Xi Chen

Angela Jen

Alice Warley

M Jayne Lawrence

Peter J Quinn

Dr Roger J Morris
email: roger.morris@kcl.ac.uk

Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition

Keywords: lipid rafts, detergent resistant membrane, non-ionic detergent, cholesterol, prion protein, Thy-1 DRM, detergent resistant membrane, DTT, dithiothreitol, EM, electron microscope, GPI, glycosylphosphatidylinositol, ISB, intracellular solubilisation buffer, L d , liquid disordered, L o , liquid ordered, MßCD, methyl-ß-cyclodextrin, SB, standard solubilization buffer, PrP, prion protein, WGA, wheat germ agglutinin

Isolation of DRMs at 37˚C

The failure of most non-ionic detergents to release patches of detergent-resistant membrane (DRM) at 37˚C undermines the claim that DRMs consist of lipid nanodomains that exist in an L o (liquid ordered) phase on the living cell surface. We show here that inclusion of cations (Mg 2+ , K + ) to mimic the intracellular environment stabilises membranes during solubilization sufficiently to allow the isolation of DRMs at 37˚C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outsideout orientation of membrane glycoproteins, have prolonged (18 hour) stability at 37˚C, and are vesicles or sheets up to 150-200 nm diameter. DRMs containing glycosylphosphatidylinositol (GPI)-anchored proteins PrP and Thy-1 can be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking on the neuronal surface. Thy-1 but not PrP DRMs are associated with actin. Electron microscope (EM) immunohistochemistry shows most PrP, and some Thy-1, to be clustered on DRMs, again maintaining their organization on the neuronal surface. For DRMs labelled for either protein, the bulk of the surface of the DRM is not labelled, indicating that the GPI-anchored protein is a minor component of its lipid domain. These 37˚C DRMs thus have properties expected of raft membrane, yet pose more questions about how proteins are organized within these nanodomains.

INTRODUCTION

Lipid rafts are postulated to be nanodomains within the cell surface membrane, formed by local condensation of cholesterol with saturated lipids to form distinct patches of L o phase within the general fluid 'sea' of liquid disordered (L d ) phase lipids. Proteins partition differentially into either of these two lipid phases, providing a lipid-based organization of surface proteins [START_REF] Simons | Functional rafts in cell membranes[END_REF].

A major biochemical aim has been to isolate these distinctive membrane nanodomains to allow their composition and structure to be determined, and hence their mechanism in directing signalling and other cellular functions to be understood. The most common approach has been to exploit the observation that the fluid L d phase of the membrane can be fully solubilized by non-ionic detergents at 4˚C, leaving L o phase membrane, whose ordered structure excludes these surfactants, to float as light detergent-resistant membrane (DRM) in density gradient ultracentrifugation [START_REF] Brown | Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface[END_REF][START_REF] Hooper | Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114[END_REF][START_REF] London | Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)[END_REF].

The extent to which DRMs represent the composition of 'rafts' on living cells has been repeatedly challenged (e.g. [START_REF] Lichtenberg | Detergent-resistant membranes should not be identified with membrane rafts[END_REF]6]). A general problem is that DRMs cannot be isolated at physiological temperature, but p 2 only at 4˚C [START_REF] London | Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)[END_REF]6]. Many membrane lipids that at 37˚C are in a fluid L d phase, on chilling to 4˚C form an ordered, detergent-resistant gel phase. DRMs isolated at 4˚C must include lipids that are in L d phase at 37˚C [START_REF] London | Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)[END_REF][START_REF] Lichtenberg | Detergent-resistant membranes should not be identified with membrane rafts[END_REF]. If exclusion of detergent from an L o phase is the criterion for selection of DRMs, it is not easy to explain why 'raft' membrane in an L o phase cannot be isolated at 37˚C on the basis of detergent insolubility.

In addition, DRMs made with the benchmark detergent Triton X-100 have been shown to contain proteins from totally different cells [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF] and to have scrambled the asymmetry of the surface membrane [START_REF] Radeva | Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukemia) cells[END_REF], results pointing to merger during solubilization of membrane fragments that are unrelated on the cell surface. This possibly results from the tendency of Triton X-100 to selectively deplete inner leaflet lipids from DRMs [START_REF] Koumanov | Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes[END_REF][START_REF] Pike | Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis[END_REF][START_REF] Schuck | Resistance of cell membranes to different detergents[END_REF], which would destabilise and presumably merge membrane patches into larger vesicles. This effect, however, is not found with all non-ionic detergents. In particular, Brij 96 used under similar conditions does not selectively deplete inner membrane lipids [START_REF] Schuck | Resistance of cell membranes to different detergents[END_REF][START_REF] Chen | The Isolation and Structure of Membrane Lipid Rafts from Rat Brain[END_REF], nor fuse fragments from different membranes into mixed DRMs [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF], nor scramble membrane symmetry [START_REF] Radeva | Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukemia) cells[END_REF].

However, the inner leaflet of the plasma membrane differs in lipid composition and phase characteristics from the outer leaflet, making it more vulnerable to disruption during membrane solubilization. Unlike the outer leaflet, whose major components (phosphatidylcholine and sphingomyelin) each form stable leaflets, the major inner leaflet lipid, PE, cannot by itself form a lamellar phase unless mixed with negatively charged PS -whose large hydrated headgroup complements the small headgroup of PE to seal the membrane-water interface [START_REF] Lewis | Surface charge markedly attenuates the nonlamellar phase-forming propensities of lipid bilayer membranes: calorimetric and (31)P-nuclear magnetic resonance studies of mixtures of cationic, anionic, and zwitterionic lipids[END_REF][START_REF] Li | Theory of lipid polymorphism: application to phosphatidylethanolamine and phosphatidylserine[END_REF][15]. This balanced mosaic of intracellular lipids is dependent upon exclusion of Ca 2+ , maintained extracellularly at 1.2mM but held within the cytoplasm at sub-µM basal levels. Sustained influx of µM Ca 2+ into the cell leads to separation of PS -into Ca 2+ -chelated PS -domains, leaving PE to either fuse with nearby membrane, or delaminate to form non-bilayer reverse hexagonal phases [16][START_REF] Duzgunes | Studies on the mechanism of membrane fusion. Role of head-group composition in calcium-and magnesium-induced fusion of mixed phospholipid vesicles[END_REF][START_REF] Tilcock | The polymorphic phase behaviour of mixed phosphatidylserinephosphatidylethanolamine model systems as detected by 31P-NMR[END_REF][START_REF] Tokutomi | Ca2+-induced phase separation in phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine mixed membranes[END_REF]. Cholesterol condensed with saturated PE and PS -in the inner leaflet [START_REF] Bakht | Effect of the structure of lipids favoring disordered domain formation on the stability of cholesterol-containing ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms[END_REF] sensitizes further the Ca 2+ chelation of PS -and separation of PE into an hexagonal phase [START_REF] Bally | Polymorphism of phosphatidylethanolaminephosphatidylserine model systems: influence of cholesterol and Mg2+ on Ca2+-triggered bilayer to hexagonal (HII) transitions[END_REF].

Uncontrolled high levels of Ca 2+ will access the cytofacial leaflet as the plasma membrane is disrupted by homogenization and the major intracellular Ca 2+ stores (ER, mitochondria and lysosomes) are lysed by detergent; if unchecked, this Ca 2+ would destabilize PE at its interface with water, the region Triton X-100, with its short, bulky hydrophobic chain will particularly perturb in contrast to the long, monolayer-spanning hydrophobic chain of Brij 96 (Supplementary Figure 1). Addition of Ca 2+ chelators such as EGTA to buffers should limit the destabilization of the inner leaflet as the cell is disrupted, but it has not prevented the selective depletion of inner leaflet lipids nor scrambling of the asymmetry of membrane proteins by Triton X100 [START_REF] Radeva | Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukemia) cells[END_REF][START_REF] Schuck | Resistance of cell membranes to different detergents[END_REF]. In order to maintain full membrane-dependent function in isolated cytosolic organelles, such as translocation of protein in RER microsomes [START_REF] Martoglio | Co-translation of proteins into microsomes derived from the rough endoplasmic reticulum of mammalian cells[END_REF], it is necessary to not only remove Ca 2+ , but also provide Mg 2+ and K + in a reducing buffer, where the effect of Mg 2+ in promoting lipid order [23] may be particularly relevant.

We have therefore investigated whether solubilization using buffers that chelate Ca 2+ , or further mimic the intracellular ionic environment, stabilize membrane domains sufficiently to allow subsets to be isolated at 37˚C as DRMs. We show this in detail for Brij 96, with which we have extensive experience in DRM isolation at 4˚C [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF][START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF][START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF]. Experiments with Triton X-100 are included to show that cytoplasmic cations affect membrane solubilization by this detergent also. Brij 98 (with a similar C 18 H 35 alkyl chain to Brij 96 but with ~20 polyoxyethylene head groups), that alone of non-ionic detergents can be used to isolate DRMs at 37˚C without using intracellular cations [START_REF] Drevot | TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts[END_REF][START_REF] Pike | Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study[END_REF], is also briefly examined with these buffers.

EXPERIMENTAL Animals and reagents

Wistar rats aged 4 weeks were killed by CO 2 inhalation, their brains immediately removed and homogenized. PrP, Thy-1 and LRP1 were detected by antibodies as described [START_REF] Parkyn | LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein[END_REF]; rabbit antibodies to Fyn (sc-434) and Src (sc-56890) were from Santa Cruz; mouse anti-actin (clone 4), anti-tubulin (clone DM1A) and rabbit antiflotillin-1 (E446) were from Chemicon. Fab antibody fragments from Ox7 anti-Thy-1 and 2S anti-PrP antibodies, and wheat germ agglutinin (WGA), were directly coupled to 5 and 10 nm gold (British BioCell Ltd) as described [START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF]. Laboratory chemicals were analytical grade from standard suppliers.

