Microvillar Membrane Microdomains Exist at Physiological Temperature

ROLE OF GALECTIN-4 AS LIPID RAFT STABILIZER REVEALED BY "SUPERRAFTS"*

Anita BracciaDagger, Maristella VillaniDagger, Lissi Immerdal, Lise-Lotte Niels-Christiansen, Birthe T. Nystrøm, Gert H. Hansen, and E. Michael Danielsen§

From the Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark

Received for publication, November 4, 2002, and in revised form, February 19, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 °C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta -galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipid rafts are ordered (lo phase) membrane microdomains consisting mainly of glycosphingolipids and cholesterol in the outer leaflet of the bilayer that serve as lateral platforms for many types of proteins in the cell membrane (1-3). The lipid raft hypothesis was originally proposed to explain how lipids and proteins are sorted to the apical surface of polarized cells (4), but in recent years the raft concept has expanded into other areas of biology, including signal transduction (5-7), pathogen invasion (8-10), cholesterol homeostasis and transport (11, 12), drug targeting (13), and biomedicine (14). However, despite their popularity, fundamental questions concerning the functional relevance of lipid rafts in vivo persist (15, 16). To complicate matters further, recent work has made it increasingly clear that the most commonly used experimental criterion for lipid rafts, detergent insolubility at low temperature combined with flotation in a density gradient (17), may define different subsets of rafts, depending on the type of detergent used (18-21). A common current view considers lipid rafts as small, dynamic assemblies of lipids and proteins with the ability to cluster into larger ordered platforms that may serve in specialized, cell type-dependent functions, such as nutrient absorption, cell-cell communication, or endocytosis (2, 14, 22, 23). These developments have turned the raft concept increasingly complex, and underline the fact that much is still unknown about lipid rafts concerning their molecular organization, dynamics of assembly/disassembly, and mechanism of function.

The brush border membrane of small intestinal enterocytes is a highly specialized cell surface involved in a multitude of digestive and absorptive functions (24). The microvilli of this membrane are particularly rich in glycosphingolipids and cholesterol (25), and several proteins, including some of the major digestive enzymes, such as aminopeptidase N and sucrase-isomaltase (26-28), and galectin-4 (29) have been shown to reside in Triton X-100-insoluble lipid rafts. Microvillar rafts may well be unique in their molecular organization, because unlike most other types of lipid raft membranes, such as caveolae (30), those prepared from microvilli are cholesterol-independent, and caveolin-1, a commonly used lipid raft marker in other cell types, is essentially excluded from microvillar rafts (31).

In the present work, we aimed first to answer the fundamental question whether microvillar rafts are purely a "low temperature" phenomenon, created by the conditions normally used for detergent extraction. To tackle this problem, microvillar rafts were prepared by using Brij 98, a detergent recently described for isolation of the T cell receptor signaling machinery at a physiological temperature (20). Second, we studied in closer detail the molecular organization of lipid rafts prepared from microvillar membrane vesicles by sequential extraction with Triton X-100 at increasing temperature. Here, a fraction of "superrafts" was isolated and characterized with galectin-4 as the predominant protein. Altogether, our data show that microvillar rafts do exist also at a physiological temperature. In addition, the results underline the importance of galectin-4 functioning as a central organizer/stabilizer of lipid rafts in the microvillar membrane with other classes of membrane proteins depending, at least partially, on this lectin for their association with these microdomains.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Rabbit polyclonal antibodies to galectin-4, lactase, and aminopeptidase N were those described previously (29, 32, 33). A rabbit antibody to alkaline phosphatase was from Biogenesis (Poole, UK), and a mouse monoclonal antibody to annexin 2 was obtained from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-coupled swine anti-rabbit IgG and rabbit anti-mouse IgG were from DAKO (Glostrup, Denmark). Gold-labeled goat anti-rabbit immunoglobulin was from Amersham Biosciences. Methyl-beta -cyclodextrin, Brij 98 (polyoxyethylene 20 oleyl ether), and lipid standards were purchased from Sigma, and DynabeadsTM M-500 Subcellular was from Dynal (Oslo, Norway).

Pig small intestines were kindly given by Letty Klarskov and Mette Olesen (Department of Experimental Medicine, The Panum Institute, Copenhagen, Denmark).

