Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells

Cameron N. Johnstone, Niall C. Tebbutt, Helen E. Abud, Sara J. White, Kaye L. Stenvers, Nathan E. Hall, Stephen H. Cody, Robert H. Whitehead, Bruno Catimel, Edouard C. Nice, Antony W. Burgess, and Joan K. Heath

Ludwig Institute for Cancer Research, Melbourne Branch, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia


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The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.

immunoglobulin superfamily; intestinal epithelium


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MOUSE MONOCLONAL ANTIBODY A33 (MAb A33) was elicited in mice injected with the human pancreatic carcinoma-derived cell line ASPC1 and found to react with a cell surface protein with a highly restricted pattern of expression. Immunohistochemical analysis of frozen sections of a range of normal human tissues revealed strong and almost exclusive expression of human A33 (hA33) antigen by intestinal epithelial cells, with a suggestion of weak staining in the salivary gland (12). In an analysis of thirty different tumor types, 95% of primary and secondary colorectal carcinomas and >50% of gastric carcinomas were found to express the hA33 antigen (12). Only a few instances of nongastrointestinal A33 antigen expression, including areas of intestinal differentiation in certain testicular and germ cell tumors, were detected (12). In view of this highly restricted pattern of expression, the hA33 antigen has attracted attention as a promising target for immunotherapeutic approaches to the treatment of metastatic colorectal carcinoma. Indeed, phase I/II studies have shown highly selective targeting of radiolabeled MAb A33 to tumors in patients with widely disseminated disease (38-40). Recently, to permit prolonged and repeated treatment regimes, a humanized version of A33 was produced and is currently undergoing phase I biodistribution studies.

The hA33 antigen was recently cloned and characterized (18). cDNAs were obtained from a cDNA library constructed from RNA extracted from a colorectal carcinoma-derived cell line, LIM1215 (18). The nucleotide sequence predicted a type 1 transmembrane protein of 298 amino acids (aa), comprising an extracellular domain with an NH2 terminal variable (V)-type Ig-like domain followed by one of the constant 2 (C2)-type (44), a single-span transmembrane domain, and a highly acidic cytoplasmic domain of 62 aa.

Here we describe the cDNA cloning of the mouse A33 (mA33) antigen, deduce its amino acid sequence, predict its molecular structure, and describe its pattern of expression and subcellular localization during development and in adulthood. On the basis of aa sequence identity and conservation of key structural residues, mA33 and hA33 antigens were found to be closely related to several recently described transmembrane proteins. These included the marker of cortical thymocytes in Xenopus (CTX) (8), its putative chicken ortholog ChT1 (22), mouse and human homologs of CTX (CTM/CTH) (7), and the receptor for group B Coxsackie viruses and adenoviruses types 2 and 5 (CAR) (1, 2, 35). These proteins appear to be founder members of a novel subfamily of molecules within the Ig superfamily (IgSF) that is conserved throughout vertebrates. Our results further suggest that the A33 antigen may provide a definitive marker of the basolateral surfaces of intestinal epithelial cells. This distribution is likely to be of significance to the currently unknown function of the A33 antigen.


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Cell culture. The LIM1863 cell line and embryonic stem (ES) cells were grown as described elsewhere (17, 44). Mouse F9 embryonal carcinoma cells were grown in tissue culture flasks coated with 0.1% gelatin in RPMI medium containing 10% FCS.

Cloning of mouse A33 antigen cDNA. Sequence similarity searching of available expressed sequence tag (EST) databases with the full-length hA33 antigen cDNA sequence identified a 249-bp EST (GenBank accession no. D28657) from undifferentiated F9 cells (28) that was 74% identical to hA33 antigen cDNA (nucleotides 551-792). In the likelihood that this EST represented part of the mouse ortholog of hA33 antigen, two primers based on the EST (sense 5'-TGACAAAGAAATACATC-3' and antisense 5'-TCTGGCTTGGAGGGTGG-3') were used to amplify, using a touchdown PCR protocol (10), a 217-bp product from a cDNA library derived from RNA extracted from purified adult mouse colonic epithelial cells (20). This product was gel purified, radiolabeled with [alpha -32P]dATP and [alpha -32P]dCTP (Geneworks, Adelaide, Australia) using the Megaprime DNA labeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden), and used to screen 1 × 106 clones of the adult mouse colonic epithelial cell cDNA library. The cDNA inserts of 20 positive clones were excised and subcloned into pBluescript (KS+).

