Lysosomal Targeting of P-selectin Is Mediated by a Novel Sequence within Its Cytoplasmic Tail*

Anastasia D. Blagoveshchenskaya, John P. Norcott, and Daniel F. CutlerDagger

From the Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

A substantial body of data indicates that post-Golgi sorting of integral membrane proteins is largely dependent upon information contained within the cytoplasmic domains of these proteins (for review, see References 1-4). This information is usually found in short stretches of amino acids, "sorting signals," which serve to direct proteins to a variety of intracellular destinations. The most common signals can be classified into two groups: Tyr-based and di-Leu signals. Conformational modeling and two-dimensional nuclear magnetic resonance spectroscopy (NMR) of peptides encoding these motifs have revealed that the first group, typically conforming to Asn-Pro-X-Tyr or Tyr-X-X-Ø sequences (where X is any amino acid and Ø is a bulky hydrophobic amino acid), form a "tight turn" (5-7), whereas di-Leu or Leu-Ile motifs may display an extended, random coil conformation within the cytoplasmic domain of that protein (8). More recently, a novel category of targeting signals that lie within an alpha -helix (9, 10) has been defined. In contrast, other sorting signals are centered on strongly hydrophilic sequences, comprising clusters of acidic residues (Glu and Asp) functioning either as independent determinants or in concert with Tyr-based or di-Leu motifs (11, 12).

The type I membrane protein P-selectin belongs to a family of adhesion molecules. It initiates leukocyte recruitment during the inflammatory response and is involved in hemostasis (13-16). Found in the membrane of regulated secretory organelles of platelets and endothelial cells (16-18), it is redistributed to the plasma membrane after stimulation of these cells with agonists (14, 19, 20). When expressed in cells lacking a regulated secretory pathway, P-selectin is constitutively transported to the cell surface (21-23). After appearance at the plasma membrane, it is rapidly internalized (20-22, 24, 25) and passes through the endocytic pathway to its final destination(s) (25, 26). When expressed in Chinese hamster ovary and PC12 cells, P-selectin is efficiently targeted to lysosomes, and its cytoplasmic tail comprising the C1 and C2 domains is both necessary and sufficient for this trafficking step mediated by the putative lysosomal targeting signal within the C1 domain (21).

Much data derived from a variety of systems suggest that LTS1 and internalization signals often overlap or are co-linear, both for proteins biosynthetically targeted to lysosomes such as resident lysosomal membrane proteins as well as for membrane proteins capable of efficient lysosomal targeting during receptor-mediated endocytosis (see Table I and references cited therein). One interesting exception is the EGFR, in which mutations inactivating lysosomal targeting were found not to affect internalization (27-29). Thus, although an overlap is often evident, there is no simple correlation between internalization and lysosomal targeting, implying that the structural requirements for interacting with the cytoplasmic machinery which facilitates these two trafficking events may be complex.

Since the short cytoplasmic tail of P-selectin has been found to contain a variety of signals for targeting to organelles along the regulated secretory pathway as well as on the endocytic pathway (21, 25, 26), distinguishing different signals from each other is a complex problem. We therefore chose to study targeting in a system that lacks some of the possible destinations. We have begun by analyzing P-selectin targeting to late endocytic compartments in cells in which the endocytic pathway has been extensively characterized (30-34) and that lack regulated secretory organelles.

In this study, we examined the effects of mutations within the cytoplasmic tail on lysosomal targeting of P-selectin in H.Ep.2 cells, which lack regulated secretory organelles. We have uncovered a novel short LTS, KCPL, which is located within the C1 domain and which has no obvious homology to LTSs identified in other proteins. The proline residue within this LTS significantly contributes to the efficiency of lysosomal sorting.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials and Reagents-- Rabbit polyclonal anti-HRP was from DAKO, Glostrup, Denmark. Fab fragments of this antibody were generated using a commercial ImmunoPure® Fab preparation kit (Pierce) according to the manufacturer's instructions. Mouse receptor-grade epidermal growth factor (EGF) and human, iron-saturated transferrin (Trn) were purchased from Sigma. Iodination of Fab fragments, EGF, and Trn has been performed according to the modified IODO-GEN method as described elsewhere (35). The specific activity of preparations were: 3 × 104, 1 × 105, and 2 × 104 cpm/ng, respectively. Protein concentration was determined using Coomassie Plus protein assay reagent (Pierce) according to the manufacturer's instructions. Other chemicals were from Sigma and Pharmacia Biotech Inc.

Constructs-- A chimeric cDNA containing the human growth hormone signal sequence, followed by HRP, and finally the transmembrane domain and cytoplasmic tail of P-selectin (Fig. 1) was generated as described previously (26). cDNAs encoding the deletion mutants, ssHRPP-selectin776, ssHRPP-selectin763, and ssHRPP-selectinDelta C1, were constructed as detailed in this report. Both the point mutation of proline 767 and the tetra-alanine changes were made using the Stratagene QuikChange site-directed mutagenesis kit. Briefly, cesium chloride-purified pRK34 plasmid containing the ssHRPP-selectin was added to two complementary synthetic oligonucleotide primers which contained the change of interest. After temperature cycling with Pfu DNA polymerase, a mutated plasmid containing a staggered nick was generated. The parental DNA was then digested away with DpnI restriction endonuclease, which is active only on methylated and hemimethylated DNA. The nicked mutated vector was transformed into supercompetent Escherichia coli. The sequences generated were confirmed by sequencing. The sequences of the oligonucleotide primers used to generate the mutants are listed below. Only the sense primer is shown; the antisense primer used is the exact complement.

The primers used are as follows: P-selectinP767A, GATGGGAAATGCgCCTTGAATCCTCAC; P-selectinKDDG, CGTTTCAGACAGGCAGCTGCTGCGAAATGCCCCTTG; P-selectinKCPL, AAAGATGATGGGGCAGCCGCCGCGAATCCTCACAGC; and P-selectinNPHS, AAATGCCCCTTGGCTGCTGCCGCCCACCTAGGAACA.

