Penetratin translocation mechanism through asymmetric droplet interface bilayers.

Penetratin is a cell penetrating peptide (CPP) that can enter cells by direct translocation through the plasma membrane. The molecular mechanism of this translocation still remains poorly understood. Here we provide insights on this mechanism by studying the direct translocation of the peptide across model membranes based on Droplet Interface Bilayers (DIBs), which are bilayers at the interface between two adhering aqueous-in-oil droplets. We first showed with symmetric bilayers made of a mix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (POPC) that the translocation of penetratin required the presence of at least 40% of POPG on both leaflets. Interestingly when replacing POPG with another anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), translocation was inefficient. To elucidate the lipid partners required at each step of the CPP translocation process, we then investigated the crossing of asymmetric bilayers. We found that POPG on the proximal leaflet and POPS on the distal leaflet allowed penetratin translocation. Translocation was not observed when POPS was on the proximal leaflet and POPG on the distal leaflet or if POPS on the distal leaflet was replaced with POPC. These observations led us to propose a three-step translocation mechanism: (i) peptide recruitment by anionic lipids, (ii) formation of a transient peptide-lipid structure leading to the initiation of translocation which required specifically POPG on the proximal leaflet, (iii) termination of the translocation process favored by a driving force provided by anionic lipids in the distal leaflet.

Amphiphilic macromolecules on cell membranes: from protective layers to controlled permeabilization.

Antimicrobial and cell-penetrating peptides have inspired developments of abiotic membrane-active polymers that can coat, penetrate, or break lipid bilayers in model systems. Application to cell cultures is more recent, but remarkable bioactivities are already reported. Synthetic polymer chains were tailored to achieve (i) high biocide efficiencies, and selectivity for bacteria (Gram-positive/Gram-negative or bacterial/mammalian membranes), (ii) stable and mild encapsulation of viable isolated cells to escape immune systems, (iii) pH-, temperature-, or light-triggered interaction with cells. This review illustrates these recent achievements highlighting the use of abiotic polymers, and compares the major structural determinants that control efficiency of polymers and peptides. Charge density, sp. of cationic and guanidinium side groups, and hydrophobicity (including polarity of stimuli-responsive moieties) guide the design of new copolymers for the handling of cell membranes. While polycationic chains are generally used as biocidal or hemolytic agents, anionic amphiphilic polymers, including Amphipols, are particularly prone to mild permeabilization and/or intracell delivery.

Incorporation of vinylogous scaffolds in the C-terminal tripeptide of substance P.

Glycine-9 and leucine-10 of substance P (SP) are critical for (NK)-1 receptor recognition and agonist activity. Propsi(Z)-CH=CH(CH3)-CONH)Leu (or Met) and Propsi((E)-CH=CH(CH3)-CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C-terminal tripeptide, Gly-Leu-Met-NH2, of SP. Propsi((Z)-CH=C(CH2CH(CH3)2)-CONH)Met-NH2, with an isobutyl substituent to mimic the Leu side-chain, was also incorporated in place of the C-terminal tripeptide. The substituted-SP analogs were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK-1 receptor. The most potent SP analogs [Pro9psi((Z)CH=C(CH3)CONH)Leu10]SP and [Pro9psi ((E)CH=C(CH3)CONH)Leu10]SP, are about 100-fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)- or (E)-CH=C(CH3)- or (Z)-CH=C(CH2CH(CH3)2) moieties hamper the correct positioning of the C-terminal tripeptide of SP within both the NK-1M- and NK-1m-specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.

Biological activity of silylated amino acid containing substance P analogues.

The need to replace natural amino acids in peptides with nonproteinogenic counterparts to obtain new medicinal agents has stimulated a great deal of innovation on synthetic methods. Here, we report the incorporation of non-natural silylated amino acids in substance P (SP), the binding affinity for the two hNK-1 binding sites and, the potency to stimulate phospholipase C (PLC) and adenylate cyclase of the resulting peptide. We also assess the improvement of their stability towards enzyme degradation. Altogether, we found that replacing glycine with silaproline (Sip) in position 9 of SP leads to a potent analogue exhibiting an increased resistance to angiotensin-converting enzyme hydrolysis.

Met174 side chain is the site of photoinsertion of a substance P competitive peptide antagonist photoreactive in position 8.

