyzed activation of factor IX and factor X under a variety of cofactor conditions that include calcium alone, calcium and phospholipids, calcium, phospholipids, and tissue factor apoprotein, and calcium and cell-surface tissue factor. Calcium alone stimulated the activation of factors IX and X by factor VIIa maximally at 1 and 2.5 mM, respectively. In the presence of 25 microM phospholipids, maximal rates of factor IX and factor X activation were achieved at 2.5-5 mM calcium. With calcium alone, or with phospholipid and calcium, the initial rates of factor IX activation by factor VIIa were significantly higher than that observed for factor X. Kinetic studies revealed that the Km for the factor VIIa catalyzed activation of factor IX was essentially constant in the presence of 5 mM calcium and 1-500 microM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 microM phospholipid. At all concentrations of added phospholipid, the kcat/Km ratio for the activation of factor IX by factor VIIa appeared to be considerably greater than that observed for the activation of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoactivation of human recombinant coagulation factor VII.

Single-chain human recombinant factor VII produced by transfected baby hamster kidney cells was purified to homogeneity in the presence of benzamidine. The amidolytic activity of single-chain recombinant factor VII with a peptidylnitroanilide substrate, methoxycarbonyl-D-cyclohexanylglycyl-L-arginine-p-nitroanilide, was less than 1% of that obtained with factor VIIa. Purified single-chain recombinant factor VII spontaneously activated in the absence of inhibitor. The activation reaction was enhanced by at least 2 orders of magnitude in the presence of a positively charged surface, provided either as an anion-exchange matrix or as poly(D-lysine). The progress curve for factor VIIa generation was sigmoidal. Benzamidine inhibits recombinant factor VIIa activity and factor VII activation with identical inhibition constants (Ki) of 11 mM. In contrast, benzamidine inhibition of bovine factor Xa and bovine factor IIa was observed at Ki values equal to 0.3 and 0.5 mM, respectively. Bovine factors Xa and IIa are known activators of factor VII and the most likely contaminants of our recombinant factor VII preparations. Single-chain recombinant factor VII purified from cells cultured in the absence of bovine serum activated at the same rate as factor VII from cells cultured in the presence of bovine serum. This also excluded the possibility that the activation reaction was caused by contaminating bovine proteases. On the basis of these observations, we propose that factor VII is autoactivated in vitro in the presence of a positively charged surface.

Binding of human factors VII and VIIa to a human bladder carcinoma cell line (J82). Implications for the initiation of the extrinsic pathway of blood coagulation.

We have studied the binding of radioiodinated human factor VII and its activated form, factor VIIa, to monolayers of a human bladder carcinoma cell line (J82) that expresses functional cell surface tissue factor. The binding of factors VII and VIIa to these cells was found to be time-, temperature-, and calcium-dependent. In addition, the binding of each protein to J82 cells was specific, dose-dependent, and saturable. The binding isotherms for factors VII and VIIa were hyperbolic, and Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for each protein with Kd values of 3.20 +/- 0.51 and 3.25 +/- 0.31 nM, respectively. Factors VII and VIIa, respectively, interacted with 256,000 +/- 39,000 and 320,000 +/- 31,000 binding sites/cell. Competition experiments suggested a common receptor for factors VII and VIIa. Binding of factor VIIa to the cells was completely blocked by preincubation of the cells with polyclonal anti-tissue factor IgG, whereas binding of factor VII was inhibited approximately 90%, suggesting the presence of a small number of tissue factor-independent binding sites specific for factor VII on this cell. Functional studies revealed that factor X activation by increasing amounts of cell-bound factor VII or VIIa was hyperbolic in nature. Half-maximal rates of factor Xa formation occurred at factor VII and VIIa concentrations of 3.7 +/- 0.47 and 3.2 +/- 0.31 nM, respectively. No factor VII- or VIIa-mediated activation of factor X was observed when cells were preincubated with anti-tissue factor IgG. Two-chain 125I-factor VIIa recovered from the cells was identical to the offered ligand as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In contrast, the offered single-chain 125I-factor VII was progressively converted to two-chain 125I-factor VIIa upon binding to the cells. When the J82 cells were pretreated with anti-tissue factor IgG, both factor VII recovered from the cells and factor VII in the supernatant were in the single-chain form, indicating that cell-surface tissue factor was essential for the activation of factor VII on these cells. These data indicate that binding of factor VII to tissue factor appears to be a prerequisite for its conversion to factor VIIa and the initiation of the extrinsic pathway of coagulation on these cells.