Cell Culture and Transfection-- The human cell line H.Ep.2 was cultured and transiently transfected as described elsewhere (36). Cells were plated on 150- and 35-mm dishes and in some experiments were grown for 24 h in presence of 100 µM each of pepstatin A and leupeptin prior to use.

HRP Proteolysis Assay-- Cells on 35-mm dishes were placed on ice, washed twice with ice-cold PBS, and subjected to Triton X-114 partitioning as described (37) followed by a standard HRP assay (see below) performed with two phases. Amounts of HRP activity present in the upper, hydrophilic and lower, hydrophobic phases were used to determine the percentage of clipped chimera as a ratio of HRP activity in the upper phase to the total activity in the lysate. To normalize for inter-experimental variations, we subtracted the fraction of HRP proteolysis of tailless ssHRPP-selectin763 chimera (typically about 25%) as background from each data set, since this chimera has been demonstrated previously to be incapable of internalization and, consequently, of delivery to lysosomes instead accumulating on the plasma membrane (26).

Cell Surface Expression Assay-- Cells on 35-mm dishes were washed with growth medium, placed on ice, and incubated with 2 µg/ml 125I-Fab fragments in growth medium at 4 °C for 1 h. Unbound ligand was removed by three rinses with growth medium. Cells were then washed with ice-cold PBS and lysed in 1 ml of HRP assaying buffer (100 mM sodium citrate, pH 5.5, 0.1% Triton X-100) at 4 °C for 10 min. Lysates were counted in a gamma -counter (Hewlett Packard) and then spun at 13,000 × g for 5 min in a refrigerated centrifuge to pellet cell debris. Supernatants were used for measurement of HRP activity (see below). Cell surface expression levels were determined as a ratio of the amount of 125I-Fab fragments to the level of HRP activity for each chimera, to normalize for variation of expression level. The ratio for ssHRPP-selectin was set at 1 for all experiments, and the data from all other chimeras have been normalized accordingly.

Endocytosis Assays-- Cells on 150-mm dishes were washed with binding medium (Dulbecco's modified Eagle's medium, 20 mM HEPES, 0.1% bovine serum albumin) and incubated with either 5 ng/ml 125I-EGF at 4 °C for 1 h or 100 ng/ml 125I-Trn at 37 °C for 1 h. Excess ligand was removed by three rinses with cold Dulbecco's modified Eagle's medium. The cells loaded with 125I-Trn were placed on ice and subsequently treated with MES buffer I (25 mM MES, pH 4.0, 150 mM NaCl) and MES buffer II (25 mM MES, pH 7.5, 150 mM NaCl) at 4 °C for 10 min each to remove any cell surface-bound ligand. The cells labeled with 125I-EGF were allowed to internalize ligand for 30 min at 37 °C, followed by a 2.5-min mild acid wash (100 mM sodium acetate, pH 4.5, 500 mM NaCl) at 4 °C to extract 125I-EGF present on the plasma membrane. In both cases, the cells were then thoroughly washed with ice-cold binding medium, washed twice with HB (320 mM sucrose, 10 mM HEPES, pH 7.3), and subjected to subcellular fractionation as detailed below.

Subcellular Fractionation and Quantitation of Data-- Following two rinses with ice-cold HB, cells were scraped into 1.5 ml of HB with a rubber policeman and passed 10 times through a ball bearing homogenizer with a 0.009-mm clearance (made at EMBL, Heidelberg, Germany). The cell homogenate was spun at 8,500 × g for 5 min and 1.3 ml of postnuclear supernatant (PNS) was layered on an 11-ml 1-16% preformed linear Ficoll gradient made in HB. The gradients were centrifuged for 45 min at 35 ,00 rpm in a SW40Ti rotor (Beckman Instruments, Palo Alto, CA) and then fractionated in 0.5-ml fractions from the top of the tube using an Autodensi-Flow IIC (Buchler Instruments, Kansas City, MO), and radioactivity of fractions was counted. Positions of lysosomes and late endosomes were identified by measurement of activity of the lysosomal marker enzyme, N-acetyl-beta -D-glucosaminidase (NAGA), as described previously (38).

The fractions containing most NAGA activity were further purified using a second centrifugation. Fractions 17-23 from the 1-16% Ficoll gradient were pooled together; 2.5 ml of this material was diluted with HB to make 4 ml and layered on a 9-ml 7-25% Ficoll gradient. Centrifugation, fractionation, and measurement of NAGA activity were then carried out as described for initial Ficoll gradients.

Targeting data were described as a lysosomal targeting index (LTI), i.e. the amount of HRP activity present in LE/Lys fractions for each mutant normalized to that for ssHRPP-selectin. Accordingly, in all the experiments, the LTI for ssHRPP-selectin was set at 1. To take into account variations of expression level, number of cells, and lysosomal yield, the amount of HRP activity present in the LE/Lys peak (HRP peak) has been corrected for the amount of NAGA activity or 125I-EGF radioactivity (NAGA(EGF) peak) within the LE/Lys fractions and for total HRP activity in the homogenate (HRP hmg). After simplifying the original equation, the LTI was defined as follows.
<UP>LTI</UP>=<FR><NU><UP>mutant HRP peak/mutant NAGA </UP>(<UP>EGF</UP>)<UP> peak</UP>×<UP>mutant HRP hmg</UP></NU><DE><UP>WT HRP peak/WT NAGA </UP>(<UP>EGF</UP>)<UP> peak</UP>×<UP>WT HRP hmg</UP></DE></FR> (Eq. 1)

Typically, the LTI for tailless ssHRPP-selectin763 was about 20% of that for ssHRPP-selectin and was subtracted from those for the other chimeras in each experiment to provide a base-line value. Thus, the LTI for ssHRPP-selectin763 was considered as 0. The LTIs of the mutants were therefore described on a scale within a range set by ssHRPP-selectin (LTI = 1) and ssHRPP-selectin763 (LTI = 0).