Numerous photoaffinity studies of the NK-1 receptor have been carried out with peptide agonist analogues of substance P (SP). However, no information is available with regard to the domain interaction of peptide antagonists within this receptor. We describe herein the photoaffinity labelling of the SP receptor with a peptide antagonist analogue, Bapa(0)[(pBzl)Phe(8),DPro(9),MePhe(10),Trp(CHO)(11)]SP. Photolabelling, enzymatic or chemical cleavage of the covalent complex, purification via streptavidin-coated beads and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis led us to show that the methyl of Met174 side chain, within the receptor's second extracellular loop, is covalently linked to the antagonist photoreactive at position 8.

Analogs of Substance P modified at the C-terminus which are both agonist and antagonist of the NK-1 receptor depending on the second messenger pathway.

The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK-1 receptor. Therefore, the photoreactive amino acid para-benzoyl-phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa0[(pBzl)Phex]SP analogs. Biotinyl sulfone-5-aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK-1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK-1 receptor-binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa0[(pBzl)Phe11]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa0[Pro9, (pBzl)Hcy(O2)11]SP is a partial agonist, whereas Bapa0[Hcy(ethylaminodansyl)11]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK-1 receptor.

Internalization of [3H]substance P analogues in NK-1 receptor transfected CHO cells.

The internalization of [3H]propionyl[Met(O2)11]SP(7-11) which binds one binding site and of [3H][Pro9]SP which binds the two binding sites associated with the NK-1 receptor has been examined in CHO cells. The quantity of [3H][Pro9]SP measured inside the cytoplasm in kinetic experiments is fully temperature-dependent. In contrast, [3H]propionyl[Met(O2)11]SP(7-11) internalization reaches the same extent whatever the temperature, although the rate slowed down with lower temperature. The extent of internalization of [3H][Pro(9)]SP relative to the total specific bound is biphasic, when the extent of internalization of [3H]propionyl[Met(O2)11]SP(7-11) remains constant. For [3H][Pro9]SP, a high-affinity high-yield component inhibited in the presence of propionyl[Met(O2)11]SP(7-11) and a low-affinity low-yield component in the internalization process could be determined. Saturation studies show that [3H][Pro9]SP-binding parameters are insensitive to both phenylarsine oxide and monensin treatment, whereas [3H]propionyl[Met(O2)11]SP(7-11) maximal binding is decreased in both cases. Altogether, these data suggest that the two radiolabeled peptides should not follow the same internalization pathway.

Calpha methylation in molecular recognition. Application to substance P and the two neurokinin-1 receptor binding sites.

Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Calpha methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Calpha methylated analogues of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Calpha methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Calpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [alphaMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Calpha methylation of SP are sequence dependent. Moreover, a single Calpha methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha-aminoisobutyric acid. Furthermore, Calpha methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other potent SP analogues.

Pharmacological characterization of tachykinin septide-sensitive binding sites in the rat submaxillary gland.

Binding studies have shown that [125I]NKA is a selective ligand of tachykinin septide-sensitive binding sites from membranes of the rat submaxillary gland. Indeed, this ligand bound with high affinity to a single population of sites. In addition, competition studies indicated that natural tachykinins and tachykinin-related compounds had a similar affinity for these sites than for those labeled with [3H]ALIE-124, a selective ligand of septide-sensitive binding sites. Moreover, selective tachykinin NK2, or NK3 agonists or antagonists exhibited weak or no affinity for [125I]NKA binding sites. As indicated by Ki values of several compounds, the pharmacological characteristics of the septide-sensitive binding sites (labeled with [125I]NKA) largely differ from those of classic NK1 binding sites, as determined on crude synaptosomes from the rat brain using [125I]Bolton-Hunter substance P (SP) as ligand. Indeed, several tachykinins including neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NKgamma), and neurokinin B, as well as some SP and NKA analogues or C-terminal fragments such as septide, ALIE-124, SP(6-11), NKA(4-10), which have a weak affinity for classic tachykinin NK1 binding sites exhibited a high affinity for the septide-sensitive binding sites. In contrast, SP, classic selective NK1 agonists, and antagonists had a high affinity for both types of binding sites. The presence of a large population of tachykinin septide-sensitive binding sites in the rat submaxillary gland may thus explain why NPK and NPgamma induce salivary secretion and may potentiate the SP-evoked response in spite of the absence of tachykinin NK2 receptors in this tissue.

