©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of a Novel Protein (p137) Which Transcytoses Bidirectionally in Caco-2 Cells (*)

(Received for publication, February 8, 1995; and in revised form, April 27, 1995)

Juliet A. Ellis J. Paul Luzio (§)

From the Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Antisera raised against detergent-extracted membrane fractions from the human intestinal epithelial cell line Caco-2 were used to screen a human colon cDNA library in a bacteriophage expression vector. This led to the identification, molecular cloning, and sequencing of a novel plasma membrane protein (p137) which was present in approximately equal amounts on the basolateral and apical surfaces of the cell. The pattern of extraction of p137 from membranes by Triton X-114 and its release from membranes after incubation with phosphatidylinositol-specific phospholipase C were consistent with it being a glycosylphosphatidylinositol-anchored membrane protein. Using antibodies raised against bacterial fusion proteins, it was shown that p137 was present on the cell surface as a reducible homodimer of 137 kDa subunits. There was constitutive release of p137 into the culture medium as a non-reducible 280-kDa entity. Pulse-chase experiments showed that newly synthesized p137 appeared at the basolateral side of a Caco-2 cell layer before appearing at the apical domain. Domain-specific surface biotinylation of Caco-2 cells at 4 °C, followed by chasing at 37 °C, demonstrated that p137 is capable of transcytosing in both directions across Caco-2 cells. The unusual plasma membrane domain distribution of this glycosylphosphatidylinositol-linked protein and its transcytosis characteristics demonstrate the existence of a previously uncharacterized apical to basolateral transcytotic pathway in Caco-2 cells.


INTRODUCTION

The plasma membrane of polarized epithelial cells consists of apical and basolateral domains of unique protein and lipid composition (reviewed in (1) ). Plasma membrane polarity is achieved by sorting newly synthesised membrane constituents, followed by targeting to the correct membrane domain. Sorting can occur either in the trans-Golgi network (TGN) (^1)prior to separate vesicular transport to the apical and basolateral domains or, after initial delivery to the basolateral surface, by transcytosis of selected proteins to the apical domain(2) . The extent to which one or other sorting site is used is dependent on cell type and the individual protein(3) . Maintenance of membrane polarity is further aided by recycling most endocytosed membrane proteins to the domain from which they are internalized, despite the existence of common endosomal compartments which can be accessed from both sides of the cell(4) .

Transcytosis not only contributes to the development and maintenance of membrane polarity, but also provides an endocytic route whereby specific membrane proteins and bound ligands internalized at one side of a polarized cell may be transported and delivered to the other. The best characterized transcytotic route is that taken by the polymeric immunoglobulin receptor (pIgR) from the basolateral to the apical domain of a variety of polarized epithelial cells(5, 6) . In liver, salivary gland and mammary tissue transcytosis of the pIgR results in release of the ectodomain of the receptor together with pIgA into bile, saliva, and colostrum, respectively, and makes a major contribution to the immune system in these secretions (reviewed in (7) ). Sequence motifs in the cytoplasmic tail of the pIgR required for transcytosis have been defined (5, 9, 10) as well as the endocytic compartments through which it travels (8) and the proteolytic events occurring when it arrives at the apical cell surface and is released(11) . In contrast, much less is known about the apical to basolateral transcytosis of any single membrane protein. Among the best described apical to basolateral transcytotic routes are those of the neonatal rat gut immunoglobulin receptor, FcRn (12) and of FcRII-B2 transfected into MDCK cells(13) . Despite the availability of much sequence and structural information, in neither case have the targeting motifs in the cytoplasmic tail of the receptor been well defined. Transcytosis of cobalamin has also been investigated in adult gut and the human colon adenocarcinoma-derived cell line Caco-2,(14) , but this involves an indirect route whereby cobalamin internalized from the apical surface complexed with intrinsic factor is transferred to endogenously synthesized transcobalamin II prior to secretion from the basolateral side. Such apical to basolateral transcytic routes in enterocytes lining the gut are of particular interest since, if better defined, they could be exploited for efficient drug delivery(15, 16) .

In the present study we attempted to identify and clone endogenous membrane proteins which would be candidates for apical to basolateral transcytosis in Caco-2 cells. We have previously shown that some antisera raised against plasma membrane fractions from Caco-2 cells react with subsets of proteins common to the apical and basolateral domains(17) . Such proteins are candidates for apical to basolateral transcytosis, since if internalized from the apical surface they would not necessarily carry signals allowing sorting back to the apical domain. To clone these proteins we screened an appropriate cDNA library in a bacterial expression vector and sorted the clones using antibodies affinity purified on individual expressed fusion proteins. This approach has been successfully used previously to identify Golgi membrane proteins when starting with an antiserum raised against Golgi membranes(18) . In the present study we have identified and cloned a novel membrane protein present in both the apical and basolateral plasma membrane domains of Caco-2 cells, and show that it is transcytosed in both directions.


