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【关键词】 Conformational
The present study characterizes the methanethiosulfonate (MTS) inhibition profiles of 26 consecutive cysteine-substituted mutants comprising transmembrane (TM) helix 6 of the human apical Na+-dependent bile acid transporter (SLC10A2). TM6 is linked exofacially to TM7 via extracellular loop 3. TM7 was identified previously as lining part of the substrate permeation path (Mol Pharmacol 70: 1565, 2006[Abstract/Free Full Text]). Most TM6 cysteine replacements were well tolerated, except for five residues with either severely hampered (I229C, G249C) or abolished (P234C, G237C, G241C) activity. Disruption of protein synthesis or folding and stability may account for lack of activity for mutant P234C. Subsequent Pro234 amino acid replacement reveals its participation in both structural and functional aspects of the transport cycle. Application of polar MTS reagents (1 mM) significantly inhibited the activity of six mutants (V235C, S239C, F242C, R246C, A248C, and Y253C), for which rates of modification were almost fully reversed (except Y253C) upon inclusion of bile acid substrates or removal of Na+ from the MTS preincubation medium. Activity assessments at equilibrative [Na+] revealed numerous Na+-sensitive residues, suggesting their proximity in or around Na+ interaction sites. In silico modeling reveals the intimate and potentially cooperative orientation of MTS-accessible TM6 residues toward functionally important TM7 amino acids, substantiating TM6 participation during the transport cycle. We conclude a functional requirement for helical flexibility imparted by Pro234, Gly237, and Gly241, probably forming a "conformational switch" requisite for substrate turnover; meanwhile, MTS-accessible residues, which line a helical face spatially distinct from this switch, may participate during substrate permeation.
By coupling bile acid movement to the passive flow of Na+ ions down their concentration gradient, the human apical Na+-dependent bile acid transporter (ASBT; SLC10A2) concentrates bile acids within the cell interior. Viewed from a physiological perspective, ASBT effectively conserves the body's recirculating bile acid pool (Trauner and Boyer, 2003) in tandem with numerous active transporters expressed along the enterohepatic pathway. Because cholesterol provides the precursor molecule in FXR- and hepatic CYP7A-mediated bile acid synthesis (Chiang et al., 2001; Pauli-Magnus et al., 2005), ASBT also constitutes a key modulator of cholesterol homeostasis. Numerous studies have recently underscored the exploitive potential of ASBT in cholesterol-lowering therapies (Oelkers et al., 1997; Izzat et al., 2000; Huff et al., 2002; Li et al., 2004) and emphasizing the usefulness of this high-capacity, high-affinity transporter in prodrug targeting (Swaan et al., 1997; Balakrishnan and Polli, 2006; Geyer et al., 2006). Consequently, ASBT's unique pharmaceutical relevance coupled to the absence of a crystal structure has provided a strong impetus toward elucidation of its structure/function relationships.
Using cysteine mutagenesis and thiol modification (SCAM), our previous studies identified transmembrane (TM) domain 7 in forming part of the putative substrate permeation pathway (Hussainzada et al., 2006) with extracellular loop (EL) 3 containing Na+ and bile acid interaction sites (Banerjee et al., 2008). We continue SCAM analysis along TM6 based on the following rationale: 1) our topology model published previously predicts that TM6 lies adjacent to and may interact with TM7 in forming a putative translocation pathway (Zhang et al., 2004); 2) EL3 amino acids link TM6 and TM7 membrane-spanning segments along the exofacial matrix; 3) the highly conserved nature of TM6 amino acids corroborate a potential role during transport; 4) presence of the charged, conserved Arg246, which could potentially participate in electrostatic interactions implicated previously during ligand binding (Banerjee et al., 2008); and finally, 5) the presence of two conserved proline residues (Pro234, Pro251), which have been shown in other membrane-bound carriers to provide cation binding sites and enable formation of conformational switches essential for substrate translocation (Deber et al., 1990; Sansom and Weinstein, 2000; Pajor and Randolph, 2005). As in our previous studies, the C270A mutant provides the scaffold for subsequent cysteine introduction as a result of its insensitivity to methanethiosulfonate (MTS) reagents. Therefore, the present study assesses MTS sensitivity of 26 consecutive cysteine mutants introduced along TM6 of hASBT, thereby providing novel insight into the molecular workings of the ASBT translocation cycle. We demonstrate a functional prerequisite for TM6 helical flexibility in global conformational changes to protein structure, leading to substrate turnover and the putative involvement of TM6 amino acids in lining portions of the permeation pathway.