5' RACE PCR. To extend the 5' nucleotide sequence, 5' rapid amplification of cDNA ends (RACE) PCR was performed using the Marathon kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions and using total RNA (14 µg) extracted from purified mouse intestinal epithelial cells (41) as the template. PCR amplification of the resultant double-stranded cDNA using the AP1 sense primer (provided with the kit) paired with a mA33 antigen-specific antisense primer (5'-GTGTGTGTCCAGGAACAGAAACGCCATGGT-3') yielded a single 1.65-kb product. The product was subcloned into the TopoTA vector (Invitrogen, Leek, The Netherlands), and the 5' sequences of two individual clones were determined on both strands.

DNA sequencing. DNA sequencing reactions were performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit, followed by analysis on an ABI-373 automated DNA sequencer, according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). DNA sequence comparisons were performed using the SeqManII program (DNASTAR, Madison, WI).

Northern blot analysis. Total RNA was prepared from various tissues of ICR mice according to the method of Chomczynski and Sacchi (6). Samples (20 µg) were electrophoresed in 1% agarose-0.4 M formaldehyde gels and transferred to nylon membranes (Hybond N; Amersham). Membranes were hybridized overnight at 42°C with a 2.1-kb mA33 antigen cDNA probe labeled with [alpha -32P]dATP (Geneworks) using the Megaprime DNA labeling kit. All images were generated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Polyclonal antisera production. A peptide containing the most COOH terminal 17 aa of the mA33 antigen, with an additional lysine residue at the NH2 terminus to facilitate conjugation (KHEDRWSSGRSTPDQPFQ), was custom synthesized (Chiron Mimotopes, Clayton, Australia) and conjugated to keyhole limpet hemocyanin. Rabbits were primed with 1.2 mg of peptide in complete Freund's adjuvant (Sigma Chemical, St. Louis, MO) and boosted with 500 µg of peptide in incomplete Freund's adjuvant every 4 wk. Ten milliliters of blood was obtained 12 days after the final boost, and the serum was collected. The IgG fraction was purified from both the antiserum and preimmune serum using protein A-Sepharose chromatography (Amersham).

Western blot analysis. Membrane proteins were extracted from purified intestinal epithelial cells (41), homogenized whole mouse bladder, and cultured mouse cells by incubation in lysis buffer [1% Triton X-100 (TX-100), 20 mM Tris · HCl, pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 15 mM sodium pyrophosphate, and 1× complete protease inhibitor cocktail (Roche Diagnostics Australia, Castle Hill, Australia)] at 4°C for 30 min. Lysates were centrifuged, and the protein concentration of the supernatant was determined using the BCA protein assay (Pierce Chemical, Rockford, IL). Protein extract (15 µg) was subjected to SDS-PAGE in 10% gels in the presence or absence of beta -mercaptoethanol, transferred to polyvinylidene difluoride membranes (Immobilon P; Millipore, Bedford, MA) and blocked overnight at 4°C in 1× PBS-3% BSA-0.05% Tween 20. Membranes were then incubated overnight at 4°C with either preimmune IgG or anti-mA33 antigen IgG (both at 1.2 µg/ml) in 1× PBS-0.1% BSA-0.05% Tween 20. These were washed 3 times and incubated at 25°C for 60 min in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG at a 1:10,000 dilution (Bio-Rad, Hercules, CA) in 1× PBS-0.1% BSA-0.05% Tween 20. Membranes were washed 3 times, and HRP activity was determined using enhanced chemiluminescence (Amersham).

Whole mount immunohistochemistry. E4.5 blastocysts were flushed from the uteri of pregnant ICR mice generated by timed matings. Blastocysts were fixed in freshly prepared 4% paraformaldehyde-PBS for 15 min at 4°C, washed in PBS, and permeabilized with 2 × 15 min washes in 1% TX-100-PBS. The blastocysts were then blocked in 2% BSA-1% TX-100-PBS for 15 min at room temperature before incubation with preimmune IgG or anti-mA33 antigen IgG (5 µg/ml) in 0.2% BSA-1× PBS overnight at 4°C. The blastocysts were then washed for 4 × 30 min in 1% TX-100-PBS before incubation in Alexa Fluor 546-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR) overnight. A final series of washes in 1% TX-100-PBS was used before imaging the fluorescent signals in a confocal microscope (1024ES; Bio-Rad, Hemel Hempstead, UK). Segments of adult small intestine and colon from ICR mice were processed for whole mount immunohistochemistry in a similar way but with longer incubation times. Fixation (4% paraformaldehyde) was performed overnight at 4°C; permeabilization and washing steps (1% TX-100-PBS) comprised 4 × 60 min washes, and incubation in primary anti-mA33 antigen IgG or preimmune IgG was for 60 h. All processing steps were performed under conditions of gentle agitation.

