MOLECULAR CHARACTERIZATION OF A TISSUE-POLYPEPTIDE-SPECIFIC-ANTIGEN EPITOPE AND ITS RELATIONSHIP TO HUMAN CYTOKERATIN-18

Tissue-polypeptide-specific antigen (TPS) from the human colon adenoca rcinoma cell line WiDr was purified using the monoclonal antibody M3 a s a probe. Upon SDS/polyacrylamide gradient gel electrophoresis, sever al TPS-positive bands were detected (corresponding to 13 kDa, 22 kDa a nd a doubler at 42 kDa). The 13-kDa moiety was purified about 30 000-f old by a 5-step protocol. The electro-phoretically homogeneous compone nt was obtained in a 7% yield of the total TPS activity of the crude e xtract. N-terminal sequence analysis showed the presence of an N-termi nally truncated molecule and identified the 13-kDa TPS component as a fragment of human cytokeratin 18, with a major form starting at positi on 284 of the parent molecule. Laser-desorption mass spectrometry show ed the presence of one major component with a molecular mass correspon ding to a C-terminal end close to position 396 (which gives 12 776 Da for the form with non-truncated N-terminus). The M3 antibody was also used to screen a human prostate cDNA lambda gt11 library. Four identic al phage clones were detected, each producing a fusion protein with be ta-galactosidase and the M3-positive component. PCR amplification show ed the presence of an approximately 1200-bp insert, and sequence analy sis revealed it to contain a 996-nucleotide fragment corresponding to residues 103-429 of human cytokeratin 18 (plus a non-coding human desm in artifact fragment). Smaller fragments, engineered by PCR and expres sed as fusion proteins using the pET3xc vector in Escherichia coli, sh owed that the M3 epitope is localized to cytokeratin 18, residues 322- 340. Two other TPS-active monoclonal antibodies were localized to cyto keratin 18 with similar techniques, ascribing an epitope (to M21) to r esidues 414-429 and another (to M24) to residues 139-297. Combined, th e results demonstrate that TPS reactivity is derived from specific epi topes of human cytokeratin 18.