Membrane interaction of ‘peptide P’ derived from the repeating motif of properdin

Stankowski, Stefan; Wey, Josef; Kalb, Edwin; Goundis, Dimitrios

doi:10.1016/0005-2736(91)90060-l

Cited by 5 publications

(3 citation statements)

References 16 publications

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“…If this is the case for cecropins, then we would expect a larger intrinsic binding affinity for negatively charged lipids than for neutral lipids, consistent with the observed dependence of the monomer binding constant on surface potential. Furthermore, arginine residues seem to have little contribution to the total interfacial charge Stankowski et al, 1991). In fact, our data (Table 1) suggest that the curvature of the binding isotherms at large Q values (Figures 4 and 7) might be due to repulsion among peptide monomers rather than to charging of the bilayer.…”

Section: Discussionmentioning

confidence: 70%

Binding and State of Aggregation of Spin-Labeled Cecropin AD in Phospholipid Bilayers: Effects of Surface Charge and Fatty Acyl Chain Length

Mchaourab¹,

Hyde²,

Feix³

1994

Biochemistry

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The binding and state of aggregation of cecropin in large unilamellar vesicles of different surface potential and varying acyl chain length were examined using a Cys-33 spin-labeled derivative of cecropin AD (CAD). Association isotherms of the peptide were measured for vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) containing 5, 15, and 30 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). The isotherms display a concentration-dependent positive cooperativity indicating the possible formation of cecropin aggregates in the lipid phase. The critical aqueous concentration for aggregation was dependent on the fraction of POPG, suggesting the involvement of acidic lipids in the formation and stabilization of the putative aggregate. Our data also indicate that cooperativity depends on the state of side-chain ionization of an acidic residue that titrates between pH 7 and 4.4. The binding of spin-labeled Cys-33 CAD was found to be influenced by the acyl chain length of the host lipid. The association isotherm of the peptide for dilaureoyl-sn-glycero-3-phosphatidylcholine vesicles containing 30 mol % dilauroyl-sn-glycero-3-phosphatidylglycerol (DLPG) differed significantly from that in POPC/POPG and could be interpreted in terms of a monomer-monomer partitioning between the aqueous and lipid phases. ESR line-shape analysis was consistent with peptide aggregation in dioleoyl-sn-glycero-3-phosphatidylglycerol vesicles but not in DLPG vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 70%

Binding and State of Aggregation of Spin-Labeled Cecropin AD in Phospholipid Bilayers: Effects of Surface Charge and Fatty Acyl Chain Length

Mchaourab¹,

Hyde²,

Feix³

1994

Biochemistry

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“…When constructing the binding isotherms from fluorescence data, the intensity at a fixed wavelength is frequently used to estimate the extent of binding. The simplest approach for evaluating the collected data is to assume that only two states exist: peptide free in solution and membrane-associated peptide ( − ). If this is the case, the fluorescence intensity increases with the lipid concentration and asymptotically approaches a final value as the lipid to peptide molar ratio is increased.…”

Section: Resultsmentioning

confidence: 99%

Application of a Novel Analysis To Measure the Binding of the Membrane-Translocating Peptide Penetratin to Negatively Charged Liposomes

et al. 2002

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The binding of penetratin, a peptide that has been found useful for cellular delivery of large hydrophilic molecules, to negatively charged vesicles was investigated. The surface charge density of the vesicles was varied by mixing zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) at various molar ratios. The extent of membrane association was quantified from tryptophan emission spectra recorded during titration of peptide solution with liposomes. A singular value decomposition of the spectral data demonstrated unambiguously that two species, assigned as peptide free in solution and membrane-bound peptide, respectively, account for the spectral data of the titration series. Binding isotherms were then constructed by least-squares projection of the titration spectra on reference spectra of free and membrane-bound peptide. A model based on the Gouy-Chapman theory in combination with a two-state surface partition equilibrium, separating the electrostatic and the hydrophobic contributions to the binding free energy, was found to be in excellent agreement with the experimental data. Using this model, a surface partition constant of approximately 80 M(-)(1) was obtained for the nonelectrostatic contribution to the binding of penetratin irrespective of the fraction of negatively charged lipids in the membrane, indicating that the hydrophobic interactions are independent of the surface charge density. In accordance with this, circular dichroism measurements showed that the secondary structure of membrane-associated penetratin is independent of the DOPC/DOPG ratio. Experiments using vesicles with entrapped carboxyfluorescein showed that penetratin does not form membrane pores. Studies of the cationic peptide penetratin are complicated by extensive adsorption to surfaces of quartz and plastics. By modification of the quartz cell walls with the cationic polymer poly(ethylenimine), the peptide adsorption was reduced to a tolerable level. The data analysis method used for construction of the binding isotherms eliminated errors emanating from the remaining peptide adsorption, which otherwise would prevent a proper quantification of the binding.