Further delineation of the two binding sites (R*(n)) associated with tachykinin neurokinin-1 receptors using [3-Prolinomethionine(11)]SP analogues.

Two binding sites are associated with neurokinin-1 substance P receptors in both transfected cells and mammalian tissues. To further delineate the interactions between the crucial C-terminal methionine of substance P and these two binding sites, we have incorporated newly designed constrained methionines, i.e. (2S, 3S)- and (2S,3R)-prolinomethionines. The potencies of these C terminus-modified SP analogues to bind both sites and to activate phosphatidylinositol hydrolysis and cAMP formation have been measured, together with those of their corresponding sulfoxides and sulfones. The molecular nature of these two binding sites and their selective coupling to effector signaling pathways are discussed in the light of current models of receptor activation. The less abundant binding site is coupled to G(q/11) proteins, whereas the most abundant one interacts with G(s) proteins in Chinese hamster ovary cells transfected with human neurokinin-1 receptors. The specific orientation of the C-terminal methionine side chain imposed by these constraints shows that macroscopically chi(1) and chi(2) angles of this crucial C-terminal residue are similar in both binding sites. However, slight but significant variations in the rotation around the Cgamma-S bond yield different either stabilizing or destabilizing interactions in the two binding sites. These results highlight the need of such constrained amino acids to probe subtle interactions in ligand-receptor complexes.

Anandamide and WIN 55212-2 inhibit cyclic AMP formation through G-protein-coupled receptors distinct from CB1 cannabinoid receptors in cultured astrocytes.

The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the evoked formation of cyclic AMP were compared in cultured neurons and astrocytes from the cerebral cortex and striatum of mouse embryos. The three compounds inhibited the isoproterenol-induced accumulation of cyclic AMP in neuronal cells, and these responses were blocked by the selective CB1 receptor antagonist SR 141716A. The three agonists were more potent in cortical than striatal neurons. Interestingly, WIN 55212-2, CP 55940 and anandamide also inhibited the isoproterenol-evoked accumulation of cyclic AMP in astrocytes but, in contrast to WIN 55212-2 and CP 55940, anandamide was much more potent in striatal than cortical astrocytes. Inhibition was prevented by pertussis toxin pretreatment, but not blocked by SR 141716A. Therefore, G-protein-coupled receptors, distinct from CB1 receptors, are involved in these astrocytic responses. Moreover, specific binding sites for [3H]-SR 141716A were found in neurons but not astrocytes. Furthermore, using a polyclonal CB1 receptor antibody, staining was observed in striatal and cortical neurons, but not in striatal and cortical astrocytes. Taken together, these results suggest that glial cells possess G-protein-coupled receptors activated by cannabinoids distinct from the neuronal CB1 receptor, and that glial cells responses must be taken into account when assessing central effects of cannabinoids.

Asymmetric synthesis of Boc-N-methyl-p-benzoyl-phenylalanine. Preparation of a photoreactive antagonist of substance P.

The asymmetric synthesis of (S)-Boc-N-methyl-p-benzoyl-phenylalanine was performed by alkylation of sultam Boc-sarcosinate. The levorotatory sultam led to (S)-Boc-N-methyl amino acids with high optical purity. This photoreactive amino acid was incorporated into the sequence of a Substance P peptide antagonist. Comparison of the affinity and antagonistic properties of Biotinyl-apa-[D-Pro9, MePhe(pBz)10, Trp11]SP for human tachykinin NK-1 receptor demonstrated that this photoreactive antagonist should be a suitable tool for photolabelling studies.

High affinity binding of [3H]propionyl-[Met(O2)11]substance P(7-11), a tritiated septide-like peptide, in Chinese hamster ovary cells expressing human neurokinin-1 receptors and in rat submandibular glands.

