Platelet-activating factor acetylhydrolases in health and disease

The platelet-activating factor (PAF) acetylhydrolases catalyze hydrolysis of the sn-2 ester bond of PAF and related pro-inflammatory phospholipids and thus attenuate their bioactivity. One secreted (plasma) and four intracellular isozymes have been described. The intracellular isozymes are distinguished by differences in primary sequence, tissue localization, subunit composition, and substrate preferences. The most thoroughly characterized intracellular isoform, Ib, is a G-protein-like complex with two catalytic subunits (alpha1 and alpha2) and a regulatory beta subunit. The beta subunit is a product of the LIS1 gene, mutations of which cause Miller-Dieker lissencephaly. Isoform II is a single polypeptide that is homologous to the plasma PAF acetylhydrolase and has antioxidant activity in several systems. Plasma PAF acetylhydrolase is also a single polypeptide with a catalytic triad of amino acids that is characteristic of the alpha/beta hydrolases. Deficiency of this enzyme has been associated with a number of pathologies. The most common inactivating mutation, V279F, is found in >30% of randomly surveyed Japanese subjects (4% homozygous, 27% heterozygous). The prevalence of the mutant allele is significantly greater in patients with asthma, stroke, myocardial infarction, brain hemorrhage, and nonfamilial cardiomyopathy. Preclinical studies have demonstrated that recombinant plasma PAF acetylhydrolase can prevent or attenuate pathologic inflammation in a number of animal models. In addition, preliminary clinical results suggest that the recombinant enzyme may have pharmacologic potential in human inflammatory disease as well. These observations underscore the physiological importance of the PAF acetylhydrolases and point toward new approaches for controlling pathologic inflammation.     

Platelet-activating factor acetylhydrolase isoforms I and II in human uterus. Comparisons with pregnant uterus and myoma

The concentrations of platelet-activating factor (PAF) that possesses the ability to stimulate myometrial contraction are partially regulated by intracellular type of platelet-activating factor acetylhydrolase (PAF-AH) in many tissues. Tissue cytosol contains at least two intracellular PAF-AH, isoforms I and II. To examine the relationship between the activity and isoforms of intracellular PAF-AH in human uterine myometrium and myoma, we assayed the PAF-AH activity and identified the PAF-AH isoforms I and II by Western blot analysis. The intense bands of the alpha2 and ss subunits of PAF-AH isoform I were detected in nonpregnant uterus; however, the specific bands of the alpha1 subunit of PAF-AH isoform I and the PAF-AH isoform II were extremely weak. The levels of the alpha2 and ss subunits and PAF-AH activity in pregnant uterus (37-39 wk gestation) were significantly lower than those in nonpregnant uterus. On the other hand, the level of ss subunit and the PAF-AH activity in myoma were significantly higher than those in nonpregnant uterus. No significant difference was found in the expression of the PAF-AH isoform II among three tissues. These results indicate that the change in the PAF-AH activity observed in pregnant uterus and myoma are due to the lower or higher protein expression of the PAF-AH isoform I, especially the alpha2 and/or ss subunits. The decrease of the uterine PAF-AH activity in the late stage of pregnancy may facilitate the action of PAF to stimulate myometrial contraction.     

Anti-apoptotic actions of the platelet-activating factor acetylhydrolase I alpha2 catalytic subunit

Platelet-activating factor (PAF) is an important mediator of cell loss following diverse pathophysiological challenges, but the manner in which PAF transduces death is not clear. Both PAF receptor-dependent and -independent pathways are implicated. In this study, we show that extracellular PAF can be internalized through PAF receptor-independent mechanisms and can initiate caspase-3-dependent apoptosis when cytosolic concentrations are elevated by approximately 15 pM/cell for 60 min. Reducing cytosolic PAF to less than 10 pM/cell terminates apoptotic signaling. By pharmacological inhibition of PAF acetylhydrolase I and II (PAF-AH) activity and down-regulation of PAF-AH I catalytic subunits by RNA interference, we show that the PAF receptor-independent death pathway is regulated by PAF-AH I and, to a lesser extent, by PAF-AH II. Moreover, the anti-apoptotic actions of PAF-AH I are subunit-specific. PAF-AH I alpha1 regulates intracellular PAF concentrations under normal physiological conditions, but expression is not sufficient to reduce an acute rise in intracellular PAF levels. PAF-AH I alpha2 expression is induced when cells are deprived of serum or exposed to apoptogenic PAF concentrations limiting the duration of pathological cytosolic PAF accumulation. To block PAF receptor-independent death pathway, we screened a panel of PAF antagonists (CV-3988, CV-6209, BN 52021, and FR 49175). BN 52021 and FR 49175 accelerated PAF hydrolysis and inhibited PAF-mediated caspase 3 activation. Both antagonists act indirectly to promote PAF-AH I alpha2 homodimer activity by reducing PAF-AH I alpha1 expression. These findings identify PAF-AH I alpha2 as a potent anti-apoptotic protein and describe a new means of pharmacologically targeting PAF-AH I to inhibit PAF-mediated cell death.     