EXPERIMENTAL PROCEDURES

Cell Culture, Radiolabeling, and Isolation of Membrane Fractions

Caco-2 cells were grown as described previously (17) and were used between passage number 90 and 110, either 7 days (filters) or 3 days (flasks) after subculture. For steady-state radiolabeling of proteins, cells in 75-cm^2 flasks were rinsed in PBS (145 mM NaCl, 7.5 mM Na(2)HPO(4), 2.5 mM NaH(2)PO(4), pH 7.4) and then incubated overnight at 37 °C with 1 mCi of [S]methionine (1205 Ci/mmol; DuPont NEN) in 5 ml of methionine-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% dialyzed fetal calf serum. Pulse-chase radiolabeling of proteins and the preparation of apical and basolateral membrane fractions from filter-grown cells were as described previously(17) . For steady-state radiolabeling of GPI anchors, cells in 75-cm^2 flasks were rinsed in PBS and then incubated overnight at 37 °C with 0.125 mCi of myo-[2-^3H]inositol (16.5Ci/mmol; Amersham International, Amersham, Bucks, United Kingdom) in 5 ml of inositol-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum.

Immunoprecipitation, SDS-PAGE, and Immunoblotting

Rabbit polyclonal antisera to Caco-2 membrane fractions (anti-apical antibody 630; anti-basolateral antibody 620)(17) , and antibodies against bacterial fusion proteins (18, 19, 20, 21) were raised and affinity purified as described previously. A mouse monoclonal antibody to caveolin (22-kDa substrate; clone 2283) (22) was from Affiniti Research Products Ltd., Nottingham, U.K. and was used at 1:1000 dilution in immunoblots. Immunoprecipitation of whole cells, membrane fractions, and tissue culture media after [S]methionine or [^3H]inositol labeling were as described(17) , except that affinity purified antibodies were used. When necessary, samples were subjected to 7 or 10% SDS-PAGE(23) . To identify [S]methionine-labeled proteins gels were exposed for 1 week at -70 °C against Kodak X-Omat film (Eastman Kodak Co.) and to identify [^3H]inositol-labeled proteins gels were exposed for 3 days against a tritium detecting image plate and analyzed on a Fujix BAS 2000 bio-imaging analyzer (Fuji Photo Film Co., Japan). Immunoblotting of non-radioactive proteins was carried out after SDS-PAGE essentially according to Burnette(24) .

Cell Surface Biotinylation

Domain-specific biotinylation followed by immunoprecipitation, SDS-PAGE, and visualization by horseradish peroxidase-streptavidin using the Amersham ECL blotting system were carried out according to Neame and Isacke(25) . To study the transcytosis of p137, domain-specific biotinylation of Caco-2 monolayers was carried out at 4 °C, and then the cells were incubated at 37 °C for different periods of time. In control experiments, the cells were kept at 4 °C. The basolateral and apical media were collected, immunoprecipitated and analyzed by SDS-PAGE followed by visualization with horseradish peroxidase-streptavidin.

Phase Partioning in Triton X-114, Sodium Carbonate Extraction, and Treatment with PI-PLC, Pronase, and N-Glycanase

Cells labeled overnight with [S]methionine were cooled on ice prior to harvesting in PBS and then solubilized in preclouded 1% Triton X-114, and the soluble supernatant was subjected to phase partitioning according to Bordier(26) . The insoluble pellet, detergent, and aqueous phases were immunoprecipitated as described above. The nature of membrane association was also assessed by the addition of sodium carbonate (0.1 M at pH 11.5), which strips peripherally bound proteins from membranes. Purified PI-PLC was kindly provided by Dr. M. Low (Columbia University, New York) and used as described previously(17) . The presence of the CRD, an epitope appearing on some GPI-linked proteins after PI-PLC treatment, was examined by immunoblotting with a rabbit polyclonal anti-CRD antibody (RP189 raised against PI-PLC solubilized pig membrane dipeptidase), kindly provided by Dr. N. Hooper (Department of Biochemistry, University of Leeds, Leeds, U.K.) and used according to Zamze et al.(27) .