DAB Cytochemistry-- A modification of the procedure, originally developed by Courtoy et al. (39) was used. A 4.5 mM solution of DAB in HB was prepared, adjusted to pH 7.3 with 1 N NaOH, and filtered through an 0.22-µm filter (Millipore Corp.). PNS or gradient fractions (700 µl) were mixed with 800 µl of DAB solution and 8 µl of 6% H2O2 and incubated for 15 min at room temperature in the dark. A 700-µl control sample from the same PNS or Ficoll fraction was incubated in parallel with 800 µl of HB alone. Subsequently, the mixtures (1.5 ml) were layered on Ficoll gradients made with HB supplemented with 1 mM imidazole. When the DAB reaction was performed with PNS, the samples were centrifuged on 1-16% Ficoll gradients. Ficoll fractions subjected to DAB reaction were recentrifuged on secondary Ficoll gradients; 7-25% Ficoll gradients were used to analyze the LE/Lys fractions, and 3-16% Ficoll gradients were used for analysis of plasma-membrane vesicles labeled by 125I-EGF.

To quantitate the degree of the density shift of each ligand, the amount of radioactivity remaining within the peak of ligand distribution as a consequence of DAB cytochemistry was divided by the radioactivity in the endosomal peak in the samples not incubated with DAB, expressed in percentages and subtracted from 100%.

HRP Assay-- HRP activity was assayed in triplicate as described previously (26).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

HRP-P-selectin Chimeras Are Sorted to the Late Endosomes and Lysosomes in H.Ep.2 Cells-- It has been demonstrated previously that the C1 domain of the cytoplasmic tail of P-selectin is involved in the targeting of this protein to lysosomes (21). To further characterize the signal(s) involved in lysosomal targeting, a series of chimeras between P-selectin and the enzymatic reporter HRP (Fig. 1) were constructed, and their lysosomal trafficking in H.Ep.2 cells following transient expression was analyzed. These chimeras contain the transmembrane and cytoplasmic domains of human P-selectin with the lumenal portion replaced by HRP and can be followed through the cell by their enzymatic activity.


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Fig. 1.   Schematic illustration of HRP-P-selectin chimeras. The top line shows the components used for construction: boxes represent the individual components; sequences outside boxes were added during construction. hGH, human growth hormone signal sequence; HRP, horseradish peroxidase; P-selectin, transmembrane (TM) and cytoplasmic domains of P-selectin. The 35 residues of the wild-type cytoplasmic domain have been assigned to the stop transfer (ST), C1 and C2 domains according to exon-intron boundaries. The bottom part shows the full amino acid sequences of the cytoplasmic domains of the chimeras starting with the wild-type P-selectin tail, ssHRPP-selectin. The carboxyl-terminal end of the TM domain is boxed. The name of each chimera is listed to the left of the diagram. The number at the end of the chimera name indicates which amino acid has been changed to a stop signal (numbering is taken from the human P-selectin sequence). ssHRPP-selectinDelta C1 is the construct in which the C1 exon has been deleted. The tetrapeptide sequence at the end of each chimera's name shows the amino acids which have been replaced by alanine. ssHRPP-selectinP767A is the chimera in which proline 767 was replaced by alanine.

We began by determining the efficiency of targeting of wild-type and mutant P-selectin chimeras to late endosomes (multivesicular bodies (MVB) in H.Ep.2 cells; see "Discussion") and lysosomes (LE/Lys) by subcellular fractionation. A mixed population of LE/Lys was deliberately analyzed since the ultimate destination of chimeras within the late endocytic system was unknown. To develop the fractionation protocol, two different markers were used: NAGA as a lysosomal marker enzyme, and 125I-EGF as a tracer, which is internalized from the plasma membrane through endosomal compartments en route to lysosomes. To establish conditions for the internalization of 125I-EGF so that it is within late endocytic compartments, the kinetics of degradation and the dynamics of compartmentalization of internalized ligand were followed by centrifugation of PNS on linear 1-16% Ficoll gradients. 125I-EGF was bound to the cells at 4 °C for 60 min and internalized at 37 °C for various times, and the cells were treated with acidic buffer to remove the plasma membrane-bound ligand. Kinetic experiments showed that after 30 min of internalization, 90% of the cell-associated radioactivity was acid-resistant, i.e. intracellular, but no significant degradation had occurred, as judged by a trichloroacetic acid precipitation of the chase medium (data not shown). When PNS obtained from these cells was centrifuged on a 1-16% Ficoll gradient, most of the NAGA and 125I-EGF sedimented in the same highest buoyant density fractions (fractions 16-20 and 21-23) (Fig. 2). In contrast, at earlier internalization times, 125I-EGF was found exclusively in the low density fractions (fractions 4-10), in which internalized 125I-Trn, a marker for early/recycling endosomes (40), was detected (Fig. 8, -DAB traces). Together, these observations strongly support the notion that the peaks of 125I-EGF in the heavy fractions of a 1-16% Ficoll gradient comprise LE/Lys.


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Fig. 2.   Distribution of internalized 125I-EGF and NAGA after centrifugation on 1-16% Ficoll gradient. H.Ep.2 cells expressing ssHRPP-selectin were incubated with 5 ng/ml 125I-EGF at 4 °C for 60 min and then for 30 min at 37 °C without ligand. Surface-bound 125I-EGF was removed by a mild acid wash. Cells were homogenized in HB, and the PNS was centrifuged on a preformed 1-16% Ficoll gradient. The radioactivity of fractions was counted and expressed as a percentage of the total radioactivity along the gradient (bullet ). The activity of the lysosomal marker enzyme NAGA was determined in each fraction as described under "Experimental Procedures" and shown as OD420 nm (open circle ).