Immunohistochemistry and in situ hybridization. Mouse tissues and embryos were fixed in 4% paraformaldehyde for 8 h, embedded in paraffin, and sectioned (4 µm) onto 3-aminopropyltriethoxy-silane-coated slides. Initial difficulties in obtaining positive immunohistochemical staining of paraffin-embedded sections were overcome by using an antigen retrieval step that involved microwaving the slides on high power for two periods of 5 min in 50 mM Tris · HCl (pH 9.5) (33). Histological sections of adult tissue were blocked in 0.2% BSA-1× PBS for 15 min and incubated in preimmune IgG, anti-mA33 antigen IgG (both at 6 µg/ml), or anti-mA33 antigen IgG (6 µg/ml) plus an excess of peptide (5 µg/ml) in 0.2% BSA-1× PBS for 1.5 h. Sagittal sections of embryos were incubated with anti-mA33 antigen IgG (25 µg/ml) or anti-mA33 antigen IgG (25 µg/ml) plus peptide (5 µg/ml). Sections were rinsed twice in 1× PBS, incubated with HRP-conjugated swine anti-rabbit IgG (DAKO, Carpinteria, CA) for 30 min, rinsed again, incubated with the chromogen 3,3'-diaminobenzidine (DAKO), and counterstained with hematoxylin. In situ hybridization histochemistry was carried out as described elsewhere with minor modifications (25). Sagittal sections described above were hybridized with a 437-bp antisense mA33 antigen cRNA probe, generated by in vitro transcription using [alpha -35S]UTP (1,250 Ci/mmol; Geneworks). Specificity of hybridization was confirmed by hybridizing adjacent tissue sections with either a radiolabeled sense mA33 antigen (437 bp) cRNA probe or a radiolabeled antisense probe for mouse insulin-like growth factor binding protein-4.


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Cloning of mouse A33 antigen cDNAs. For cloning of cDNAs encoding the mouse ortholog of the hA33 antigen, we used a cDNA library constructed from poly(A)+ RNA extracted from purified normal adult mouse colonic epithelial cells (20) that had been used previously to clone cDNAs encoding other intestine-enriched proteins, for example, Cdx1 (20). A specific probe was identified as a result of sequence similarity searching of all available databases with the full-length hA33 antigen cDNA sequence. This identified a 249-bp EST generated from a low-abundance transcript from the mouse teratocarcinoma-derived cell line F9 (28) that was 74% identical to hA33 antigen cDNA in the corresponding region (nucleotides 551-792). Translation of the F9 EST predicted a protein sequence that shared 55% aa identity with hA33 antigen in the region of overlap. In the likelihood that this EST represented part of the mouse ortholog of the human A33 antigen, two oligonucleotide primers based on the EST were synthesized and used to amplify a 217-bp product from the normal adult mouse colonic epithelial cDNA library described above. The PCR product was sequenced, found to share 95% nucleotide identity with the F9 EST, and used to screen the normal adult mouse colonic epithelial cDNA library. Of the 20 putative mA33 antigen cDNA clones obtained, the longest were ~2.1 kb in size. The sequences of two clones were determined completely on both strands, and two more clones were sequenced partially. All four clones encoded the same molecule.

The nucleotide sequence of mA33 antigen cDNA and the deduced protein sequence are depicted in Fig. 1.1 Compared with the full-length hA33 antigen cDNA, the mouse cDNA clones lacked all of the 5' untranslated region (UTR) and the first two nucleotides of the codon for initiation of translation (ATG). The remainder of the 5' sequence was determined by 5' RACE PCR, which yielded a fragment containing the entire coding region, including the putative initiation methionine and 104 bp of the 5' UTR. The mouse 3' UTR was 254 bp shorter than the human 3' UTR, with a polyadenylation signal starting at position 2270 followed 17 nucleotides downstream by a run of adenines. The overall nucleotide sequence identity between human and mouse A33 antigen cDNAs was 62% (77% identity in the coding region). The deduced mA33 antigen protein sequence shared 66% identity (77% similarity) with the product encoded by hA33 antigen cDNA (18). This degree of sequence identity compares favorably with interspecies variation observed between other IgSF orthologs, for example, mouse and human CD2 (54%) and mouse and human CD22 (62%) (32, 24).