Membrane preparation and solubilization

Standard solubilization buffer (SB) was 250 mM sucrose, 10 mM Tris-HCl pH 7.4 and 1 mM EGTA with protease inhibitor mini-tablets (Roche) and 1 mM PMSF added before use. Intracellular solubilization buffer (ISB) was 200 mM sucrose, 10 mM Hepes-KOH (pH 7.6), 50 mM KOCOCH 3 , 1 mM Mg(OCOCH 3 ) 2 and 1 mM EGTA with protease inhibitor mini-tablets, 1 mM PMSF and 1 mM DTT (dithiothreitol) added before use. Homogenization (5ml buffer per brain) was with a Dounce homogenizer with the tight-fitting pestle on ice. When particulate membrane fragments were no longer observed (~30 strokes), nuclei were pelleted at 5,000 g for 5 mins (Beckman Coulter Avanti J25 centrifuge, JA18.1 rotor) then membranes at 30,000 g av for 40 min (Beckman Optima Ultracentrifuge, TLA120.2 rotor). The membrane pellet was resuspended in half the original volume, repelleted as before, then the resuspended pellet twice depleted of myelin by ultracentrifugation through a 0.5-2.0 M sucrose gradient, with myelin (fractions lighter than 0.8 M sucrose) discarded while Thy-1 and PrP were recovered in membrane fractions from 1.0 to 1.5 M sucrose. Protein concentration was determined (on aliquots solubilized in 0.1% SDS) by the Bradford method (BioRad Detergent Compatible Protein Assay Reagents Package); final concentrations were adjusted for solubilization to 10 mg/ml of membrane protein. Detergent solutions at double strength were mixed with equal volume of membrane homogenate. Solubilization was at 4°C for 15 min, or membranes were preequilibrated at 37°C for 5 min and detergent added for another 5 min. Where indicated, membranes in ISB were pre-incubated with or without 5 mM MßCD (methyl-ß-cyclodextrin) for 30 min at 37˚C, then detergent added. Solubilized membranes were diluted 1:1 with 80% sucrose at 4˚C and loaded at the bottom of the centrifuge tubes. Gradients of 5~30% sucrose prepared in the relevant buffer with detergent (1% Triton X-100, 0.5% Brij 96) were layered on top to a total volume of 12 ml and centrifuged to equilibrium (170,000 g av for 18 hr at 4°C'; SW40Ti rotor in a Beckman XL90 ultracentrifuge). Serial 1 ml aliquots were aspirated from the top of gradients. Pellets were taken up in 1 ml and resuspended by sonication (30 kHz, 1 min; Ultrasonic Homogenizer 4710, Cole-Parmer Instrument, USA).

Sensory neurons, cultured for 5-7 days from the dorsal root ganglia of 4-6 week old rats [START_REF] Parkyn | LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein[END_REF] were harvested with a teflon scraper and collected at 800 g for 5 min. Cultured neuroblastoma-derived N2a cells were similarly harvested. Both cell pellets were resuspended in ISB and fragmented in a 45 ml nitrogen bomb (PS152BAR, Parr Instrument, USA) at 1200 to 1300 psi after equilibrating for 5 min at 4˚C. Nuclei were pelleted as above, DNAse (Promega) added at 10 unit/ml to postnuclear supernatant and protein concentration determined. Sensory neuronal membranes were adjusted to 1 mg protein/ml to which an equal volume of 0.1% Brij 96 in ISB was added; N2a membranes were adjusted to 10 mg protein/ml to which 1.0-0.1% Brij 96 was added. Sucrose gradients (5 ml total) were centrifuged to equilibrium (300,000 g av for 18 hr at 4˚C; TLS-55 rotor, Beckman Optima ultracentrifuge).

Immunoprecipitation (IP)

Purified IgG (Ox7 for Thy-1, 2S for PrP), coupled at 1 mg/ml to Dynabeads M-280 Tosyl activated, blocked with ethanolamine according to manufacturers instructions (Dynal Biotech), was incubated with gradient fractions at 4°C with gentle rotation from 1 hr to overnight. Beads retained with a magnet were washed with 1 ml SB plus 0.1% Brij 96, then twice with 0.01% Brij 96 before bound protein, recovered by boiling for 5 min in non-reducing sample buffer [START_REF] Laemmli | Cleavage of structural proteins during the assembly of bacteriophage T4[END_REF] was electrophoresed and immunoblotted.

EM (electron microscopy)

For evaluation of glycoprotein asymmetry, DRMs were fixed by 1% paraformaldehyde and 0.1% glutaraldehyde in their gradient buffer at 4°C overnight, then spun down by centrifugation at 30,000 g for 1 hr (Beckman Coulter Avanti J25, JA18.1 rotor). The pellet was resuspended in 5 µl 10% gelatine that was allowed to set by placing the tube on ice for 30 mins. Small pieces of the pellet were cryo protected by exposure to 2.3 M sucrose overnight at 4ºC, placed onto a pin and cryofixed by plunging into liquid nitrogen. Sections (80 nm thick) were cut at -80˚C using an RMC cryo-ultramicrotome, and transferred to Pioloform coated Ni EM grids where they were washed with PBS then remaining aldehyde groups blocked with 50 mM glycine in PBS pH 7.2. Sections were incubated with 0.1 µg WGA/ml in PBS for 4 hr, washed in PBS then fixed in 2% glutaraldehyde in PBS for 5 min. Grids were contrasted in a mixture of 1 part 3% uranyl acetate in 9 parts 2% methylcellulose for 10 min and viewed in a Technai 12 transmission EM.

4

Immediately before use, immunogold preparations were precleared by centrifugation for 1 hr to remove any aggregates that may have formed: 5 nm gold was spun at 25,000g (the force used to pellet 10 nm gold) and 10 nm gold was spun at 15,000g (the force used to pellet 15 nm gold; Beckman Coulter Avanti J25 centrifuge, JA18.1 rotor). For Thy-1 and PrP immunolabelling, DRMs were incubated with Fab antibody fragments of OX7/2S directly coupled to 5/10 nm gold respectively (0.1 µg protein/ml for each antibody) at 37˚C for 0.5 hour. In the control experiment, R194 anti-mouse Thy-1.2 and 3F4 anti-human PrP antibodies, directly coupled to gold as before, were used. Unbound gold was removed by centrifugation (14,000g, 5 min) through 40% sucrose; labelled DRMs at the interface were pelleted by centrifugation (14,000g, 2 min) and the pellet fixed overnight in 2% paraformaldehyde/0.1% glutaraldehyde for 1 hr. Samples were postfixed with OsO 4 (pH7.3) at 4°C for 1.5 hr, dehydrated through ascending ethanol solutions into TAAB epoxy resin (TAAB Laboratory Equipment Ltd, Aldermaston UK). The resin blocks were cut with diamond blades on a Leica Ultra-Cut microtome to 80~120 nm ultra-thin sections and picked up on to 150 mesh Guilder Grids with a support film of Pioloform. Grids, stained with uranyl acetate (25% solution in absolute methanol) and lead citrate (4.4% (w/v), in CO 2 -free H 2 O), were viewed under TEM (Hitachi H7600, Japan).

The 5 and 10 nm gold particles were initially confirmed to be of that diameter using Gatan on-screen measuring software, and thereafter identified by eye using the 4 fold difference in their cross-sectional areas, and difference in contrast, as a guide. Quantitation was done manually on high power prints by overlaying a transparency on which were printed bars of 20 and 150 nm; particles present on different membrane fragments were not counted as being adjacent on the membrane.

RESULTS

DRMs were isolated from myelin-depleted brain membranes by detergent solubilization followed by ultracentrifugation to equilibrium in a sucrose density gradient [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF][START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF] using either Solubilization Buffer (SB) containing 1mM EGTA to chelate Ca 2+ , or Intracellular Solubilization Buffer (ISB) to reproduce aspects of the intracellular ionic environment (Mg 2+ , K + and a reductive agent) during homogenization and solubilization. Preliminary experiments using dynamic light scattering found very little difference in the hydrodynamic size of micelles formed by either Triton X-100 or Brij 96 in the two buffers (Supplementary Figure 2), in keeping with the low susceptibility of non-ionic surfactants to buffer composition [START_REF] Warisnoicharoen | Light scattering investigations on dilute nonionic oilin-water microemulsions[END_REF].

Effect of buffer composition on isolation of 'raft' proteins as DRMs at 4˚C

As a preliminary control, the migration of membrane proteins in the density gradient prior to adding detergents was determined (Figure 1A). Both GPI-anchored Thy-1 and PrP remained within the loading fractions (11 and 12) of 40% sucrose at the bottom of the gradient, with some PrP also pelleting at the bottom of the gradient. Actin, the structural protein of the cortical cytoskeleton that is anchored via adaptor proteins to transmembrane protein complexes, was also recovered in the loading fractions, although a small fraction of actin and PrP rose higher in the gradient, suggesting their association with low density, lipid-rich membrane fragments. Recovery of proteins in the loading fractions after solubilization is often interpreted as their being fully solubilized; to test whether this was the case here, the 40% sucrose fractions were diluted to 8% sucrose and centrifuged at 45,000g for 3 hr (Figure 1A, right panel). All three proteins were recovered in the pellet and so were in membranes rather than fully solubilized.

Brain membranes were solubilized for 15 min at 4˚C, in both SB and ISB, in Triton X-100 (Figure 1B) and Brij 96 (Figure 1C), at concentrations (1% and 0.5% detergent respectively with 5 mg/ml membrane protein) found previously to give optimal release of PrP and Thy-1 DRMs [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF]. The distribution of Thy-1 and PrP on the gradient after solubilization in SB was very similar to that previously described [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF], with the proteins moving to lighter density in Brij 96 compared to Triton X-100. Both detergents in SB caused a major part of actin to pellet at the bottom of the gradient, with the rest primarily in the loading fractions.

In ISB, however, the major fraction of Thy-1 and PrP remained either in the loading fractions, or (Thy-1 in Brij 96) in fractions 7-10 of intermediate density. Relatively little of these proteins floated at the low density found with solubilization in SB. In addition, in the presence of ISB, some actin floated further up the gradient in fractions 2-7 that would normally contain DRMs. When the loading fractions (11+12) recovered from gradients on which membranes solubilized in either detergent in ISB were diluted to 8% sucrose and recentrifuged, virtually all Thy-1 and PrP was pelleted, indicating they remained in unsolubilized membrane (Figure 1B, C, right panels). This centrifugation test to determine whether protein was membrane-associated or fully solubilized was routinely applied hereafter to the loading fractions.

Solubilization at 37˚C

Since ISB protected membranes against solubilization at 4˚C, the effect of the buffer upon solubilization at 37˚C was investigated. Figure 2 shows the effect of ISB compared to SB upon solubilization of brain membranes for 5 min at 37˚C in Brij 96 and Triton X-100. Brij 98 in both buffers was also tested, using 15 min solubilisation at 37˚C as described by Drevot et al [START_REF] Drevot | TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts[END_REF].

