Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones.

Several proteases require propeptides for the correct folding of their own protease domain. We have recently found that the propeptide from a thermostable subtilisin homolog aqualysin I can refold subtilisin BPN' when added in trans. Here, we constructed chimeric genes with subtilisin E and aqualysin I to attempt the in cis folding of subtilisin E by means of the propeptide of aqualysin I. Our results indicate that the propeptide of aqualysin I can to some extent chaperone the intramolecular folding of the denatured subtilisin E. These results suggest that propeptides in the subtilisin family, despite their sequence diversity, have similar functions. Further, some enzymatic properties of some chimeras in which the subtilisin mature domain is partly swapped with that of aqualysin I were shown to be more similar to those of aqualysin I.

Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin.

Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.

Folding pathway mediated by an intramolecular chaperone: the structural and functional characterization of the aqualysin I propeptide.

Aqualysin I, a thermostable homologue of subtilisin, requires its propeptide (ProA) to function as an intramolecular chaperone (IMC). To decipher the mechanisms through which propeptides can initiate protein folding, we characterized ProA in terms of its sequence, structure and function. Our results show that, in contrast to ProS (propeptide of subtilisin), ProA can fold spontaneously, reversibly and cooperatively into a stable monomeric alpha-beta conformation, even when isolated from its cognate protease-domain. ProA displays an indiscernible amount of tertiary structure with a considerable solvent-accessible hydrophobic surface, but is not a classical molten-globule folding intermediate. Moreover, despite showing only 21 % sequence identity with ProS, ProA can not only inhibit enzymatic activity with a magnitude tenfold greater than ProS, but can also chaperone subtilisin folding, albeit with a lower efficiency. The structure of ProA complexed with subtilisin is different from that of isolated ProA. Hence, additional interactions seem necessary to induce ProA into a compact structure. Our results also suggest that: (a) propeptides that are potent inhibitors are not necessarily better IMCs; (b) propeptides within the subtilase family appear polymorphic and; (c) the intrinsic instability within propeptides may be necessary for rapid activation of the cognate protein.

Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli.

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.

Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses.

We studied the response of each ascorbate peroxidase (APX) isoenzyme in spinach leaves under stress conditions imposed by high light intensity, drought, salinity, and applications of methyl viologen and abscisic acid. The steady-state transcript level of cytosolic APX remarkably increased in response to high-light stress and methyl viologen treatment, but not in response to the other stress treatments. The transcript levels of the chloroplastic (stromal and thylakoid-bound) and microbody-bound APX isoenzymes were not changed in response to any of the stress treatments. To explore the responses of the APX isoenzymes to photooxidative stress, the levels of transcript and protein and activities of each isoenzyme were studied during high-light stress and following its recovery. The cytosolic APX activity increased in parallel with transcript abundance during high-light stress, while the protein level was not altered. The other isoenzymes showed no significant changes in transcript and protein levels and activities, except for the gradual decrease in chloroplastic isoenzyme activities.

Exacerbation of herpes simplex encephalitis after successful treatment with acyclovir.

Herpes simplex encephalitis (HSE) in children sometimes exacerbates after successful treatment; yet the frequency, etiology, and clinical features of exacerbation remain unclear. We report data for 27 children with HSE confirmed by polymerase chain reaction (PCR) analysis; all were successfully treated with acyclovir, but 7 (26%) had a relapse of encephalitic illness. In 2 of those 7, serial examination with a PCR assay showed that herpes simplex virus (HSV) DNA reappeared temporarily in the cerebrospinal fluid (CSF). For 5 of the 7 patients, a second course of acyclovir therapy was effective. Coxsackievirus A9 was isolated from CSF of 1 case patient during subsequent exacerbation. The total dose during initial acyclovir therapy was significantly lower in the relapse group than in the control group (P=.027). In conclusion, exacerbation of HSE in children may be more common than previously recognized. It is suggested that the replication of HSV or another viral pathogen caused a second encephalitic illness (HSE) in some cases.

Prevalence of maternal cytomegalovirus (CMV) antibody and detection of CMV DNA in amniotic fluid.

The prevalence of cytomegalovirus (CMV) IgG antibody was determined in 573 pregnant women in the first trimester. The overall prevalence of CMV IgG antibody was 77.5%. The rate of seropositivity was 67.7% in women < 25 yr, and increased with age to 85.7% in women 40 yr. These results imply that young women in Japan are at increased risk for primary CMV infection during pregnancy and that congenital CMV infection rates might increase in the future. We conducted a prospective study of 75 pregnant women who underwent amniocentesis for various indications to determine if CMV DNA could be detected in the amniotic fluid. None had symptoms associated with CMV infection, CMV IgM antibody, or seroconversion to CMV IgG antibody during pregnancy. CMV DNA was not detected in the amniotic fluid using a polymerase chain reaction assay. The 65 fetuses, including 3 sets of twins, were followed through birth. CMV DNA was not detected in urine samples obtained within the first 2 weeks of life. In conclusion, CMV DNA was not detected in the amniotic fluid of women who did not have CMV infection. These results, however, suggest that the negative predictive value of prenatal amniotic fluid analysis is high and that the presence of CMV DNA in the amniotic fluid has clinical significance for the diagnosis of congenital CMV infection if detected in pregnant women.

Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves.

We have previously shown that stromal and thylakoid-bound ascorbate peroxidase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing in the C-terminus of the isoenzymes [Ishikawa, Yoshimura, Tamoi, Takeda and Shigeoka (1997) Biochem. J. 328, 795-800]. To explore the production of mature, functional mRNA encoding chloroplast APX isoenzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analysis were performed with poly(A)+ RNA or polysomal RNA from spinach leaves. As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identified. The sAPX-I and sAPX-III mRNA species were generated through the excision of intron 11; they encoded the previously identified sAPX protein. Interestingly, the sAPX-II mRNA was generated by the insertion of intron 11 between exons 11 and 12. The use of this insertional sequence was in frame with the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the last residue of the previously identified sAPX. The recombinant novel enzyme expressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAPX-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is functional as a soluble APX in vivo. The S1 nuclease protection analysis showed that the expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions. The present results demonstrate that the expression of chloroplast APX isoenzymes is regulated by a differential splicing efficiency that is dependent on the 3'-terminal processing of ApxII, the gene encoding the chloroplast APX isoenzymes.

Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay.

To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 10(7) copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 10(3.7) copies/microg of DNA in patients with EBV-related lymphoproliferative disorders, 10(4.1) copies/microg of DNA in patients with chronic active EBV infections, and 10(2.2) copies/microg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.