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Fig. 1.   Nucleotide and deduced amino acid (aa) sequence of mouse A33 (mA33) antigen cDNA. The sequence obtained by 5' RACE PCR is indicated by the dotted underline. The longest open reading frame encodes a protein of 319 aa, including a putative leader sequence, indicated by the open-boxed area. The molecule is predicted to contain an extracellular domain, a transmembrane domain, and a 62-aa cytoplasmic domain. The spans of the two immunoglobulin (Ig)-like domains [one variable (V) type followed by one constant 2 (C2) type] in the extracellular region are each enclosed by parentheses, with residues conserved in Ig superfamily (IgSF) members shown in circles. The transmembrane domain is underlined, and the 4 adjacent consecutive cysteine residues are shown in bold. The stop codon (TGA) is highlighted in black. The polyadenylation signal starting at position 2270 (underlined) lies 17 nucleotides upstream from a run of adenines.

The longest open reading frame in the mouse cDNA was 957 bp (319 aa), as was found for the hA33 antigen cDNA (18). Starting with a codon for methionine, in a favorable context for initiation of translation (23), the open reading frame next encoded a hydrophobic region most likely representing a leader sequence. This region terminated with a putative signal sequence cleavage site (37) (Ala21,Leu22) which corresponded to the cleavage site of the hA33 antigen translation product, determined by NH2 terminal aa sequencing of the native molecule (27). Thus mature mA33 antigen is predicted to contain 298 aa (estimated molecular weight, 33,550 Da) with leucine at the NH2 terminus. Many features of the mouse and human A33 antigens are conserved. Both contain an extracellular region containing one V-type and one C2-type Ig-like domain, a transmembrane domain, and a 62-aa hydrophilic intracellular tail. The extracellular domain of mA33 antigen contains four potential N-linked glycosylation sites (two in the V-type and two in the C2-type domain; Fig. 2). Three of these (Asn91, Asn179, and Asn202) are present in analogous positions in hA33 antigen; the extra site (Asn78) in the mouse protein is found in the V-type domain (Fig. 2). Like hA33 antigen, mA33 antigen contains four contiguous cysteines immediately COOH-terminal of the transmembrane domain (Fig. 2). These are thought to be a target for palmitoylation in hA33 antigen and may provide further tethering of the molecule to the plasma membrane (31).


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Fig. 2.   Relationship of A33 antigen and other marker of cortical thymocytes in Xenopus (CTX) family members with junctional adhesion molecule (JAM) and human CD2. The aa sequences of the extracellular, transmembrane, and membrane-proximal cytoplasmic domains of mA33 antigen, human A33 (hA33) antigen, Xenopus CTX (xCTX), its putative chicken ortholog ChT1 (cChT1), human and mouse receptors for Coxsackie group B and adenoviruses types 2 and 5 (hCAR and mCAR, respectively), mouse and human homologs of CTX (mCTM and hCTH, respectively), mouse JAM (mJAM), and human CD2 were aligned using the MegAlign program (DNAStar) with manual adjustment. The cleavage site yielding mature hA33 antigen and CTX is indicated with an arrow. The positions of the beta strands within the V-type and C2-type Ig-like domains of CD2 that were deduced from its crystal structure (3) are highlighted in black and allow us to assign the putative positions of the corresponding beta strands (A-G) within the V-type and C2-type Ig-like domains in CTX family members. Conserved residues are shaded, and potential N-linked glycosylation sites are circled. Cysteine residues involved in the formation of disulfide bonds within the Ig-like domains are boxed, and the putative transmembrane domain of mA33 antigen is underlined. The numbering above the alignment indicates the aa position of mature hA33 antigen.

The A33 antigen is related to CTX/ChT1, CAR, CTM/CTH, and JAM. The domain structure of mA33 antigen shows striking similarity to that of the T lymphocyte cell adhesion molecule CD2, for which the crystal structure of the extracellular domain is available (3). However, the primary sequences of their V-type and C2-type domains are only 12% identical, and more marked sequence identity was noted with four recently cloned IgSF members named CTX (7, 8, 11) (25% identity in the V-type domain; 33% identity in the C2-type domain), ChT1 (22) (23% identity in the V-type domain, 26% identity in the C2-type domain), hCAR and mCAR (1, 2, 35) (23% identity in the V-type domain; 29% identity in the C2-type domain), CTM and CTH (7) (21% in the V-type domain, 31% in the C2-type domain), and junctional adhesion molecule (JAM) (26) (22% identity in the V-type domain; 24% identity in the C2-type domain). Meanwhile, the molecules share only weak sequence similarity in their intracellular domains (data not shown).