cDNA Library Screening and DNA Sequencing

All DNA manipulations were carried out according to Sambrook et al.(28) . A human colon cDNA library constructed in gt11 was purchased from Clontech (Palo Alto, CA) and used for immunological screening (21) with antisera 620 and 630 mixed equally, at a dilution of 1:100. Following affinity purification of the mixed antisera on individual fusion proteins (18, 20, 21) and subsequent immunoblotting of apical and basolateral membrane fractions, one clone (clone C), containing a cDNA insert (insert C) encoding a protein (p137) found in both membrane fractions, was selected for further analysis. Insert C (1076 bp) was P-labeled by random priming and used to screen a human colon 5'STRETCH gt11 library (Clontech) in order to obtain additional cDNA sequence. Positive cDNA clones from these screens were assessed for the presence of additional sequence by polymerase chain reaction using appropriately designed primers within the insert sequence and the vector(21) . Using these procedures a second clone was isolated containing a cDNA insert (insert 15; 1885 bp) which overlapped insert C with an extra 596 bp at the 5` end and 214 bp at the 3` end, and a third clone containing a cDNA insert (insert 40; 2200 bp) which overlapped with the 3` end of insert 15 by 1029bp, and confirmed the identity of the stop codon. To find the 5` end of the predicted ORF, advantage was taken of the internal EcoRI site 239 bp downstream of the 5` end of insert 15. The 239-bp EcoRI fragment was isolated, radiolabeled, and used to rescreen the human colon 5` STRETCH cDNA gt11 library, identifying a clone containing an insert (insert 9; 1615 bp) with a predicted start codon. Cloning of a partial EcoRI digest of clone 9 allowed sequencing across the internal EcoRI site in the cDNA insert. The final DNA sequence was determined throughout from both strands after subcloning the inserts into pBluescript KS± (Stratagene Cloning Systems, San Diego, CA), constructing appropriate nested deletions, and sequencing by the dideoxy chain termination method (29) using Sequenase version 2.0 with Universal and Reverse primers (United States Biochemical Corp.).

In Vitro Transcription and Translation

pBluescript clones containing bases 1-2252 and 129-2252 of the full-length contiguous cDNA encoding p137 were linearized with BamHI and mRNA transcribed from the T7 promoter and translated using an mCAP RNA capping kit (Stratagene) and nuclease-treated rabbit reticulocyte lysate translation kit (Promega) according to the manufacturer's instructions. Incorporation into microsomes was assessed by inclusion of canine pancreatic microsomal membranes (Promega) in the translation mix as described by the manufacturer. Proteins were judged to be translocated into microsomes if protected from incubation with proteinase K, 0.1 mg/ml, 1 h, 4 °C. Addition of 1% Triton X-100 resulted in complete proteolysis. Proteinase K was inactivated by addition of 1 mM phenylmethylsulfonyl fluoride prior to analysis by SDS-PAGE.

Northern Blotting

A human Northern blot II (Clontech) with tracks containing 2 µg of poly(A) RNA from each of colon, small intestine, testis, ovary, prostate, pancreas, spleen, and peripheral blood leukocytes was probed according to the manufacturer's instructions, using a P-random primed, labeled 1647 bp EcoRI fragment of insert 15 and washing at increasingly high stringency.

Computer Analysis of DNA Sequence

The cDNA sequence was analyzed using the computer programmes of Staden(30) . Hydropathy analysis was performed in accordance with Kyte and Doolittle(31) , with a window size of 11 amino acids and a plot interval of 3 amino acids. To identify an ORF within the DNA sequence, use was made of nucleotide interpretation program for the detection of uneven base frequencies, with a window size of 11(30) . Secondary structure predictions were made using the Robson prediction program in nucleotide interpretation program (32) and similarity investigation program(33) .


RESULTS

A human colon carcinoma gt11 cDNA library (1.5 10^6 independent recombinants) was screened with a mixture of two antisera (coded 620 and 630), raised against isolated apical and basolateral plasma membrane fractions prepared from Caco-2 cells. Seventeen positive plaques were picked and plaque-purified. Antibodies were affinity purified from the original antisera on individual gt11 fusion proteins and used to immunoblot Caco-2 plasma membrane fractions and to sort clones into epitope groups. In this way, clones encoding one apical plasma membrane protein (80 kDa; identified by three affinity purified antibodies), one basolateral membrane protein (64 kDa; identified by two affinity purified antibodies), and two common proteins (105 and 137 kDa; each recognized by one affinity purified antibody) were identified (data not shown). Antibodies affinity purified on 10 of the original 17 positive clones did not give clear, unique immunoblotting signals with membrane fractions.