Propionyl-[Met(O2)11]substance P(7-11) [ALIE-124 or propionyl-[Met(O2)11]SP(7-11)] has been designed as a septide-like ligand adequate for tritiation and, therefore, adequate for binding studies. In Chinese hamster ovary (CHO) cells expressing human tachykinin neurokinin (NK)-1 receptors, ALIE-124 displaced [3H][Pro9]substance P (SP) from its binding site at micromolar concentrations. However, ALIE-124 stimulated phosphatidylinositol hydrolysis, as previously shown for septide-like peptides. With [3H]ALIE-124 (95 Ci/mmol), we have been able to reveal a high affinity binding site in CHO cells (Kd = 6.6 +/- 1.0 nM), with a low maximal binding capacity. [3H]ALIE-124 specific maximal binding represented only 15-20% of that observed with [3H][Pro9]SP in CHO cells. Septide-like peptides, including septide and NKA, were potent competitors (in the nanomolar range) of [3H]ALIE-124 specific binding site. Interestingly, SP and [Pro9]SP were also potent competitors, with 10-fold greater potency for sites labeled with [3H]ALIE-124 than for sites labeled with [3H][Pro9]SP. The NK-1 antagonist RP 67580 also showed a higher potency for [3H]ALIE-124 than for [3H][Pro9]SP-specific binding sites. NKB and [Lys5,methyl-Leu9,Nle10]NKA(4-10) displaced [3H]ALIE-124 binding but with lower potency, whereas senktide had no affinity. The existence of [3H]ALIE-124 specific binding sites was also demonstrated in rat submandibular gland. In this tissue, [3H]ALIE-124 specific maximal binding was higher, reaching 40-50% of that achieved with [3H][Pro9]SP.

(R)-methanandamide inhibits receptor-induced calcium responses by depleting internal calcium stores in cultured astrocytes.

The effect of (R)-methandamide, a chiral analog of the endogenous cannabimimetic anandamide, was investigated on calcium signalling in cultured rat astrocytes loaded with Indo-1. Pretreatment of astrocytes with (R)-methandamide resulted in the inhibition of calcium responses induced by endothelin-1 or glutamate. Test of the filling level of internal calcium stores and biochemical assays of phospholipase C activity suggested that this inhibition resulted from the depletion of internal pools. This effect occurred in a reversible time- and dose-dependent manner, and was prevented by treating the cells with pertussis toxin (PTX) but was not reproduced by similar concentrations of arachidonic acid. Altogether, these observations demonstrate that this stable analog of anandamide controls calcium signalling in astrocytes through a PTX-sensitive mechanism which leads to the depletion of fast mobilizable internal stores.

Tachykinin NK-1 receptor probed with constrained analogues of substance P.

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. L-tetrahydroisoquinoleic acid (Tic), L-fluorenylglycine (Flg), L-diphenylalanine (Dip), the diastereoisomers of L-1-Indanylglycine (Ing) and L-benz[f]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (delta ZPhe, delta EPhe, delta ZNal, ENal) and L-O,O'-dimethylphenylalanine (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S7 and S8 subsites, corresponding to residues Phe7 and Phe8 of substance P. According to the binding potencies of these substituted-SP analogues, the S7 binding subsite is smaller than the S8 subsite: the S7 subsite accepts only one aromatic nucleus, while the S8 can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S8 binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu6, Pro9]SP(6-11), discrepancies are observed between affinity (K1) and activity (EC50) values for IPs production. While a weak correlation between K1 and EC50 values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K1) and their potencies (EC50) for cAMP production (r = 0.97). The high potency (EC50) observed for "septide-like' molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC50 values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).

Topographic analysis of the S7 binding subsite of the tachykinin neurokinin-1 receptor.

Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2-for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S. 3R)- and (2S, 3S)-indanylglycines, E- and Z-alpha, beta-dehydrophenylalanines, and 2(S)-alpha, beta-cyclopropylphenylalanines [delta E Phe. delta Z Phe. inverted delta E2 (S) Phe, and inverted delta Z 2 (S) Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (delta E Phe, delta Z Phe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (-) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is small since the other rotamer can be detected in [(2S, 3R)Ing7]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe7 substituted analogues of SP must come from steric hindrance in the S7 binding site, which drastically restricts the space filling around the C alpha-C beta bond of residue 7.

The use of photolabelled peptides to localize the substance-P-binding site in the human neurokinin-1 tachykinin receptor.