Materials. [3H]Taurocholic acid (0.2 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc., (St. Louis, MO); Taurocholic acid (TCA) and glycodeoxycholic acid (GDCA) were from Sigma (St. Louis, MO); and sulfosuccinimidyl-2 (biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) was from Pierce Chemical Co. (Rockford, IL). MTS reagents (2-aminoethyl)-methanethiosulfonate (MTSEA), [2-(trimethylammonium) ethyl] methanethiosulfonate (MTSET), and methanethiosulfonate ethylsulfonate (MTSES) were from Toronto Research Chemicals, Inc. (North York, ON, Canada). Cell culture media and supplies were obtained from Invitrogen (Carlsbad, CA). All other reagents and chemicals were of highest purity available commercially.
Cell Culture and Transient Transfections. COS-1 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 4.5 g/l glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere with 5% CO2. Transient transfections were performed as described previously (Banerjee et al., 2005).
Site-Directed Mutagenesis. Site-directed mutations were incorporated into hASBT cDNA using the Quik Change site-directed mutagenesis kit from Stratagene (La Jolla, CA) and mutagenesis primers custom-synthesized and purchased from Sigma Genosys (St. Louis, MO). Plasmid purifications were performed using a kit from QIAGEN (Valencia, CA) and amino acid substitutions confirmed via DNA sequencing using an ABI 3700 DNA analyzer (Applied Biosystems, Foster City, CA) at the Plant-Microbe Genomics Facility of the Ohio State University (Columbus, OH).
Uptake Assay and Protein Membrane Expression. Initial rates of transport for each mutant were determined in transiently transfected COS-1 cells incubated in modified Hanks' balanced salt solution (MHBSS), pH 7.4, uptake buffer containing 5.0 µM[3H]TCA at 37°C for 12 min. We have demonstrated that this uptake period ensures linear steady-state kinetics in conjunction with an optimal signal-to-noise ratio for subsequent [3H]TCA analysis via liquid scintillation counting (Banerjee et al., 2005; Banerjee and Swaan, 2006; Hussainzada et al., 2006). Uptake was halted by a series of washes with ice-cold Dulbecco's phosphate-buffered saline, pH 7.4, containing 0.2% fatty acid free bovine serum albumin and 0.5 mM TCA. Cells were lysed in 350 µl of 1 N NaOH and subjected to liquid scintillation counting using an LS6500 liquid scintillation counter (Beckmann Coulter, Inc., Fullerton, CA) and total protein quantification using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Uptake activity was calculated as picomoles of [3H]TCA internalized per minute per milligram of protein.
Protein expression was determined by washing transiently transfected COS-1 cells in PBS followed by lysis in 0.2 ml of lysis buffer B (25 mM Tris, pH 7.4, 300 mM NaCl, 1 mM CaCl2, 1% Triton X-100, and 0.5% Sigma Protease Inhibitor Cocktail). Cell lysates were separated on a 12.5% SDS-polyacrylamide gel and transferred onto an Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories). Blots were probed with rabbit anti-ASBT primary antibody (1:1000) and visualized using goat anti-rabbit IgG/horseradish peroxidase-conjugated secondary antibody with chemiluminescent detection (ECL Plus Western Blot kit; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Levels of cell surface protein expression were measured via biotin labeling, wherein transiently transfected COS-1 cells were incubated with sulfo-NHS-SS-biotin for 30 min at room temperature (Wong et al., 1995; Mitchell et al., 2004). After several washes with PBS containing 0.1 mM CaCl2 and 1.0 mM MgCl2, cells were disrupted with lysis buffer B at 4°C for 20 min (Zhang et al., 2004), and biotinylated proteins were recovered overnight at 4°C using 100 µl of streptavidin agarose beads. Samples were eluted with SDS-polyacrylamide gel electrophoresis buffer and immunoblotting performed as described above. Blots were probed for positive and negative controls, the plasma membrane marker -integrin (150 kDa) and the endoplasmic reticulum membrane protein calnexin (90 kDa), respectively, to assess the integrity of the biotinylation procedure (calnexin; data not shown). Relative hASBT membrane expression was standardized to integrin expression and quantified via densitometry as described previously (Hussainzada et al., 2006).