The sequences of the extracellular and transmembrane domains of the related molecules were aligned (Fig. 2) and compared with human CD2, which allowed us to predict the sequences likely to contribute to each of the beta -strands in the related proteins (3). Several residues, in addition to those conserved in Ig-like domains, were invariable among A33 antigen, CTX/ChT1, CAR, and CTM/CTH. These included Ala79, Cys125, Gly129, Glu145, and Cys201 as well as a conserved membrane-proximal potential N-linked glycosylation site in the C2-type domain (Asn179). Conservation of these features as well as a similar organization of the genes encoding CTX, ChT1, CTM, CTH, and hA33 antigen led Chrétien and colleagues (7) to propose that these molecules (along with CAR) were founder members of a novel subfamily, the CTX family, within the IgSF. On the other hand, JAM lacks all of the signature residues of the CTX family and appears to be more distantly related (Fig. 2).

Localization of mA33 antigen in adult mouse tissues. The expression of mA33 antigen mRNA was examined in multiple mouse tissues by Northern blot analysis (Fig. 3A). Of the 23 tissues studied, only small intestine and colon were strongly positive, producing a single intense band corresponding to a transcript of ~2.4 kb. Mouse stomach and bladder produced weak signals (~10% the intensity of that obtained from intestine). This is in contrast to the pattern of expression reported in humans (12), in which expression of hA33 antigen was absent in 13 samples of normal human stomach and several samples of normal and neoplastic human bladder. However, 14 of 24 gastric carcinomas expressed hA33 antigen in >80% of cells (12). All other human and mouse tissues were negative except for very weak staining in human salivary gland, which was not seen in mice. Differences in expression pattern between mouse and human have also been observed for other members of the CTX family (2, 35). For instance, mCAR was strongly expressed in liver, lung, and kidney, whereas hCAR expression appeared to be minimal or absent in these tissues. Northern blot analysis of mA33 antigen mRNA levels throughout the rostrocaudal axis of the intestine revealed uniformly high expression from duodenum to distal colon, with a significantly lower level of expression in stomach epithelium (Fig. 3B).


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Fig. 3.   A: expression of mA33 antigen mRNA in multiple mouse tissues. Total RNA was extracted from indicated tissues of ICR mice and subjected to Northern blot analysis as described in MATERIALS AND METHODS. Mouse A33 antigen mRNA was detected by hybridizing the membrane with a radiolabeled 2.1-kb mA33 antigen cDNA (top rows). The signals obtained when the membranes were rehybridized with an oligonucleotide probe designed to hybridize to 18S rRNA are shown in the bottom rows. B: expression of mA33 antigen mRNA along the rostrocaudal axis of the mouse gastrointestinal tract. The intestine was dissected into the regions indicated, and purified populations of epithelial cells were prepared (41) before RNA extraction. Total RNA was subjected to Northern blot analysis using a radiolabeled 2.1-kb mA33 antigen cDNA probe. The filter was reprobed with a radiolabeled oligonucleotide designed to hybridize to 18S rRNA.

The expression of mA33 antigen protein was found to correlate closely with mA33 antigen mRNA expression. Western blot analysis and immunohistochemistry were performed using a polyclonal rabbit anti-mA33 antigen antiserum. A highly abundant protein was detected in membrane extracts from purified mouse colonic epithelial cells (Fig. 4A). mA33 antigen migrated on SDS-PAGE as a single band of ~49 kDa under reducing conditions and ~45 kDa under nonreducing conditions. An increase in apparent molecular weight on reduction was also observed for hA33 antigen and CTX (5, 7, 8). This behavior implies the presence of disulfide bonds in unreduced mA33 antigen, which cause the protein to migrate anomalously on SDS-PAGE. The formation of disulfide bonds between all three pairs of cysteine residues in the extracellular domain of hA33 antigen has been now been demonstrated empirically [Refs. 5, 27, and B. Catimel, unpublished observations]. The higher apparent molecular weight of mA33 antigen on SDS-PAGE, compared with that predicted from the deduced sequence (33,550 Da), is most likely explained by N-linked glycosylation of the extracellular domain, as previously demonstrated for hA33 antigen (31).


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Fig. 4.   Analysis of mA33 antigen protein expression in mouse tissues and cell lines. A: Western blot analysis of mA33 antigen from purified colonic epithelial cells conducted under reducing (lane 1) or nonreducing (lane 2) conditions using the anti-mA33 antigen IgG. B: Western blot analysis of mA33 antigen expression performed under nonreducing conditions using anti-mA33 antigen IgG. Triton X-100 extracts were prepared from each tissue or cell line (see MATERIALS AND METHODS), and 15 µg of protein were loaded in each lane. Lanes 1 and 7, small intestinal epithelium; lane 2, colonic epithelium; lane 3, whole bladder; lane 4, F9 embryonal carcinoma cells; lane 5, embryonic stem cells; lane 6, LIM1863 human colorectal carcinoma cells. MW, molecular weight.

