MAb E4 recognizes a 66-kDa glycoprotein, pE4, which is a member of the immunoglobulin gene superfamily. This protein is expressed at the cell surface in rat colon and mammary carcinomas, but only in trace amounts in normal adult rat tissues. Since expression of aberrant carbohydrate structures is often associated with malignant transformation, glycosylation of pE4 was analyzed. Reactivity of lectins with pE4 suggested the absence of N-acetylneuraminic acid, terminal galactose and O-linked glycan, and the presence of N-linked glycans. Tunicamycin treatment reduced the binding of MAb E4 to cancer cells suggesting that the E4 epitope is at least partially glycosylated. Digestions with neuraminidases, O-glycosidase and peptide-N-glycosidase F confirmed these results. Pronase treatment abolished the binding of MAb E4, indicating that E4 epitope involves not only a carbohydrate determinant but also a peptide moiety. Mild periodate oxidation abolished the binding of MAb E4, indicating that non-reducing terminus carbohydrates are part of the E4 epitope. Neutral sugar analysis revealed the absence of galactose and the presence of fucose. Since fucose is sensitive to periodate oxidation, this sugar could be the carbohydrate part of the determinant recognized by MAb E4. Reactivity of lectins specific for fucose indicated the presence of alpha(1-6)-fucose on pE4.
In a hope to isolate genes whose expression was elevated in colon cancer cells, we used a plus/minus screening of a rat colon carcinoma cDNA library. We were thus able to isolate a cDNA clone (B9) encoding the rat S13 ribosomal protein (1). The corresponding mRNA is-30-fold more abundant in the cancer cells than in normal colon, and 50-fold more important in lung metastasis than in normal lung. The expression level of this gene was found to be closely correlated with the growth rate of rat cell lines (1). Unfortunately, the B9 probe was short, and did not hybridize to RNA prepared from human tissues. In order to obtain longer clones encoding the rat S13 ribosomal protein we first rescreened the PROb cDNA library. A longer insert was then used to screen a cDNA library prepared from the T-84 colonic epithelial cell line constructed in the Uni-ZAP XR vector (Stratagene, La Jolla, CA). Inserts were excised with helper phage R408 to generate subclones in the pBluescript plasmid and sequenced by the dideoxy-chain termination method (2) using T7 DNA polymerase. The final sequences were determined from both strands. This cDNA contains 548 nucleotides and includes a 5' noncoding sequence of 32 nucleotides, an open reading frame of 456 nucleotides, a 3' noncoding sequence of 60 nucleotides, followed by a 19 bp polyA tail (Figure 1). The presumed polyadenylation signal, AATAAA, is located at bases 509-514. The nucleotide sequence in the coding region differs from the rat S13 ribosomal protein sequence (3) in the first position in 6 codons and in the third position in 52 codons. It encodes a sequence of 151 aminoacids which is identical to the rat protein. This highly conserved primary structure of ribosomal proteins across species has been observed for other ribosomal proteins (4, 5). Northern blot analysis revealed that the size of the human S13 ribosomal protein mRNA is approximately 600 bp. This probe hybridized to 8-10 genomic DNA fragments, probably representing one (or a few) functional S13 gene and a family of nonfunctional pseudogenes, as it has been shown for many other ribosomal protein genes (6, 7).
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