MTS Inhibition Studies. Sensitivity of mutants to charged, membrane-impermeant MTS reagents was determined by preincubation of transiently transfected COS-1 cells with either 1 mM MT-SES, MTSET, or MTSEA for 10 min at room temperature. After MTS treatment, cells were washed twice in modified Hanks' balanced salt solution (Sigma) followed by [3H]TCA uptake as described above. All MTS solutions were freshly prepared before each study because of the short aqueous half-life of these MTS reagents.
Cation and Substrate Protection Assays. To determine whether the presence or absence of Na+ and/or bile acid substrates alters MT-SEA labeling, transiently transfected COS-1 cells were washed twice in 1x PBS, pH 7.4, followed by coincubation with equal concentrations of MTSEA and GDCA (1 mM) prepared either in MHBSS or Na+-free buffer (MHBSS except choline chloride entirely substitutes NaCl) for 10 min at room temperature. After preincubation treatments, cells were washed twice in either MHBSS, pH 7.4, or Na+-free buffer and additionally equilibrated for 15 min at 37°C in these buffers followed by determination of [3H]TCA uptake as described above. All control wells were treated identically. For each mutant transporter, uptake values were determined by taking a ratio of mutant uptake at each experimental condition versus mutant uptake for its respective unmodified control. We normalize mutant ratios to C270A by expressing mutant ratios for each condition as a percentage of C270A ratios calculated in the same manner.
Sodium Activation. Measurement of [3H]TCA uptake at equilibrative extracellular Na + concentrations (12 mM; i.e., at equilibrium with cytosolic [Na+]) was performed (uptake conducted as described above; choline chloride used as equimolar NaCl replacement) and expressed as a ratio of uptake at physiological (137 mM) Na+ concentrations to determine overall sensitivity of each mutant to the presence/absence of Na+. In theory, Na+ ratios equal to 1 imply little measurable difference in transporter activity despite the scarcity of Na+ ions, whereas fractions less than 1 indicate a greater necessity for physiological Na+ concentrations for proper transport function of a mutant transporter.
Data Analysis. For each mutant, data are represented as mean ± S.D. of at least three different experiments with triplicate measurements. Data analysis was performed with Prism 4.0 (GraphPad Software, Inc., San Diego, CA) using analysis of variance with Dunnett's post hoc test. Data were considered statistically significant at p 0.05.
Fig. 1. Multiple sequence alignment of TM6 amino acids. A, secondary structure model of the last three transmembrane domains (TMDs) of hASBT according to the 7TM model. Roman numerals indicate flanking TMD, whereas TMD 6 amino acids are represented by gray circles inscribed with amino acid identity and position. Phospholipids of plasma membrane represented by circle (polar phosphate head group) with two tails (hydrophobic lipids). Top, exofacial; bottom, cytosolic. B, sequence alignment of amino acids 227 to 253, putatively forming TM6, for all known ASBT paralogs. Sequences were retrieved from GeneBank and aligned via the MULTALIN routine with annotation performed via the MPSA program. Shaded regions denote complete amino acid conservation among all species. Amino acid positioning relative to human ASBT is indicated by numbering on top. Bottom line indicates primary consensus for the TM6 region.
Cysteine Scan of TM6. Based on our topology model published previously (Zhang et al., 2004; Banerjee and Swaan, 2006), residues spanning Trp227 to Tyr253 are predicted to constitute TM6 of hASBT (Fig. 1A). High-sequence homology is observed among various evolutionarily diverse species for this protein region (Fig. 1B), which lies in intimate proximity to critical protein regions described previously (Hallén et al., 2000, 2002; Kramer et al., 2001; Hussainzada et al., 2006). Systematic cysteine substitutions were incorporated along TM6 followed by structural and functional analysis of mutant transporters.