Under nonreducing conditions, Western blot analysis detected a single, highly abundant protein of ~45 kDa in membrane extracts from small intestinal and colonic epithelial cells (Fig. 4B) and a less abundant protein of the same size in extracts of bladder. Since mA33 antigen was originally identified as an EST from undifferentiated F9 embryonal carcinoma cells, we also investigated the expression of mA33 antigen protein in F9 cells and in ES cells. Low levels of the protein were detected in both undifferentiated F9 cells and undifferentiated ES cells. No signal was obtained with protein extracted from hA33 antigen-positive LIM1863 cells, indicating that the antiserum did not cross-react with the human protein (5, 18). Incubation of a duplicate membrane with preimmune IgG showed that the signals observed with the anti-mA33 antigen IgG were not due to nonspecific interactions (data not shown).

The subcellular localization of mA33 antigen was determined by immunohistochemistry of both whole mount tissue preparations and histological sections. Whole mount immunohistochemistry of mouse colon followed by optical sectioning using the confocal microscope demonstrated strong and even staining of the membranes of all epithelial cells throughout the length of the crypt (Fig. 5A). mA33 antigen was restricted to the basolateral surfaces, with no staining of the apical membranes. The surrounding lamina propria and smooth muscle were negative. We did not notice any crypt epithelial cells that appeared to be negative for mA33 antigen expression, providing evidence that mA33 antigen is expressed by all three cell lineages found in the colonic crypts (columnar, goblet, and enteroendocrine). In histological sections of duodenum, mA33 antigen staining was also restricted to the epithelial layer, with uniformly strong staining extending from the base of the crypt to the villus tip (Fig. 6A). mA33 antigen was again localized to basolateral surfaces and appeared to be especially concentrated in the lateral membranes and absent from the apical membranes (Fig. 6A). This was demonstrated clearly with a maximum intensity projected image of a whole mount preparation of a small intestinal villus examined in the confocal microscope (Fig. 5B). Again, the apparent absence of negative cells indicates that cells of all four lineages found in the small intestine (enterocyte, goblet, enteroendocrine, and Paneth) most likely express mA33 antigen. Histological sections showed that Brunner's glands, nestled beneath the duodenum, also gave positive staining (Fig. 6A). Incubation of an adjacent section of duodenum with anti-mA33 antigen IgG preincubated with the COOH terminal peptide failed to produce a specific signal (Fig. 6B), indicating that the signals observed with the anti-mA33 antigen IgG were specific for mA33 antigen. The expression of mA33 antigen extended into the pyloric region of the stomach, with staining of all epithelial cells in the gastric pits at levels roughly comparable to that in intestinal epithelium (Fig. 6C). Meanwhile, the rest of the stomach was negative (data not shown). The strong staining of the pyloric epithelium, although somewhat unexpected in view of the relatively weak mRNA signals we obtained on Northern blot analysis of whole stomach epithelium (Fig. 3B), can be reconciled with one region of the stomach expressing mA33 antigen and all other regions being negative. The bladder expression of mA33 antigen was restricted to the transitional epithelium, in which staining was observed throughout the plasma membrane in all cell layers (Fig. 6D). Interestingly, the transitional epithelial cells of the urinary bladder are derived from primitive gut endoderm during embryogenesis (13).


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Fig. 5.   A: confocal optical section through a whole mount preparation of mouse colon incubated with anti-mA33 antigen IgG (5 µg/ml) followed by an Alexa Fluor 546-conjugated goat anti-rabbit secondary antibody (1:200). The antigen is distributed to the basolateral surfaces of epithelial cells. The arrow indicates absence of mA33 antigen staining in the apical membrane. Scale bar = 20 µm. B: maximum-intensity projection (extended focus image) of a whole mount preparation of a mouse small intestinal villus. Scale bar = 20 µm. Inset: confocal optical section through the tip of a villus. Again, the mA33 antigen is distributed to the basolateral surfaces of the epithelial cells and is especially concentrated on the lateral membranes.



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Fig. 6.   Immunohistochemical analysis of mA33 antigen expression in mouse tissues. Immunohistochemistry was performed on sections of the following tissues after antigen retrieval: A and B, duodenum; C, stomach pylorus; D, bladder; E, dissected E14.5 hindgut; F, sagittal section of E15.5 embryo showing hindgut expression. Sections were incubated with anti-mA33 antigen IgG (6 µg/ml), except B, which was incubated with anti-COOH terminal A33 antigen antiserum (6 µg/ml) preincubated with the peptide used for inoculation (5 µg/ml) and E and F, incubated with anti-mA33 antigen IgG (25 µg/ml). Scale bars = 50 µm.

