Date of Award
Doctor of Philosophy (PhD)
Dr. S. K. Liao
The studies contained in this thesis describe physical, biochemical and immunochemical characterization of human malignant melanoma cell surface antigens defined by a monkey antiserum and a mouse monoclonal antibody (MoAb).
The monkey anti-CaCL 73-36 antiserum identified, melanoma-associated surface antigens (MAAs) that were shed from cultured melanoma cells. The shed MAAs appeared to be similar to shed HLA-A,B,C antigens in terms of flotation on KBr, sedimentation in sucrose gradient, and association with B2-microglobulin.
The MoAb 140.240, which reacted with a melanoma-specific oncofetal antigen identified an epitope on an 87kd molecule that was integrally associated with melanoma cell plasma membrane. This molecule was a monomeric sialoglycoprotein that originated from a 77kd precursor polypeptide (p77). The precursor was converted to an intermediate 83kd giycopolypeptide (gp83) which in turn was further glycosylated to yield the mature 87kd glycopolypeptide (gp87).
Both gp87 and p77 were detectable in the chase medium of melanoma cells metabolically labelled in the presence of tunicamycin (TM), but had molecular weights slightly larger than their cellular counterparts. Comparison of tryptic peptide maps of cellular and shed gp87 showed complete overlapping, except that the shed form contained two additional methionine-containing peptides. Surface radioiodination of TM-treated cells showed that p77 was also expressed on the cell surface.
The purification of gp87 from membrane lysate and spent medium of cultured melanoma cells was achieved by a procedure involving ion-exchange, gel filtration, and antibody affinity column chromatography. Compared with the starting materials, the procedure yielded a purification factor of >3,300 and 28.6% recovery for cellular gp87, and a purification factor of >1,200 and 41.5% recovery for shed gp87. On SDS-PAGE, the purified preparations gave a single band of 87kd.
Immunochemical analysis of MoAb 96.5 to a 97kd MAA (p97) developed in another laboratory showed that gp87 and p97 were identical molecules, although the epitope recognized by MoAb 140.240 was distinct from that recognized by MoAb 96.5.
The biological and clinical significance of these findings in relation to the reports of other investigators on human melanoma-associated antigens are discussed.
Khosravi, Mohammad Javad, "Physical and Molecular Characterization of Human Malignant Melanoma Antigens Defined by a Monkey Antiserum and a Mouse Monoclonal Antibody" (1984). Open Access Dissertations and Theses. Paper 1404.