Regulating inflammation through the anti-inflammatory enzyme platelet-activating factor-acetylhydrolase

Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.     

Biochemical characterization of various catalytic complexes of the brain platelet-activating factor acetylhydrolase.

Brain intracellular platelet-activating factor acetylhydrolase (PAF-AH) isoform I is a member of a family of complex enzymes composed of mutually homologous alpha(1) and alpha(2) subunits, both of which account for catalytic activity, and the beta subunit. We previously demonstrated that the expression of one catalytic subunit, alpha(1), is developmentally regulated, resulting in a switching of the catalytic complex from alpha(1)/alpha(2) to alpha(2)/alpha(2) during brain development (Manya, H., Aoki, J., Watanabe, M., Adachi, T., Asou, H., Inoue, Y., Arai, H., and Inoue, K. (1998) J. Biol. Chem. 273, 18567-18572). In this study, we explored the biochemical differences in three possible catalytic dimers, alpha(1)/alpha(1), alpha(1)/alpha(2), and alpha(2)/alpha(2). The alpha(2)/alpha(2) homodimer exhibited different substrate specificity from the alpha(1)/alpha(1) homodimer and the alpha(1)/alpha(2) heterodimer, both of which showed similar substrate specificity. The alpha(2)/alpha(2) homodimer hydrolyzed PAF and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylethanolamine (AAGPE) most efficiently among 1-O-alkyl-2-acetyl-phospholipids. In contrast, both alpha(1)/alpha(1) and alpha(1)/alpha(2) hydrolyzed 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoric acid more efficiently than PAF. AAGPE was the poorest substrate for these enzymes. The beta subunit bound to all three catalytic dimers but modulated the enzyme activity in a catalytic dimer composition-dependent manner. The beta subunit strongly accelerated the enzyme activity of the alpha(2)/alpha(2) homodimer but rather suppressed the activity of the alpha(1)/alpha(1) homodimer and had little effect on that of the alpha(1)/alpha(2) heterodimer. The (His(149) to Arg) mutant beta, which has been recently identified in isolated lissencephaly sequence patients, lost the ability to either associate with the catalytic complexes or modulate their enzyme activity. The enzyme activity of PAF-AH isoform I may be regulated in multiple ways by switching the composition of the catalytic subunit and by manipulating the beta subunit.     

Direct association of LIS1, the lissencephaly gene product, with a mammalian homologue of a fungal nuclear distribution protein, rNUDE

LIS1 is a product of the causative gene for type I lissencephaly characterized by a smooth brain surface due to a defect in neuronal migration during brain development and a regulatory subunit of platelet-activating factor acetylhydrolase (PAF-AH). It is also a mammalian homologue of the fungal nuclear distribution (nud) gene, nudF, which controls the migration of fungal nuclei. Using the two-hybrid system, we identified a novel LIS1-interacting protein, rat NUDE (rNUDE), and found that it is a mammalian homologue of another fungal nud gene product, NUDE, and Xenopus mitotic phosphoprotein 43 which is phosphorylated in a cell cycle-dependent manner. rNUDE and the catalytic subunits of PAF-AH interact with the N- and C-termini of LIS1, respectively. However, these proteins, instead of simultaneously binding to LIS1, appeared to bind to LIS1 in a competitive manner. These results suggest that LIS1 functions in nuclear migration by interacting with multiple intracellular proteins in mammals.     