Identification and Properties of p137

The cDNA inserts (insert C) encoding part of the 137-kDa membrane protein (p137) was subcloned into pGEX-1N(19) , and an antiserum (coded 803) prepared against, and immunoaffinity purified on, the resulting gluthathione S-transferase fusion protein. Affinity purifed antiserum 803 reacted with p137 on immunoblots of Caco-2 cell plasma membrane fractions in agreement with the data from blots with antibodies from antisera 620/630 affinity purified on the clone C fusion protein (data not shown). A subsequently prepared, affinity purified antiserum (coded 660), raised against a pGEX-1N fusion protein encoded by part of an overlapping clone (the large EcoRI fragment of insert 15), also reacted with p137 in membrane fractions, but was of higher avidity than antiserum 803 and therefore was used in subsequent experiments. The reaction of affinity purified antiserum 660 with p137 on immunoblots of reduced samples of both apical and basolateral membrane fractions from Caco-2 cells is shown in Fig. 1. The immunoblots suggested that the concentration of p137 in each membrane fraction was approximately equal. This distribution of p137 could not be accounted for by cross-contamination of the two membrane fractions, since this had previously been estimated as <10% by analysis of marker enzymes(17) . When non-reduced samples of membrane fractions were immunoblotted with antiserum 660, a major band of 280 kDa was observed in each membrane fraction suggesting that p137 normally exists in the plasma membrane as a disulfide bonded dimer (Fig. 1). p137 was found to be in the pellet following Na(2)CO(3) treatment and centrifugation, showing that it was neither a soluble nor peripheral membrane protein (Fig. 2a). Extraction of [S]methionine-labeled Caco-2 cell membranes with Triton X-114, followed by temperature-induced phase separation (26) and immunoprecipitation with antibody 660, gave anomolous results. p137 was found to partition mostly into the insoluble pellet formed at 0 °C and the aqueous phase formed at 30 °C with negligible amounts in the detergent phase (Fig. 2b). Such anomalous distribution, upon Triton X-114 phase separation, is characteristic of some GPI-anchored proteins(34, 35) . Further evidence for the presence of a GPI anchor on p137 was obtained by incubating [S]methionine-labeled Caco-2 cell membranes with PI-PLC for 1 h at 37 °C, which resulted in the release of most of the protein into a soluble fraction (Fig. 2c). No change in molecular weight on SDS-PAGE was seen after removal of the acylglycerol moeity, but this is a frequently observed phenomenon(36) . The small amount of p137 seen in the soluble phase after control incubation in the absence of exogenous PI-PLC (Fig. 2c) may be explained by the presence of endogenous PI-PLC or other enzymes cleaving p137 from the membrane (see also below). This may also explain the observation of some p137 in the aqueous phase after Triton X-114 extraction and phase separation. No reaction with the anti-CRD antibody (27) was observed after PI-PLC treatment in the present experiments. However, the antiserum used does not react with all PI-PLC-cleaved membrane proteins(37) . Confirmation of the presence of a GPI anchor was obtained by [^3H]inositol labeling followed by immunoprecipitation (Fig. 2d). Neither incubation of Caco-2 cells with tunicamycin prior to [S]methionine labeling nor treatment of membrane fractions with Pronase under the conditions of PI-PLC treatment or N-glycanase had any apparent effect on the molecular mass of p137 determined by SDS-PAGE (data not shown).


Figure 1: Immunoblots of apical (A) and basolateral (B) membranes of Caco-2 cells using affinity purified antibody 660. 5-µg membrane samples were run on 7% SDS-PAGE in the presence or absence of 10 mM DTT, blotted, and incubated with affinity purified antibody 660 at 1:500 and bands visualized by ECL. The lower molecular weight bands visible in the left-hand lane appear to be proteolytic degradation products of p137, the amounts of which increased considerably after repeated cycles of freezing and thawing of membrane fractions.




Figure 2: Properties of p137. a, a Caco-2 apical membrane fraction was treated with 0.1 M Na(2)CO(3) as described under ``Experimental Procedures,'' the resulting soluble (S) and insoluble (P) phases subjected to 7% SDS-PAGE, blotted, and incubated with affinity purified antibody 660. p137 was visualized by ECL (basolateral membranes gave the same result). In b and c, Caco-2 cells were labeled with [S]methionine and after treatment as described below were immunoprecipitated with affinity purified antibody 660 followed by SDS-PAGE and autoradiography. b, cells were subjected to differential partitioning in Triton X-114 to produce an insoluble pellet (P), soluble (S), and detergent-rich (D) fractions; c, membranes were incubated in the presence (+) or absence(-) of bacterial PI-PLC, and separated into soluble (S) and insoluble (P) fractions. In d, Caco-2 cells were labeled with [S]methionine (lanes 1 and 3) or [^3H]inositol (lanes 2 and 4) and after immunoprecipitation of solubilized cells (lanes 1 and 2) or culture medium (lanes 3 and 4) with affinity purified antibody 660, were subjected to SDS-PAGE followed by analysis on a Fujix BAS 2000 bio-imaging analyzer. All samples were treated with DTT prior to SDS-PAGE. Only the portion of the gel containing p137 is shown in each case.