Transport Activity and Membrane Expression of Cysteine Mutants. Because of its low background levels of bile acid transport (Hussainzada et al., 2006), the COS-1 cell line was used to transiently express all TM6 mutant transporters. Surface biotin labeling of membrane-expressed proteins was accomplished using the membrane-impermeant sulfo-NHS-SS-biotin and quantified via densitometry of protein bands (Fig. 2B). ASBT bands for each sample were standardized to an internal control (-integrin) and expressed as a percentage of C270A intensity (Fig. 2C). Initial transport activities (Fig. 2A) were then normalized to relative membrane expression for each mutant transporter.
Fig. 2. [3H]TCA uptake activity and membrane expression of TM6 cysteine mutants. A, uptake of [3H]TCA was measured in COS-1 cells as described under Materials and Methods and expressed as a percentage of the parental transporter C270A. B, intact transfected COS-1 cells were treated withsulfo-NHS-SS-biotin as described under Materials and Methods followed by Western blot processing. Blots were probed with the anti-hASBT antibody (1:30,000 dilution) followed by horseradish peroxide-linked anti-rabbit immunoglobin (1:2000 dilution). Each blot was probed for the internal plasma membrane marker -integrin (150 kDa) and the absence of calnexin (90 kDa) (data not shown), an endoplasmic reticulum membrane protein representing the negative control in the biotinylated fractions. Marker lanes are shown on the left side of the individual blots. Mature glycosylated hASBT visualizes as the 41-kDa band, whereas the lower 38-kDa band (not shown) represents the unglycosylated species. C, densitometric analysis for cysteine mutants normalized to internal marker (-integrin) and represented as a percentage of C270A parent. D, [3H]TCA uptake activity normalized to relative cell surface expression. Bars represent mean ± S.D. of three separate experiments with ***, p 0.001; **, p 0.01; and *, p 0.05, respectively, using analysis of variance with Dunnett's post hoc analysis.
After data normalization (Fig. 2D), most TM6 mutants retained appreciable levels of activity, except for five mutants either severely hampered (I229C, G249C) or inactivated (P234C, G237C, G241C) upon cysteine substitution. Only P234C lacked expression in biotinylated (Fig. 2B) and whole-cell (data not shown) extracts. This may be due to disruptions in protein synthesis, but more likely, alterations in protein folding and stability occur that induce rapid protein degradation via endoplasmic reticulum-associated machinery. It is interesting that all five residues are conserved among known species of ASBT (Fig. 1B), suggesting primary roles in transport function that necessitate preservation of these amino acids. Because of their low activity levels, these mutants were excluded from further studies.
TM6 Mutants Demonstrated Substantial Na+ Sensitivity. AsaNa+ cotransporter, ASBT activity relies on proper recognition, binding, and translocation of two Na+ ions per one bile acid molecule (Weinman et al., 1998). Thus, we examined the consequences of equilibrative extracellular Na+ concentrations upon mutant activity. For each mutant, the ratio of transport rates at equilibrative (12 mM) versus physiological (137 mM) Na+ concentrations was calculated and expressed as a percentage of the C270A Na+ ratio. This experimental scheme may uncover hidden functional defects in mutants otherwise unaffected by cysteine mutation. Therefore, the C270A parental construct displays a Na+ ratio of 0.70 ± 0.04 (data not shown), indicating minimal consequences to transporter function upon alanine substitution at the native cysteine residue. In contrast, significant decreases in activity were observed for the majority (64%) of TM6 cysteine mutants. Of 21 mutants assayed, 14 demonstrated hampered uptake rates at equilibrative [Na+] (Fig. 3). EL3 cysteine mutants from our earlier study also exhibited similarly extensive Na+ sensitivity (Banerjee et al., 2008), wherein uptake activities of 90% of assayed mutants were susceptible to equilibrative [Na+]. Because EL3 residues putatively form Na+ interaction sites (Banerjee et al., 2008), the widespread Na+-dependence observed for TM6 mutants implies their close proximity to such Na+ interaction sites and lends credence to TM6 participation during Na+ permeation.