Following solubilisation in Brij 96 or Triton X-100, a substantial portion of the GPI-anchored proteins, and the intracellular 'raft' protein flotillin-1 (not shown for Triton X-100), floated at the top of the gradient (fractions 2-4) irrespective of whether SB or ISB was used. Upon dilution to 5% sucrose, Thy-1 and PrP in these light fractions could all be pelleted by centrifugation (45,000g, 3 hr), confirming their membrane nature (not shown; see Figures 6,7 below). LRP1, the transmembrane endocytic partner for PrP on neurons [START_REF] Parkyn | LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein[END_REF] remained within the loading fractions. Pre-treatment of the membranes with the cholesterol chelator, methylß-cyclodextrin (MßCD), depleted this low density fraction as expected for cholesterol-dependent DRMs [6]. These results together argue that it is possible to isolate DRMs from neuronal membranes by solubilization at 37˚C using either SB or ISB.

Solubilization in ISB differed from that using SB in delivering Thy-1 and PrP DRMs in a smaller number of fractions; in addition, a small proportion of the intracellular diacylated kinase Fyn, and even some of the monoacylated kinase Src, floated within the DRM fraction when ISB but not SB was used (Figure 2A). A small proportion of the cytoskeletal proteins actin and tubulin also floated at low density in ISB but not SB.

Similar results to those shown in Figure 2 were obtained with shorter (30 & 60 second) solubilization times although 5 min proved more reproducible in the proportion of Thy-1 and PrP released to float at low density (not shown). With longer solubilization (30-60 min) at 37˚C, sphingomyelinase degradation of raft sphingomyelin becomes evident [START_REF] Chen | The Isolation and Structure of Membrane Lipid Rafts from Rat Brain[END_REF]. Brij 98 in SB showed a pattern of solubilization similar to that seen using ISB with Triton X-100 or Brij 96, in that much of the GPI-anchored proteins, and some Fyn and Src, floated at low density. ISB, however, appeared to stabilize the membrane too strongly to allow effective solubilization by Brij 98, since most of the GPI-anchored proteins and cytoplasmic kinases remained in the loading fractions (Figure 1C), from which they could be pelleted by centrifugation (not shown) and so were membrane associated.

Stability of DRMs at 37˚C and above

Membranes solubilized as above were at 37˚C for just 10 min: 5 min to pre-equilibrate, then 5 min more with detergent. Subsequent gradient preparation and 18 hr ultracentrifugation was at 4˚C, during which detergent was also present in the gradient. To determine whether DRMs isolated at 37˚C remain stable at this temperature, samples processed at 37˚C for gradient formation and ultracentrifugation were compared with those centrifuged in a gradient at 4˚C. As shown in Figure 3 for Triton X-100, both Thy-1 and PrP on the external surface, and flotillin-1 on the inner surface, floated at 2-3 fractions lighter density when ultracentrifuged at 37˚C. This indicates a higher lipid to protein ratio at the higher temperature, and longterm stability of the DRMs at 37˚C.

Since L o domains can be stable up to ~50-60˚C [31,[START_REF] Chachaty | Building up of the liquid-ordered phase formed by sphingomyelin and cholesterol[END_REF] it should be possible to obtain DRMs after solubilization at temperatures higher than 37˚C. This was explored with solubilization for 5 min using Triton X-100 in ISB at higher temperatures (followed by ultracentrifugation at 4˚C). DRMs of Thy-1 and PrP remained resistant to solubilization at temperatures up to 50-55˚C (Figure 4).

Separate immunoisolation of PrP and Thy-1 DRMs in ISB/Brij 96 at 37˚C

Since Thy-1 and PrP DRMs could be immunopurified largely as separate DRMs after solubilization using Brij 96 at 4˚C [START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF], immunoaffinity separation of DRMs was undertaken after solubilization at 37˚C. Since Thy-1 is the more abundant protein on the neuronal surface [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF], PrP was first immunoisolated then Thy-1 was purified from the PrP-depleted fraction. Each protein was recovered almost entirely free of the other A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. p 6 (Figure 5). Actin was co-eluted with Thy-1 DRMs but not PrP DRMs, but not when the DRMs were fully solubilized, suggesting that actin does not bind directly to Thy-1 but to some other component of its DRMs.

Preservation of asymmetry of glycoproteins in DRMs isolated at 37˚C

DRMs isolated from fractions 3+4 of sucrose gradients, after solubilization in Brij 96 in ISB, were fixed, snap-frozen, cryo-sectioned and labelled with WGA coupled to 5 nm gold, to identify the extracellular glycan chains of glycoproteins that are present only extracellularly [START_REF] Tartakoff | Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distal maturation of oligosaccharides[END_REF]. This method allows equal access of the lectin to both sides of the DRM membrane, so that any scrambling of the natural asymmetry of the plasma membrane should be evident as scrambling of WGA-gold labelling on both sides of the DRM membrane. Typical results for Brij 96 DRMs are shown in Figure 6. Interpretation of results is complicated because the cryo-sections are ~80 nm thick, whereas many of the labelled DRMs are smaller so that their membrane is often seen en face as it curves within the plane of the section (e.g. Figure 6, top left). However, whenever a clear cross-section of membrane was seen, the WGA label was always on the external surface only, indicating that DRMs preserve an outside-out orientation of membrane, and do not randomly scramble the in vivo asymmetry of the plasma membrane.

Morphology of individual Thy-1 and PrP DRMs isolated at 37˚C

The ultrastructure of individual Brij 96/ISB DRMs taken from gradient fractions 3+4 was examined in the EM by immunolabelling DRMs at 37˚C with anti-PrP Fab on 5 nm gold, and anti-Thy-1 Fab on 10 nm gold (Figure 7). The overall vesicle population varied considerably in size and shape, with ~30% of the vesicles immunolabelled for either Thy-1 or PrP. En face and glancing side views of membranes predominate.

Virtually all PrP label occurred in large clusters, often in linear arrays (e.g. Figure 7C-F) as found on the surface of cultured neurons [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF][START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF][START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF]. At the magnification reproduced here, overlapping 5 nm grains within a cluster could be mistaken for 10 nm label (e.g. Figure 7C, E), although overlapping particles lack the symmetry and contrast of single 10 nm gold particles. With overlapping label, individual grains were resolved at high power under the microscope, and their size and number noted for quantitation. PrP clusters only occasionally had Thy-1 label near them (e.g. Figure 7F; applying the same criterion as [START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF] only 3.9 ±2.0% (mean ±s.e.m.) of PrP label was within 20 nm of Thy-1). However, a single DRM that contained a large PrP cluster could also contain a relatively distant label for Thy-1 (e.g. Figure 7C,G).

Thy-1 label most frequently occurred at low density upon thin tubular structures (Figure 7I-K) that were possibly derived from axons, although larger clusters were also found (Figure 7H,L). Most Thy-1 was not associated with PrP immunolabelling (3.2 ±1.4% Thy-1 label was within 20 nm of PrP label, and even extending the range, only 13.1 ±4.5% of Thy-1 label was within 150 nm of labelled PrP).

The striking feature of these images, however, is that the labelled GPI-anchored proteins occupy only a small fraction of the DRM surface; the major part of DRMs isolated under these conditions was not occupied by our 'marker' proteins. This was also found with DRMs isolated in Brij 96 at 4˚C [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF].

Isolation of DRMs at 37˚C from primary cultured adult sensory neurons

To test whether DRMs could be isolated at 37˚C from cultured cells, sensory neurons were chosen as they can uniquely be cultured from adult animals [START_REF] Scott | Adult mouse dorsal root ganglia neurons in cell culture[END_REF]. Neurons were fragmented by N 2 cavitation, their membranes collected by centrifugation and run on a sucrose density gradient where colloidal gold staining showed their proteins were recovered in the loading fractions and pellet, and not in the low density fractions (Supplementary Figure 3A). The small amounts of material limited the membrane protein concentration to 0.5 mg/ml, so Brij 96 was proportionately diluted to 0.05% for solubilization for 5 min at 37˚C, releasing membrane protein to float at low density (Supplementary Figure 3B). Virtually all Thy-1 floated as low density DRMs (fractions 1-3; Figure 8), whereas only ~10% of PrP floated as somewhat heavier DRMs (fractions 3-5), the majority remaining in the loading fraction (where it was fully solubilized by the criterion of not being pelleted by ultracentrifugation; not shown). This difference between the two GPI-anchored proteins matches the almost exclusive location of Thy-1 on the surface, and of 90% of PrP in intracellular, membranes on these neurons [START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF][START_REF] Parkyn | LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein[END_REF]. Flotillin-1 behaved very similarly to PrP, the majority being fully solubilized but some floated in fractions 3-5. The intracellular diacylated kinase Fyn was largely recovered in the pellet (fraction 12), probably as part of larger protein complexes; but a proportion was recovered in the Thy-1-rich fraction 1. Two 'non-raft' proteins, monoacylated Src and transmembrane LRP1, remained

Isolation of DRMs at 37˚C from N2a Cells

N2a cells were similarly fragmented by N 2 cavitation and solubilized with Brij 96 in ISB at 4˚C and 37˚C at the same protein concentration (5 mg/ml) used with brain membranes. It was found that 0.5% Brij 96 solubilized most of the PrP at 37˚C (not shown), and lowering the detergent concentration to 0.05% was required to obtain PrP and flotillin-1 in low density fractions; no actin was recovered in DRM fractions (Figure 9). Thy-1 was not assessed as N2a cells do not express it.

The distinctive difference between solubilization at 4 and 37˚C was in the size of the DRMs obtained, which were smaller and more homogeneous at 37˚C, irrespective of whether ISB or SB was used (Supplementary Table 1).

DISCUSSION

This paper examines the hypothesis that ionic stabilization of the lamellar phase of inner leaflet lipids enables L o nanodomains on the cell surface to resist detergent solubilization at physiological temperature. Experimentally convenient immunoassays of 'raft' proteins have been used to explore relevant variables; lipid and structural analyses will be reported elsewhere (Chen et al, in preparation; [START_REF] Chen | The Isolation and Structure of Membrane Lipid Rafts from Rat Brain[END_REF]).