Cloning and Sequencing of p137

Starting with the initially identified partial cDNA clone (insert C), a strategy was developed to obtain cDNA clones encoding the complete sequence of p137 ( Fig. 3and ``Experimental Procedures''). DNA sequencing of overlapping clones (Fig. 3) resulted in the contiguous cDNA sequence and deduced amino acid sequence shown in Fig. 4. Comparison of the nucleotide sequence with the EMBL, GenBank and OWL data bases, showed it to be novel, although seven regions showed identity with human expressed sequence tags to unknown genes recorded in the EMBL data base. These expressed sequence tags were as follows: accession no. T24120, ID HS1207, 100% identity to nucleotides 125-471; accession no. M85418, ID HSXTO1933(38) , 100% identity to nucleotides 389-603 when reversed and complemented; accession no. T23613, ID HS6137, (^2)100% identity to nucleotides 1258-1591 when reversed and complemented; accession no. T32790, ID HS79013, (^3)97% identity to nucleotides 1326-1587; accession no. T10644, ID T10644 (39) , 94% identity to nucleotides 2456-2818; accession no. T31552, ID HS55215,^3 97% identity to nucleotides 3017-3268; accession no. T 10313, ID HS313,^2 95% identity to nucleotides 3034-3268. In addition, a mouse-expressed sequence tag, accession no. Z36353, ID MM49 (^4)showed 80% identity to nucleotides 2604-2848.


Figure 3: Summary of partial cDNA clones encoding p137 and of antisera raised against pGEX proteins. The initial clone isolated from the human colon cDNA library in gt11 was clone C. Subsequently, overlapping clones 15, 40, and 9 were isolated as described under ``Experimental Procedures.'' Positions of the clones relative to the final contiguous sequence (see Fig. 4) are indicated by double-headed arrows. Fragments of cDNA were subcloned into pGEX-1N to prepare fusion proteins for immunization, and the predicted ORF sequences to which antisera were raised are indicated by arrows labeled with the numbers given to the resulting antisera (660, 803, and 1002). The open bar represents the 5`-UTR (1-202 bp); the closed bar represents the predicted ORF (203-2148 bp); and the stippled bar represents the 3`-UTR (2149-3268 bp).




Figure 4: Nucleotide and derived amino acid sequence of p137. The numbering of the amino acids is based on the position of the first predicted methionine residue. The underlined sequence represents the moderately hydrophobic region which may signal GPI addition. The two broken underlines indicate the regions which, relative to each other, show 23% identity of amino acids. Amino acids typed in bold represent potential sites for attachment of N-linked oligosaccharides. indicates the position of the proposed GPI cleavage/attachment site. The stop codon limiting the open reading frame is indicated by an asterisk.



An ORF of 649 amino acids was evident within the nucleotide sequence, commencing with an initiating methionine codon 202 bases from the 5` end of the contiguous cDNA sequence flanked by a Kozak consensus sequence(40) . It should be noted that this region of sequence agreed exactly with that of the first of the expressed sequence tags described above providing independent confirmation of its accuracy. Analysis of the derived primary amino acid sequence showed it to be enriched overall in glutamate (9.6%) and glutamine (10.9%). There are two regions enriched in proline and glutamine residues, which show 23% identity to one another (amino acid residues 310-370 and 431-488; broken underline in Fig. 4) using similarity investigation program. The derived protein sequence contains three potential N-glycosylation sites, NXS/T (where X cannot be P).

A single moderately hydrophobic region (residues 583-613; underlined in Fig. 4), occurred toward the carboxyl terminus. This moderately hydrophobic region is interrupted by polar residues and two charged His residues and does not conform to a conventional transmembrane region. Analysis of this region and surrounding residues based on the sequences of known GPI-anchored membrane proteins(41, 42) , suggests the possibility of a GPI anchor cleavage/attachment site,``''; at the Ala residue 565. Taken together with secondary structure predictions this sequence analysis suggests that the protein can be divided into three structural domains; residues 1-275 containing several potential alpha-helices, 275-469 containing the proline/glutamine-enriched repeats, and 469-601 containing the potential GPI anchor site.

The predicted molecular mass of the derived amino acid sequence without post-translational modification was 72,751 Da, considerably smaller than the observed molecular mass of p137 on SDS-PAGE. The possibility that the protein was running anomalously was suggested from the observed size of the glutathione transferase fusion proteins obtained after subcloning inserts C and 15 into pGEX-1N. Each of these fusion proteins appeared to have an apparent molecular weight on SDS-PAGE approximately double that predicted from the encoded sequence (data not shown). To resolve this issue, a cDNA construct covering nucleotides 1-2252 was prepared and expressed after transcription and translation in reticulocyte lysate. On SDS-PAGE, the molecular mass of the protein produced was close to 137 kDa, considerably higher than predicted from the derived ORF (data not shown). Immunoblotting of the translation product showed that it reacted with affinity purified antiserum 660 (data not shown). To show that initiation did not start upstream of the predicted initiating methionine, a cDNA construct missing bases 1-128 of the predicted 5`-UTR was transcribed and translated in the reticulocyte lysate. This also resulted in the production of a protein of the same size (137 kDa), which could be immunoblotted with antibody 660 (Fig. 5). Translation in the reticulocyte lysate in the presence of dog pancreatic microsomes showed that the newly synthesized protein was incorporated into the microsomes such that protease treatment only fully destroyed the protein when detergent was present (compare Fig. 5, lanes 6 and 7). In this experiment, a positive control, beta-lactamase, was translocated into microsomes and processed from a 31.5-kDa precursor to the 28.9-kDa mature form as expected (data not shown), demonstrating the functional integrity of the microsomes.


