Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.     

Human Kallikrein 2 (hK2) and prostate-specific antigen (PSA): two closely related, but distinct, kallikreins in the prostate

Recent studies on human kallikrein 2 (hK2) have revealed striking similarities and significant differences with the closely related kallikrein PSA. Both PSA and hK2 are primarily localized to the prostate and share close structural similarities. Although both kallikreins are produced by the same secretory epithelial cells in the prostate, hK2 is associated more with prostate tumors than PSA and is highly expressed in poorly differentiated cancer cells. The potent trypsin-like activity of hK2 contrasts with the weak chymotrypsin-like activity of PSA. The inactive precursor form of PSA, proPSA, is converted rapidly to active PSA by hK2, suggesting an important in vivo regulatory function by hK2 on PSA activity. The high homology between hK2 and PSA results in significant cross-reactivity to hK2 by polyclonal and some monoclonal antibodies to PSA. Future studies on both PSA and hK2 need to take into account this potential for cross-reactivity. Specific monoclonal antibodies to hK2 have now demonstrated that serum levels of hK2, like PSA, are correlated with prostate cancer. The production of hK2 protein in active protease form and specific monoclonal antibodies to the hK2 antigen will allow extensive future studies delineating the physiological and clinical utility of this new prostate antigen.     

The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.     

Expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale

We have expressed human prostate-specific antigen (PSA) on a pilot-scale in Spodoptera frugiperda Sf9 insect cells using recombinant baculovirus system. Infected cells secreted PSA into culture medium at a concentration of 2-4 mg per liter. PSA was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. The N-terminus of active PSA was correctly cleaved; two amino acids of the propeptide remained, however, at the N-terminus of the inactive PSA. Purified recombinant PSA showed a chymotrypsin-like activity with the synthetic substrate MeO-Suc-Arg-Pro-Tyr-pNA, but did not have a trypsin-like activity when Pro-Phe-Arg-pNA was used. The molecular mass of active PSA was 31.0 kDa in reduced SDS-PAGE, 26.0 kDa in nonreduced SDS-PAGE and 26.5 kDa in ion spray mass spectrometry. The active protein formed complexes with alpha 1-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M) in vitro similar to the commercial PSA purified from human seminal fluid.     

Contamination of purified prostate-specific antigen preparations by kallikrein hK2

This paper ascertained the contamination by hK2 of two types of PSA preparation, that of Sensabaugh and Blake (J. Urol., 144: 1523, 1990) and that of Deperthes et al (J. Androl., 17: 659, 1996). In the first procedure, the free forms of hK2 co-migrated with PSA during the CM-Sephadex and the Sephacryl S-200 steps. By contrast, in the second procedure a very high proportion of hK2 was separated from PSA. In two different Sensabaugh and Blake procedures, the hK2 contamination per microg. of PSA was found to be respectively 0.3 and 1.0 ng. We conclude that hK2 is a quantitatively minor contaminant of some PSA preparations. That contamination is probably of little consequence for PSA standardization but it could lead to erroneous conclusions in enzymatic studies of PSA.     

The importance of human glandular kallikrein and its correlation with different prostate specific antigen serum forms in the detection of prostate carcinoma