Preparation of Microvillar Membranes-- Right-side-out microvillar vesicles were prepared from small intestinal mucosa by the divalent cation precipitation technique (34). Briefly, mucosal scrapings were homogenized in 2 mM Tris-HCl, 50 mM mannitol, pH 7.1, containing 10 µg/ml aprotinin and leupeptin, using a manually operated Potter-Elvehjem homogenizer. The homogenate was cleared by centrifugation at 500 × g for 10 min, and MgCl2 was added to a final concentration of 10 mM. After incubation at 15 min on ice, the preparation was centrifuged at 1500 × g for 10 min to pellet intracellular and basolateral membranes. Finally, the supernatant was centrifuged at 48,000 × g for 1 h to obtain a pellet of microvillar membrane vesicles. One common preparation was used in all subsequent comparative lipid raft experiments with the two nonionic detergents Brij 98 and Triton X-100 as well as in preparation of superrafts.

Lipid Raft Analysis-- A lipid raft analysis by detergent extraction followed by sucrose gradient centrifugation (17) was performed essentially as previously described (26), with the modification that the extract was placed in a cushion of 60% sucrose with a gradient of 50-25% sucrose on top. Extraction with Triton X-100 and with Brij 98 was carried out on ice and at 37 °C, respectively. Solutions of each detergent (10% (w/v) dissolved in distilled water) were added to the microvillar membrane vesicles to a final detergent concentration of 1% (w/v). After centrifugation overnight, the gradients were collected in 12 1-ml fractions for subsequent analysis by SDS-PAGE and Western blotting. Alternatively, the floating fractions were carefully collected by a pipette, diluted five times with 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, and centrifuged at 48,000 × g, 1 h, to obtain a pellet of lipid rafts for further biochemical and electron microscopic analyses.

Preparation of Superrafts-- Lipid rafts were prepared by extraction with Triton X-100 followed by sucrose density gradient centrifugation as described above. Rafts contained in the upper six fractions of the gradient were harvested; resuspended in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1; and extracted with 1% Triton X-100 for 10 min at room temperature. The extract was centrifuged at 20,000 × g for 30 min, and the resulting pellet was resuspended in the same buffer and re-extracted with 1% Triton X-100 for 10 min at 37 °C. After centrifugation at 20,000 × g for 30 min, the resulting pellet, termed "superrafts," was collected.

Lipid Analysis-- For lipid analysis of membranes, microvillar vesicles and lipid rafts were extracted with chloroform/methanol (25, 35) as previously described (31). Aliquots of the lipid extracts (120-130 mg of lipid) were subjected to thin layer chromatography analysis together with appropriate lipid standards on 0.25-mm silica gel 60 plates (Merck). Cholesterol was separated from other neutral lipids in petroleumsether/diethylether/acetic acid (50:50:1). Glycolipids and phospholipids were separated in chloroform/methanol/water (64:24:4). After chromatography, cholesterol was detected with a CuSO4/H3PO4 reagent and glycolipids with a alpha -naphtol spray reagent.

Gel Electrophoresis and Western Blotting-- SDS-PAGE electrophoresis in 10% polyacrylamide gels was performed according to Laemmli (36). After electrophoresis and transfer of proteins onto ImmobilonTM, Western blotting was performed with antibodies to galectin-4 (29), lactase (33), aminopeptidase N, alkaline phosphatase, and annexin 2. Blots were developed by an electrochemoluminescence detection reagent kit according to the protocol supplied by the manufacturer (Amersham Biosciences).

Immunoisolation of Lipid Rafts-- Superrafts and lipid rafts, prepared by extraction either with Triton X-100 on ice or Brij 98 at 37 °C, were isolated on magnetic beads, coated with antibodies to galectin-4 and aminopeptidase N, by a previously described procedure (31).