In summary, the A33 antigen expression pattern in the adult mouse encompasses epithelial cells of the small intestine, colon, stomach pylorus, and bladder. In the intestine, mA33 antigen is expressed uniformly along the rostrocaudal axis in both proliferating cells at the base of the crypts and differentiated cells at the top of crypts and on the small intestinal villi.

Mouse A33 antigen expression is regulated during embryonic development. Murine A33 antigen expression was not detected in embryos between E5.5 and E13.5 (H. E. Abud, unpublished observations) through immunohistochemistry. Intestinal mA33 antigen expression was first detected in the hindgut endoderm at E14.5 with cytoplasmic as well as cell surface expression (Fig. 6E). Interestingly, immunoreactivity along the rostrocaudal axis of the intestine was polarized at this stage, with the distal end of the intestine being strongly positive for mA33 antigen expression and the proximal end being only barely positive. A signal was not observed in the midgut at this stage of development (data not shown) nor in any other tissue in the developing embryo (Fig. 7B). Notably, the initial appearance of mA33 antigen expression was therefore in the opposite direction to the proximal-to-distal wave of morphogenesis and cytodifferentiation that mediates substantial remodeling of the intestine between E15 and E19 of mouse development (14). By E15.5, strong mA33 antigen expression was found throughout the intestine and was restricted to the plasma membranes of endoderm cells (Fig. 6F). At this stage, the endoderm had yet to convert from a multilayered stratified structure to a simple columnar epithelium (36). By E16.5, the endoderm had thinned to a columnar epithelium, which maintained strong membranous expression of mA33 antigen (data not shown). Northern blot analysis confirmed that mA33 antigen mRNA was highly expressed at E16.5 in both small intestine and colon (Fig. 7A). The mA33 antigen mRNA levels increased gradually until adulthood with no dramatic change in expression after birth and weaning (24 days postnatal). In contrast, expression in bladder appeared to be induced between 1 and 4 days after birth but was always present at much lower levels than in intestine (Fig. 7A). At E18.5, in situ hybridization histochemistry of sections of whole embryo torsos detected mA33 antigen mRNA specifically in the intestine (Fig. 7B), indicating that the highly restricted pattern of expression observed in the adult was essentially established at this stage of development.


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Fig. 7.   A: expression of mA33 antigen mRNA in intestine and bladder during embryonic and postnatal development. Mice were killed at the stages of development indicated, and samples of total RNA prepared from whole colon, small intestine, and bladder were subjected to Northern blot analysis using a radiolabeled 2.1-kb mA33 antigen cDNA probe (top). The membrane was reprobed with a radiolabeled oligonucleotide designed to hybridize to 18S rRNA (bottom). B: intestine-specific expression of mA33 antigen mRNA at E18.5. Adjacent sagittal sections of mouse embryos (torsos) were subjected to in situ hybridization histochemistry with a mA33 antigen cRNA probe (left) and a probe specific for insulin-like growth factor binding protein-4 (IGFBP-4), which is widely expressed at this stage of mouse development (right). Positive signals with the mA33 antigen cRNA probe were observed in the intestine only. C: confocal optical section through a whole mount preparation of an isolated E4.5 blastocyst processed for immunofluorescence using anti-mA33 antigen IgG (5 µg/ml) followed by an Alexa Fluor 546-conjugated goat anti-rabbit secondary antibody (1:200). All cells of the inner cell mass stained positively for mA33 antigen.

