(Received for publication, February 8, 1995; and in revised form, April 27, 1995)
From the
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.
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) ()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.
A human colon carcinoma gt11 cDNA library (
1.5
10
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.
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 NaCO
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 [
H]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.
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
-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,
-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).
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.
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;
, 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;
, B to A, basolateral to
apical). Data from representative experiments are
shown.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]and ID HSGPI137.