BACKGROUND: Human glandular kallikrein (hK2), the prostate specific antigen (PSA) close homologue, possesses approximately 80% structure identity with PSA. The identification of PSA was an important step in the detection of prostate carcinoma (PCa). Thus, hK2 measurement in the serum has the potential to become another important diagnostic test for PCa. In the current study, the authors measured the serum concentrations of the hK2 with "in-house" immunofluorometric assays in different patient groups. The correlation between serum hK2 and different PSA forms was investigated. METHODS: The prospectively collected serum samples were obtained preoperatively on admission from 311 consecutive male patients. Sixteen patients did not fulfill inclusion criteria; the remaining patients were divided into four groups (Groups I-III confirmed histologically): Group I: patients with PCa (n = 56); Group II: patients with benign prostatic hyperplasia (BPH) (n = 163); Group III: patients with BPH with a chronic in-dwelling catheter (BPH cat) (n = 44); and Group IV-control group (n = 32). The patients in Group IV had urolithiasis, varicocele, or kidney or bladder tumors). An experimental immunofluorometric assay with an analytic sensitivity of 0.01 ng/mL and a functional sensitivity of 0.05 ng/mL was used to determine serum hK2 concentrations. Total PSA, free PSA, and PSA complexed to alpha-1-antichymotrypsin (PSA-ACT) also were measured. hK2 concentrations equal to or above the functional sensitivity limit were correlated with each of these PSA serum forms. Free to total PSA, hK2 to total PSA, and hK2 to free PSA ratios were calculated and compared in different patient groups. RESULTS: The hK2 concentrations were equal to or above the functional sensitivity limit in 179 of 311 samples (57.6%). In these samples, hK2 correlated best with free PSA (correlation coefficient [r] = 0.79) and correlated well with total PSA (r = 0.72) and PSA-ACT (r = 0.74). Similar correlations also could be observed when each clinical group was analyzed separately. The median proportion of hK2 in relation to total PSA was 2.1%, 1.8%, and 1.4%, respectively, for PCa, BPH, and BPH cat patients. Both the free to total PSA ratio and the hK2 to free PSA ratio discriminated well between PCa and BPH patients. Within the range of total PSA of 4-10 ng/mL (PCa [n = 11] and BPH [n = 41]) the hK2 to free PSA ratio had a specificity of 63.4% and 90.9% sensitivity (area under the receiver operating characteristic [ROC] curve = 0.85) whereas the free to total PSA ratio had a 34.1% specificity at the same sensitivity level (area under ROC curve = 0.74). CONCLUSIONS: The hK2 serum level correlates well with all PSA serum forms in all clearly defined clinical groups. The preliminary finding that the hK2 to free PSA ratio appeared to improve the detection of PCa compared with the free to total PSA ratio in patients with total PSA within a 4-10 ng/mL range is of clinical interest. Combining human serine proteases in the multivariate regression analysis will be a tool to improve cancer detection. Further investigations with more sensitive hK2 assays and in larger patient populations are needed to confirm this finding.     

Cross-reactivity of ten anti-prostate-specific antigen monoclonal antibodies with human glandular kallikrein

OBJECTIVES: Prostate-specific antigen (PSA) is commonly used as a marker for prostate disease. Prostate epithelium expresses both PSA and human glandular kallikrein (hK2) proteins, which share 80% sequence homology. The immunologic cross-reactivity of these two proteins could potentially interfere with determination of PSA levels in diagnoses of prostate cancer. We set out to determine the extent of this cross-reactivity for a panel of 10 anti-PSA monoclonal antibodies (mAbs). METHODS: Enzyme-linked immunosorbent assay (ELISA), sandwich assays, and western transfer techniques were used to assess the PSA/hK2 cross-reactivity of the anti-PSA mAbs. RESULTS: We did not detect the hK2 protein with any of the 10 anti-PSA mAbs under western transfer conditions. In ELISA experiments, 8 of 10 mAbs exhibited hK2 cross-reactivity under certain conditions. However, no combination of mAbs tested in sandwich assays exhibited a signal in hK2 cross-reactivity experiments greater than 0.1% of the PSA signal. CONCLUSIONS: We have evaluated 10 anti-PSA mAbs and determined that despite the 80% homology between PSA and hK2 proteins, cross-reactivity with hK2 by these antibodies would not significantly affect the determination of PSA levels by means of sandwich assays.     

Acceptance of digital image distribution in a "filmless hospital"

Human glandular kallikrein (hK2) is a possible new marker for prostate cancer that is homologous to prostate specific antigen. Purified hK2 added to serum or plasma reacted with endogenous protease inhibitors to form complexes of >350, 135, and 80 kDa, and some hK2 remained free, as judged by immunoblotting. The former two complexes could be removed by specific antibodies to alpha2-macroglobulin and to C1- inactivator, respectively, and they comigrated on SDS-PAGE with complexes formed between hK2 and purified alpha2-macroglobulin or C1-inactivator. hK2 complexes of 80 kDa could not be completely removed with any anti-serpin antibody used. Thus, these may consist of more than one type of hK2 complex. In contrast, essentially all hK2 complexes were removed from seminal plasma by antibody to protein C inhibitor, demonstrating that protein C inhibitor is the only significant inhibitor of hK2 in semen. hK2 reacted more rapidly with alpha2-macroglobulin than with any other inhibitor in plasma or serum. Divalent metal ions and heparin did not appreciably affect the rate of formation of any of the hK2 complexes in serum or plasma or with purified alpha2-macroglobulin or C1-inactivator. Measurement of one or more of the hK2 forms identified here may have diagnostic or prognostic potential for prostate cancer.     