Phosphorylation of casein kinase II

Casein kinase II from rabbit reticulocytes is a tetramer with an alpha,alpha' beta 2 or alpha 2 beta 2 structure; the alpha subunits contain the catalytic activity, and the beta subunits are regulatory in nature [Traugh, J.A., Lin, W. J., Takada-Axelrod, F., & Tuazon, P. T. (1990) Adv. Second Messenger Phosphoprotein Res. 24, 224-229]. When casein kinase II is isolated from rabbit reticulocytes by a rapid two-step purification of the enzyme, both the alpha and beta subunits are phosphorylated to a significant extent. In vitro, purified casein kinase II undergoes autophosphorylation on the beta subunit. In the presence of polylysine and polyarginine, phosphorylation of the beta subunits is inhibited, and the alpha subunits (alpha and alpha') become autophosphorylated. The effectiveness of polylysine coincides with the molecular weight. With basic proteins, including a number of histones and protamine, autophosphorylation of both subunits is observed. With histones, autophosphorylation of each subunit can be greater than that observed with the autophosphorylated enzyme alone or with a basic polypeptide. Thus, the potential exists for modulatory proteins to alter the autophosphorylation state of casein kinase II. Taken together, the data suggest that phosphorylation of the alpha subunit of casein kinase II in vivo may be due to an unidentified protein kinase or due to autophosphorylation. In the latter instance, casein kinase II could be transiently associated with specific intracellular compounds, such as basic proteins, with a resultant stimulation of autophosphorylation.     

Quaternary and domain structure of glycoprotein processing glucosidase II

Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, alpha and beta. The alpha subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic beta subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the beta subunit in glucosidase II activity has not been established. Furthermore, a direct interaction between alpha and beta subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (alpha(1)beta(1)) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the alpha(1)beta(1) complex was confirmed by its high susceptibility to proteases. The beta subunit could be proteolytically removed from the alpha(1)beta(1) complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the alpha subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase II resides in a globular domain of the alpha subunit, which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the beta subunit may retain the complete alpha(1)beta(1) complex in the ER.     

Platelet-activating factor acetylhydrolases: An overview and update

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique members of the phospholipase A₂ family that can hydrolyze the acetyl group of PAF, a signaling phospholipid that has roles in diverse (patho)physiological processes. Three types of PAF-AH have been identified in mammals, one plasma type and two intracellular types [PAF-AH (I) and PAF-AH (II)]. Plasma PAF-AH and PAF-AH (II) are monomeric enzymes that are structurally similar, while PAF-AH (I) is a multimeric enzyme with no homology to other PAF-AHs. PAF-AH (I) shows a strong preference for an acetyl group, whereas plasma PAF-AH and PAF-AH (II) also hydrolyze phospholipids with oxidatively modified fatty acids. Plasma PAF-AH has been implicated in several diseases including cardiovascular disease. PAF-AH (I) is required for spermatogenesis and is increasingly recognized as an oncogenic factor. PAF-AH (II) was recently shown to act as a bioactive lipid-producing enzyme in mast cells and thus could be a drug target for allergic diseases. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

Coupling PAF signaling to dynein regulation: structure of LIS1 in complex with PAF-acetylhydrolase

Mutations in the LIS1 gene cause lissencephaly, a human neuronal migration disorder. LIS1 binds dynein and the dynein-associated proteins Nde1 (formerly known as NudE), Ndel1 (formerly known as NUDEL), and CLIP-170, as well as the catalytic alpha dimers of brain cytosolic platelet activating factor acetylhydrolase (PAF-AH). The mechanism coupling the two diverse regulatory pathways remains unknown. We report the structure of LIS1 in complex with the alpha2/alpha2 PAF-AH homodimer. One LIS1 homodimer binds symmetrically to one alpha2/alpha2 homodimer via the highly conserved top faces of the LIS1 beta propellers. The same surface of LIS1 contains sites of mutations causing lissencephaly and overlaps with a putative dynein binding surface. Ndel1 competes with the alpha2/alpha2 homodimer for LIS1, but the interaction is complex and requires both the N- and C-terminal domains of LIS1. Our data suggest that the LIS1 molecule undergoes major conformational rearrangement when switching from a complex with the acetylhydrolase to the one with Ndel1.     

