The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A

We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F and Y248A mutants to find that the K(M) values were increased by 4.5-11-fold and the k(cat) values were reduced by 4.5-10.7-fold by the replacement of Tyr248 with Phe for the hydrolysis of hippuryl-L-Phe (HPA) and N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In the case of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, the K(M) value was increased by 2.5-fold, and the k(cat) was reduced by 20-fold. The replacement of Tyr248 with Ala decreased the k(cat) values by about 18- and 237-fold for HPA and ClCPL, respectively, demonstrating that the aromatic ring of Tyr248 plays a critical role in the enzymic reaction. The increases of the K(M) values were only 6- and 5-fold for HPA and ClCPL, respectively. Thus, the present study indicates clearly that Tyr248 plays an important role not only in the binding of substrate but also in the enzymic hydrolysis. The kinetic results may be rationalized by the proposition that the phenolic hydroxyl of Tyr248 forms a hydrogen bond with the zinc-bound water molecule, causing further activation of the water molecule by reducing its pK(a) value. The pH dependency study of k(cat) values and the solvent isotope effects also support the proposition. A unified catalytic mechanism is proposed that can account for the different kinetic behavior observed in the CPA-catalyzed hydrolysis of peptide and ester substrates.     

Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta

We produced mice lacking the sperm surface protein cyritestin (ADAM 3) and found mutant males are infertile. Similar to fertilin beta (ADAM 2) null sperm (C. Cho et al., 1998, Science 281, 1857-1859), cyritestin null sperm are drastically deficient in adhesion to the egg zona pellucida (0.3% of wild type) and to the egg plasma membrane (9% of wild type). Thus sperm from male mice with a gene deletion of either ADAM have a loss of adhesive function in at least two steps of fertilization. We found deletion of either ADAM gene resulted in the loss of multiple gene products. This loss of multiple gene products (sperm membrane proteins) appears to result from a novel, developmental mechanism during sperm differentiation. Because the altered sperm protein expression must be responsible for the fertilization defects, our data suggest new models for the molecular basis of the affected steps in fertilization.     

Generation of macrophages from early T progenitors in vitro

Early T progenitors in the thymus have been reported to have the capacity to develop into B cells, thymic dendritic cells, and NK cells. Here we describe conditions that induce early T progenitors to develop into macrophages. Initially, we observed that early T progenitors could be induced to develop into macrophages by cytokines produced from a thymic stromal cell line, TFGD, and later we found that the cytokine mixture of M-CSF plus IL-6 plus IL-7 also induced macrophage differentiation from pro-T cells. M-CSF by itself was unable to induce macrophage differentiation from early T progenitors. To correlate this observation with the developmental potential of early T progenitors, mouse embryonic thymocytes were sorted into four populations, pro-T1 to pro-T4, based on the expression of CD44 and CD25, and then cultured with TFGD culture supernatant. We found that pro-T1 and pro-T2 cells, but not pro-T3 and pro-T4 cells, generate macrophages. Limiting dilution analysis of the differentiation capability of sorted pro-T2 cells also confirmed that pro-T2 cells could generate macrophages. These results suggest that T cells and thymic macrophages could originate from a common intrathymic precursor.     

A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression

ST2/ST2L, a member of the IL-1R gene family, is expressed by fibroblasts, mast cells, and Th2, but not Th1, cells. It exists in both membrane-bound (ST2L) and soluble forms (ST2). Although ST2L has immunoregulatory properties, its ligand, cellular targets, and mode of action remain unclear. Using a soluble ST2-human IgG fusion protein, we demonstrated that ST2 bound to primary bone marrow-derived macrophages (BMM) and that this binding was enhanced by treatment with LPS. The sST2 treatment of BMMs inhibited production of the LPS-induced proinflammatory cytokines IL-6, IL-12, and TNF-alpha but did not alter IL-10 or NO production. Treatment of BMMs with sST2 down-regulated expression of Toll-like receptors-4 and -1 but induced nuclear translocation of NF-kappaB. Administration of sST2 in vivo after LPS challenge significantly reduced LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-alpha. Conversely, blockade of endogenous ST2 through administration of anti-ST2 Ab exacerbated the toxic effects of LPS. Thus, ST2 has anti-inflammatory properties that act directly on macrophages. We demonstrate here a novel regulatory pathway for LPS-induced shock via the ST2-Toll-like receptor 4 route. This may be of considerable therapeutic potential for reducing the severity and pathology of inflammatory diseases.     

Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases

The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.     

Central beta-amyloid peptide-induced peripheral interleukin-6 responses in mice

beta-Amyloid peptides (Abetas) share with lipopolysaccharide, a potent pro-inflammatory agent, the property of stimulating glial cells or macrophages to induce various inflammatory mediators. We recently reported that central administration of lipopolysaccharide induces peripheral interleukin-6 responses via both the central and peripheral norepinephrine system. In this study, the effect of intracerebroventricular injection of various synthetic Abetas on plasma interleukin-6 levels was examined in mice. Abeta(1-42) dose-dependently increased plasma interleukin-6 levels: 'aged' Abeta(1-42) was more effective than fresh, whereas Abeta(42-1) had no effect. 'Aged' Abeta(1-42) (205 pmol/mouse i.c.v.)-induced plasma interleukin-6 peaked at 2 h post injection, which is earlier than the peak time of the Abeta(1-42)-induced brain interleukin-6, tumor necrosis factor-alpha and interleukin-1beta levels, which was 4, 4 and 24 h, respectively. Among various peripheral organs, Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased interleukin-6 mRNA expression in lymph nodes and liver. Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased norepinephrine turnover in both hypothalamus and spleen. Either central or peripheral norepinephrine depletion effectively inhibited the Abeta(1-42)-induced peripheral interleukin-6 response. Pretreatment with prazosin (alpha(1)-adrenergic antagonist), yohimbine (alpha(2)-adrenergic antagonist), and ICI-118,551 (beta(2)-adrenergic antagonist), but not with betaxolol (beta(1)-adrenergic antagonist), inhibited Abeta(1-42)-induced plasma interleukin-6 levels. These results demonstrate that centrally administered Abeta(1-42) effectively induces the systemic interleukin-6 response which is mediated, in part, by central Abeta(1-42)-induced activation of the central and the peripheral norepinephrine systems.     

Characteristics of an alpha-galactosidase associated with grape flesh

alpha-galactosidase activity in grape flesh (Vitis venifera L. Muscat of Alexandria) was characterized by a marked increase in its activity 4 weeks after fruit bearing. After 12 weeks the specific activity of the enzyme had increased 15-fold. Several other glycosidases were measured at different stages of fruit development but none showed the increased levels of activity displayed by this alpha-galactosidase. alpha-Galactosidase activity (unit/g.fresh wt) increased by 52% during postharvest storage, whereas the unripe grape showed a "stagnancy" for 10-15 days prior to the increase. An alpha-galactosidase was partially purified ca. 103-fold from grape flesh of Vitis labruscana Honey black, by a procedure involving ammonium sulfate fractionation, Biogel P-60, melibiose-agarose, and Sephacryl S-200 chromatographic separations. The enzyme was effectively separated by affinity chromatography on melibiose-agarose, and was a monomer of 40-45 kDa as determined by SDS-PAGE and Sephacryl S-200 chromatographic analysis. The hydrolysis rate of p-nitrophenyl-alpha-D-Gal (PNP-alpha-D-Gal) was 4.2 times higher than that of PNP-beta-D-Gal, implying an apparent alpha-anomer specificity, and natural oligosaccharides such as melibiose, stachyose, and raffinose were also considerably hydrolyzed. The enzyme was active over a narrow pH range with an optimal hydrolysis of stachyose and PNP-alpha-D-Gal at pH 6.0 and 7.0, respectively. EDTA or 1,10-phenanthroline did not substantially affect enzyme activity.