Demyelinated membranes from adult rat brain have been used as starting material, providing fully differentiated plasma membranes, albeit of mixed cellular origin (neurons, glia and vasculature). Two GPIanchored 'raft' proteins, Thy-1 and PrP, are expressed only on neurons in normal adult brain [START_REF] Ford | A marked disparity between the expression of prion protein and its message by neurons of the central nervous system[END_REF][START_REF] Morris | Thy-1, the enigmatic extrovert on the neuronal surface[END_REF], and occupy topographically, biochemically and functionally different nanodomains on the neuronal surface [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF][START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF][START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF][START_REF] Morris | Traffic of prion protein between different compartments on the neuronal surface, and the propagation of prion disease[END_REF]. Their behaviour during solubilization monitors the detergent resistance of distinct sub-domains of the surface of mature neurons. To monitor the integrity of the inner leaflet, flotillin, that inserts into this leaflet via a polypeptide loop, and Fyn, that inserts via two acyl chains, have been used [START_REF] Morris | Rafts, little caves and large potholes: how lipid structure interacts with membrane proteins to create functionally diverse membrane environments[END_REF]. Src, similar to Fyn but with only one acyl chain inserting into the lipid bilayer, is found at 4˚C not to associate with DRMs [START_REF] Morris | Rafts, little caves and large potholes: how lipid structure interacts with membrane proteins to create functionally diverse membrane environments[END_REF]. The cytoskeletal structural proteins actin and tubulin were included, since although the vast majority of these proteins are in cytoplasmic and cytoskeletal pools, a very minor proportion contacts and organizes the topography of transmembrane proteins. Finally, LRP1 has been used as an example of a transmembrane protein not normally found within rafts [START_REF] Parkyn | LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein[END_REF].

DRMs could be isolated from brain membranes and cultured adult sensory neurons at 37˚C using the same detergent:protein ratio; however, with the N2a cell line, this ratio had to be lowered 10-fold. The source of the higher detergent resistance of the adult neuronal membrane is unknown, but correlates with the high resistance (compared to cell lines) to transfection of DNA vectors into primary cells in general, and into adult neurons in particular [START_REF] Buchser | 96-well electroporation method for transfection of mammalian central neurons[END_REF]. These differences in permeability of surface membranes, whether to detergent solubilization or large DNA complexes, means that no single protocol for DRM isolation applies to all cell types. The basic approach needs to be fine tuned to match the membranes studied.

Surprisingly, both Thy-1 and PrP floated within low density membrane fragments after solubilization at 37˚C in either Triton X-100 or Brij 96, irrespective of the buffer (SB or ISB) used. However, incorporation of cytofacial leaflet proteins in DRMs was improved using ISB, so ISB/Brij 96 DRMs were characterised both biochemically and ultrastructurally.

The association of proteins with ISB/Brij 96 DRMs isolated at 37˚C agrees with known differences in their biochemistry. Functionally, Thy-1 inhibits signalling of Src-family kinases [START_REF] Hueber | Thymocytes in Thy-1-/-mice show augmented TCR signaling and impaired differentiation[END_REF], of which Fyn is the main diacylated member in neurons and floated in the same light DRM fractions as Thy-1. The actual proportion of total Fyn in the DRM fraction was low, with most in the pellet or solubilized fractions. We find a similar low proportion of Fyn in brain DRMs isolated at 4˚C (not shown), presumably because in neurons most Fyn associates with adhesion and postsynaptic density fractions that are not within L o phase membrane. Neurons differ from the immortalized cell lines typically used for raft studies in being post-mitotic and nontransformed, primary cultured cells, which may account for their different compartmentalization of Fyn. Actin is involved in raft signalling (e.g. [START_REF] Holowka | Lipid segregation and IgE receptor signaling: A decade of progress[END_REF][START_REF] Viola | Tether and trap: regulation of membrane-raft dynamics by actin-binding proteins[END_REF]), which may explain its selective association with Thy fractions 1-3), confirming independently of immunoaffinity isolation or immunolabelling, the separate identity of PrP and Thy-1 nanodomains on these neurons. The DRM component of flotillin-1 peaked with PrP, in keeping with its involvement in PrP signalling [START_REF] Stuermer | PrPc capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction[END_REF].

One notable benefit of solubilization at 37˚C was the almost complete immunoseparation of PrP from Thy-1 DRMs compared to the presence of 20% of PrP in Thy-1 DRMs at 4˚C [START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF]. This separation, seen also at the level of immunogold labelled vesicles, argues that merger of neuronal surface nanodomains in detergent has not occurred since it would inevitably merge Thy-1 and PrP domains that are often within µm of each other on the neuronal surface [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF][START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF][START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF]. Further, the distinctive clustering of PrP on the neuronal surface [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF][START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF][START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF] is retained in isolated DRMs. WGA labelling of cryo-sections of DRMs showed labelling only on their outer surface, again indicating that arbitrary merger of rafts had not occurred. Were raft membrane to curl arbitrarily upon solubilization, glycan labelling would be found equally on the inner or outer surface of individual vesicles. Label was only found on the outer surface, possibly because the Brij 96 causes lipid rafts to vesiculate from the surface, similar to Triton X-100-induced budding of Lo domains from giant unilamellar vesicles [START_REF] Staneva | Detergents induce raft-like domains budding and fission from giant unilamellar heterogeneous vesicles: a direct microscopy observation[END_REF]. However, Triton X-100 scrambles membrane orientation in individual DRMs isolated from cells [START_REF] Radeva | Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukemia) cells[END_REF], arguing that solubilization of cell membranes is more complex than that of pure lipid bilayers. A factor that could confer intrinsic convex pressure in raft membrane is the extraordinarily large glycoprotein headgroup on the GPI lipid, which should act like other disproportionately large lipid headgroups to impose convex curvature on raft membrane [START_REF] Morris | Rafts, little caves and large potholes: how lipid structure interacts with membrane proteins to create functionally diverse membrane environments[END_REF]45].

The larger clusters of 20-40 grains of monovalent immunogold label for Thy-1 and PrP that we find on isolated DRMs and on the neuronal surface [START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF][START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF] are contrary to findings that GPI-anchored proteins are not confined to recognisable domains until they are cross-linked by ligand [START_REF] Paar | Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI[END_REF][START_REF] Suzuki | Dynamic recruitment of phospholipase C gamma at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2[END_REF][START_REF] Suzuki | GPI-anchored receptor clusters transiently recruit Lyn and G alpha for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1[END_REF] or multivalent antibody [START_REF] Harder | Lipid domain structure of the plasma membrane revealed by patching of membrane components[END_REF]. The GPI-anchored proteins in these fluorescence-based studies do not include Thy-1 or PrP. Fluorescence correlation spectroscopy shows Thy-1 to be confined to 40-120 nm domains on living cells [START_REF] Lenne | Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork[END_REF][START_REF] Wenger | Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization[END_REF] similar to those seen in the EM. We therefore suggest that the expectation that all GPI-anchored proteins are not clustered until cross-linked reflects the range of molecules and cell types examined, and does not apply to neuronal rafts of Thy-1 and PrP.

Immunolabelled PrP and Thy-1 occupy an unexpectedly small part of their DRMs, suggesting that both proteins are clustered due to properties additional to their partitioning into L o domains, and occupy only a small proportion of their rafts. Studies show that the L o phase can be the major lipid phase on some cell surfaces (e.g. [START_REF] Ge | Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study[END_REF][START_REF] Swamy | Coexisting domains in the plasma membranes of live cells characterized by spin-label ESR spectroscopy[END_REF]) whereas our fluorescent and EM immunolabelling indicate that Thy-1 and PrP occupy at most a few % of the neuronal surface [START_REF] Madore | Functionally different GPI proteins are organised in different domains on the neuronal surface[END_REF][START_REF] Brügger | The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition[END_REF][START_REF] Sunyach | The mechanism of internalisation of GPI anchored prion protein[END_REF]. Although the mechanism is currently unclear, clustering of PrP and Thy-1 on the neuronal surface into sub-regions of larger, L o phase lipid domains is possible.

Overall, the use of a buffer that reproduces aspects of the intracellular environment during solubilization enables the isolation of DRMs at 37˚C that have properties compatible with their being isolated lipid 'rafts'. It is now possible to identify their composition and determine whether these components associate at the surface membrane of the living cell. Our results further indicate the need to examine the topographical relationship on neurons between raft proteins and lipids. Cell membranes could only be obtained at 0.5 mg/ml of membrane protein, and so were solubilized in ISB at 37˚C in 0.05% Brij 96, to maintain the same detergent:protein ratio as used with brain membranes. Immunoblots are of fractions along the gradient as before, fraction 12 is the pellet. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 03 Oct 2008 as manuscript BJ20081385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081385A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 03 Oct 2008 as manuscript BJ20081385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081385A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  p

  Biochemical Journal Immediate Publication. Published on 03 Oct 2008 as manuscript BJ20081385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081385A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 03 Oct 2008 as manuscript BJ20081385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081385

Biochemical

  Journal Immediate Publication. Published on 03 Oct 2008 as manuscript BJ20081385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081385A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  ) or largely (Src) in the loading fractions with no trace in DRM fractions 1-5. Actin was primarily in the pellet, but a very small proportion floated as high as fraction 1.

  -1 DRMs. With cultured sensory neurons, PrP occurred in denser DRMs (peak fractions 4/5) than Thy-1 (peak Biochemical Journal Immediate Publication. Published on 03 Oct 2008 as manuscript BJ20081385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081385A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

p 11 Figure 1 .

 111 Figure 1. Immunoblots of protein distribution on sucrose density gradients following solubilisation of brain membranes at 4˚C(A) shows Thy-1, PrP and actin on membranes without solubilization, with the top (fraction 1) and bottom (fraction 12) of the gradient indicated; the pellet at the bottom of the gradient (P) was resuspended in 1 ml of buffer (ISB). The panel to the right shows an immunoblot of supernatant (S') and pellet (P') fractions after the loading fractions (11+12) were combined, diluted to 8% sucrose and re-centrifuged at 45,000 g for 3 hr. (B) and (C) show the distribution of these proteins following solubilization of the brain membranes for 15 min in Triton X-100 (B) or Brij 96 (C), with the right hand panels showing the supernatant and pellet fractions after dilution and centrifugation as above.

p 12 Figure 2 . 13 Figure 3 .

 122133 Figure 2. Immunoblots of protein distribution on sucrose density gradients following solubilisation of brain membranes at 37˚C (A) shows the distribution of a range of proteins (fraction 1, top, fractions 11,12 the bottom loading fractions; P, resuspended pellet) following solubilization in Brij 96 for 5 min. (B) Effect of depleting the membranes of cholesterol by chelation with 5 mM MßCD for 30 min at 37˚C prior to solubilization in Brij 96, shown by immunoblots for Thy-1 and PrP of pooled DRM fractions 2-4 and pooled solubilised (Sol) fractions 11+12. (C) shows the distribution of Thy-1 and PrP after solubilization of membranes in 1% Triton X-100 for 5 min at 37˚C, and (D) the distribution of the proteins shown after solubilization in 1% Brij 98 for 15 min at 37˚C.