Coexpression of both alpha and beta subunits is required for assembly of regulated casein kinase II

Casein kinase II is an ubiquitous serine-threonine kinase whose functional significance and regulation in the living cell are not clearly understood. The native enzyme has an oligomeric structure made of two different (alpha and beta) subunits with an alpha 2 beta 2 stoichiometry. To facilitate the study of the structure-activity relationship of the kinase, we have expressed its isolated subunits in a baculovirus-directed insect cell expression system. The resulting isolated recombinant alpha subunit exhibited a protein kinase catalytic activity, in agreement with previous observations [Cochet, C., & Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406]. Coinfection of insect cells with recombinant viruses encoding the two kinase subunits resulted in the biosynthesis of a functional enzyme. Active recombinant oligomeric kinase was purified to near homogeneity with a yield of about 5 mg of enzymatic protein per liter, showing that, in coinfected host cells, synthesis was followed, at least in part, by recombination of the two subunits with an alpha 2 beta 2 stoichiometry. The catalytic properties of the recombinant enzyme appeared highly similar to those previously observed for casein kinase II purified from bovine tissue. Access to the isolated subunits and to their alpha 2 beta 2 association disclosed that the beta subunit is required for optimal catalytic activity of the kinase. In addition, the beta subunit is suggested to play an essential role in the regulated activity of the native casein kinase II. This is clearly illustrated by the observation of the effect of spermine which requires the presence of the beta subunit to stimulate the kinase catalytic activity which is borne by the alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle.

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.     

Biosynthesis and processing of the alpha subunit of the voltage-sensitive sodium channel in rat brain neurons

The sodium channel from rat brain is a complex of alpha (260 kd), beta 1 (36 kd), and beta 2 (33 kd) subunits. The alpha and beta 2 subunits are linked by disulfide bonds. The earliest biosynthetic precursor of the alpha subunit is a 203 kd core polypeptide with sufficient high-mannose carbohydrate chains to increase its apparent size to 224 kd. It is processed to 224 kd and 249 kd precursor forms containing complex carbohydrate chains before it achieves the mature size of 260 kd. Most newly synthesized alpha subunits are not disulfide-linked to beta 2 subunits, but remain as a metabolically stable pool of intracellular subunits. alpha subunits disulfide-linked to beta 2 are found preferentially at the cell surface. A possible role for this intracellular pool as a rate-limiting step in the regulation of the cell surface density and localization of sodium channels in developing neurons is proposed.     

Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme

Platelet-activating factor acetylhydrolases (PAF-AHs) are a group of enzymes that hydrolyze the sn-2 acetyl ester of PAF (phospholipase A(2) activity) but not phospholipids with two long fatty acyl groups. Our previous studies showed that membrane-bound human plasma PAF-AH (pPAF-AH) accesses its substrate only from the aqueous phase, which raises the possibility that this enzyme can hydrolyze a variety of lipid esters that are partially soluble in the aqueous phase. Here we show that pPAF-AH has broad substrate specificity in that it hydrolyzes short-chain diacylglycerols, triacylglycerols, and acetylated alkanols, and displays phospholipase A(1) activity. On the basis of all of the substrate specificity results, it appears that the minimal structural requirement for a good pPAF-AH substrate is the portion of a glyceride derivative that includes an sn-2 ester and a reasonably hydrophobic chain in the position occupied by the sn-1 chain. In vivo, pPAF-AH is bound to high and low density lipoproteins, and we show that the apparent maximal velocity for this enzyme is not influenced by lipoprotein binding and that the enzyme hydrolyzes tributyroylglycerol as well as the recombinant pPAF-AH does. Broad substrate specificity is also observed for the structurally homologous PAF-AH which occurs intracellularly [PAF-AH(II)] as well as for the PAF-AH from the lower eukaryote Physarum polycephalum although pPAF-AH and PAF-AH(II) tolerate the removal of the sn-3 headgroup better than the PAF-AH from P. polycephalum does. In contrast, the intracellular PAF-AH found in mammalian brain [PAF-AH(Ib) alpha 1/alpha 1 and alpha 2/alpha 2 homodimers] is more selectively operative on compounds with a short acetyl chain although this enzyme also displays significant phospholipase A(1) activity.     