Figure 5: In vitro transcription and translation of DNA encoding for p137. pBluescript containing bases 129-2252 of the full-length contiguous cDNA encoding p137 was transcribed and translated as described under ``Experimental Procedures.'' Approximately 1 µg of mRNA was translated per lane. Lane 1, Caco-2 apical plasma membranes; lane 2, translated product, blotted with affinity purified antibody 660. Lanes 3-7, autoradiographs of [S]methionine-labeled translation products. Lane 3, no mRNA; lane 4, mRNA encoding p137; lane 5, mRNA encoding p137 in the presence of microsomes (M); lane 6, mRNA encoding p137 plus microsomes followed by incubation in 0.1 mg/ml proteinase K (PK) for 1 h, 4 °C; lane 7, as for lane 6 but in the presence of 1% Triton X-100. Only the portion of the gel containing p137 is shown.



Northern blot analysis showed that transcripts for p137 were present in all the tissues examined and in each case, except testis, two transcripts of 3.4 and 2.7 kb were observed (Fig. 6). In testis, two additional transcripts of 5.3 and 2.0 kb were also present.


Figure 6: Northern blot of various human tissues. The Northern blot contained 2 µg of poly(A) RNA/lane from eight different tissues. The blot was probed with P-random primed labeled fragment of insert 15 (bases 605-2252 of the contiguous sequence shown in Fig. 4).



Biosynthesis of p137

The discovery of a GPI-anchored protein present in approximately equal amounts on the apical and basolateral surfaces of Caco-2 cells raised questions about its biosynthesis and route of delivery to each cell surface domain. Prior to performing a pulse-chase experiment, the extent to which p137 was released into the culture medium was determined, since if there was substantial release this would have to be taken into account when considering delivery of newly synthesized protein to the cell surface. Culture medium from flask-grown Caco-2 cells radiolabeled overnight with [S]methionine was collected and shown to contain p137 by immunoprecipitation with affinity purified antibody 660 (Fig. 7a). The released protein was recovered as a 280 kDa entity, which could not be reduced by treatment with dithiothreitol, in contrast to the 280-kDa dimer found on the cell surface (Fig. 7a). The 280-kDa released protein also contained inositol (Fig. 2d, lanes 3 and 4). Biosynthetic labeling experiments showed the [S]methionine/[^3H]inositol ratio to be the same in the membrane-bound and released forms of the protein (Fig. 2d and data not shown). Immunoprecipitation of both plasma membrane and released forms of p137 was also carried out with a further antibody (coded 1002, Fig. 3) prepared against, and immunoaffinity purified on, a glutathione transferase fusion protein containing the fragment of p137 encoded by HindIII-EcoRI fragment (nucleotides 263-605). This showed that both amino-terminal and carboxyl-terminal epitopes were present in the immunoprecipitable protein (Fig. 7b), providing additional evidence that the predicted ORF in the contiguous cDNA sequence encodes p137.


Figure 7: Shedding of p137 into the medium. a, [S]methionine-labeled, flask grown Caco-2 cells (lanes 1 and 2) and surrounding media (lanes 3 and 4) were immunoprecipitated with affinity purified antibody 660 and analyzed by 7% SDS-PAGE and autoradiography after sample preparation in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of DTT. b, [S]methionine-labeled, flask grown Caco-2 cells (lanes 1 and 2) and surrounding media (lanes 3 and 4) were immunoprecipitated with either affinity purified antibodies 1002 (lanes 1 and 3) or 660 (lanes 2 and 4) and analyzed by 7% SDS-PAGE and autoradiography after sample preparation in the presence of DTT.



The post-synthetic delivery of p137 to the cell surface was examined by pulse-chase labeling followed by preparation of apical and basolateral plasma membrane fractions and immunoprecipitation(17) , using affinity purified antibody 660. In addition, the tissue culture media in the apical and basolateral chambers present during the chase were also immunoprecipitated. It was found that newly synthesized p137 was first detected as a 280-kDa entity in the basolateral culture medium within 1 h of the start of the chase (Fig. 8). p137 could just be detected in the apical plasma membrane domain within 1 h of the start of the chase. By 2 h from the start of the chase fairly equal amounts of radiolabeled p137 were present in both apical and basolateral membranes, but there was still a considerable excess of the non-reducible 280-kDa protein in the basolateral culture medium compared with the apical culture medium (Fig. 8).


Figure 8: The post-synthetic transport of p137 in Caco-2 cells. Caco-2 cells grown on filters for 7 days were pulse-chased with [S]methionine, subcellularly fractionated, and membranes and chase media were immunoprecipitated with antisera 660. a, time course of appearance of p137 in apical (A) and basolateral (B) membranes; b, time course of appearance of secreted p137 in apical (A) and basolateral (B) chase media.



Transcytosis and Shedding of p137

To investigate transcytosis of p137, filter-grown Caco-2 cells were cell surface biotinylated in a domain-specific manner, essentially according to Neame and Isacke (25) . First, both the apical and basolateral surface of Caco-2 cells were biotinylated, the membrane proteins immunoprecipitated, separated by SDS-PAGE, and biotinylated p137 detected using horseradish peroxidase-streptavidin (Fig. 9a). This confirmed that p137 could be biotinylated and demonstrated its presence on both surface domains of the cell monolayer by a technique other than subcellular fractionation. The cell surface biotinylated form of p137 was detected as a 280 kDa entity which could not be reduced by the addition of dithiothreitol. This biotinylated protein was released into the culture medium from the cell surface when cells were incubated at 37 °C. Transcytosis of p137 was studied by immunoprecipitating the culture medium from the opposite chamber to the cell surface that had been biotinylated and visualizing the biotinylated proteins by SDS-PAGE followed by horseradish peroxidase-steptavidin detection. The time courses of shedding of biotinylated p137 (Fig. 9b) and transcytosis in the basolateral to apical and apical to basolateral directions (Fig. 9c) were followed by measuring the appearance of the 280-kDa biotinylated protein in the appropriate chambers. It was found that basolateral to apical transcytosis was detectable by 3 h and proceeded at a steady rate for 24 h by which time 50% of basolaterally labeled p137 had been transcytosed compared with 25% shed from the basolateral surface. Apical to basolateral transcytosis could not be detected until after 4 h, but by 24 h 25% of apically labeled p137 had been transcytosed and 20% shed from the apical surface.


Figure 9: Domain specific biotinylation and transcytosis. Caco-2 monolayers grown on filter supports were biotinylated either on the apical or basolateral surface and analyzed for the release of p137 into the media as described under ``Experimental Procedures.'' Medium from three filters was used for tracks 1-8 on the gel and 25% of the cells from three filters for tracks 9-12. a, Caco-2 monolayers were biotinylated on the apical (A, lanes 1, 2, 5, 6, 9, and 10) or basolateral (B, lanes 3, 4, 7, 8, 11, and 12) surface. p137 dimer was detected in the membranes after sample preparation in the presence (lanes 9 and 11) or absence (lanes 10 and 12) of DTT. Shedding of biotinylated p137 dimer was examined after incubation at 4 °C for 24 h (lanes 1-4) or 37 °C for 24 h (lanes 5-8). Apical (A) medium was immunoprecipitated for lanes 2, 4, 6, and 8 and basolateral (B) medium for lanes 1, 3, 5, and 7. b, p137 shed from the same surface domain that was biotinylated is expressed as a percentage of the total p137 that was originally biotinylated (, Ato A, apical; bullet, B to B, basolateral). c, p137 transcytosed from one membrane domain to the other was quantified by expressing the amount secreted from the opposite domain as a percentage of the total p137 that was originally biotinylated ( A to B, apical to basolateral; bullet, B to A, basolateral to apical). Data from representative experiments are shown.




DISCUSSION

In the present study we have developed a strategy to identify and clone membrane proteins capable of transcytosis in polarized epithelial cells. This strategy was based on the availability of antisera to plasma membrane fractions which recognized a subset of common proteins present on opposite cell surface domains. The antisera were used to identify and sort clones from a cDNA library in a bacterial expression vector, in much the same way as has previously been used in the molecular cloning of membrane proteins in the Golgi complex(18, 43) . The only general strategy previously available for identifying transcytosed membrane proteins involved the use of a ricin-resistant MDCK cell line(44) , in which it was subsequently shown that there was a sorting defect which resulted in abnormal surface distribution of at least one of the identified transcytosed proteins(45) . The present strategy avoids this difficulty since it does not require a mutant cell line.