Figure 4 . 14 Figure 5 .

 4145 Figure 4. Effect on increasing temperatures of solubilization by Triton X-100 Immunoblots show the distribution of Thy-1 and PrP along sucrose gradients after solublization of brain membranes in 1% Triton X-100 in ISB for 5 min at the temperature shown,

Figure 6 . 15 Fig 7 . 16 Figure 8 .

 6157168 Figure 6. Glycoproteins are only on the outer face of DRMs isolated at 37˚C in Brij 96/ISB Cryo-EM sections labelled with WGA-5nm gold. Scale bar is 100 nm.

Figure 9 .

 9 Figure 9. Immunoblots to demonstrate the release of DRMs from N2a cell membranes Solublization was of 5 mg protein/ml by 0.05% Brij 96 in ISB, at 37˚C for 5 min, or 4˚C for 15 min; P indicates the pellet fraction.

© 2008 The Authors Journal compilation © 2008 Biochemical Society

This work was aided by a grant from the Human Frontier Science Program (RGP0016/2005C) to PJQ. XC was supported by a King's College post-graduate studentship and an ORS award.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society


Meghan C Brown

Johannes A Eble

Juan J Calvete

Cezary Marcinkiewicz

Structural Requirements of KTS-Disintegrins for Inhibition of α 1 β 1 integrin

Keywords: KTS-disintegrin, obtustatin, viperistatin, integrin α 1 β 1 inhibitor, recombinant disintegrin, structure-function correlation

Obtustatin and viperistatin represent the shortest known snake venom monomeric disintegrins. We have produced recombinant full-length wild-type and site-directed mutants of obtustatin to assess the role of the 21 KTS 23 tripeptide and C-terminal residues for specific inhibition of the α 1 β 1 integrin. Threonine-22 appeared to be the most critical residue for disintegrin activity, whereas substitution of flanking lysine or serine for alanine resulted in less pronounced decreased of the disintegrin´s anti-α 1 β 1 integrin activity. The triple mutant 21 AAA 23 was devoid of blocking activity towards α 1 β 1 integrin-mediated cell adhesion. The potency of recombinant KTS-disintegrins also depended on the residue C-terminally adjacent to the active motif. Substitution of wild-type obtustatin´s leucine-24 for alanine slightly decreased the inhibitory activity of the mutant, whereas arginine in this position enhanced by 6-fold the potency of the mutant over wild type obtustatin. In addition, the replacements leucine-38/valine and proline-40/glutamine may account for a further 25-fold increase in α 1 β 1 inhibitory potency of viperistatin over KTSR-obtustatin.

INTRODUCTION

Integrins are αβ heterodimeric type-I transmembrane glycoproteins that mediate a range of regulated and dynamic interactions between cells and extracellular adhesion molecules. In humans, 18 α and 8 β integrin subunits combine in a restricted manner to form at least 24 dimers, each of which exhibits a distinct ligand-binding profile [START_REF] Calvete | Platelet integrin GPIIb/IIIa: structure-function correlations. An update and lessons from other integrins[END_REF][START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF][START_REF] Takagi | Integrin activation and structural rearrangement[END_REF]. Integrins transduce bidirectional signals across the plasma membrane, between the extracellular matrix (ECM) outside a cell and the cytoskeleton inside the cell [START_REF] Berrier | Cell-matrix adhesion[END_REF][START_REF] Luo | Structural basis of integrin regulation and signaling[END_REF], and integrin-mediated cell attachment to the ECM is a basic requirement to build a multicellular organism. Among the ligands of integrins are fibronectin, vitronectin, laminin, and collagen. The collagen-binding integrins (α 1 β 1 , α 2 β 1 , α 10 β 1 , α 11 β 1 ) contain an approximately 200-amino-acid-residue (I-or A-) domain inserted between the second and the third Ca 2+ -binding motifs within the N-terminal part of the α subunit that plays a central role in ligand binding [START_REF] Eble | Collagen-binding integrins as pharmaceutical targets[END_REF], and play an important role in in the physiology of many organs and in innate immunity, inflammation and autoimmunity [START_REF] Mccall | Collagen receptor integrins: rising to the challenge[END_REF]. The αI-domain-containing integrins that mediate direct adhesion to collagens seem to have a relatively recent history, since they have been found only in the vertebrate clade [START_REF] Hughes | Evolution of the integrin α and β families[END_REF]. Among them, integrin α 1 β 1 has been recognized as a high affinity receptor for collagen type IV, and binds also, albeit with less avidity, to other collagens such as type I, XIII and XVI [START_REF] Leitinger | Integrin I domains and their function[END_REF][START_REF] Dickeson | Determinants of ligand binding specificity of the α 1 β 1 and α 2 β 1 integrins[END_REF]. Integrin α 1 β 1 is highly expressed on the microvascular endothelial cells, and blocking of its adhesive properties significantly reduces the vascularization ratio and tumor growth in the mouse [START_REF] Marcinkiewicz | Obtustatin: a potent selective inhibitor of α 1 β 1 integrin in vitro and angiogenesis in vivo[END_REF][START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF][START_REF] Calvete | KTS and RTS-disintegrins: antiangiogenic viper venom peptides specifically targeting the α 1 β 1 integrin[END_REF], suggesting potential stategies to therapeutically target these receptors to alleviate or treat disease.

Viperidae snakes have developed in their venoms an efficient and almost complete repertoire of β1and β3-integrin receptor antagonists [START_REF] Sanz | Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system[END_REF]. Thus,with the exception of the α 2 β 1 integrin,which is targeted by a number of C-type lectin-like proteins [START_REF] Ogawa | Molecular diversity and accelerated evolution of C-type lectin-like proteins from snake venom[END_REF], inhibitory motifs towards β1 and β3 integrins have evolved in different members of the disintegrin family [START_REF] Niewiarowski | Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors[END_REF][START_REF] Marcinkiewicz | Functional characteristic of snake venom disintegrins: potential therapeutic implication[END_REF][START_REF] Calvete | Snake venom disintegrins: evolution of structure and function[END_REF][19]. Among them, RGD blocks integrins α 8 β 1 , α 5 β 1 , α v β 1 , α v β 3 and α IIb β 3 ; MLD targets the α 3 β 1 , α 4 β 1 , α 4 β 7 , α 7 β 1 and α 9 β 1 integrins; VGD and MGD impair the function of the α 5 β 1 integrin; KGD inhibits the α IIb β 3 integrin with a high degree of selectivity;WGD has been reported to be a potent inhibitor of the RGD-dependent integrins α 5 β 1 , α v β 3 and α IIb β 3 ; and KTS and RTS represent selective α 1 β 1 inhibitors [START_REF] Sanz | Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system[END_REF][START_REF] Scarborough | Barbourin. A GPIIb-IIIa-specific integrin antagonist from the venom of Sistrurus m. barbouri[END_REF][START_REF] Marcinkiewicz | Structural and functional characterization of EMF10, a heterodimeric disintegrin from Eristocophis macmahoni venom that selectively inhibits α 5 β 1 integrin[END_REF][START_REF] Calvete | The presence of the WGD motif in CC8 heterodimeric disintegrin increases its inhibitory effect on α IIb β 3 , α v β 3 , and α 5 β 1 integrins[END_REF].

KTS/RTS-disintegrins, 41-residue monomeric polypeptides crosslinked by 4 conserved disulfide bonds, represent the shortest snake venom disintegrins described to date [START_REF] Calvete | KTS and RTS-disintegrins: antiangiogenic viper venom peptides specifically targeting the α 1 β 1 integrin[END_REF]. Disintegrin obtustatin, isolated from the venom of Vipera lebetina obtusa, was the first described α 1 β 1 antagonist [START_REF] Marcinkiewicz | Obtustatin: a potent selective inhibitor of α 1 β 1 integrin in vitro and angiogenesis in vivo[END_REF][START_REF] Moreno-Murciano | Amino acid sequence and homology modeling of obtustatin, a novel non-RGDcontaining short disintegrin isolated from the venom of Vipera lebetina obtusa[END_REF]. It blocked the adhesion of α 1 β 1 -expressing K562 cells to immobilized collagens IV and I with an IC 50 of 2 nM and 0.5 nM, respectively, but was inactive against a other integrin receptors, including the α 1 β 1 structurally closely-related collagen receptor α 2 β 1 integrin. Obtustatin (5 µg/disk) inhibited 84% of FGF2-induced angiogenesis in vivo in the chicken [START_REF] Marcinkiewicz | Obtustatin: a potent selective inhibitor of α 1 β 1 integrin in vitro and angiogenesis in vivo[END_REF] and the quail [START_REF] Brown | Angiostatic activity of obtustatin as α1β1 integrin inhibitor in experimental melanoma growth[END_REF] chorioallantoic membrane assay. Moreover, in the Lewis lung [START_REF] Marcinkiewicz | Obtustatin: a potent selective inhibitor of α 1 β 1 integrin in vitro and angiogenesis in vivo[END_REF] and B16F10 melanoma [START_REF] Brown | Angiostatic activity of obtustatin as α1β1 integrin inhibitor in experimental melanoma growth[END_REF] syngenic mouse models, this disintegrin reduced tumor growth by about 50% and 65%, respectively, when administered intraperitoneally every other day at a dose of 5 mg/Kg. Subsequently, the KTS-disintegrins viperistatin (from Vipera palestinae venom) [START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF] and lebestatin (from Macrovipera lebetina transmediterranea venom [START_REF] Bazaa | Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina[END_REF][START_REF] Olfa | Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis[END_REF], and the RTS-disintegrin jerdostatin (recombinantly expressed by [START_REF] Sanz | cDNA cloning and functional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the α 1 β 1 integrin[END_REF] from a Protobothrops jerdonii cDNA library) have been proven to be also very potent and highly selective inhibitor of the α 1 β 1 integrin. NMR studies of the solution structure [START_REF] Moreno-Murciano | NMR solution structure of the non-RGD disintegrin obtustatin[END_REF] and internal motions [START_REF] Monleón | Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin[END_REF] revealed that in contrast to all known disintegrin structures in which the RGD adhesion motif is located at the apex of an eleven residue hairpin loop, the active 21 KTS 23 tripeptide is oriented towards a side of a nine residue integrin-binding loop. The integrin binding loop of obtustatin exhibits lateral local motions within a range of approximately 35º and with maximal displacement of about 5Å, and the integrin binding loop and the C-terminal tail of the disintegrin display concerted motions in the 100-300 picosecond timescale [START_REF] Monleón | Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin[END_REF]. The shape and size of its short integrin-binding loop along with its composition, flexibility, and the distinct orientation of the KTS tripeptide may underlay the structural basis of obtustatin´s unique selectivity and specificity for integrin α 1 β 1 .