Electron Microscopy-- Lipid rafts, isolated from microvillar membrane vesicles using either Triton X-100 or Brij 98, were fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 2 h at 4 °C. After a wash in 0.1 M sodium phosphate buffer, the lipid rafts were treated with osmium tetroxide in 0.1 M sodium phosphate buffer for 1 h at 4 °C, dehydrated in graded concentrations of acetone, and finally embedded in Epon. The superrafts and lipid rafts isolated on magnetic beads were fixed in glutaraldehyde, treated with osmium tetroxide, dehydrated, and embedded in Epon as described above, with the modification that the entire fixation and embedding procedure was performed in Eppendorf tubes. Ultrathin sections were cut on an LKB Ultrotome III ultramicrotome, stained in 1% uranyl acetate in water and lead citrate, and finally examined in a Zeiss EM 900 electron microscope equipped with a Mega View camera system.

Immunoelectron Microscopy-- Postembedding immunogold labeling using an antibody against aminopeptidase N was performed on ultrathin Epon sections of lipid rafts and superrafts, as previously described (37).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipid Rafts from Microvillar Membranes Exist at Physiological Temperature-- The brush border of small intestinal enterocytes is well suited for studies on the molecular organization of a specialized cell membrane. Upon tissue homogenization, microvilli spontaneously form a homogeneous population of right-side-out vesicles that are easily isolated from other cell membranes by the divalent cation precipitation technique (34). We have previously used flotation in a density gradient after Triton X-100 extraction on ice for isolation of lipid rafts and observed that several microvillar proteins partition differently into the rafts. Thus, the glycosylphosphatidylinositol-anchored alkaline phosphatase is predominantly (about 90%) and the transmembrane aminopeptidase N and sucrase-isomaltase partially (40-50%) raft-associated, whereas lactase is essentially excluded from microvillar rafts (26). In addition, galectin-4, a member of the galectin family of beta -galactoside-binding proteins (38, 39), has been identified as a microvillar protein that resides exclusively in rafts and forms clusters with some of the brush border enzymes (29).

In the present work, these microvillar proteins were used as probes for lipid rafts, prepared using either Triton X-100 or Brij 98. Fig. 1 shows a comparison of the flotation properties in a sucrose density gradient of microvillar membrane proteins extracted by Triton X-100 on ice or Brij 98 at 37 °C. For both detergents, distinct subsets of proteins floated into the upper fractions, indicative of their association with lipid rafts. An overall comparison of the profiles of lipid raft-associated proteins isolated with the two detergents showed many common prominent bands, but some minor quantitative differences in the relative intensity of several bands were observed (Fig. 1). In addition, the densities of the rafts differed. Thus, rafts extracted with Triton X-100 were relatively homogeneous and of low density, whereas those extracted with Brij 98 were more heterogeneous in density.


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Fig. 1.   Comparison of microvillar rafts using Triton X-100 and Brij 98. A, microvillar membrane vesicles prepared by the divalent cation precipitation method were resuspended in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, and 0.9-ml samples of membranes were solubilized for 10 min by 1% Triton X-100 on ice or 1% Brij 98 at 37 °C, respectively, before isolation of lipid rafts by sucrose gradient centrifugation in the same rotor as described under "Experimental Procedures." After centrifugation, the gradients were fractionated, and samples of equal volume of the fractions were subjected to SDS-PAGE. After electrophoresis and electrotransfer onto ImmobilonTM, total protein was visualized by staining with Coomassie Brilliant Blue. Molecular mass values (kDa) are indicated, and fractions containing soluble proteins and lipid rafts are indicated by the arrows. B, densitometric scanning of fraction 10 from the two gradients shown in A. Molecular mass values (kDa) are indicated.

The specific distribution in the density gradient of the four microvillar proteins described above was revealed by Western blotting (Fig. 2). As shown in Fig. 2A, galectin-4 was exclusively present in the raft fractions of Triton X-100-extracted microvillar membranes and also predominantly raft-associated in the Brij 98 gradient. Contrary to galectin-4, essentially all lactase was seen in the heavy fractions of the gradient that contains solubilized proteins, irrespective of the detergent used for the membrane extraction (Fig. 2B), confirming lactase being a "non-raft" marker among microvillar proteins. The glycosylphosphatidylinositol-anchored alkaline phosphatase was exclusively present in the floating lipid raft fractions with both detergents (Fig. 2C), whereas the transmembrane aminopeptidase N was only partially raft-associated with both detergents (Fig. 2D).


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Fig. 2.   Distribution of microvillar raft markers. The density gradient fractions, shown in Fig. 1A were analyzed by Western blotting for the distribution of different microvillar proteins. A, galectin-4; B, lactase; C, alkaline phosphatase; D, aminopeptidase N. Fractions containing soluble proteins and lipid rafts are indicated by the arrows.

The lipids of microvillar rafts, isolated after extraction with the two detergents, were analyzed by thin layer chromatography (Fig. 3). Previously, rafts, prepared using Triton X-100, have been shown to contain cholesterol and to be particularly enriched in glycosphingolipids (31). When approximately equal amounts of raft lipids were analyzed in parallel by thin layer chromatography, the lipid profiles of those isolated by use of Brij 98 and Triton X-100 were essentially similar, indicating that the overall lipid-solubilizing properties of the two detergents were alike (Fig. 3). However, rafts obtained with Brij 98 contained relatively higher amounts of phospholipids.


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Fig. 3.   Lipid analysis of microvillar rafts. The lipid composition of native microvillar membrane vesicles (1), rafts isolated with Triton X-100 (2), rafts isolated with Brij 98 (3), and superrafts (4) was analyzed by thin layer chromatography as described under "Experimental Procedures." The experiment shown is a representative of five independently performed analyses. Cholesterol was analyzed separately in a petroleumsether/diethylether/acetic acid solvent system and glycolipids and phospholipids in a chloroform/methanol/water solvent system. Lipid markers (M) were chromatographed in parallel. Chol, cholesterol; Gal-cer, galactocerebrosides; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; asialo-GM1, asialoganglioside-GM1 (Cer-Glc-Gal(NANA)-GalNAc-Gal). The percentage values show the amounts of cholesterol, phosphatidylethanolamine, and asialoganglioside-GM1 in the raft and superraft fractions relative to the amounts of the respective lipids in the native microvillar membrane vesicles, as determined by densitometric scanning. *, an unidentified lipid that was heavily stained by the alpha -naphtol reagent, indicating that it is a glycolipid. It was enriched in the raft and superraft fractions relative to the amount in the native microvillar membrane vesicles.

Taken together, the above experiments with Brij 98 demonstrate that microvillar lipid raft microdomains isolated at a physiological temperature were quite similar to those prepared using Triton X-100 at low temperature. Importantly, it can therefore be concluded that microvillar rafts are not "low temperature artifacts."

Morphology of Lipid Rafts-- As shown in Fig. 4, lipid rafts, prepared as described above, had a strikingly different morphology, depending on the type of detergent used for extraction. Those isolated after treatment with Triton X-100 appeared as a homogeneous population of closed, spherical vesicle-like structures with an average diameter in the range of 200-300 nm. In addition, some more complex conglomerate structures of fused vesicles were seen. By immunogold electron microscopy, aminopeptidase N was predominantly seen lining the membranes. This localization was also observed in intact microvilli as well as microvillar membrane vesicles (31), but in some of the more complex multivesicular structures, labeling was also observed along the interior membranes. Single raft structures, immunocaptured onto magnetic beads revealed a distinct bilayer composition. Lipid rafts isolated using Brij 98 as detergent were seen predominantly as nonvesiculated, pleiomorphic membrane sheets with an approximate length of 200-300 nm, but some vesicle-like structures were also present (Fig. 4). Aminopeptidase N labeling was observed both in membranes of the vesicles and the sheets.


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Fig. 4.   Morphology and immunogold labeling of lipid rafts. Lipid rafts, isolated by extraction with either Triton X-100 (A-D) or Brij 98 (E-H). A and E, sections of pellets of lipid rafts; B, C, F, and G, similar sections, immunogold-labeled for aminopeptidase N. D and H, single lipid raft structures, immunocaptured on magnetic beads coated with antibodies to aminopeptidase N and galectin-4. Bars, 0.5 µm (A, B, E, and F), 0.1 µm (C and G), and 0.05 µm (D and H).

In conclusion, Triton X-100 extraction on ice generally resulted in preservation of vesicle-like structures, whereas extraction with Brij 98 at 37 °C largely broke up the microvillar vesicles into open membrane fragments. The more heterogeneous morphology of the latter parallel the heterogeneity in lipid raft density indicated by Figs. 1 and 2. Taken together, the results suggest that raft morphology is quite sensitive to the rather small differences observed with regard to protein and lipid composition.

Microvillar Rafts Are Resistant to Cholesterol Depletion-- We have previously reported that microvillar rafts, isolated by extraction with Triton X-100, are cholesterol-independent, because unlike, for instance, caveolae in other cell types, they are resistant to treating the membranes with methyl-beta -cyclodextrin prior to the addition of detergent (31). To test whether this characteristic property was temperature- and/or detergent-related, microvillar membrane vesicles were cholesterol-depleted by treatment with methyl-beta -cyclodextrin (31, 40) before extraction with Brij 98 at 37 °C. About 70% of the microvillar cholesterol is removed by this treatment (31), but as shown in Fig. 5, a profile of polypeptides in the floating fractions was obtained almost identical to that observed with Brij 98 without methyl-beta -cyclodextrin treatment. Only small amounts of aminopeptidase N, alkaline phosphatase, and galectin-4 were shifted to the heavy fractions containing soluble protein. Thus, it can be concluded that the cholesterol independence of microvillar rafts reflects a bona fide membrane microdomain organization that exists at physiological temperature and is not a phenomenon that can be attributed to low temperature extraction with Triton X-100.


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Fig. 5.   Cholesterol depletion with methyl-beta -cyclodextrin. Methyl-beta -cyclodextrin was added (1% final concentration) to microvillar membrane vesicles, resuspended in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1. After incubation for 30 min at 37 °C, the vesicles were extracted with Brij 98 and analyzed as described in the legend to Fig. 1A. Molecular mass values (kDa) are indicated, and a densitometric scanning of fraction 10 is shown. ApN, aminopeptidase N; Alk P, alkaline phosphatase; Gal-4, galectin-4.

Superrafts, Lipid Rafts Insoluble by Triton X-100 at 37 °C-- For preparation of lipid rafts using Triton X-100, it is essential to perform the extraction at low temperature to preserve the lipid-lipid and protein-lipid interactions defining these microdomains, because at higher temperatures, Triton X-100 solubilizes completely both lipids and proteins of microvillar membrane vesicles (data not shown). However, when microvillar lipid rafts, prepared by Triton X-100 extraction on ice, were subsequently re-extracted with the same detergent, first at 20 °C and then at 37 °C, a fraction of the original lipid raft membranes remained insoluble in Triton X-100 even at the latter temperature (Fig. 6). A comparison of these high temperature Triton X-100-insoluble membranes (termed "superrafts") with the "normal" lipid raft from which they were derived showed that superrafts are relatively enriched in a small subset of proteins. A further analysis by Western blotting revealed a hierarchy of the different microvillar lipid raft proteins with regard to their partition into the superrafts (Fig. 6). First, the most prominent protein of the superrafts was galectin-4, which completely resisted extraction with Triton X-100, both at 20 and 37 °C. Second, the glycosylphosphatidylinositol-anchored alkaline phosphatase was partially solubilized at 20 and 37 °C but predominantly resisted detergent extraction. Third, the major part of the transmembrane protein aminopeptidase N was solubilized, but a significant fraction of this abundant microvillar protein remained associated with the superrafts. Finally, most of the lipid raft-associated annexin 2, a peripheral membrane protein, was largely solubilized by Triton X-100 at 20 °C, and only small amounts were present in the superraft fraction.


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Fig. 6.   Isolation of superrafts. Lipid rafts were prepared from microvillar membrane vesicles by extraction with 1% Triton X-100 on ice and sucrose gradient centrifugation as described in the legend to Fig. 1. After centrifugation, the lipid rafts were carefully collected from the gradient by a pipette, diluted five times in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, and pelleted by centrifugation at 48,000 × g for 1 h. After resuspension in the above buffer, the lipid rafts were re-extracted with 1% Triton X-100, first at room temperature and then at 37 °C, as described under "Experimental Procedures." Samples of lipid rafts (R), proteins solubilized at room temperature (S1), proteins solubilized at 37 °C (S2), and superrafts (SR) were subjected to SDS-PAGE. After electrophoresis and electrotransfer onto ImmobilonTM, total protein was visualized by staining with Coomassie Brilliant Blue. Molecular mass values (kDa) are indicated. Aminopeptidase N (160 kDa), alkaline phosphatase (67 kDa), galectin-4 (36 kDa), and annexin 2 (36 kDa) were detected by Western blotting.