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Diphtheria on Skid Road, Seattle, Wash., 1972-75.

From July 1972 to December 1975, an unusual outbreak of diphtheria in Seattle, Wash., resulted in a total of 558 cases and carriers, mostly among heavy alcohol users. Skin infections were predominant. Four white men died. The highest attack rate was among native American Indians. Environmental contamination and poor personal hygience were believed to be important in continuation of the epidemic, but could not be proved. Control measures included casefinding, isolation and quarantine, sanitizing dwelling units and mass immunization with Td toxoid. The high-risk geographic area was the city's Skid Road. This area continues to be the reservoir of continuing infection, but not all population subgroups there have been at equal risk. Spread to other geographic areas of the city and county has been minimal and remains under control.

Venereal transmission of shigellosis in Seattle-King county.

In the past, shigellosis in Seattle-King County has been primarily a disease of children, their parients, and foreign travelers. During the 18 months beginning in July 1975, an outbreak of shigellosis in Seattle's community of gay men involved both Shigella flexneri and Shigella sonnei. They accounted for nearly 30% of all cases of shigellosis reported to the health department. Fellatio and/or oral-anal contact was reported by 90% of the infected homosexual men; this was probably the mechanism of transmission of most infections. Intercity spread was determined by case histories and by the finding of S. flexneri 3, a previously unusual organism in Seattle.

Spectinomycin versus tetracycline for the treatment of gonorrhea.

Spectinomycin and tetracycline are alternative drugs to penicillin in the treatment of gonorrhea. To compare the efficacy of these agents and their propensity to select resistant gonococci, we treated 4043 patients randomly with either 2 or 4 g of spectinomycin once or 9 g of oral tetracycline for four days. Minimum cure rate for anogenital gonorrhea was 94 per cent with either drug. Oropharyngeal infection responded poorly to spectinomycin in men, with failure of therapy in six of 11. Postgonococcal urethritis in men was less common after tetracycline than after spectinomycin (P less than 0.005). Spectinomycin failure was not related to drug resistance. Tetracycline failure correlated with resistance (P less than 0.0002); one fifth of the isolates resistant to 1.0 mug per milliter of tetracycline were not eradicated. For several reasons, including the appearance of beta-lactamase-producing gonococci, it is no longer clear that penicillin G is the "drug of choice" for gonorrhea. Spectinomycin and tetracycline are equally acceptable alternatives, each with distinct advantages and disadvantages.

A field trial of the Clinicult system for detection of asymptomatic gonorrhea in women.

Clinicult, a selective medium for culturing Neisseria gonorrhoeae, was field-tested in a gonorrhea screening program in Seattle, Wash., in 1973. The results with this medium and with the Transgrow and Thayer-Martin culture systems were compared as to sensitivity and specificity. A total of 5,141 women from three patient groups were included in the study. Group 1 consisted of 720 female patients of the venereal disease clinic of the Seattle-King County Health Department, who served as the control group. When this group was screened with the Clinicult and Thayer-Martin culture media, the Thayer-Martin medium proved superior in identifying positive carriers. Group 2 was composed of approximately 2,000 patients from five different facilities, including family planning clinics and hospital out patient services. No statistical difference in accuracy was found between the two culture systems used for this group-Clinicult and Transgrow. Group 3 was comprised of approximately 2,500 female patients who were screened with the Clinicult and Transgrow cultures by their own private physician or his staff. The Clinicult system proved significantly less effective than the Transgrow cultures in identifying infected females in group 3. The physicians varied greatly in their ability to use the Clinicult system successfully. Possible reasons for their errors may have been (a) lack of motivation and of care by their office personnel in conducting the necessary additional procedures required with Clinicult, (b) the inhibitory nature of the medium, and (c) the failure of the medium to produce colonies of adequate size. The staffs of communitywide screening programs for gonorrhea need to be highly selective in choosing the medical facilities in which to use the Clinicult culture system. When laboratory facilities are available for the full utilization of the Thayer-Martin medium, this system is preferable. When, however, standard culture procedures are not readily available, Clinicult, properly used, can reduce the central laboratory load by eliminating the need for processing negative cultures.