Anchor lipids

Figure 13.2 Activated G protein receptors, here represented as seven red transmembrane helices, catalyze the exchange of GTP for GDP on the Gapy trimer. The then separated Ga-GTP and Gpy molecules activate various effector molecules. The receptor is embedded in the membrane, and Ga, Gpy and G py are attached to the membrane by lipid anchors, and they all therefore move in two dimensions. (Adapted from D. Clapham, Nature 379 297-299, 1996.)...

Airother interesting facet of lipid anchors is that they are transient. Lipid anchors can be reversibly attached to and detached from proteins. This provides a switching device for altering the affinity of a protein for the membrane. Reversible lipid anchoring is one factor in the control of signal transduction pathways in eukaryotic cells (Chapter 34). 

Four different types of lipid-anchoring motifs have been found to date. These are amide-linked myristoyl anchors, thioester-linked fatty acyl anchors, thioether-linked prenyl anchors, and amide-linked glycosyl phosphatidylinosi-tol anchors. Each of these anchoring motifs is used by a variety of membrane proteins, but each nonetheless exhibits a characteristic pattern of structural requirements. 

Fig. 8 Immobilization of urokinase on the surfaces of islet cells, (a) Surface modification (/) chemical structure of ssDNA-PEG-lipid, and (2) ssDNA-PEG-lipid anchoring to the cell membrane. (b) Introduction of a complementary ssDNA onto urokinase, which was first modified with a madeimide group by a cross-linker, EMCS. (c) Urokinase-immobilization through DNA...

All proteins of the Ras superfamily (Table 1) - with the exception of the Ran protein - undergo a posttranslational modification which introduces at least one hydrophobic modification. These lipid anchors qualify the members of the Ras family as peripheral membrane proteins, which stick into membrane structures from the cytoplasmic side only. 

Ras is strictly localized to the inner side of the plasma membrane. A lipid anchor covalently attached to the C-terminus of Ras penetrates into the lipid bilayer. This membrane anchorage is essential for the biological activity of Ras. Hence, the inhibition of anchor attachment has become an attractive pharmacological target [ 13]. See Waldmann H, Thutewohl M,Ras-Farnesyltransferase-inhibitors as promising anti-tumor drugs, this volume. 

As soon as Ras sticks to the plasma membrane another lipid anchor is attached to it. A putative palmitoyl transferase which is assumed to reside in the... 

Fig. 5. Schematic representation of lipid anchors. The arrow heads and tails represent the N termini and the C termini of mature proteins, respectively, (a) Palmitoylation, (b) Ahnyristoylation, (c) Prenylation, (d) GPI anchor... 

The structure of a cationic lipid can be broken-down into three structural elements a lipophilic lipid anchor comprising one or—mostly—two long alkyl chains or Choi, a spacer, and a polar, positively charged head group consisting of one or more quaternised or protonatable amino groups. Figure 2 shows a few of the well-known, older cationic lipids, which can be classified as either monocationic or polycationic lipids. A series of recently synthesized cationic lipids will be discussed later. 

Cholesterol as well as unsaturated or saturated hydrocarbon chains are used as lipophilic lipid anchors. Although Cl 8-hydrocarbon chains (oleoyl or oleyl unit) are only used in unsaturated compounds, structural variations with C14-, C16-, or even Cl 8-hydrocarbon chains in saturated compounds are known (27). The lipophilic units are linked with a parent structure (usually glycerol) via ether (e.g., DOTMA) or ester bridges (e.g., DOTAP). Ester bridges are often used to create the linkage to avoid cytotoxicity, because ether bonds are more difficult to break down biologically than ester bonds (58). Substances that are easy to decompose and are therefore often used as a spacer are carbamate units (29) [e.g., 3p-[A-(A, A -dimethylaminoethyl)carbamoyl]-cholesterol (DC-Chol)], amide units, or phosphate esters. However, a direct correlation between toxicity and the... 

Figure 2 Examples of cationic lipids, differing in the head group structure (mono/ poly cationic) and the nonpolar lipid anchor (Chol/hydrocarbon chains). Abbreviations DOTAP, A-[l-(2,3-dioleoyloxy)propyl]-A,A,7V-trimethyl-ammoniumchloride DOTMA, A-[l -(2,3-dioleyloxy)propyl]-A A, A-trimethylammoniumchloride DC-Chol, 3 P"[A-(A, A -dimethylaminoethyl)carbamoyl]-cholesterol DOGS, A, A-dioctodecyl-amidoglycylspermin DORI, A-(l, 2-dioleoyloxypropyl)-A,A-dimethyl-A-hydroxyethyl-ammoniumbromide SpdC, spermidin-cholesterol.

Fichert T, Regelin A, Massing U. Synthesis and transfection properties of novel non-toxic monocationic lipids. Variation of lipid anchor, spacer and head group structure. Bioorg Med Chem Lett 2000 10(8) 787-791. 

Our laboratory mostly works with PEG-cholesterol. It is easily obtained in one step by addition of cholesteryl chloroformate and amino-methoxy-PEG (31). Introduction of a linker between the cholesterol and the PEG part would induce higher membrane fluidity and reduce more efficiently protein interactions as compared to PEG-cholesterol. A diaminobutane spacer was shown to improve significantly the sustained release of calcein from lipoplexes incubated in 30% serum (32). The spacer effect on bicatenar PEG-lipid has not been intensively studied because it can be expected that it would induce less effect on PEG-dioleoyl than on PEG-cholesterol, the lipidic anchor being predominant in the bilayer stabilization (Fig. 2). 

Using N-terminus modified polylysine, we developed a synthesis for an amphiphilic polychelator, A,a-(DTPA-polylysyl)glutaryl phosphatidyl ethanolamine (DTPA-PL-NGPE). This polychelator was incorporated into the liposomal membrane and micelle core during liposome or micelle preparation. This system sharply increased the number of chelated Gd atoms attached to a single lipid anchor. This increased the number of bound reporter metal atoms per vesicle and decreased the dosage of an administered... 

Targeting Hgands can be attached to the microbubble directly (Fig. 12 A) or via a flexible polymer spacer arm (e.g., polyoxyethylene. Fig. 12 C). Use of the spacer arm may allow for tighter binding in some situations. When the detachment force of the targeted microbubble removal from the receptor-coated surface was measured, microbubbles with spacer-ligand construct demonstrated superior retention as compared with the bubbles where the ligand was connected directly to the lipid anchor, without a spacer [92]. 

Isoprene chains are sometimes used as lipid anchors to fix molecules to membranes (see p. 214). Chlorophyll has a phytyl residue (1 = 4) as a lipid anchor. Coenzymes with isoprenoid anchors of various lengths include ubiquinone (coenzyme Q 1 = 6-10), plastoqui-none (1 = 9), and menaquinone (vitamin K 1 = 4-6). Proteins can also be anchored to membranes by isoprenylation. 

Type V and Vi proteins carry lipid anchors. These are fatty acids (palmitic acid, myristic acid), isoprenoids (e.g., farnesol), or glycoli-pids such as glycosyl phosphatidylinositol (GPi) that are covalently bound to the peptide chain. 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

Often the cell accomphshes the association of signal proteins with the membrane by post-translationaUy affixed lipid anchors composed of hydrophobic residues, such as fatty acids, isoprenoids or complex glycolipids (see fig. 3.11). These lipid moieties of lipidated proteins favor membrane association by inserting themselves into the phospholipid bilayer. 

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