Structural diversity among the α 1 β 1 -specific disintegrins appears to be segregated within their C-terminal halves (see Fig. 3 in [START_REF] Calvete | KTS and RTS-disintegrins: antiangiogenic viper venom peptides specifically targeting the α 1 β 1 integrin[END_REF]). Viperistatin differs from obtustatin by only three amino acids at positions 24, 38 and 40, and is about 25-fold more potent than obtustatin inhibiting the interaction of human α 1 β 1 integrin with collagen IV in vitro [START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF]. Here we report the production and structure/function correlations of recombinant KTS-disintegrins.

EXPERIMENTAL

Antibodies, Extracellular Matrix and Cell Adhesion Proteins

Polyclonal antiserum against the snake venom disintegrin eristostatin was developed commercially (Chemicon, Temecula, CA), and the IgG fraction was purified using protein Abased purification kit (Pierce Biotechnology, Rockford, IL). Mouse anti-human β1 integrin subunit monoclonal antibodies B44 and Lia1/2 were purchased from Chemicon and Beckman Coulter (Fullerton, CA), respectively. Goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) and alkaline phoshatase (AP) were purchased from Cell Signaling Inc. (Beverley, MA) and Sigma Inc. (St. Louis, MO), respectively. Bovine collagens type IV and type I were purchased from Chemicon and Chrono-Log Corp. (Havertown, PA), respectively. Human plasma fibronectin was from Sigma. Human VCAM-1 Ig domain was kindly provided by Dr. P. Wainreb (Biogen Inc., Cambridge, MA).

Cell Lines K562 and Jurkat cell lines were purchased from ATCC (Manassas, VA) and cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA). K562 cells lines transfected with α1 and α2 integrins were kindly provided by Dr. M. Hemler (Dana-Farber Cancer Institute, Boston, MA) and were cultured in RPMI 1640 containing 500 µg/mL G418 Sulfate (Mediatech Inc, Herdon, VA).

Purification of Anti-adhesive Proteins from Snake Venoms

The following snake venom proteins were purified on a C18 column eluted with a linear acetonitrile gradient using the previously described two-step reverse phase HPLC protocol [START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF][START_REF] Bazan-Socha | Structural requirements of MLD-containing disintegrins for functional interaction with α 4 β 1 and α 9 β 1 integrins[END_REF]. Disintegrins obtustatin, VLO4 and VLO5 were purified from the venom of Vipera lebetina obtusa purchased from Latoxan (Valence, France). Disintegrin eristostatin was isolated from Eristocophis macmahoni venom (Latoxan). Disintegrin viperistatin and the C-type lectin-like protein VP12 were purified from the venom of Vipera palestinae kindly provided by Dr. A. Bdolah (Zoology Department of Tel-Aviv University, Israel).

Cloning and Expression of Recombinant Obtustatin and its Mutants

A synthetic gene for obtustatin was designed based on the 41 amino acid sequence of obtustatin [START_REF] Moreno-Murciano | Amino acid sequence and homology modeling of obtustatin, a novel non-RGDcontaining short disintegrin isolated from the venom of Vipera lebetina obtusa[END_REF] and optimal codons for E. coli expression, and was commercially synthesized by Genscript Corp. (Piscataway, NJ). The obtustatin gene was PCR-amplified and ligated into the pGex-4-T1 vector for expression as a thrombin-cleavable gluthatione S-transferase-fusion protein using the pUC57 plasmid containing the synthetic gene as a template. Forward (5'-GTTCCGCGTGGATCC TGTACGACGGGTCCTTGTTGTCGT-3') and reverse (5'-TCGACCCGGGAATTCTTAGCCGGGGTAAAGTGGACAATC-3') primers (Oligos Etc., Wilsonville, OR) contained restriction sites (underlined) for BamHI and EcoRI, respectively. Recombinant obtustatin molecules contained the additional N-terminal dipeptide (Gly-Ser).

Site-directed mutagenesis was performed by PCR using the pGex-4-T1 vector containing obtustatin as a template and the following primers (Oligos Etc., Wilsonville, OR): mutant (KAS): forward primer 5'-GCTGGCACGACGTGTTGGAAGGCTTCTCTTACATCC-3' and reverse primer 5'-CTTCCAACACGCGTGCCAGCTGGCTTAAGTTT-3'; mutant (ATS): forward primer 5'-CCAGCTGGCACGACGTGTTGGGCTACATCTCTTACA-3' and reverse primer 5'-CCAACACGTCGTGCCAGCTGGCTTAAGTTTGCA-3'; mutant (KTA): forward primer 5'-GGCACGACGTGTTGGAAGACAGCTCTTACATCCCAT-3' and reverse primer 5'-TGTCTTCCAACACGTCGTGCCAGCTGGCTTAAG-3'; mutant (AAA): forward primer 5'-CCAGCTGCCACGACGTGTTGGGCTGCTGCTCTT ACATCCCAT-3' and reverse primer 5'-CCAACA CGTCGTGCCAGCTGGCTTAAGTTTGCA-3'; mutant (KTSR): forward primer 5'-ACGACGTGTTGGAAGACATCTAGAACATCCCATTAC-3' and reverse primer 5'-AGATGTCTTCCAACACGTCGTGCCAGCTGGCTT-3'; mutant (KTSA): forward primer 5'-ACGACGTGTTGGAAGACATCTGCTACATCCCATTAC-3' and reverse primer 5'-AGATGTCTTCCAACACGTCGTGCCAGCTGGCTT-3'.

The sequences of cDNAs of obtustatin and its mutants were verified at the Kimmel Cancer Center (Thomas Jefferson University, Philadelphia, PA) and used to transform E. coli BL21 (DE3) strain. The gluthatione S-transferase fusion protein was induced and isolated as described [START_REF] Smith | Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase[END_REF]. After digestion with thrombin, recombinant obtustatin and its mutants were purified by reverse-phase HPLC as described [START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF]. The purity of the recombinant proteins was verified by SDS-PAGE, and Western blot using an anti-eristostatin polyclonal antibody which cross-react with native obtustatin and HRP-based chemiluminescence detection kit (Cell Signaling Inc.).

Cell Adhesion Studies

Cell adhesion studies were performed using an assay developed previously in our laboratory [START_REF] Marcinkiewicz | Significance of RGD loop and C-terminal domain of echistatin for recognition of α IIb β 3 and α v β 3 integrins and expression of ligand-induced binding site[END_REF]. Briefly, integrin ligands and monoclonal antibody B44 were immobilized on a 96-well plate in PBS (or in 0.02 M acetic acid for collagens I and IV) overnight at 4 0 C . Wells were blocked for 1h at room temperature with 1% bovine serum albumin (BSA) in Hanks' balanced salt solution (HBSS) buffer containing 3 mM MgCl 2 . Cells were labeled with 12.5 µM of 5chloromethylfluoresceine diacetate (CMFDA) (Invitrogen, Carlsbad, CA) in HBSS buffer containing 3 mM MgCl 2 for 30 min at 37 0 C. Cells were freed from unbound reagent by washing in the same buffer. Labeled cells (1 x 10 6 /ml) suspension were premixed with increasing concentrations of disintegrins, incubated for 15 min at room temperature and added to the wells of the ligand-coated plate. The plate was incubated for 30 min. at 37 0 C and unbound cells were removed by washing. Bound cells were lysed by adding 0.5% Triton X-100. In parallel, the 5 A c c e p t e d M a n u s c r i p t standard curve was prepared in the same plate using known concentrations of labeled cells. Plates were read using an FLx800 (BioTek, Winooski, VT) fluorescence plate reader selecting the bottom reading option, and an excitation wavelength of 485 nm using a 530 nm emission filter.

ELISA

Soluble α 1 β 1 integrin was recombinantly produced in an insect cell expression system and purified as described [START_REF] Eble | Collagen XVI harbors an integrin α 1 β 1 recognition site in its C-terminal domains[END_REF]. Integrin interaction experiments were carried out using an ELISAtype protocol according to [START_REF] Eble | Collagen XVI harbors an integrin α 1 β 1 recognition site in its C-terminal domains[END_REF]. Briefly, the CB3 [IV] collagen fragment was immobilized on a 96-well plate overnight at 4ºC in PBS at a concentration of 20 µg/ml. The plate was washed three times with TBS, pH 7.5 containing 2 mM MnCl 2 (TBS/Mg) and blocked with 1% BSA in TBS/Mg at room temperature for 1 hour. Then 30 nM of soluble α 1 β 1 integrin, dissolved in the same buffer supplemented with 1 mM MnCl 2 , was mixed with different concentrations of recombinant disintegrins. After incubation for 15 min at room temperature the mixtures were added to the plate and incubated for 2 hours at room temperature. The plate was then washed twice with 50 mM HEPES (pH 7.5) containing 150 mM NaCl 2 , 2 mM MgCl 2 , and 1 mM MnCl 2 , and bound integrin was fixed with 2.5% glutardialdehyde (Sigma Inc.). The α 1 β 1 integrin was detected using a rabbit anti-β1 antiserum. After washing, goat anti-rabbit IgG conjugated with alkaline phosphatase (AP) was added. 4-Nitrophenyl phosphate disodium salt hexahydrate (Sigma Inc.) was added as a substrate for AP and color was developed at room temperature. The plate was read using ELISA plate reader at 405 nm.