Our finding that mA33 antigen protein was present in murine ES cells (Fig. 4B) suggested the possibility that mA33 antigen was also expressed very early during development. ES cells are considered to be the in vitro counterpart of the inner cell mass of E3.5/E4.5 blastocysts (9). Accordingly, we used immunohistochemistry of whole mount preparations of blastocysts to investigate mA33 antigen expression. Striking mA33 antigen expression was detected on the cell surface of apparently all cells of the inner cell mass (ICM) (Fig. 7C). ICM cells are pluripotent stem cells that give rise to all the tissues of the developing embryo. Whether the physiological roles of the mA33 antigen are similar in the ICM and the intestinal epithelium is not known at present. A33 antigen-deficient mice have been developed to explore the indispensable/nonredundant functions of mA33 antigen and so far have shown enhanced immune responses of the intestinal mucosa (N. C. Tebbutt, S. Bao and M. Ernst, unpublished observations).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The characteristics of mA33 antigen expression appear to provide us with a definitive marker for all intestinal epithelial cells irrespective of their cell lineage and proliferative/differentiation status. Although a related molecule, JAM, was localized to tight junctions in endothelial cells and intestinal epithelial cells (26), mA33 antigen appears to be distributed specifically to the basolateral membranes. The subcellular localization of mA33 antigen combined with the structure of its extracellular domain, which is highly reminiscent of CD2, strongly suggest that mA33 antigen may be involved in cell-cell adhesion within the gastrointestinal tract. Here the A33 antigen could be involved in cell-cell adhesion between adjacent epithelial cells or between epithelial cells and the resident population of intraepithelial T lymphocytes (21). The presence or absence of mA33 antigen at tight junctions and other epithelial cell-cell adhesion structures such as desmosomes and adherens junctions (4, 15) must be addressed in the future with the use of electron microscopy.

The exquisite, almost intestine-specific, pattern of mA33 antigen expression strongly suggests that the regulatory elements of the gene encoding mA33 antigen would be perfectly suited for directing expression of gene products implicated in the regulation of epithelial cell growth and differentiation and colorectal tumorigenesis to the intestinal epithelium in transgenic mice. Other promoters have been investigated for these purposes with varying degrees of success (16, 19, 42, 45). Two of the more promising ones, the promoters of the rat liver fatty acid-binding protein gene (Fabpl) (34) and the mouse villin gene (29), were recently described. An artificial Fabpl promoter, containing four extra copies of a heptad repeat inserted in its normal position at nucleotide -132, directed strong transgene expression to colonic, cecal, and distal small intestinal epithelium and minimal expression to kidney epithelium (34). Unlike expression of the endogenous gene, expression of the transgene extended to the base of the crypt in the large and small intestine. Similarly, a relatively large region of the mouse villin gene (-3.5 kb to +5.5 kb) was sufficient to drive expression of a beta -galactosidase reporter gene in a manner that essentially recapitulated endogenous villin gene expression in transgenic mice (29). The transgene was strongly expressed by differentiated colonic and small intestinal epithelial cells, with reduced activity in the lower crypt region where some beta -galactosidase-negative cells were found.

One important consideration for this type of animal model is whether transgene expression can be induced in intestinal epithelial stem cells or not. The ability to deliver expression to stem cells would provide a particularly powerful system since stem cell anchorage and self renewal would ensure continual expression of the transgene within the proliferative cell compartment (30). At present, it is impossible to identify intestinal stem cells accurately and determine their patterns of gene expression. Our data, although not absolutely conclusive, demonstrated that mA33 antigen-negative cells were conspicuously absent in the region around the base of crypts and raise the intriguing possibility that stem cells express mA33 antigen. Accordingly, we propose that transgenic mice based on the mA33 antigen gene promoter may provide significant new opportunities to investigate the genetic basis of colorectal cancer.


    ACKNOWLEDGEMENTS

We thank Dr. Robert James for the adult mouse colonic epithelial cell cDNA library, Dr. Guo-Fen Tu for DNA sequencing, Dr. Stacey Gabriel, Dianne Grail, and Melissa Inglese for collection of mouse tissues, Valerie Feakes for expert tissue sectioning and immunohistochemistry, and Janna Stickland for photography.


    FOOTNOTES

C. N. Johnstone and N. C. Tebbutt were supported by an Australian Postgraduate Award and a National Health and Medical Research Council (NH&MRC) Medical Postgraduate Research Scholarship, respectively. This study was funded in part by a project grant from the NH&MRC (Australia).

Present address for R. H. Whitehead: G. I. Cancer Program, Vanderbilt Medical Center, Nashville, TN 37232-2583.

Address for reprint requests and other correspondence: J. K. Heath, Ludwig Institute for Cancer Research, Post Office Royal Melbourne Hospital, Parkville, Victoria 3050, Australia (E-mail: joan.heath{at}ludwig.edu.au).

1 The GenBank accession number for the novel murine A33 antigen sequence described in this study is AF24765. Other relevant GenBank accession numbers are hA33 antigen cDNA (U79725), CTX cDNA (U43330), ChT1 cDNA (AF061023), mCAR cDNA (Y10320), hCAR cDNA (U90716), CTM cDNA (AF061024), CTH cDNA (AF061022), JAM cDNA (U89915), and hCD2 cDNA (M16445).

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. §1734 solely to indicate this fact.

Received 28 December 1999; accepted in final form 30 March 2000.


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