To further purify late endocytic organelles, a secondary centrifugation of the fractions containing most of the NAGA and 125I-EGF was performed. As shown in Fig. 3A, 125I-EGF and NAGA co-sediment within the same single symmetrical peak with a low surrounding background after this treatment. An estimation of organelle yield within this peak was carried out using the endogenous lysosomal marker NAGA. Thus, the ratio of NAGA to total protein within the equilibrium peak divided by that for PNS demonstrated a 90-100-fold enrichment for the marker over starting material while still recovering 26% of the NAGA activity from total activity present in PNS. This two-gradient procedure therefore provides an appropriate preparation for quantitation of targeting of our chimeras to LE/Lys.


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Fig. 3.   Purification of LE/Lys-containing fractions by centrifugation on a secondary 7-25% Ficoll gradient. Cells expressing ssHRPP-selectin or ssHRPP-selectinDelta C1 were labeled with 125I-EGF and then subjected to centrifugation on initial 1-16% Ficoll gradients as described in the legend for Fig. 2. Fractions 17-23 were pooled and recentrifuged on secondary 7-25% Ficoll gradients. Fractionation was followed by gamma -counting and assaying for HRP and NAGA activities. Radioactivity of fractions is expressed in percentages (bullet ), calculated as for Fig. 2. NAGA activity is shown in the absolute values of OD420 nm (open circle ) and HRP activity (OD450 nm) for ssHRPP-selectin and ssHRPP-selectinDelta C1 (× and black-square, respectively) is expressed in arbitrary units calculated as a ratio of HRP activities present in each fraction to that of the total homogenate. A, distribution of 125I-EGF and NAGA; B, distribution of HRP activity.

To determine whether significant levels of ssHRPP-selectin are found within the LE/Lys equilibrium peak, cells were transfected either with ssHRPP-selectin or with ssHRPP-selectinDelta C1 and subjected to subcellular fractionation according to the two-gradient protocol. As shown in Fig. 3B, significant amounts of the ssHRPP-selectin chimera were recovered in the purified LE/Lys peak. Approximately 21% of cellular HRP activity was found within the LE/Lys fractions. Given the high levels of P-selectin biosynthesis 2 days after transfection on the one hand and the expected clearance of degraded HRP from lysosomal compartments on the other (Fig. 6), this percentage shows that targeting to the degradative compartments is a major pathway for HRP-P-selectin chimeras. Although detectable, much less targeting to LE/Lys of ssHRPP-selectinDelta C1 was seen, in line with expectations.

One advantage of using HRP chimeras is that the vulcanizing effect of the DAB reaction product within organelles containing HRP can be exploited to determine whether co-distribution on gradients is matched by co-occupation of the same membrane-bound organelle. If the chimeras are within the same membrane-bound organelle where the marker is present, then a shift in density of the marker following the DAB reaction will occur. In previous studies, this approach was successfully applied to establishing the sorting kinetics of two distinct endocytic tracers at various time points after internalization and, in particular, determining the duration of two tracers remaining within the same compartment (41, 42). Accordingly, cells were transfected to express ssHRPP-selectin or ssHRPP-selectinDelta C1, labeled with 125I-EGF (see above and "Experimental Procedures"), followed by centrifugation of PNS on 1-16% Ficoll gradients. Fractions 17-23 were pooled, and this was then split into two aliquots. One of them was incubated with DAB and H2O2 and the another one with HB alone. Samples were then layered on secondary 7-25% Ficoll gradients supplemented with imidazole, followed by equilibrium centrifugation as described under "Experimental Procedures." As shown in Fig. 4, both chimeras caused 125I-EGF to shift to denser fractions when LE/Lys-containing fractions were incubated with DAB and H2O2. However, the degree of DAB shift of 125I-EGF for ssHRPP-selectinDelta C1 chimera was much lower (Fig. 4B) than that for ssHRPP-selectin: 17% and 71% of original radioactivity in the endosomal peak before incubation with DAB, respectively. As one final control, the DAB reaction was carried out with plasma-membrane vesicles in which the 125I-EGF and the HRP catalytic domain of HRP-P-selectin chimeras are exposed outside the vesicular lumen. To isolate these organelles, 125I-EGF was bound to cells expressing ssHRPP-selectin at 4 °C for 60 min, followed by centrifugation of PNS on 1-16% Ficoll gradients. Only one peak of 125I-EGF (fractions 7-10) was found (data not shown), which was then pooled, incubated with or without DAB plus H2O2, and subjected to subcellular fractionation using 3-16% Ficoll gradients. As seen in Fig. 4C, no shift of 125I-EGF after the DAB reaction was detected. These experiments indicate that targeting of HRP-P-selectin chimeras to the LE/Lys compartment of H.Ep.2 cells can be quantified by subcellular fractionation.


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Fig. 4.   DAB-induced density shift of 125I-EGF-containing organelles by HRP-P-selectin chimeras. A and B, cells expressing ssHRPP-selectin and ssHRPP-selectinDelta C1 were homogenized and centrifuged on 1-16% Ficoll gradients. Fractions 17-23 were collected and split into two aliquots. One portion was subjected to the DAB reaction (open circle ), and the other was incubated with HB alone (bullet ), as described under "Experimental Procedures." Following the DAB reaction, samples were recentrifuged on secondary 7-25% Ficoll gradients and the radioactivity of fractions was counted and expressed in percentages of total radioactivity along the gradient. C, cells expressing ssHRPP-selectin were incubated with 5 ng/ml 125I-EGF at 4 °C for 60 min, homogenized in HB, and centrifuged on a 1-16% Ficoll gradient. The peak (fractions 7-10) was collected and split into two parts. One aliquot was incubated with DAB/H2O2 (open circle ), and the other with HB alone (bullet ). Recentrifugation was performed on secondary 3-16% Ficoll gradients, radioactivity of fractions was measured and expressed in percentages as described in Fig. 2.

Effect of Different Mutations within the C1 Domain of the Cytoplasmic Tail on Targeting to LE/Lys-- To quantitate the targeting to LE/Lys of wild-type and mutant ssHRPP-selectin chimeras, cDNAs encoding the various chimeras (Fig. 1) were transiently transfected into H.Ep.2 cells. To make sure that the data on targeting for different mutants were comparable, we determined the expression level for each chimera as a ratio of HRP activity to milligrams of total protein. In two independent experiments (Fig. 5A), the greatest difference between expression levels of any mutant and that of ssHRPP-selectin was 3.215 ± 0.025 and 2.17 ± 0.05 (± S.E.).


