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Human phosphodiesterase 4A: characterization of full-length and truncated enzymes expressed in COS cells.

ABSTRACT: The type 4 phosphodiesterase (PDE) family comprises four enzymes (4A, 4B, 4C and 4D) that are characterized by their specificity for cAMP and selective inhibition by the anti-depressant drug rolipram (4-[3-(cyclopentoxyl)-4-methoxyphenyl]2-pyrrolidone). In common with other PDEs, they consist of a central conserved domain associated with catalytic activity in addition to two N-terminal upstream conserved regions (UCR1 and UCR2) that are unique to the type 4 enzymes. We have isolated a 2 kb cDNA encoding a full-length type 4A PDE{HSPDE4A4B[Bolger, Michaeli, Martins, St.John, Steiner, Rodgers, Riggs, Wigler and Ferguson (1993) Mol. Cell. Biol. 13, 6558-6571]} from a human frontal cortex cDNA library. Northern blot analysis showed that the major PDE4A mRNA of 4.5 kb was widely distributed in different human tissues. The recombinant PDE4A expressed in COS cells had a molecular mass of approx. 117 kDa as revealed by SDS/PAGE/Western blotting with a PDE4A-specific antibody and was specific for cAMP with a Km of 4.8 microM. The enzyme activity was potently inhibited by R-rolipram (IC50 204 nM) and showed a 2.7-fold stereoselectivity over the S enantiomer. Analysis of the kinetics of inhibition indicated that R-rolipram did not behave as a simple competitive inhibitor. Dixon replots suggested that there was more than one mode of interaction consistent with the detection in the enzyme of a high-affinity binding site for R-rolipram with a Kd of 2.3 nM. Truncation of the PDE4A enzyme by deletion mutagenesis showed that neither of the UCRs was required for catalytic activity and identified an approx. 71 kDa core enzyme with a K(m) for cAMP of 3.3 microM. In contrast with the full-length PDE4A, R-rolipram behaved as a simple competitive inhibitor of this form of the enzyme with decreased potency (IC50 1022 nM) and no stereoselectivity. In addition, no high-affinity rolipram-binding site was detected in the truncated enzyme, indicating that this interaction involves sequences upstream of the catalytic domain of the enzyme.

SUBMITTER: Owens RJ

PROVIDER: S-EPMC1218636 | biostudies-other | 1997 Aug

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: ?-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing ?-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of ?-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel ?-1,3-glucanases with high catalytic efficiency and stability is of particular interest. RESULTS: A ?-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the ?-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes ?-1,3-glucan. Insoluble ?-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains. CONCLUSIONS: This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of ?-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble ?-1,3-glucan, and could be classified into CBM families.

Project description:Accumulation of amyloid-? peptides is a dominant feature in the pathogenesis of Alzheimer's disease; however, it is not clear how individual amyloid-? species accumulate and affect other neuropathological and clinical features in the disease. Thus, we compared the accumulation of N-terminally truncated amyloid-? and full-length amyloid-?, depending on disease stage as well as brain area, and determined how these amyloid-? species respectively correlate with clinicopathological features of Alzheimer's disease. To this end, the amounts of amyloid-? species and other proteins related to amyloid-? metabolism or Alzheimer's disease were quantified by enzyme-linked immunosorbent assays (ELISA) or theoretically calculated in 12 brain regions, including neocortical, limbic and subcortical areas from Alzheimer's disease cases (n = 19), neurologically normal elderly without amyloid-? accumulation (normal ageing, n = 13), and neurologically normal elderly with cortical amyloid-? accumulation (pathological ageing, n = 15). We observed that N-terminally truncated amyloid-?42 and full-length amyloid-?42 accumulations distributed differently across disease stages and brain areas, while N-terminally truncated amyloid-?40 and full-length amyloid-?40 accumulation showed an almost identical distribution pattern. Cortical N-terminally truncated amyloid-?42 accumulation was increased in Alzheimer's disease compared to pathological ageing, whereas cortical full-length amyloid-?42 accumulation was comparable between Alzheimer's disease and pathological ageing. Moreover, N-terminally truncated amyloid-?42 were more likely to accumulate more in specific brain areas, especially some limbic areas, while full-length amyloid-?42 tended to accumulate more in several neocortical areas, including frontal cortices. Immunoprecipitation followed by mass spectrometry analysis showed that several N-terminally truncated amyloid-?42 species, represented by pyroglutamylated amyloid-?11-42, were enriched in these areas, consistent with ELISA results. N-terminally truncated amyloid-?42 accumulation showed significant regional association with BACE1 and neprilysin, but not PSD95 that regionally associated with full-length amyloid-?42 accumulation. Interestingly, accumulations of tau and to a greater extent apolipoprotein E (apoE, encoded by APOE) were more strongly correlated with N-terminally truncated amyloid-?42 accumulation than those of other amyloid-? species across brain areas and disease stages. Consistently, immunohistochemical staining and in vitro binding assays showed that apoE co-localized and bound more strongly with pyroglutamylated amyloid-?11-x fibrils than full-length amyloid-? fibrils. Retrospective review of clinical records showed that accumulation of N-terminally truncated amyloid-?42 in cortical areas was associated with disease onset, duration and cognitive scores. Collectively, N-terminally truncated amyloid-?42 species have spatiotemporal accumulation patterns distinct from full-length amyloid-?42, likely due to different mechanisms governing their accumulations in the brain. These truncated amyloid-? species could play critical roles in the disease by linking other clinicopathological features of Alzheimer's disease.

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Human phosphodiesterase 4A: characterization of full-length and truncated enzymes expressed in COS cells.

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