Fig. 3. Sodium sensitivity of cysteine mutants. COS-1 cells expressing mutant transporters were incubated in uptake medium (5 µM[3H]TCA) containing low (12 mM) or physiological (137 mM) Na+ concentrations as described under Materials and Methods. Sodium ratios were calculated for each mutant as the quotient of activity at 12 versus 137 mM [Na+] and expressed as a percentage of C270A. Bars represent mean activity ± S.D. (n = 3). *, p 0.05.
Substrate and Cation Binding Modulate Accessibility of Cysteine Mutants to MTS Modification. Both positively and negatively charged MTS reagents were used to probe the solvent accessibility of 21 cysteine mutants demonstrating measurable uptake activity. Intact monolayers of COS-1 cells expressing mutant transporters were preincubated with 1.0 mM concentration of either MTSES, MTSET, or MTSEA followed by uptake assessments. MTSET (109 Å3) and MTSES (90 Å3) exhibited similar inhibition profiles, in which activities of only mutants V235C, S239C, F242C, and R246C were significantly reduced (data not shown), suggesting minimal electrostatic effects in accessibility at those sites. The remainder of the TM6 mutants assayed were either inaccessible to these MTS reagents, or their modification was functionally silent. Incubation with the relatively smaller MTSEA (69 Å3) inhibited uptake at sites accessed by the larger MTS reagents (i.e., V235C, S239C, F242C, and R246C; Fig. 4) and at two additional sites (A248C and Y253C; Fig. 4).
Fig. 4. Cation and substrate protection of TM6 cysteine mutants. Transiently transfected COS-1 cells expressing TM6 cysteine mutants were preincubated in buffer, pH 7.4, containing 1 mM MTSEA and either 137 mM NaCl (), 137 mM NaCl and 1 mM GDCA (gray bar); 137 mM choline chloride (dark gray bar); or 137 mM choline chloride and 1 mM GDCA () and followed by [3H]TCA uptake as described under Materials and Methods. Choline chloride does not activate the transporter and provides equimolar replacement for NaCl. All control wells were treated identically. Bars represent mean ± S.D. of at least three separate measurements. Data are expressed as a percentage of C270A values for each condition as described under Materials and Methods. Student's t test analysis performed with *, p < 0.05, and **, p < 0.01.
Because MTSEA (1 mM) application produced the most pronounced inhibition of the three MTS reagents used (similar to our previous study with TM7; Hussainzada et al., 2006), the effects of Na+ and bile acid substrate on MTSEA accessibility of mutants V235C, S239C, F242C, R246C, A248C, and Y253C was evaluated. Our previous studies with EL3 and TM7 have shown that coincubation of MTS reagents with bile acids and/or removal of Na+ from the preincubation buffer caused a reversal of the inhibitory effect observed with MTS incubation alone (Hussainzada et al., 2006; Banerjee et al., 2008). Likewise, in the present study, all TM6 mutants inhibited by MTSEA (1 mM) demonstrated significant uptake recovery when MTSEA incubation was performed in the absence of Na+ and/or presence of 1.0 mM glycodeoxycholic acid (GDCA) (Fig. 4). In particular, the removal of Na+ from the MTSEA preincubation medium significantly restored transport activity for mutants V235C, S239C, F242C, R246C, and A248C, whereas coincubation with GDCA (Km = 2.0 ± 0.4 µM) significantly protected mutants F242C and A248C. Concurrent removal of Na+ and the addition of the high-affinity substrate GDCA (1 mM) resulted in significant MTSEA protection for mutants V235C, S239C, F242C, and R246C. In all cases, mutant activities were restored to control (C270A) levels (within standard deviation; Fig. 4).