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Fig. 5.   Lysosomal targeting of HRP-P-selectin chimeras. H.Ep.2 cells were transiently transfected with a variety of cDNAs encoding HRP-P-selectin chimeras as shown in Fig. 1.  A, expression level of chimeras. Cells were scraped in HB, followed by measurement of protein concentration and HRP activity. Filled and hatched bars correspond to two independent experiments. For comparative purposes, the ratio of HRP activity to milligrams of protein for each chimera is expressed relative to that for ssHRPP-selectin, which was set at 1. B, lysosomal targeting indexes. Cells were treated and fractionated as described in the legend for Fig. 3. Targeting to LE/Lys fractions was quantitated by calculating LTIs allowing for HRP activity in the LE/Lys peak to be normalized by the chimera expression level, the number of cells, and the organelle recovery as independently judged by NAGA activity (filled bars) and 125I-EGF radioactivity (hatched bars). Each bar represents the mean ± S.E. of 3-10 independent determinations with each chimera. C, HRP proteolysis. Cells expressing different HRP-P-selectin chimeras were lysed with 1% Triton X-114 in PBS, followed by phase separation and assaying for HRP activity in each phase. The extent of proteolysis was expressed as a percentage of HRP activity in the soluble phase compared with the total activity in the lysate. Each bar represents the mean ± S.E. of at least four independent determinations.

Transfected cells were subjected to subcellular fractionation and targeting indexes to LE/Lys for each chimera were then calculated as described under "Experimental Procedures." Fig. 5B shows that the efficiency of accumulation of HRP activity within LE/Lys varied significantly between different chimeras. Two of the tetrapeptide alanine substitutions within the C1 domain (ssHRPP-selectinKDDG and ssHRPP-selectinNPHS) and the C2-truncated chimera (ssHRPP-selectin776) displayed lysosomal targeting indexes similar to that of ssHRPP-selectin. In contrast, the ability of ssHRPP-selectinDelta C1 as well as ssHRPP-selectinKCPL to reach lysosomes was about 7-fold less than that for wild type. The targeting of ssHRPP-selectinDelta C1 and ssHRPP-selectinKCPL (0.16 ± 0.07 and 0.13 ± 0.03 (± S.E.), respectively) was barely above the level of ssHRPP-selectin763. Since the internalization of P-selectin lacking either the C1 or the C2 domains is not abrogated (21, 24, 26), one could envisage two scenarios accounting for the failure of ssHRPP-selectinDelta C1 and ssHRPP-selectinKCPL to accumulate in LE/Lys. The first is that these chimeras are capable of trafficking to lysosomes but could not be detected there due to rapid degradation, causing a loss of HRP activity from this compartment. Alternatively, sorting of these mutants to lysosomes does not occur at all, which would presumably result in rerouting to the recycling pathway coupled to a transient appearance of protein at the plasma membrane (see below). A similar situation has been described previously for mutants of the EGF-receptor (27, 29), influenza virus hemagglutinin (43), and beta -chain of the interleukin-2 receptor (9), which have had their degradation signals disrupted.

To test the efficiency of proteolysis of the chimeras, Triton X-114 partitioning (44) was exploited for separation of membrane-bound and soluble HRP activity. The extent of HRP proteolysis of ssHRPP-selectinDelta C1 and ssHRPP-selectinKCPL (2.97 ± 0.9% and 0.5 ± 0.2%, correspondingly) was significantly lower than that for ssHRPP-selectin, ssHRPP-selectinKDDG, ssHRPP-selectinNPHS, and ssHRPP-selectin776 (on average, 12-17%), although ssHRPP-selectinDelta C1 displayed a slightly higher percent than ssHRPP-selectinKCPL (Fig. 5C). The latter observation presumably reflects the effect(s) of the larger mutation.

Absence of HRP activity of ssHRPP-selectinKCPL within the LE/Lys fractions or in the soluble phase in the proteolysis assay might be explained by either a failure of targeting to LE/Lys or increased proteolytic degradation within those fractions followed by rapid loss of the enzyme activity. We therefore quantified the lysosomal targeting indexes in cells pretreated with a mixture of 0.1 mM pepstatin A and leupeptin for 24 h before fractionation. These inhibitors, which block the enzymatic activity of major lysosomal hydrolases, have been shown previously to prevent the degradation of proteins delivered to lysosomes (45). Importantly, the selective action of these inhibitors does not interfere with membrane traffic, as is the case for other agents such as weak bases and proton ionophores. Thus, following pretreatment with a mixture of pepstatin A and leupeptin, the extent of HRP proteolysis in cells expressing ssHRPP-selectin was reduced more than 4-fold (data not shown), strongly suggesting that degradation was indeed lysosomal. We therefore employed the same treatment to examine the lysosomal fate of chimeras through subcellular fractionation. The amount of HRP activity recovered in the LE/Lys fraction of cells expressing ssHRPP-selectin rose by up to 2.2 times compared with control untreated cells, as would be expected when degradation is blocked (Fig. 6). The other mutants (ssHRPP-selectinKDDG and ssHRPP-selectinNPHS), which behaved similarly to ssHRPP-selectin as judged by lysosomal targeting indexes and proteolysis data in nontreated cells, also exhibited an elevated level of lysosomal HRP activity after treatment. Thus, ssHRPP-selectinKDDG and ssHRPP-selectinNPHS are delivered to LE/Lys and degraded as efficiently as ssHRPP-selectin. In contrast, we could not detect any increase in the level of HRP activity for ssHRPP-selectinKCPL: 0.2 ± 0.08 for treated cells and 0.13 ± 0.03 for nontreated cells, indicating that ssHRPP-selectinKCPL is neither routed to LE/Lys nor degraded. Altogether, these results support the contention that KCPL is a novel LTS within the C1 domain that is required for efficient targeting of P-selectin to the LE/Lys fractions of H.Ep.2 cells.


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Fig. 6.   The effect of protease inhibitors on LTIs of HRP-P-selectin chimeras. Cells were transiently transfected with cDNAs encoding the ssHRPP-selectin, ssHRPP-selectin763, ssHRPP-selectinKCPL, ssHRPP-selectinNPHS, and ssHRPP-selectinKDDG and grown for 24 h without inhibitors and then for 24 h in growth medium supplemented with 100 µM each of pepstatin A and leupeptin. H.Ep.2 cells expressing ssHRPP-selectin and ssHRPP-selectin763, analyzed in parallel as controls, were not pretreated with inhibitors. Cells were labeled with 125I-EGF, homogenized in HB, and subjected to the two-step centrifugation on Ficoll gradients followed by calculation of LTIs as described under "Experimental Procedures." Each bar represents the mean ± S.E. of three independent determinations. Filled bars, data are normalized by NAGA recovery; hatched bars, data are normalized by 125I-EGF recovery. - or + above bars indicates control or treated cells, respectively.

Contribution of Proline 767 to Lysosomal Targeting-- The sequence KCPL is different from motifs known to be involved in sorting or thought to be critical for internalization. However, it contains a proline (number 767 within the human sequence; Ref. 15) followed by leucine. This Pro-Leu pairing is present within tyrosine-based sorting signals mediating the transport of CD3gamma to lysosomes (46) and of HLA-DMb to antigen-processing, lysosome-like compartments (47). In addition, proline has been identified as a key amino acid within NPXY-based motifs required for internalization (5). We have therefore engineered a chimera in which proline 767 is replaced by alanine; ssHRPP-selectinP767A. Subcellular fractionation demonstrated that the efficiency of lysosomal targeting of this mutant was about 35% of the level of ssHRPP-selectin and was only 2-fold higher then that for ssHRPP-selectinKCPL (Fig. 5B), suggesting that proline does indeed make a major contribution to lysosomal targeting. Surprisingly, this mutation did not result in a dramatic effect on HRP proteolysis since the clipping of ssHRPP-selectinP767A was only half that of ssHRPP-selectin (Fig. 5C). These data suggest the possibility of differential targeting and degradation within the late endocytic system.

Cell Surface Expression of HRP-P-selectin Chimeras-- Since we had established that ssHRPP-selectinDelta C1 and ssHRPP-selectinKCPL are not sorted to LE/Lys, we next asked where in the cell these chimeras reside. One possibility was that mutants which failed to be targeted to the LE/Lys would enter a recycling pathway between the plasma membrane and an early endocytic compartment. This would be in agreement with previous experiments suggesting that ssHRPPselectinDelta C1 co-localizes with the classic marker for a recycling itinerary, the transferrin receptor, when expressed in PC12 cells (26). One prediction for such behavior was that a significant proportion of the mutant chimeras should appear on the plasma membrane during recycling. The level on the surface should be intermediate between a protein that is efficiently targeted to lysosomes, e.g. ssHRPP-selectin and a protein that fails to be internalized, e.g. ssHRPP-selectin763. To determine whether this is the case, we employed an 125I-Fab anti-HRP binding assay to detect the chimeras on the cell surface. As seen in Fig. 7, ssHRPP-selectin763 showed the highest level at the plasma membrane, twice that of ssHRPP-selectin. Analysis of the other chimeras revealed that ssHRPP-selectinDelta C1, ssHRPP-selectinKCPL, and ssHRPP-selectinP767A displayed an intermediate level between ssHRPP-selectin and ssHRPP-selectin763, whereas ssHRPP-selectinNPHS and ssHRPP-selectinKDDG were present at the same level as ssHRPP-selectin. Since substitution of KCPL, deletion of the C1 domain, or substitution for proline 767 did not affect or had only a little effect on internalization rates (24),2 the appearance of these chimeras at the plasma membrane strongly suggests an increased pool of recycling protein.


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Fig. 7.   Cell surface expression of HRP-P-selectin chimeras. H.Ep.2 cells expressing ssHRPP-selectin, ssHRPP-selectin763, ssHRPP-selectinKCPL, ssHRPP-selectinP767A, ssHRPP-selectinDelta C1, ssHRPP-selectinKDDG or ssHRPP-selectinNPHS were incubated with 2 µg/ml 125I-Fab fragments at 4 °C for 60 min, then lysed in HRP assay buffer, and radioactivity in each fraction was counted. Cell surface expression is expressed as a ratio of 125I-Fab radioactivity to the total level of HRP activity for each chimera and then expressed in arbitrary units relative to the level of ssHRPP-selectin set at 1.

ssHRPP-selectinDelta C1 and ssHRPP-selectinKCPL Are Localized in Trn-positive Endosomes-- The recycling pathway of H.Ep.2 cells has previously been characterized in detail, and Trn has been found to be the best marker of this itinerary (31, 32, 41). To determine whether ssHRPP-selectinDelta C1, ssHRPP-selectinKCPL, and Trn are present within the same endosomes, cells expressing ssHRPP-selectin, ssHRPP-selectinDelta C1, or ssHRPP-selectinKCPL were loaded with 125I-Trn, treated with MES buffer to remove the plasma membrane-bound ligand, and then the co-localization of HRP activity and iodinated ligand was examined by exploiting the DAB-induced density shift procedure. PNS obtained from these cells was split in two equal parts. One of them was incubated with DAB and H2O2, and the another one with HB alone. Samples were layered on 1-16% Ficoll gradients and centrifuged to equilibrium as described under "Experimental Procedures." In control cells expressing ssHRPP-selectin, the DAB reaction shifted 24% of the total 125I-Trn radioactivity, whereas in cells expressing ssHRPP-selectinDelta C1 or ssHRPP-selectinKCPL, significantly larger amounts of ligand were shifted: 54% and 52%, respectively. Based on these data, we assume that ssHRPP-selectin moves transiently through the Trn-positive endosomes en route to the lysosome. Importantly, both ssHRPP-selectinDelta C1 and ssHRPP-selectinKCPL reveal significant co-localization with Trn, strongly suggesting a recycling itinerary for these chimeras.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present work, we have identified a novel LTS in the cytoplasmic tail of P-selectin. Green et al. (21) had found previously that a putative LTS of P-selectin is located within the membrane-proximal C1 domain of the cytoplasmic portion of this protein, the amino acid sequence of which is shown in Fig. 1. Using site-directed mutagenesis, we have engineered a series of tetrapeptide substitutions within HRP-P-selectin chimeras to further characterize lysosomal targeting information within the C1 domain. The assays introduced in the present study have allowed us to quantify even minor differences in endosomal/lysosomal sorting. Summarizing the results of both lysosomal targeting and HRP proteolysis experiments, we have uncovered a novel determinant within the C1 domain of P-selectin: KCPL. This LTS appears to be the sole element of the cytoplasmic tail necessary for lysosomal targeting since neither the disruption of the two other tetrapeptides within the C1 domain, KDDG and NPHS, nor the removal of the C2 domain affected lysosomal targeting. Whether this sequence is also sufficient for lysosomal targeting has not yet been directly established. Carrying out such experiments may not provide simple answers, since by placing it into another context, KCPL may well operate differently, as has been observed elsewhere. For example, the transfer of the Leu-Ile signal of LIMP2 to CD36 and CD8 plasma membrane proteins did not induce diversion of these proteins to lysosomes (8). In addition, the spacing of an LTS relative to the transmembrane domain in proteins possessing a short cytoplasmic tail has been found to be critical for proper functioning of the signal (45).

The HRP proteolysis data are more complex than those obtained by subcellular fractionation, since we observed some difference between ssHRPP-selectin and mutants in which KCPL has not been inactivated. We suspect that this may reflect some differential targeting between subcompartments of the late endocytic pathway. It is striking, however, that mutation of KCPL to tetra-alanine resulted in levels of HRP proteolysis similar to those seen for tailless ssHRPP-selectin763, strongly suggesting that KCPL contains all the information needed to accomplish the lysosomal delivery of P-selectin.

The effect of tetra-alanine substitution of KCPL was very marked: lysosomal targeting and HRP clipping were reduced by 7- and 15-fold, respectively, of the levels found for ssHRPP-selectin (Fig. 3). By contrast, inactivation of the LTSs of other transmembrane proteins and receptors caused no more than a 3-fold reduction of targeting compared with that of the intact protein, suggesting that additional sequences may be involved in those cases (9, 27, 29, 43). The existence of multiple lysosomal sorting signals has indeed been demonstrated for gamma  and delta  chains of CD3 (48), for the invariant chain (Ii) of class II major histocompatibility complex (49) and EGFR (27-29). In principal, by acting in concert, multiple LTSs could provide for a targeting activity equivalent to that of KCPL within P-selectin.

Many LTSs also operate to mediate internalization at the plasma membrane, as is the case for the interleukin-6 signal transducer gp130 (50), LAMP1 (45, 51, 52), Ii (49), lysosomal acid phosphatase (53, 54), LIMP2 (8), and CD 3 gamma  and delta  chains (48). These data indicate that in most cases the LTS and internalization signal are co-linear (see Table I). The relationship between internalization and lysosomal targeting is generally acknowledged, although its functional significance remains to be investigated. One of the sources of confusion is that there is no simple correlation between internalization and degradation rates for proteins targeting to lysosomes via the plasma membrane (9, 27-29, 43, 45). Thus, whether the multiple signals function to increase the overall efficiency of targeting by acting together at one rate-limiting step or operate at different stages is not clear.

                              
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Table I
An alignment of the LTS in P-selectin with other lysosomal targeting determinants
The targeting sequences within cytoplasmic tails are oriented amino- to carboxyl-terminal and represent the minimal consensus determined to be necessary to function. Bold residues indicate the critical residues for targeting activity. 1022-1123 in the row for EGFR corresponds to the sequence locolizing between indicated amino acid residues of cytoplasmic tail. The references for the work on each signal are listed in the final column. Abbreviations used are as follows: WT-P-selectin, P-selectin of human origin; CD3gamma and CD3delta , two subunits of the T cell receptor complex; LAP, lysosomal acid phosphatase; LAMP1, -2, -3, three families of lysosomal associated membrane proteins; LIMP2, a member of the family of lysosomal integral membrane proteins; HA+8, a mutant influenza hemagglutinin with an 8-amino acid extension; EGFR, epidermal growth factor receptor; TK, tyrosine kinase domain of EGFR; HLA-DMbeta , beta -chain of heterodimeric molecule associated with class II molecules of the major histocompatibility complex; IL-2beta , beta -chain of interleukin 2 receptor; IL-6 gp130, signal transducing component gp130 of interleukin-6 receptor complex; Ii, invariant chain which associates noncovalently with alpha  and beta  chains of major histocompatibility complex II.

Does KCPL operate at more than one step in directing the protein to late endocytic compartments? A detailed search for internalization signals in the cytoplasmic tail of P-selectin did not succeed in identifying any potential motifs despite the presence of an obvious candidate sequence within the C2 domain (YGVFTNAAF) that provides a good match with known internalization signals. Instead, mutation of most amino acids throughout the cytoplasmic tail affects the efficiency of internalization (24). This finding, coupled to internalization experiments using HRP-P-selectin chimeras2 firmly supports the view that KCPL is a signal that mediates endosome-to-lysosome trafficking without affecting internalization.

As illustrated in Table I, the LTS of P-selectin, KCPL, has no obvious similarity to a large number of lysosomal targeting determinants, most of which also act as internalization signals in other proteins. Our data also indicate that the proline 767 provides a major contribution to the LTS. Interestingly, proline located within sorting signals that center on the NPXY motif was shown to be a key amino acid, thought to be involved in stabilizing the predicted "tight turn" (1, 5), whereas in other signals it does not seem to be important.

P-selectin has been shown previously to be efficiently endocytosed in many cell lines (20, 24, 25). If inactivation of KCPL has no effect on the internalization ability of this chimera but prevents degradation, then where in the cell is HRPP-selectinKCPL likely to accumulate?

In H.Ep.2 cells, recycling and lysosomally directed tracers have been found to be internalized into the same compartment (31, 33, 34) where their sorting occurs. These endosomes were morphologically identified as MVBs (30, 32-34). The MVBs undergo a gradual maturation process involving removal of recycling receptors such as Trn receptor (TrnR), and retention of lysosomally directed proteins such as EGFR. By contrast, subsequent transfer of endocytosed proteins from late or mature endosomes to lysosomes is believed to be a discontinuous process requiring heterotypic fusion between fully matured but LAMP1-negative MVB and pre-existing LAMP1-positive, acid hydrolase active lysosomes (32, 55).

Our findings suggest that KCPL is most likely to operate where the segregation of recycling and degradative pathways occurs. Electron microscopy revealed that, during the maturation of MVBs, EGFR are recruited from the outer membrane of MVB onto internal vesicles while TrnR remains on the perimeter membrane (34, 56). This recruitment onto inner vesicles is critical for the lysosomal degradation of the EGFR. Felder et al. (27) have documented that the tyrosine kinase of the EGFR controls sorting of internalized receptor through spatial segregation within the MVB. Thus, wild-type EGFR was shown to be recruited to internal vesicles of MVBs and subsequently degraded, whereas kinase-negative receptor underwent increased recycling and was localized to the outer membrane of the MVBs followed by removal to small tubulovesicles (27).

We speculate therefore that KCPL promotes the retention of P-selectin within the maturing MVBs, possibly by promoting inclusion in inwardly budding vesicles along with the EGFR. When KCPL-dependent retention within MVBs is abolished, then P-selectin normally destined for degradation is likely to be diverted to the recycling pathway which may occur by default (4, 57). This scheme is supported by experiments (Fig. 8) showing that the level of ssHRPP-selectinKCPL on the plasma membrane is greater than that of ssHRPP-selectin, as would be expected if the mutant protein efficiently recycles. Moreover, DAB cytochemistry experiments in the present work demonstrate that, although the wild-type HRP-P-selectin chimera is found within 125I-EGF-containing endosomes and lysosomes, the distribution of ssHRPP-selectinKCPL and ssHRPP-selectinDelta C1 overlapped with 125I-TrnR-containing compartments. Other studies have also revealed increased levels of recycling for an EGFR lacking a degradation signal (29), as well as for a LAMP1 mutant in which the LTS was displaced within the cytoplasmic tail (45).


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Fig. 8.   DAB-induced density shift of internalized 125I-Trn in H.Ep.2 cells transfected with ssHRPP-selectin, ssHRPP-selectinDelta C1, or ssHRPP-selectinKCPL. Cells expressing ssHRPP-selectin, ssHRPP-selectinDelta C1, or ssHRPP-selectinKCPL were incubated with 100 ng/ml 125I-Trn at 37 °C for 60 min. Surface-bound ligand was removed by MES buffer wash. Cells were then homogenized in HB and a PNS split in two aliquots. One part was incubated with DAB/H2O2 (open circle ), and the other with HB alone (bullet ). The samples were centrifuged on 1-16% Ficoll gradients following the fractionation. Radioactivity of fractions is shown in counts/min. Percentage of DAB shift was determined as described under "Experimental Procedures." A, ssHRPP-selectin; B, ssHRPP-selectinDelta C1; C, ssHRPP-selectinKCPL.

The data obtained in the present study coupled with published observations from other laboratories demonstrate an abundance of different sorting signals involved in lysosomal targeting. What are the reasons for such a heterogeneity? One possibility is the need to provide for the different physiological and signaling functions of proteins routed to lysosomes. For example, some receptors of hormones, growth factors, and cytokines utilize the degradative pathway to desensitize the cell during down-regulation. In these cases, rapid clearance of these proteins may require efficient internalization and degradation. P-selectin transiently appearing on the plasma membrane of activated endothelial cells during inflammatory response was also found to be quickly cleared (58). A prolonged appearance on the plasma membrane might lead to over-recruitment of leukocytes with a subsequent uncontrolled inflammation. A similar rapid trafficking has been observed in nonendothelial cells in which P-selectin is rapidly transported to the plasma membrane and then efficiently delivered to lysosomes for degradation (21-23). On the other hand, resident lysosomal membrane proteins, traveling from the plasma membrane to their final destination, may not have the same need for rapid clearance or degradation. Such different trafficking requirements would explain the variety of signals, but, clearly, further studies will be needed to investigate the basis for LTS diversity.

    ACKNOWLEDGEMENTS

We thank Prof. C. R. Hopkins, Dr. C. E. Futter, Dr. M. Marsh, Dr. S. Moss, Dr. K. Römisch, Dr. E. Hewitt, and Dr. M. Arribas for discussion and critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a Wellcome Trust fellowship (to A. B.).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.

Dagger To whom correspondence should be addressed. Tel.: 44-171-380-7808; Fax: 44-171-380-7805; E-mail: d.cutler{at}ucl.ac.uk.

1 The abbreviations used are: LTS, lysosomal targeting signal; EGF, epidermal growth factor; EGFR, EGF receptor; HRP, horseradish peroxidase; Trn, transferrin; TrnR, Trn receptor; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; PNS, postnuclear supernatant; Lys, lysosome; NAGA, N-acetyl-beta -D-glucosaminidase; LTI, lysosomal targeting index; LE, late endosome; DAB, 3,3'-diaminoBenzidine; MVB, multivesicular bodies.

2 A. D. Blagoveshchenskaya and D. F. Cutler, unpublished observations.

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Abstract
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Results
Discussion
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