The amino acid p-benzoyl-L-phenylalanine, (p-Bz)Phe, has been incorporated into substance P (SP), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, to localize the agonist-binding domains of the human neurokinin-1 (NK-1) receptor overexpressed in a transfected mammalian cell line. The NK-1-specific agonist [Pro9]SP was modified at position 8 by (p-Bz)Phe and acylated at the N-terminus by a biotinyl sulfone reporter via a 5-aminopentanoyl spacer. After photolysis, the biotinyl sulfone moiety allowed easy and efficient removal of biotinylated fragments from the complex incubation mixture with streptavidin-coated beads. Direct elution from the beads with the matrix used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS), which was facilitated by saturation of streptavidin sites with biotin, and subsequent MALDI-TOF mass spectrometry analysis allowed identification of the NK-1 fragments obtained after photolysis and proteolytic digestion. Trypsin digestion and combined trypsin/Staphylococcus aureus V8 protease enzymatic cleavage established that the site of covalent attachment of the photolabelled SP resides in the second extracellular loop Thr173-Arg177. Cyanogen bromide cleavage shows that the probe is covalently attached to the methyl group of a methionine residue from human NK-1. These experiments identified Met174 as the modified residue.

Tachykinin peptides affect differently the second messenger pathways after binding to CHO-expressed human NK-1 receptors.

The human NK-1 receptor transfected in Chinese hamster ovary (CHO) cells was studied with use of different tachykinin analogs: Substance P, [Pro9]SP, [Sar9, Met(O2)11]SP, [Gly9 psi (CH2CH2) Leu10]SP, Ac-Arg-septide, septide, [Gly9 psi (CH2CH2) Gly10]SP, NKA, [pGlu6]SP(6-11) and [Lys5]NKA(4-10). Binding experiments with [3H][Pro9]SP discriminated two classes of peptides with either high affinity (K iota in the nanomolar range) for the human NK-1 receptor or with low affinity (K iota in the micromolar range); this second group of peptides included NKA and [pGlu6]SP(6-11). In spite of these differences, both peptide families evoked potent stimulation of phosphatidylinositol hydrolysis (EC50 in the nanomolar range). In contrast, only NK-1 agonists, with high affinity, stimulated with great potency cyclic AMP formation (EC50 from 8 to 50 nM), whereas the second family of peptides were only weak agonists (EC50 in the micromolar range). RP 67580, CP 96345 and GR 94800, a NK-2 antagonist, were either competitive or uncompetitive inhibitors of inositol phosphates or cyclic AMP formations induced by [Pro9]SP, septide or NKA, independently of the agonist or the response studied. Thus, NKA, the presumed NK-2 endogenous peptide that may be co-released with SP, and the enzymatically produced C-terminal fragment of SP, [pGlu6]SP(6-11), may trigger specific pharmacological responses via the NK-1 receptor, at nanomolar concentrations, and thus regulate the action of SP at the NK-1 receptor.

Mechanism of delta-opioid receptor selection by the address domain of dermenkephalin.

Dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-AspNH2) is a highly potent and selective delta-opioid peptide isolated from frog skin. It was recently recognized that the C-terminus His4-Leu5-Met6-Asp7NH2 of dermenkephalin was responsible for the addressing of the peptide towards the delta-opioid receptor. In order to investigate the role played by residues 4, 5 and 6 in this 'delta address', we synthesized and evaluated 20 new analogues for their ability to displace tritiated ligands from mu- and delta-opioid sites. Results showed that position 4 of dermenkephalin contributes to delta selectivity independently of delta-opioid receptor binding by preventing a high affinity mu binding. Position 5 requires a hydrophobic side chain to enhance delta affinity. A high delta affinity was obtained with any amino acids introduced in position 6 suggesting that residue 6 serves as a neutral spacer. Thus, the main features responsible for the high delta-opioid selectivity of dermenkephalin are electrostatic repulsions with the mu-opioid receptor, additional hydrophobic interactions with the delta-opioid receptor and folding of the C-terminal domain.

The human sperm protein PH-20 has hyaluronidase activity.

The PH-20 protein present on the membrane of guinea pig sperm was characterized using a monoclonal antibody [(1991) J. Cell Biol. 111, 2939-2949]. We have isolated the cDNA encoding the human PH-20 protein from a testis library. This cDNA was expressed in RK 13 cells using a vaccinia virus expression system. Cells expressing the human PH-20 protein possess hyaluronidase activity. Treatment with PI-PLC releases the hyaluronidase into the the medium with a concomitant large increase in enzymatic activity. These results demonstrate that the human PH-20 protein has hyaluronidase activity.