The overall lipid composition of superrafts showed a profile qualitatively similar to native microvillar membranes and rafts, isolated by Triton X-100 or Brij 98 (Fig. 3). However, the enrichment of glycolipids and, to a lesser degree, cholesterol, relative to phospholipids, was larger than for the other two raft fractions. Native microvillar membranes from pig enterocytes are rich in glycosphingolipids (>30% of total membrane lipid), indicating that they constitute more than half the amount of lipid in the exoplasmic leaflet (25). The relative glycolipid enrichment seen for the superrafts thus suggests that very little phospholipid remained in the outer leaflet of the membranes after the sequential extractions at increasing temperature. The phospholipids remaining in the superraft fraction most likely constitute the cytoplasmic leaflet of the core structure of these lipid rafts.

By electron microscopy, superrafts had a surprisingly well preserved membrane morphology, consisting of spherical vesicle-like structures of about 150-200 nm in diameter (Fig. 7). Multilamellar structures were visible in the preparation, and immunogold labeling with an anti-aminopeptidase N antibody revealed that the enzyme lined both exterior and interior membranes. In comparison with lipid rafts isolated by extraction with Triton X-100 (Fig. 4, A-D), the immunogold labeling of superrafts was more sparse, in agreement with the observation that a substantial fraction of aminopeptidase N was released from the membranes during preparation of the superrafts (Fig. 6).


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Fig. 7.   Morphology and immunogold labeling of superrafts. A and C, sections of pellets of superrafts. B, D, and E, similar sections immunogold-labeled for aminopeptidase N. Bars, 0.5 µm (A and B) and 0.1 µm (C-E).

Although the superrafts described above must be considered membrane artifacts created by the sequential detergent extraction at increasing temperature, the protocol used for their preparation represents a method for probing the relative strength of the molecular interactions that exist between the various lipid and protein constituents that define lipid rafts. Along this line, the protein composition of superrafts suggests that galectin-4 is the major raft stabilizer among the proteins of the microvillar membrane. To test this hypothesis, superrafts were re-extracted with Triton X-100 at 37 °C after a preincubation in the absence or presence of lactose, a soluble ligand for galectin-4. As shown in Fig. 8, the presence of lactose caused the release of some, but not all, galectin-4 from the superrafts, indicating that at least some of this lectin is raft-associated via its cognate ligand. However, in addition to galectin-4, markedly increased amounts of alkaline phosphatase and aminopeptidase N were also released by lactose, indicating that their raft association is at least partially galectin-4-dependent. Lactose also released galectin-4, alkaline phosphatase, and aminopeptidase N from lipid rafts, isolated by Brij 98-extraction and even from microvillar membranes, untreated with detergent (Fig. 8), showing that the association between these microvillar proteins is not entirely induced by detergent extraction.


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Fig. 8.   Release of proteins from superrafts, Brij 98 rafts, and microvillar membranes. Superrafts, Brij 98 rafts, and microvillar membranes, each prepared as described under "Experimental Procedures," were resuspended in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, in the absence (-) or presence (+) of 100 mM lactose. After incubation for 10 min at room temperature, Triton X-100 was added (final concentration 1%), and incubation at room temperature continued for 10 min before centrifugation at 20,000 × g for 30 min (the addition of Triton X-100 was omitted in the experiment with the microvillar membranes). The pellet (P) and supernatant (S) fractions were collected and analyzed by SDS-PAGE, followed by Western blotting. ApN, aminopeptidase N; Alk P, alkaline phosphatase; Gal-4, galectin-4.