Direct activation of human CD8+ cytotoxic T lymphocytes by interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8+ T cells present after 28 days in the induction culture. When purified CD8+ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8+ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.     

Purification and characterization of a soluble form of mammalian adenylyl cyclase

An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 micromol/min/mg, corresponding to a turnover number of 720 min-1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by Gsalpha and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein betagamma subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.     

Diamine oxidase induces neurite outgrowth in chick dorsal root ganglia by a nonenzymatic mechanism

The enzyme diamine oxidase (DAO) catalyzes the oxidative deamination of histamine, diamines, and polyamines. DAO has been localized to several tissues, including thymus, kidney, intestine, seminal vesicles, placenta, and pregnancy plasma. DAO is not constitutively expressed in the mammalian brain, but it becomes detectable following focal injury. Although the physiologic role of DAO remains unknown, the observation that it is present at the interface between rapidly dividing and quiescent cells in several tissues suggests that it might be involved in regulating cell division or differentiation at tissue boundaries. In addition, the observation that DAO is expressed in the brain following injury suggests that the protein might play a role in the CNS response to focal neuronal damage. To test that hypothesis, we assessed the ability of purified DAO to alter the pattern of neuronal differentiation and nerve growth in vitro. In chick dorsal root ganglion explant cultures, purified porcine DAO induced neurite outgrowth in the low nanomolar range. Addition of aminoguanidine, which inhibits DAO enzyme activity, did not inhibit the protein's neurotrophic activity. These findings suggest that DAO can function as a neurotrophic ligand independent of its enzymatic activity.     

A soluble form of Wnt-1 protein with mitogenic activity on mammary epithelial cells

The proto-oncogene Wnt-1 plays an essential role in fetal brain development and causes hyperplasia and tumorigenesis when activated ectopically in the mouse mammary gland. When expressed in certain mammary epithelial cell lines, the gene causes morphological transformation and excess cell proliferation at confluence. Like other members of the mammalian Wnt family, Wnt-1 encodes secretory glycoproteins which have been detected in association with the extracellular matrix or cell surface but which have not previously been found in a soluble or biologically active form. We show here that conditioned medium harvested from a mammary cell line expressing Wnt-1 contains soluble Wnt-1 protein and induces mitogenesis and transformation of mammary target cells. By immunodepletion of medium containing epitope-tagged Wnt-1, we show that at least 60% of this activity is specifically dependent on Wnt-1 protein. These results provide the first demonstration that a mammalian Wnt protein can act as a diffusible extracellular signaling factor.     

Mammalian transcobalamin II metabolism. The immunological and the biological cross-reactivity of mammalain transcobalamin II

Assay of the reactivity between the chicken anti-rabbit transcobalamin II antiserum and the sera of 19 vertebrate species was carried out by both immuno-diffusion and Sephadex G-200 gel filtration column chromatography. Mammalian transcobalamin II cross-reacted with the antiserum whereas the serum vitamin B-12 binders of the bird, amphibian reptile and fish did not. The biological activity of the purified rabbit transcobalamin II was assessed using reticulocytes or erythrocytes of human, rabbit, guinea pig and rat. The purified rabbit transcobalamin II promoted the uptake of vitamin B-12 by the cells but showed a great variation in its activity. It is suggested that the rabbit transcobalamin II is immunologically and biologically similar to the serum transcobalamin II of the mammalian species studied.     