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“…Like most of the above-mentioned proteins, properdin also acts at cell surfaces and is capable of proteinprotein (10,11,(51)(52)(53), protein-sulfatide (54), and direct protein-membrane interactions (55).…”

Section: Fig 4 Esimsms Of the [M ؉ 2h]mentioning

confidence: 99%

Properdin, the Positive Regulator of Complement, Is HighlyC-Mannosylated

Hartmann

Hofsteenge

2000

Journal of Biological Chemistry

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Properdin is the positive regulator of the alternative pathway of complement activation. The 53-kDa protein is essentially composed of six thrombospondin type 1 repeats, all of which contain the WXXW motif, the recognition sequence for C-mannosylation. C-Mannosylation is a post-translational modification of tryptophan residues in which, in contrast to the well known N-and O-glycosylation, the carbohydrate is attached via a C-C bond to C-2 of the indole moiety of tryptophan. C-Mannosylation was first found in human RNase 2 and interleukin-12. The terminal complement proteins C6 -C9 also carry this modification as part of their thrombospondin type 1 repeats. We studied the C-mannosylation pattern of human properdin by mass spectrometry and Edman degradation. Properdin contains 20 tryptophans of which 17 are part of a WXXW motif. Fourteen tryptophans were found to be modified 100%. This is the first example of a protein in which the majority of tryptophan residues occurs in the C-mannosylated form. These results show that C-mannosylated proteins occur at several steps along the complement activation cascade. Therefore, this system would be ideal to investigate the function of C-mannosylation.Glycosylation is one of the most abundant and widespread post-translational modifications of proteins. In the common cases of N-and O-glycosylation the glycan is attached via an amide or a hydroxyl group of an amino acid side chain to the protein. In human RNase 2 a fundamentally different type of glycosylation was discovered (1-3). An ␣-mannosyl residue was found to be linked via a C-C bond to the C-2 of the indole ring of tryptophan (Fig. 1). It was shown that this modification is enzyme-catalyzed and that dolichylphosphate mannose is the sugar donor (4). In RNase 2 the recognition signal for the transferase was determined to be WXXW (or less efficiently WXXF), in which the first tryptophan becomes modified (5). Meanwhile a total of 22 tryptophans in 7 proteins have been shown to be C-mannosylated, i.e. RNase 2 (1), interleukin-12 (6), and terminal complement proteins C6, C7, C8␣ and ␤, and C9 (7). The terminal complement proteins showed a more complex pattern of C-mannosylated tryptophans than RNase 2 and interleukin-12. They contain as a part of their thrombospondin repeats (TSRs) 1 WXXWXXW motifs, in which both of the first two tryptophans and, surprisingly, also the last one can be C-mannosylated. In addition, in TSRs of C6 and C7, tryptophans were found to be modified although they were not even part of a WXXW motif (7).The complement system is an innate first line host defense mechanism against microbes. Its activation pathways are composed of three proteolytic cascades, which merge at the cleavage step of (inactive) C3, converting it into active C3b ( Fig. 2; reviewed in Ref. 8). Properdin (or factor P) is a positive regulator of the complement system. It stabilizes the C3 convertase (C3bBb) in the feedback loop of the alternative pathway (Fig. 2), protecting it from rapid inactivation (9 -11). In addition, the C5 con...

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Membrane interaction of ‘peptide P’ derived from the repeating motif of properdin

Cited by 5 publications

References 16 publications

Binding and State of Aggregation of Spin-Labeled Cecropin AD in Phospholipid Bilayers: Effects of Surface Charge and Fatty Acyl Chain Length

Binding and State of Aggregation of Spin-Labeled Cecropin AD in Phospholipid Bilayers: Effects of Surface Charge and Fatty Acyl Chain Length

Application of a Novel Analysis To Measure the Binding of the Membrane-Translocating Peptide Penetratin to Negatively Charged Liposomes

Properdin, the Positive Regulator of Complement, Is HighlyC-Mannosylated

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