Cell-free disintegrin binding assay for LIBS expression

Disintegrins were labeled with FITC as described [START_REF] Marcinkiewicz | Significance of RGD loop and C-terminal domain of echistatin for recognition of α IIb β 3 and α v β 3 integrins and expression of ligand-induced binding site[END_REF]. Anti-LIBS monoclonal antibody B44 (0.5 µg/ml) or the blocking, conformational insensitive anti-β1 integrin subunit monoclonal antibody Lia1/2 (0.5 µg/ml) were immobilized on 96-well plate. Wells were blocked with 1% BSA as described above (Cell Adhesion Studies). α1K562 cells were harvested and washed with HBSS containing 3 mM Mg 2+ . Cell pellet was lysed with 0.5% Triton X-100 in HBSS containing 3 mM Mg 2+ and protein concentration in the lysate was estimated using the BCA assay kit (Pierce Inc., Rockford, IL). FITC-disintegrin was mixed with cell lysate (2 mg/ml protein) to a total sample volume of 100 µl. Samples were incubated for 30 min at 37 0 C and transferred to a 96-well plate with immobilized B44 antibody. To measure unspecific binding of FITC-disintegrin, the same experiment was carried out but using non-α1-transfected K562 cells. After incubation for 30 min at 37 0 C, the plates were washed three times with HBSS containing 3 mM Mg 2+ and 1 % BSA. Finally, plates were read (bottom reading option) using a fluorescent plate reader FLx800 (BioTek) at an excitation wavelength of 485 nm and a 530 nm emission filter. In parallel, a standard curve of bound disintegrin was prepared on the same plate using known concentrations of FITC-disintegrin.

Molecular modeling

Structure of obtustatin mutants were calculated using NOE information and disulfide bond restraints of the NMR solution structure of obtustatin [1MPZ] and an energy minimization protocol supplied with the program CNS [START_REF] Brunger | Crystallography & NMR system: A new software suite for macromolecular structure determination[END_REF]. For the final ensembles, a set of 50 structures was calculated and the converged structures selected corresponded to a plateau in the values of the overall calculated energy. For the analysis of the mutants the lowest energy structure was used as 6 representative model, and the mutations were introduced using the protocol supplied with the program PyMOL [START_REF] Delano | The PyMOL Molecular Graphics System[END_REF] available at http://www.pymol.org.

RESULTS

Antagonistic profile of native and recombinant obtustatin

Obtustatin recombinantly produced in E. coli exhibited the same chromatographic behavior on reverse phase HPLC as obtustatin purified from viper venom and showed the same inhibitory saturation pattern and IC50 on α1K562 cell adhesion to immobilized collagen IV as the native (venom-isolated) disintegrin (Fig. 1A, Table 1). The selectivity of recombinant and native obtustatins was also indistinguishable from one another (Fig. 1B). Particularly, neither native nor recombinant obtustatin blocked the adhesive functions of the α 2 β 1 integrin that is structurally and functionally highly related to α 1 β 1 integrin. The recombinant production of fully active obtustatin allowed us to assess the previouly suggested relevance of synthetic KTS-bearing peptides for specific α 1 β 1 integrin inhibition [START_REF] Marcinkiewicz | Obtustatin: a potent selective inhibitor of α 1 β 1 integrin in vitro and angiogenesis in vivo[END_REF][START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF][START_REF] Moreno-Murciano | Amino acid sequence and homology modeling of obtustatin, a novel non-RGDcontaining short disintegrin isolated from the venom of Vipera lebetina obtusa[END_REF] employing the site-directed mutated molecules displayed in Fig. 2. Specifically, substitutions within KTS tripeptide motif showed significant impairment of the α 1 β 1 integrin inhibitory ability of the recombinant proteins (Fig. 3). Using a cell adhesion assay, the IC 50 s of the single alanine mutants 21 ATS 23 , KAS, and KTA showed, respectively, 25-fold, 82-fold, and 13-fold decrease inhibiting the α1K562 cell adhesion to immobilized collagen IV (Fig. 3A, Table 1). The triple mutant, 21 AAA 23 , was devoid of inhibitory activity up to a concentration of 10 µM (Fig. 3A, Table 1). Similar features were observed when the single and triple obtustatin mutants were tested for their ability to inhibit the binding of a recombinant soluble form of the α 1 β 1 integrin to the CB3 fragment of collagen IV in a cell-free ELISA-type assay (Fig. 3C).

Next, we checked the hypothesis that the residue immediately C-terminal to the KTS motif may be partly responsible for the increased inhibitory potential of viperistatin over obtustatin [START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF]. To this end, leucine-24 (obtustatin) was replaced for alanine and for arginine (as in viperistatin). The 21 KTSA 24 mutant (Fig. 2G) showed only a slight decrease in IC50 in both ELISA-type and cell adhesion assays. On the other hand, the L 24 /R mutant (Fig. 2F) exhibited a 6-fold enhancement in its α 1 β 1 inhibitory potential (Table 1) (Fig. 3, panels B and C). However, in the adhesion but not in the ELISA-type assay, this latter effect was concentration-dependent since the difference in activity of the KTSR mutant disappeared at disintegrin concentrations higher than 10 nM (compare Figs. 3B and3C).

KTS-disintegrins induced LIBS epitope upon binding to α1β1 integrin

Ligand engagement to integrins may trigger conformational changes leading to the exposure of cryptic epitopes (LIBS, Ligand-Induced Binding Sites) recognized by monoclonal antibodies [START_REF] Humphries | Monoclonal antibodies as probes of integrin priming and activation[END_REF]. The ability of KTS-disintegrins to induce LIBS was assessed using two different assay systems. Using a solid-phase adhesion assay, we show that both obtustatin and viperistatin significantly induce conformational alterations in α 1 β 1 integrin reported by expression of a neoepitope on the β1 subunit recognized by the B44 monoclonal antibody (Fig. 4A). However, the ability to induce LIBS was significantly lower for viperistatin than for obtustatin. On the other hand, wild-type obtustatin and its KTSR-mutant had similar effects on mAbB44 epitope exposure suggesting that the distinct LIBS-inducing activity of obtustatin and viperistatin may not reside in their integrin-binding loops. On the other hand, using a cell-free binding assay, in 7 which FITC-labeled disintegrins complexed with α 1 β 1 integrin in the cell lysate were detected following binding to the immobilized B44 antibody, obtustatin, viperistatin and the mutated recombinant KTSR-obtustatin showed similar ability inducing LIBS epitope on α1β1 integrin (Fig. 4B) as in the adhesion assay (Fig. 4A). Binding of FITC-disintegrins to the anti-β1 blocking antibody Lia1/2, used as a control, was negligible and was substracted as background from each reading. The outcome of this experimental setup ruled out the possible controversy concerning the possibility that internalization of the ligand/integrin complex influenced the results gathered through the adhesion assay.

DISCUSSION

The α 1 β 1 integrin is widely considered to represent a target for anti-antiangiogenic, and thus anticancer, therapeutical intervention. Delineating structure-function correlations of specific α 1 β 1 inhibitors is therefore of major pharmaceutical relevance. Our studies using linear synthetic peptides indicated a role for the KTS and RTS tripeptide motifs of obtustatin, viperistatin and jerdostatin in the selective inhibition of integrin α 1 β 1 binding to collagens I and IV in vitro [START_REF] Marcinkiewicz | Obtustatin: a potent selective inhibitor of α 1 β 1 integrin in vitro and angiogenesis in vivo[END_REF][START_REF] Kisiel | Structural determinants of the selectivity of KTS-disintegrins for the α 1 β 1 integrin[END_REF][START_REF] Olfa | Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis[END_REF] and angiogenesis in vivo [START_REF] Marcinkiewicz | Obtustatin: a potent selective inhibitor of α 1 β 1 integrin in vitro and angiogenesis in vivo[END_REF]. Although not so widely spread among integrin ligands as the RGD tripeptide, the KTS motif is present in human and mouse ADAM-9 disintegrin-like domain at a topologically equivalent position in snake venom disintegrins [START_REF] Weskamp | MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains[END_REF] [UniProtKB/Swiss-Prot entry Q13443]. This motif is also localized in the C-terminal part of VP3, a structural protein of human polyomavirus JC, which after activation induces a multifocal leukoencephalopathy in brain [START_REF] Trowbridge | Identification of three new JC virus proteins generated by alternative splicing of the early viral mRNA[END_REF] . These findings strongly suggest that the KTS motif may play an important role in certain human pathophysiological conditions. To establish structure-function correlations of KTS-bearing proteins, we have set up a bacterial system for the recombinant expression of fully active obtustatin. A major conclusion from comparison of the α 1 β 1 inhibitory assays using point mutations which gradually converted obtustatin into viperistatin is that substitution of leucine for arginine at position 24, immediately C-terminal to the KTS motif, did not compromise the α 1 β 1 selectivity but accounted for only 40% of the 25-fold enhanced activity of viperistatin over obtustatin. A corollary of this conclusion is that the amino acid adjacent to the KTS motif confers inhibitory potency rather than receptor specificity. Furthermore, the partial enhancement of the inhibitory activity of the KTSR-obtustatin mutant indicated that the other two different residues between viperistatin and obtustatin (L38/V and P40/Q, Fig. 2) may contribute to the distinct inhibitory features of KTS-disintegrins.

To provide a structural ground for the functional role of the KTSX motif residues, and to investigate structural determinants of the enhanced inhibitory activity of viperistatin over obtustatin, we generated molecular models of obtustatin and viperistatin (Fig. 5). Among the KTS motif residues, mutation of the central threonine-22 for alanine had the major functional impact (Table 1). This effect can be rationalized due to the critical role of this residue to maintain the conformation of the integrin binding loop [START_REF] Moreno-Murciano | Amino acid sequence and homology modeling of obtustatin, a novel non-RGDcontaining short disintegrin isolated from the venom of Vipera lebetina obtusa[END_REF][START_REF] Moreno-Murciano | NMR solution structure of the non-RGD disintegrin obtustatin[END_REF]. A network of interactions between the solvent-shielded H γ2 of T22 and residues T25 and S26 explains the restrained mobility of threonine-22 [START_REF] Monleón | Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin[END_REF]. On the other hand, the 6-fold gain-of-activity conferred by the Leu 24 /Arg mutation indicates that the positively charged, extended side-chain of arginine is better suited than the solvent-exposed alkyl side-chain of leucine for establishing productive interactions with residues within the ligand-binding site of the α 1 β 1 integrin. The size and shape of the integrin-binding loop has been proven to be a determinant for conferring integrin specificity to cyclic RGD peptides with a known conformation [START_REF] Pfaff | Selective recognition of cyclic RGD peptides of NMR defined conformation by α IIb β 3 , α v β 3 and α 5 β 1 integrins[END_REF] and to the short-sized RGD-disintegrins echistatin and eristostatin [START_REF] Mclane | Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of α IIb β 3 and α v β 3 integrins[END_REF]. Thus, the critical importance of T22 may be linked to a structure-stabilizing mechanism of the integrin-binding loop, which supports the correct conformational presentation of the KTS sequence to be recognized by α 1 β 1 integrin, rather than to a direct role in receptor binding.