Although mechanistic details of the ASBT translocation cycle are as yet unresolved, ordered binding of ligands followed by translocation probably occurs, similar to many other Na+-coupled transporters (Quick and Jung, 1997; Jung, 2001; Pajor and Randolph, 2005; Zhang and Rudnick, 2005). In this scenario, the ordered binding of Na+ and bile acids would trigger the protein to assume various discrete structural conformations, eventually leading to carrier reorientation within membrane leaflets and substrate turnover. Within TM6, the lack of Na+ binding events (simulated by substitution of Na+ with choline+ in preincubation buffers) significantly decreased MT-SEA modification rates for all sites (Fig. 4), suggesting that protein conformational states assumed before the binding of Na+ occlude these thiol groups from subsequent modification. Furthermore, the binding of bile acid substrate (GDCA) in either the presence or absence of Na+ also triggers protein conformations that significantly decrease MTSEA access to all sites (Fig. 4). Because of the close association between TM6 and protein regions previously implicated during ligand binding and translocation (i.e., TM7 and EL3), the observed substrate protection may result from 1) occlusion of these sites via conformational changes; 2) the physical presence of substrates preventing access; or 3) a combination of both scenarios. The alternating accessibility of TM6 sites to thiol modification suggests the first scenario, whereas the restoration of mutant activity to control levels via substrate protection infers the second scenario. It is likely that a union of both situations prevails, in which TM6 residues may line portions of the permeation pore and also transduce conformational changes resulting from ligand interactions at adjacent protein regions (TM7, EL3), although further studies are needed to unequivocally conclude the origin of substrate protection. However, it is noteworthy that our data highlight a trend toward protection from MTS modification in the absence of Na+, which is entirely plausible given that EL3 residues contain putative Na+ interaction sites (Banerjee et al., 2008). It may be that the lack of Na+ binding at EL3 regions prevents downstream conformational changes that "open" TM6 residues to MTS modification. Overall, we conclude that TM6 amino acids probably line portions of the substrate permeation route due to their spatial proximity with EL3 and TM7 residues and their solvent-accessibility profile.
Mutation of Pro234 Affects Both Transporter Expression and Function. Because the P234C double mutant (C270A/P234C) lacked expression both at the plasma membrane (Fig. 2B) and in whole-cell extracts (data not shown), additional replacements were made at this site to determine whether Pro234 makes functional contributions during the hASBT transport cycle. Using the wild-type (WT) species as the scaffold, alanine, glycine, and cysteine replacements were incorporated and analyzed with respect to uptake activity and membrane expression. As expected from our results using the kinetically similar C270A template (Banerjee et al., 2005), the P234C mutant constructed against the WT background lacked expression both in membrane (Fig. 5B) and whole-cell (Fig. 5C) extracts, confirming that cysteine replacement at this position affects transporter expression levels irrespective of the mutational template used. Glycine replacement (P234G) also seems to disrupt protein expression, resulting in minimal transporter expression in membrane (Fig. 5B) and whole-cell (Fig. 5C) extracts. In contrast, the alanine mutant (P234A) displayed plasma membrane expression, albeit at reduced levels (30% of C270A levels; Fig. 5A). After normalization to cell surface expression levels, uptake function of the P234A mutant remained severely inhibited (Fig. 5A), suggesting functional and structural impairments to transporter function. Defective trafficking to the plasma membrane may account for the lowered membrane expression of the P234A mutant, because the ratio of its plasma membrane expression to whole-cell expression is approximately half of the similar ratio for the WT species (i.e., 0.452 versus 0.814); however, further studies are needed for unequivocal evidence. We conclude that Pro234 participates in both protein expression and transporter function, confirming the overall importance of this atypical amino acid.
Fig. 5. [3H]TCA uptake and membrane expression of Pro234 mutants. A, the initial [3H]TCA uptake (gray bars), relative intensity of immunoblotting (dark gray bars), and normalized uptake activities (black bars) for Pro234 single and double mutants and their parental templates are depicted. Bars represent mean ± S.D. of at least three separate measurements. Data are expressed as a percentage of C270A values for each condition. Student's t test analysis was performed with *, p < 0.05, and **, p < 0.01. Activities of the Pro234 constructs are statistically different from both WT and C270A (p < 0.01); however, asterisks (**) have been omitted for visual clarity. B, intact transfected COS-1 cells were treated with sulfo-NHS-SS-biotin as described under Materials and Methods followed by Western blot processing. Marker lanes are shown on the left side of the individual blots. Mature glycosylated hASBT visualizes as the 41-kDa band, whereas the lower 38-kDa band (not shown) represents the unglycosylated species. C, immunoblotting of whole-cell extracts from transfected COS-1 cells as described under Materials and Methods showing glycosylated (41 kDa) and unglycosylated (38 kDa) hASBT species.
作者单位：Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland