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Förderbeginn 01.07.2003

Biophysikalische Untersuchung der Membran-permeabilisierenden Aktivität von Pentraxinen

Prof. Dr. Matthias Leippe
Universität Würzburg
Molekulare Parasitologie, Zentrum für Infektionsforschung

Prof. Dr. Peter Armstrong
University of California, Davis
Department of Molecular and Cellular Biology

Wir werden die mechanistische und strukturelle Basis untersuchen, die es Pentraxinen erlaubt Membranen zu permeabilisieren und so den Tod von Zielzellen hervorzurufen. Bei Pentraxinen handelt es sich um eine wichtige Klasse von Proteinen des Immunsystems (z.B. C-reaktives Protein), die in dem langlebigen wirbellosen Limulus und im Menschen zu finden ist. Diese Wirkungsweise spiegelt eine wichtige Strategie des angeborenen Immunsystems wider - der Angriff auf Membranen von fremden Zellen inklusive der von potentiellen Pathogenen. Wir haben ein Modellsystem etabliert, bei dem die im Blut frei löslichen Pentraxine in die künstlich hergestellten Membranen von Lipidvesikeln inserieren und dort Transmembranporen ausbilden, welche den freien Fluss von kleinen Molekülen und Wasser über die Lipidmembran erlauben. Wir werden dieses System dazu benutzen, um die strukturellen Veränderungen in den Pentraxinen bei der Membraninsertion zu analysieren, die physikalischen und physiologischen Charakteristika der entstehenden Membranpore zu beschreiben, und letztendlich die Rolle dieses Prozesses innerhalb des Immunsystems zu verstehen.

Abschlussbericht
Pore formation in lipid bilayers by pentraxin proteins from the invertebrate Limulus polyphemus

The pentraxins are a family of proteins characterized by cyclic oligomeric symmetry (Gewurz et al. 1995). The pentraxins are components of the innate immune systems of vertebrates (reference) and arthropods (Nguyen et al. 1986a,b; Armstrong et al. 1966). Human pentraxins include the classical acute phase protein C-reactive protein, named for its Ca²⁺-dependent ability to bind and precipitate pneumococcal C-polysaccharide, and serum amyloid P component, a constituent of amyloid plaques. The American horseshoe crab, Limulus polyphemus, has a set of plasma proteins (LPx) that are are homologues of mammalian C-reactive protein (Robey and Liu 1981, Nguyen et al. 1986a,b) and serum amyloid P component (Shrive et al 1999, Tharia et al. 2002) (L-CRP and L-SAP respectively) and a third member limulin (Roche and Monsigny 1974, Kaplan et al. 1977, Armstrong et al. 1996). The LPx are constitutively expressed at high levels (1-5 mg/mL as reported by Robey and Liu 1981) and as a group represent the second most abundant protein in the hemolymph behind hemocyanin (Armstrong et al. 1993). Human and invertebrate pentraxins can be separated by their differing affinities for phosphoamino compounds (Schwalbe et al. 1992). The LPx share an affinity for phosphorylethanolamine. Phosphorylcholine binding is a characteristic of limulin and L-CRP but not L-SAP (Armstrong et al. 1996, Shrive et al. 1999). It is known that limulin and two pentraxins from the Japanese horseshoe crab Tachypleus C-reactive protein 2 and 3 lyse mammalian red blood cells in a Ca²⁺-dependent manner (Armstrong et al. 1993, Iwaki et al. 1999). In this regard a role for these proteins as cytolytic effecter molecules operating in immune defense has been proposed. Limulin targets erythrocytes through binding sialic acid residues at the surface of the erythrocyte (Armstrong et al. 1996). The non-hemolytic LPx, L-CRP and L-SAP, lack sialic acid recognition and it is hypothesized that the failure of these molecules to “dock” at the surface of the erythrocyte precludes any lytic activity in such a system. Thus, a biological function for these abundant plasma proteins has remained elusive.

Cytolytic attack against potential pathogens is a major mechanism of host defense. The vertebrate complement system (Müller-Eberhard 1988) and a plethora of antimicrobial peptides from virtually all forms of life have been shown to exert their immune protective effect through the disruption of pathogens’ membrane integrity (Yeaman and Yount 2003). Increase in membrane permeability rapidly induces death of the target cell through the collapse of electrochemical gradients and/or osmotic shock. Two general mechanisms of non-enzymatic membrane permeabilization are recognized, each with its own variations: (1) the barrel-stave mechanism entails an oligomerization and orientation of the effecter molecule(s) that results in a membrane-spanning proteinaceous pore, (2) the carpet mechanism describes a detergent like disruption of lipid packing such that high protein to lipid ratios results in micellization of targeted membranes (Bechinger 1999, Shai and Oren 2001). Limulin, as well as tCRP-3, appears to operate as a hemolysin through the former process, forming stable transmembrane pores. This idea is based on the ability of externally presented macromolecular osmolites to reversibly inhibit limulin’s hemolytic effect (Iwaki et al. 1999, Swarnakar et al 2000). Formation of a transmembrane pore entails insertion across the lipid bilayer of the target cell and it is not understood how such a large, highly soluble protein such as limulin might accomplish this.

We have addressed both points by investigating the ability of the LPx to depolarize liposomes (see Loew et al. 1983. Leippe et al. 1991) or to induce entrapped calcein release (see Bruhn et al. 2003) from model liposomes. Our data show that (1) the LPx, as preparations of limulin-free LPx or individually, are capable of permeabilizing liposomes in a Ca²⁺-dependent fashion and (2) liposome permeabilization occurs in a manner consistent with stable transmembrane pore formation.

Each individual member of LPx is capable, under the proper conditions, of permeabilizing lipid bilayers. We monitored permeabilization of the liposome membrane both by the dissipation of a valinomycin induced K⁺-diffusion potential and by the release of entrapped calcein from liposomes constructed of azolectin. Liposomes were depolarized or rendered incapable of retaining calcein when exposed to LPx in acidic conditions. Purified L-CRP, L-SAP and limulin each showed potent dose-dependent and Ca²⁺-dependent permeabilizing activity at nanomolar concentrations. For all the LPx, activity decreased with increasing pH, being greatest at pH 5.2, the lowest pH tested, and insignificant at pH 7.0. Liposomes alone support a stable transmembrane potential or retain calcein for time periods in excess of 15 min.

We employed a variation of the depolarization assay where the LPx were added prior to the induction of a K⁺-diffusion potential by valinomycin. Incubation of liposomes with the LPx over a time period that results in 100% fluorescence recovery under normal assay procedures (i.e. induction of a diffusion potential followed by addition of LPx) leaves the liposomes capable of supporting a full, yet transient, quenching of dye fluorescence. Dye quenching is dependent on the presence of intact bilayers for two reasons. First, an intact bilayer is necessary for the formation of a diffusion potential. Secondly, the particular dye used in these depolarization assays intercalates into the outer leaflet of the bilayer by means of two short acyl chains, thus aggregating and ultimately self-quenching.

Surprisingly, the LPx do not exhibit significant permeabilizing activity against liposomes constructed from purified egg phosphatidylcholine (PC) or mixtures of PC/phosphatidylethanolamine, PC/phosphatidylglycerol, PC/phosphatidylserine or PC/cardiolipin (3:1 by mass). Indeed, of the lipid mixtures employed, only those consisting of azolectin were found to result in liposomes susceptible to permeabilization by the LPx. This activity remained robust at salt concentrations approximating and exceeding physiological conditions of Limulus plasma, i.e. 750 mM NaCl. These data argue against a situation where the LPx might initially bind lipid bilayers through electrostatic attraction and suggest that a specific component present in azolectin provides a unique binding site.

We will complement the data obtained during the course of this investigation with an analysis of the membrane-binding activities of the LPx and an assessment of their ability to kill microorganisms. Do the LPx bind membranes in a Ca²⁺-dependent fashion? Does binding occur at neutral or more basic pH? Are the LPx sufficient to kill microorganisms such as E. coli or Candida? With continuing investigation we hope to define not only the mechanism by which the LPx permeabilize lipid bilayers but also a physiological role for the LPx.

References

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