Though a tripeptide at the tip of a mobile loop represents the primary integrin inhibitory determinant of RGD-disintegrins, both the potency and receptor selectivity of disintegrins appears to be finely tuned by residues flanking the active sequence [16 ,40, 41]. Similarly, the presence of arginine at the C-terminal side of the primary KTS integrin-recognition motif of viperistatin enhances its inhibitory activity towards α 1 β 1 integrin by approximately 4-fold in comparison to obtustatin, which contains leucine in this position. However, this non-conservative mutation does not affect the integrin specificity of KTS-disintegrins, whereas structural evidence gathered on synthetic peptides and RGD-disintegrins showed that the amino acid immediately Cterminal to the RGD sequence influence the disintegrin´s integrin-recognition selectivity through modulation of the RGD loop conformation [START_REF] Pfaff | Selective recognition of cyclic RGD peptides of NMR defined conformation by α IIb β 3 , α v β 3 and α 5 β 1 integrins[END_REF].

It is also worth noticing that substitution of leucine for arginine at position 24 (sequence F in Fig. 2) was not enough for switching the inhibitory activity of obtustatin to that of viperistatin (Table 1 and Fig. 3B,C). The partial (40%) gain-of-function of recombinant KTSRobtustatin is in the range of inhibitory activity of lebestatin (IC 50 , 0.4 nM), another α 1 β 1 -specific KTSR-disintegrin isolated from the venom of Macrovipera lebetina transmediterranea [START_REF] Bazaa | Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina[END_REF][START_REF] Olfa | Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis[END_REF]. Besides position 24, obtustatin and lebestatin sequences differ only at position 38, which contains Leu and Ser, respectively. In line with structural studies on obtustatin showing that its integrin-binding loop and C-terminal tail displays concerted motions [START_REF] Monleón | Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin[END_REF] our results suggest that these structural elements act as a secondary determinant of integrin-binding specificity/potency. Relevant to this point, obtustatin and viperistatin have different amino acids at positions 38 and 40, which are L 38 /V and P 40 /Q, respectively, (Fig. 2). We argue that these two substitutions, along with the active site L 24 /R mutation, may create a distinct chemical environment responsible for the increased inhibitory activity towards α1β1 integrin of viperistatin over obtustatin. Furthermore, here we show that obtustatin, and to a lower extent viperistatin, elicit the exposure of ligand-induced binding sites (LIBS) in α 1 β 1 recognized by monoclonal antibody B44 (Fig. 4). Our working hypothesis is that proline at position 40 may contribute to the better exposure of conformational changes in the α 1 β 1 -obtustatin complex.

CONCLUDING REMARKS AND SIGNIFICANCE

The α 1 β 1 integrin is an attractive target for the development of novel therapical strategies against pathological angiogenesis in cancer and certain inflammatory diseases. The KTSX sequence found in viper venom disintegrins represents a specific motif targeting the α 1 β 1 integrin. The NMR solution structure of the KTS-disintegrin obtustatin along with the structure-function correlations reported here may provide the ground for designing a new generation of synthetic lead compounds with potential application for anti-angiogenic intervention.

A further novel finding of our research is the demonstration that ligand engagement elicits in α 1 β 1 the exposure of cryptic epitopes recognized by monoclonal antibody B44. Ligandinduced binding sites had been reported in β3 and α 4 β 1 integrins. Our results suggest that 9 Finally, although our research focused on the structural determinants of snake venom disintegrins for specific inhibition of α 1 β 1 integrin, the occurrence of the KTS tripeptide in certain proteins, such as human ADAM-9 and VP3 capsid protein of the human polyomavirus JC, suggests a hitherto unrecognized role for this motif in physiology and pathophysiology. Functions of ADAM family proteins are not fully characterized. Interactions with collagen receptors may regulate their adhesive and/or enzymatic functions. On the other hand, the presence of KTS sequence on a capsid protein of a virus attacking the human nervous system, suggests that the α 1 β 1 integrin may serve as part of the pathogen´s host cell entry strategy. Further investigations on novel KTS-bearing proteins are needed for expanding our understanding of the basic principles underlying its functionality in diverse biological contexts. with wild-type or mutated obtustatin molecules and with viperistatin (p < 0.05). In panel B, experiments were performed using a cell-free binding assay. α1K562 and cells were lysed with HBSS containing 3 mM Mg 2+ and 0.5% Triton X-100. FITC-disintegrins were mixed with 100 µl of cell lysate (2mg/ml) for 30 min at 37 ºC followed by incubation for another 30 min at 37 ºC with monoclonal antibody B44 immobilized on the wells of a 96-well plate. The same experiment was carried out using the lysate of non-transfected K562 cells as a control of the specific binding of the FITC-disintegrin to integrin α 1 β 1 (♦). After washing with HBSS containing 3 mM Mg 2+ fluorescence was read using a fluorescence plate reader. Error bars represent S.D. from three independent experiments.

of obtustatin have 3 Biochemical

 3 Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403 A c c e p t e d M a n u s c r i p t

4

  Biochemical Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403

  Biochemical Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403 A c c e p t e d M a n u s c r i p t conformational regulation may represent a general feature of integrins for transducing ligandspecific signals. LIBS offer further possibilities for developing immunochemical weapons to fight undesired upregulation of the α 1 β 1 integrin in pathological conditions.

BiochemicalFigure 1 .

 1 Figure 1. Comparison of the effect of recombinant and native obtustatin on integrinmediated cell adhesion to different ECM proteins.ECM proteins (2 µg/ml) were immobilized overnight at 4°C on a 96-well plate in 0.02 M acetic acids for collagen and in PBS for VCAM-1 and fibronectin. After blocking the plate with BSA, CMFDA-labeled cells preincubated in the presence or absence of snake venom proteins were added to the wells, and incubated for 30 min at 37°C. Non-binding cells were washed out, adhered cells were lysed, and the plate was read using a fluorescence plate reader. (Panel A) Effect of different concentrations of recombinant and native obtustatin on inhibition of adhesion of α1K562 cells to collagen IV. (Panel B) Effect of recombinant (a) and native (b) obtustatin, VP12 (c), VLO5 (d) and VLO4 (c) on adhesion of the indicated cell lines to their respective immobilized ligands. Venom proteins were applied at a concentration of 20 µg/ml. VP12 belongs to subgroup of snake venom C-type lectin-like proteins that specifically target the α 2 β 1 integrin [50]. VLO5 and VLO4 are dimeric disintegrins from V.l. obtusa which antagonize the function of integrins α 4 β 1 and α 4 β 1 , respectively [30, 51]. The observed small inhibition activity of native obtustatin on α 4 β 1 integrin binding to VCAM-1 was due to a slightly contamination with VLO5 which has almost identical reverse-phase retention time as obtustatin. Error bars represent S.D. from three independent experiments.

Figure 2 .

 2 Figure 2. Amino acids sequences of obtustatin and its mutants recombinantly expressed in E. coli. Mutated residues are in boldface. A, wild-type obtustatin; sequences B -E, mutants affecting the KTS motif; sequences F -H, mutations for converting obtustatin into viperistatin.

Figure 3 .

 3 Figure 3. Effect of recombinant obtustatin and its mutants on the interaction of α1β1 integrin with collagen IV. (Panel A) Effect of wild-type obtustatin and its KTS motif mutants on the adhesion of α1K562 cells to immobilized collagen IV. (Panel B) Effect of wild-type viperistatin and obtustatin and the KTSA and KTSR mutants on the adhesion of α1K562 cells to immobilized collagen IV. (Panel C) Effect of wild-type obtustatin and its mutants on the binding of soluble α 1 β 1 integrin to immobilized CB3 fragment of collagen IV using the ELISA-type assay described in the Experimental Procedures section. Error bars represent S.D. from three experiments.

Figure 4 .

 4 Figure 4. Effect of wild-type viperistatin and obtustatin and the KTSR-obtustatin mutant on LIBS expression.In panel A, experiments were performed using cell adhesion assays. To this end, monoclonal antibody B44 recognizing a LIBS epitope on the β1 subunit was immobilized on a 96-well plate (0.5 µg/ml) by overnight incubation at 4 0 C in PBS. After blocking the plate with BSA, CMFDA-labeled α1K562 cells preincubated in the presence (1 mM in HBSS containing 3 mM Mg 2+ ) or absence of disintegrins were added and incubated at 37°C for 30 min. Unbound cells were removed by washing, adhered cells were lysed with 0.5% Triton X-100, and fluorescence was read using a fluorescence plate reader. The error bars represent S.D. from three experiments. (*) Represents statistical difference between control and samples incubated with disintegrins, as determined by unpaired T-test (p < 0.05). (**), Statistical difference between samples incubated

Figure 5 . 15 Biochemical

 515 Figure 5. Molecular models of obtustatin and viperistatin. (Panel A) NMR solution structure of obtustatin (1MPZ) showing the locations of residues (rendered in stick representation) which were sequentially mutated in the different recombinant proteins shown in Fig. 2 to convert recombinant obtustatin into viperistatin. (Panel B) Computer modelling of the transformation of obtustatin into viperistatin. Figures were rendered using PyMol.

Figure 1 BiochemicalFigure 1 Biochemical

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Figure 2 BiochemicalFigure 3 BiochemicalFigure 3 BiochemicalFigure 3 BiochemicalFigure 4 Biochemical

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Figure 5 Biochemical

 5 Figure 5

TABLE 1 Inhibition of α1K562 cell adhesion to immobilized collagen IV by wild-type obtustatin and viperistatin and the different obtustatin mutants.

 1 Biochemical Journal Immediate Publication. Published on 05 Sep 2008 as manuscript BJ20081403 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081403

	Type of mutation	IC 50 (nM) †
	Wild-type recombinant/native obtustatin	2.00
	Wild-type native viperistatin	0.08
	ATS-obtustatin	51.30
	KAS-obtustatin	164.18
	KTA-obtustatin	26.08
	AAA-obtustatin	>10,000
	KTSR-obtustatin	0.33
	KTSA-obtustatin	2.56
	† Data represent mean from three independent experiments

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ACKNOWLEDGMENTS

This work was supported in part by NIH grant CA100145 (C.M.) and AHA grant 0230163N (C.M.), grant EB177/4-2 from the German Research Council (J.A.E.), and grant BFU2007-61563 from the Ministerio de Ciencia e Innovación , Madrid, Spain (J.J.C.).