The functional implications of the dimerization of the catalytic subunits of the mammalian brain platelet-activating factor acetylhydrolase (Ib)

The mammalian brain contains significant amounts of the cytosolic isoform Ib of the platelet-activating factor acetylhydrolase (PAF-AH), a unique type of PLA2. This oligomeric protein complex contains three types of subunits: two homologous (63% identity) 26 kDa catalytic subunits (alpha(1) and alpha(2)) which harbor all the PAF-AH activity, and the 45 kDa beta-subunit (LIS1), a product of the causal gene for Miller-Dieker lissencephaly. During fetal development, the preferentially expressed alpha(1)-subunit forms a homodimer, which binds to a homodimer of LIS1, whereas in adult organisms alpha(1)/alpha(2) and alpha(2)/alpha(2) dimers, also bound to dimeric LIS1, are the prevailing species. The consequences of this "switching" are not understood, but appear to be of physiological significance. The alpha(1)- and alpha(2)-subunits readily associate with very high affinity to form homodimers. The nature of the interface has been elucidated by the 1.7 A resolution crystal structure of the alpha(1)/alpha(1) homodimer (Ho et al., 1997). Here, we examined the functional consequences of the dimerization in both types of alpha-subunits. We obtained monomeric protein in the presence of high concentrations (>50 mM) of Ca2+ ions, and we show that it is catalytically inactive and less stable than the wild type. We further show that Arg29 and Arg22 in one monomer contribute to the catalytic competence of the active site across the dimer interface, and complement the catalytic triad of Ser47, Asp192 and His195, in the second monomer. These results indicate that the brain PAF-acetylhydrolase is a unique PLA2 in which dimerization is essential for both stability and catalytic activity.     

The sodium pump needs its beta subunit

The sodium pump Na,K-ATPase, located in the plasma membrane of all animal cells, is a member of a family of ion-translocating ATPases that share highly homologous catalytic subunits. In this family, only Na,K-ATPase has been established to be a heterodimer of catalytic (alpha) and glycoprotein (beta) subunits. The beta subunit has not been associated with the pump's transport or enzymatic activity, and its role in Na,K-ATPase function has been, until recently, a puzzle. In this review we describe what is known about the structure of beta and summarize evidence that expression of both alpha and beta subunits is required for Na,K-ATPase activity, that inhibition of glycosylation causes a decrease in accumulation of both alpha and beta subunits, and we provide evidence that pretranslational up-regulation of beta alone can lead to increased abundance of sodium pumps. These findings are all consistent with the hypothesis that the beta subunit regulates, through assembly of alpha beta heterodimers, the number of sodium pumps transported to the plasma membrane.     

Plasma platelet activating factor-acetylhydrolase (PAF-AH)

The platelet-activating factor-acetylhydrolase (PAF-AH) is an enzyme which catalyzes the hydrolysis of acetyl ester at the sn-2 position of PAF. The family of PAF-AHs consists of two intracellular isoforms (Ib and II), and one secreted isoform (plasma). These PAF-AHs show different biochemical characteristics and molecular structures. Plasma PAF-AH and intracellular isoform, II degrade not only PAF but also oxidatively fragmented phospholipids with potent biological activities. Among these PAF-AHs, plasma PAF-AH has been the target of many clinical studies in inflammatory diseases, such as asthma, sepsis, and vascular diseases, because the plasma PAF-AH activity in the patients with these diseases is altered when compared with normal individuals. Finding a genetic deficiency in the plasma PAF-AH opened the gate in elucidating the protecting role of this enzyme in inflammatory diseases. The most common loss-of-function mutation, V279F, is found in more than 30% of Japanese subjects (4% homozygous, 27% heterozygous). This single nucleotide polymorphism in plasma PAF-AH and the resulting enzymatic deficiency is thought to be a genetic risk factor in various inflammatory diseases in Japanese subjects. Administration of recombinant plasma PAF-AH or transfer of the plasma PAF-AH gene improves pathology in animal models. Therefore, substitution of plasma PAF-AH would be an effective in the treatment of the patients with the inflammatory diseases and a novel clinical approach. In addition, the detection of polymorphisms in the plasma PAF-AH gene and abnormalities in enzyme activity would be beneficial in the diagnosis of the inflammatory diseases.     

Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.     

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.