Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing.

The colicin A lysis protein (Cal) is required for the release of colicin A to the medium by producing bacteria. This protein is produced in a precursor form that contains a cysteine at the cleavage site (-Leu-Ala-Ala-Cys). The precursor must be modified by the addition of lipid before it can be processed. The maturation is prevented by globomycin, an inhibitor of signal peptidase II. Using oligonucleotide-directed mutagenesis, the alanine and cystein residues in the -1 and +1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs. Both substitutions prevented the normal modification and cleavage of the protein. The marked activation of the outer membrane detergent-resistant phospholipase A observed with wild-type Cal was not observed with the Cal mutants. Both Cal mutants were also defective for the secretion of colicin A. In one mutant, the signal peptide appeared to be cleaved off by an alternative pathway involving signal peptidase I. Electron microscope studies with immunogold labeling of colicin A on cryosections of pldA and cal mutant cells indicated that the colicin remains in the cytoplasm and is not transferred to the periplasmic space. These results demonstrate that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of colicin A.     

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors.

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.     

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.     

Loss of Multi-Epitope Specificity in Memory CD4+ T Cell Responses to B. Pertussis with Age

Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4⁺ T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4⁺ T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4⁺ T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.     

Applications of a new subspace clustering algorithm (COSA) in medical systems biology

A novel clustering approach named Clustering Objects on Subsets of Attributes (COSA) has been proposed (Friedman and Meulman, (2004). Clustering objects on subsets of attributes. J. R. Statist. Soc. B 66, 1–25.) for unsupervised analysis of complex data sets. We demonstrate its usefulness in medical systems biology studies. Examples of metabolomics analyses are described as well as the unsupervised clustering based on the study of disease pathology and intervention effects in rats and humans. In comparison to principal components analysis and hierarchical clustering based on Euclidean distance, COSA shows an enhanced capability to trace partial similarities in groups of objects enabling a new discovery approach in systems biology as well as offering a unique approach to reveal common denominators of complex multi-factorial diseases in animal and human studies. Doris Damian, Matej Orešič, and Elwin Verheij contributed equally to this work.     

Interfacial binding of cutinase rather than its catalytic activity determines the steady state interfacial tension during oil drop lipid hydrolysis

Hydrolysis of triglycerides by cutinase from Fusarium solani pisi causes in oil drop tensiometer experiments a decrease of the interfacial tension. A series of cutinase variants with amino acid substitutions at its molecular surface yielded different values of the steady state interfacial tension. This tension value poorly correlated with the specific activity as such nor with the total activity (defined as the specific activity multiplied by the amount of enzyme bound) of the cutinase variants. Moreover, it appeared that at activity levels above 15% of that of wild type cutinase the contribution of hydrolysis to the decrease of the tension is saturating. A clear positive correlation was found between the interfacial tension plateau value and the interfacial binding of cutinase, as determined with attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR). These results indicate that the interfacial steady state level is not determined by the rate of hydrolysis, but mainly by the interfacial binding of cutinase.     

Control of the chemical step by leucine-31 of pancreatic phospholipase A2

A well-defined region of pancreatic and other secreted phospholipase A2 (PLA2), which we call the i-face, makes a molecular contact with the interface to facilitate and control the events and processivity of the interfacial catalytic turnover cycles. The structural features of the i-face and its allosteric relationship to the active site remain to be identified. As a part of the calcium binding (26-34) loop, Leu-31 is located on the surface near the substrate binding slot of PLA2. Analysis of the primary rate and equilibrium parameters of the Leu-31 substitution mutants of the pig pancreatic PLA2 shows that the only significant effect of the substitution is to impair the chemical step at the zwitterionic interface in the presence of added NaCl, and only a modest effect is seen on kcat at the anionic interface. Leu-31 substitutions have little effect on the binding of the enzyme to the interface; the affinity for certain substrate mimics is modestly influenced in W3F, L31W double mutant. The fluorescence emission results with the double mutant show that the microenvironment of Trp-31 is qualitatively different at the zwitterionic versus anionic interfaces. At both of the interfaces Trp-31 is not shielded from the bulk aqueous environment as it remains readily accessible to acrylamide and water. The NaCl-induced change in the Trp-31 emission spectrum of the double mutant on the zwitterionic interface is similar to that seen on the binding to the anionic interface. Together, the kinetic and spectroscopic results show that the form of PLA2 at the zwitterionic interface (Ez) is distinguishably different from the catalytically more efficient form at the anionic interface (Ea). This finding provides a structural basis for the two-state model for kcat activation by the anionic interface. In conjunction with earlier results we suggest that neutralization of certain cationic residues of PLA2 exerts a control on the calcium loop through residue 31.     

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.