The membrane protein, p137, identified and characterized in the present study is a surprising example of a transcytosed protein in that it appears to be attached to the plasma membrane by a GPI anchor. In some polarized epithelial cell lines such as MDCK, it is well established that newly synthesized GPI-anchored membrane proteins are co-sorted in the TGN, together with glycosphingolipids, before delivery to the apical surface(46) . Indeed the GPI anchor has been identified as an apical targeting signal in these cells(3, 47) , although the situation is complicated by it acting as a basolateral targeting signal in Fischer rat thyroid epithelial cells(48) . In yet other cells, all apically targeted newly synthesized membrane proteins are first delivered to basolateral cell surface(2) . This is true in hepatocytes where it has been shown that even GPI-anchored proteins such as 5`-nucleotidase first appear at the blood sinusoidal surface before transcytosis to the bile canalicular surface(49) . In Caco-2 cells both biosynthetic delivery pathways to the apical surface exist(50, 51) , and there is evidence for the presence of some GPI-anchored proteins such as alkaline phosphatase on the basolateral as well as the apical cell surface domain(17, 51) . In the present study it was found that newly synthesized p137 was clearly present on the basolateral side of the cell in the culture fluid as what appeared to be a 280-kDa dimer at early time points when it was only just possible to detect it at the apical surface. The simplest explanation of this data is that newly synthesized p137 is first delivered basolaterally from where it may be shed or transcytosed to the apical surface.

The derived amino acid sequence of p137 was in agreement with the biochemical data in suggesting that the protein may be GPI anchored to the membrane with the predicted anchor attachment site at the Ala residue 565. Although all endogenous GPI-anchored membrane protein precursors cloned to date possess a cleavable amino-terminal hydrophobic signal sequence, such a signal sequence is not absolutely required for translocation across the endoplasmic reticulum phospholipid bilayer(52) . The predicted open reading frame for p137 encodes no such signal, yet in vitro transcription/translation experiments have shown that the protein can be incorporated into microsomes.

At the cell surface p137 is present as a disulfide bonded dimer. It is most likely that an additional covalent bond must also be formed between the subunits since p137 is constitutively released into the culture medium as a non-reducible 280 kDa entity, presumably a non-reducible dimer. There are many examples of GPI-linked proteins which are secreted including decay accelerating factor(53) , carcinoembryonic antigen(54) , GP2(55) , and melanotransferrin(56) . Within the GPI attachment domain of p137, there are a number of sites that are potential substrates for GPI anchor-degrading enzymes including PI-PLC and phospholipase D. Although p137 was shown to be sensitive to PI-PLC, there is no evidence that this enzyme has an extracellular location. It is not yet clear which enzymic mechanism results in shedding of p137.

The endocytosis and transcytosis of GPI-anchored proteins is not well understood. GPI-anchored proteins interact poorly with clathrin-coated pits, due to the absence of the necessary cytoplasmic tail, and for this reason were thought to be endocytosed poorly or not at all. Recently it has been suggested that they may be clustered in caveolae, flask-shaped invaginations characterized by the presence of the integral membrane protein, caveolin(57) , although there is some dispute as to whether caveolae act as a route of internalization from the cell surface(58) . In Caco-2 cells there have been no reports of caveolae, and we found no evidence for caveolin being present by immunoblotting membrane fractions (data not shown). Irrespective of the route of endocytic entry, the present experiments have demonstrated that p137 may be transcytosed in both the basolateral to apical and reverse directions. While the former direction is consistent with the route of delivery of some apical plasma membrane proteins in Caco-2 cells, the latter suggests the existence of a novel apical to basolateral transcytosis route. The initial lag and slow time course of apical to basolateral transcytosis of p137 is similar to the transcytosis of cobalamin(14) , although this molecule has to be transferred intracellularly from one carrier protein (intrinsic factor) to another (transcobalamin II). A small amount of apical to basolateral transcytosis of nerve growth factor occurs in rat ileum (59) and non-selective transcytosis of epidermal growth factor in this direction has been reported in MDCK cells(60) . Some apical to basolateral transcytosis of ricin has also been observed in Caco-2 cells(61) , although the toxicity of the lectin precludes experiments lasting longer than 2-3 h. It is not clear whether the apical to basolateral transcytosis of p137 in Caco-2 cells is non-selective, for example as a result of failing to be correctly recycled from a common subapical early endosome(4) , or whether it identifies a new selective route. The fact that so much apically labeled p137 can be transcytosed (25% in 24 h), is consistent with the latter. Further biochemical and morphological experiments will be required to characterize this novel membrane traffic route.


FOOTNOTES

*
This work was supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]and ID HSGPI137.

§
To whom correspondence should be addressed. Tel.: 1223-336780; Fax: 1223-330598.

(^1)
The abbreviations used are: TGN, trans-Golgi network; CRD, Cross Reacting Determinant; DTT, dithiothreitol; ECL, enhanced chemiluminescence; GPI, glycosylphosphatidylinositol; MDCK, Madin-Darby canine kidney; ORF, open reading frame; PI-PLC, phosphatidylinositol specific phospholipase C; UTR, predicted untranslated region; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).

(^2)
M. B. Soares, unpublished results.

(^3)
M. D. Adams, unpublished results.

(^4)
R. W. Davies, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Nigel Hooper for the gift of the anti-CRD antibody, Dr. Martin Low for the gift of PI-PLC, Sally Gray for some sequencing plus a pGEX construct, and Dr. C. G. Tate for help with several molecular biology techniques.


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