Expression of functional mammal flavocytochrome b(558) in yeast: comparison with improved insect cell system.

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b(558) (Cytb(558)) which consists of a heterodimer of the gp91(phox) and p22(phox) subunits. The Cytb(558) is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb(558) (rCytb(558)). We expressed the gp91(phox) and p22(phox) subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91(phox) undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCytb(558) displayed comparable spectral properties to neutrophil Cytb(558). In contrast to rCytb(558) produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein-protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.

Neutrophils stimulated by apolipoprotein(a) generate fragments that are stronger inhibitors of plasmin formation than apo(a).

Apolipoprotein(a), the plasminogen-like component of lipoprotein(a), is transformed into fragments by polymorphonuclear neutrophils (PMNs) elastase. Since stimulated PMNs express urokinase-type plasminogen activator (uPA), we sought to investigate the relevance of apo(a) fragmentation on plasminogen activation by neutrophils. Freshly isolated human PMNs stimulated by a 10 kringle recombinant apo(a), r-apo(a), activate plasminogen in a specific and saturable manner (Km = 476 +/- 42 nM, Vmax = 896 +/- 18 pmol min(-1)). This activation is prevented by amiloride, an inhibitor of u-PA, and epsilon-aminocaproic acid, epsilon-ACA, a lysine analogue that blocks plasminogen binding to PMNs. Stimulation of PMNs by apo(a) results in the formation of elastase-derived apo(a) fragments. These fragments produce a concentration-dependent decrease in the formation of plasmin. Addition of elastase inhibitors to PMNs prevented degradation of apo(a) and partially restored the formation of plasmin. In a similar manner, isolated r-apo(a) fragments were able to produce a 100% decrease in plasmin generation as compared to intact r-apo(a). These data indicate that apo(a) fragments produce a more pronounced inhibition in the generation of cell-bound plasmin by uPA than the parent apo(a). These effects of apo(a) and its fragments were neutralised by a monoclonal antibody directed against the lysine-binding site of apo(a). This mechanism may be of biological relevance to the effects of Lp(a) in conditions where PMNs accumulate and release elastase, i.e. thrombus lysis and inflammatory lesions.

Interaction of fibrin(ogen) with apolipoprotein(a): further characterization and identification of a novel lysine-dependent apolipoprotein(a)-binding site within the gamma chain 287-411 region.

Interaction of lipoprotein(a) with fibrin associated with atherosclerotic lesions promotes its accumulation in the lesions, thereby contributing to the development of atherothrombosis. Numerous studies revealed that this interaction occurs through the apolipoprotein(a) [apo(a)] component of lipoprotein(a) and COOH-terminal Lys residues generated by partial degradation of fibrin with plasmin (a COOH-Lys-dependent mechanism). At the same time, the mechanism of the interaction of apo(a) with intact fibrin(ogen) remained unclear. Our recent study identified the Lys-independent apo(a)-binding sites within the fibrin(ogen) alphaC domains which contribute to an alternative Lys-independent mechanism. In this study, we performed direct measurements of the interaction between apo(a) and various fibrin(ogen) fragments representing the whole fibrin(ogen) molecule except the alphaC regions. The experiments revealed that the apo(a)-binding site, identified previously within fibrinogen gamma chain residues 207-235 [Klose, R., et al. (2000) J. Biol. Chem. 275, 38206-38212], is a high-affinity site and mainly Lys-independent, suggesting that it should also contribute to the Lys-independent mechanism. The experiments also identified a novel Lys-dependent high-affinity apo(a)-binding site within the sequence of gamma chain residues 287-411. This site may provide interaction of apo(a) with intact fibrin(ogen) through another alternative mechanism, which depends on internal Lys residues. Thus, apo(a) may interact with intact fibrin through the Lys-independent and Lys-dependent mechanisms, while the COOH-Lys-dependent mechanism may prevail in the presence of fibrinolytic activity.