Kininogen-derived fluorogenic substrates for investigating the vasoactive properties of rat tissue kallikreins--identification of a T-kinin-releasing rat kallikrein

Peptide substrates with intramolecularly quenched fluorescence that reproduce the rat kininogen sequences at both ends of the bradykinin moiety were synthesized and used to investigate the kinin-releasing properties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Substrates derived from rat H- and L-kininogen were cleaved best by rK1, especially that including the N-terminal insertion site of bradykinin, Abz-TSVIRRPQ-EDDnp(Abz = O-aminobenzoyl, EDDnp = ethylenediamine 2,4-dinitrophenyl), which was cleaved at the R-R bond with a k(cat)/Km of 12400 mM(-1) s(-1). Replacement of the P2' residue Pro by Val in Abz-TSVIRRPQ-EDDnp gave a far less specific substrate that was rapidly hydrolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl-N-methyl coumarylamide substrates, which lack prime residues, also had low specificities. The importance of the P2' residue for rK1 specificity was further demonstrated using a human-kininogen-derived substrate that included the N-terminal insertion site of bradykinin (Abz-LMKRP-EDDnp). This was cleaved at the M-K bond by hK1 (kallidin-releasing site), but at the K-R bond (bradykinin-releasing site) by rK1. Competition experiments with Abz-TSVIRRPQ-EDDnp, which is resistant to most kallikreins, and Abz-TSVIRRVQ-EDDnp, a general kallikrein substrate, demonstrated that the former competitively inhibited hydrolysis by rK9 and hK1, with Ki values similar to the Km values for the substrate. Thus Pro in P2' does not prevent the peptide binding to the enzyme active site, but impairs cleavage of the scissile bond. The T-kininogen-derived substrate with the T-kinin C-terminal sequence (Abz-FRLVR-EDDnp) was cleaved by rK10 (k(cat)/Km = 2310 mM(-1) s(-1)) and less rapidly by rK1, rK7 and hK1, at the R-L bond, while that corresponding to the N-terminal (Abz-ALDMMISRP-EDDnp) of T-kinin was resistant to all five kallikreins used, suggesting that none has T-kininogenase activity. But this substrate was hydrolysed by a semipurified sample of submandibular gland extract. Another kallikrein, identified as kallikrein rK3, was isolated from this fraction and shown to hydrolyze Abz-ALDMMISRP-EDDnp; rK3 also specifically released T-kinin from purified T1/T2-kininogen after HPLC fractionation. Injection of purified rK3 and of Abz-ALDMMISRP-EDDnp-cleaving fractions into the circulation of anesthesized rats caused transient falls in blood pressure, as did purified rK1 but none of the other purified rat or human kallikreins. This effect occurred via activation of the kinin system since it was blocked by Hoe140, a kinin receptor antagonist.     

Expression, purification, and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase

We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.     

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.     

A mechanism for valine-resistant growth of Escherichia coli K-12 supported by the valine-sensitive acetohydroxy acid synthase IV activity from ilvJ662

Acetohydroxy acid synthase (EC 4.1.3.18; AHAS) isozymes I and III are expressed in Escherichia coli strain K-12 and, when inhibited by L-valine, cannot support cell growth. AHAS IV, expressed from mutation at ilvJ662, exhibits valine-sensitivity similar to that of AHAS III, yet AHAS IV does support cell growth in valine minimal medium. Rate equations were derived for AHAS III and AHAS IV reaction in crude extracts and for partially purified AHAS IV. Values of kinetic constants in these equations were determined in order to model a probable reaction mechanism. Computer modeling of initial velocity reactions at physiological substrate concentrations simulated consequences of valine-inhibition and revealed that AHAS IV synthesized AHB at a maximal rate over four times faster than AHAS III under these conditions. The simulations predicted that cells depending upon AHAS III for growth in valine minimal medium would accumulate higher levels of 2-ketobutyrate than cells using AHAS IV. Experiments on growth inhibition by valine revealed more than a five-fold difference in 2-ketobutyrate accumulation, thus confirming these predictions. These data support the hypothesis that valine inhibition of growth is a consequence of 2-ketobutyrate accumulation to toxic levels. We propose that the valine-inhibited AHAS IV activity prevents growth inhibition by keeping 2-ketobutyrate accumulation to a lower level than resulting from AHAS III activity.     

The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow substrate specificity compared to the herpes simplex virus type 1 thymidine kinase

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK- cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK+ phenotype on 143B TK- cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK- cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N-, a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2'-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.