Taken together, the results obtained with the superrafts imply that galectin-4 can be characterized as an organizer/stabilizer within microvillar lipid rafts. If the lectin thus defines the core of a raft, the glycosylphosphatidylinositol-anchored alkaline phosphatase and the transmembrane aminopeptidase N may then represent more peripheral raft proteins with decreasing affinity for these microdomains.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite the rapid proliferation of the lipid raft concept into many diverse fields of biology, fundamental questions concerning the functional relevance of these lipid-based microdomains in vivo persist. To a large extent, this has to do with the most frequently used biochemical criterion for raft association, detergent insolubility at low (0-4 °C) temperature. Thus, given the temperature-dependent phase behavior of membrane lipids (2), lipid rafts could well be a phenomenon entirely related to the commonly used method for their isolation. Addressing this problem, Brij 98, a member of the polyoxyethylene ether (Brij) series of nonionic detergents, was recently used for preparation of lipid rafts at 37 °C from T cells (20). These rafts had a lipid composition expected of normal lipid rafts and harbored the components of a functional T cell receptor machinery. In the present work, four microvillar proteins were used as markers, each of which had a characteristic lipid raft profile, using Triton X-100 and differing with regard to type of membrane attachment. Brij 98 extraction at 37 °C defined microvillar rafts having a marker protein composition essentially similar to lipid rafts, isolated by use of Triton X-100 at low temperature. The fact that Brij 98 at 37 °C generates authentic lipid rafts from two so very different membrane systems as T cell membranes and microvillar membrane vesicles lends strong credibility to the bona fide existence and functional relevance of these lipid-based microdomains.

Admittedly, the Triton X-100-resistant superrafts characterized in this work are membranes artifactually created by the sequential detergent extraction at increasing temperature, but importantly, they revealed the potential of galectin-4 to act as a strong organizer/stabilizer of these membrane microdomains. Like other members of the galectin family, galectin-4 is a cytosolic protein synthesized without a signal for membrane translocation (29, 39). This implies that galectin-4 is secreted by a "nonclassical" pathway. beta -Galactosyl residues present on microvillar membrane lipids as well as glycoproteins are likely to serve as ligands for this bivalent lectin and enable it to form detergent-resistant cross-linked lattices. The resulting dependence of microvillar raft stability on beta -galactosyl residues may explain their characteristic resistance to cholesterol depletion. Such lattices have been described in the clustering and separation of distinct glycoprotein receptors on the surface of T cells by galectin-1, leading to apoptosis (41). At the microvillar surface, galectin-4-driven clustering of digestive enzymes may help to preserve these at the cell surface by preventing them from release into the lumen of the gut and maybe also from undergoing endocytosis. This function may be physiologically important, because the intestinal lumen is a harsh working environment, and many microvillar enzymes are prone to release from the membrane by the action of pancreatic enzymes. We imagine these galectin-4-stabilized microvillar rafts as being relatively stable and large microdomains (31), contrary to the current view that lipid rafts are typically transient and small (23). In a recently proposed model, the molecular address for proteins targeted to rafts is a so-called lipid shell (22). Theoretically, these shells have an estimated diameter of 7 nm and contain about 80 lipid molecules that act as a local solvent in the lipid bilayer for the single protein they harbor. Larger raft structures, such as caveolae, form in a subsequent step by fusion of numerous shells. However appealing and applicable to other, more dynamic raft-based microdomains, such as assembly of signal transduction complexes, the lipid shell hypothesis hardly describes the relatively stable molecular organization of microvillar rafts. We conclude that the lipid raft concept needs to be further developed to accommodate the great variety of membrane functions that rely on lipid-based microdomains. In this process, we believe the protocol for making superrafts should be applicable to other raft membrane systems and might be a useful approach for probing the strength of molecular interactions within these microdomains.

    FOOTNOTES

* This work was supported by grants from the Danish Medical Research Council and the Novo-Nordic Foundation and was part of the Danish Biotechnology program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

G. H. H. and E. M. D. dedicate this work to the memory of Ove Norén, mentor and longtime friend, who died on December 17, 2001.

Dagger Recipient of a Socrates/Erasmus travel stipend from the European Union. Present address: Universitá Di Perugia, Dipartimento di Scienze e Biotecnologie Molecolari, Sezione di Patologia Generale, Policlinico Monteluce, 06100 Perugia, Italy.

§ To whom correspondence should be addressed. Tel.: 45-3532-7786; Fax: 45-3536-7980; E-mail: midan@imbg.ku.dk.

Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M211228200

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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