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Biochemical characterization of human carcinoma surface antigen associated with protein kinase activity
Author(s) -
Seehafer Jutta,
Longenecker B. Michael,
Shaw Andrew R. E.
Publication year - 1984
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.2910340613
Subject(s) - glycoprotein , glycosylation , tunicamycin , biochemistry , mannose , biology , cell culture , methionine , gel electrophoresis , membrane glycoproteins , polyacrylamide gel electrophoresis , microbiology and biotechnology , amino acid , enzyme , unfolded protein response , endoplasmic reticulum , genetics
Abstract MAb 50H.19 immunoprecipitates two proteins from lysates of human carcinoma cell lines, and embryonic fibroblasts intrinsically labelled with 3 H‐leucine, 35 S‐methionine, or a 3 H‐amino acid mixture; a major component of M, = 22,000 (22 kd component) and a minor component of M, = 24,000 (24 kd component). Oligomeric forms of the proteins are not observed under reducing or non‐reducing conditions. Both proteins are expressed on the plasma membrane, and are glycoproteins. We investigated the relationship between the proteins in terms of their glycosylation and derivation from precursors. The 22 kd component is O‐glycosylated as demonstrated by 3 H‐galactose incorporation, insensitivity to tunicamycin (TM), and its stepwise generation from a 20.5 kd precursor. The 24 kd protein is N‐glycosylated, as shown by 3 H‐mannose incorporation, and by the total inhibition of its synthesis in the presence of TM. Further evidence for its N‐glycosylation is provided by the appearance of a 23 kd precursor in lysates from the osteogenic sarcoma cell line SKOSC pulse‐labelled for 5 min, a time preceding O‐glycosylation of the 20.5 kd protein. Furthermore, mild alkali treatment of the immune complex leads to a loss of approximately 1,000 daltons in each glycoprotein confirming the O‐glycosylated nature of the 22 kd component, and suggesting that the 24 kd component is additionally O‐glycosylated. Both glycoproteins undergo an apparent increase of molecular weight of about 500 daltons when run in the non‐reduced form on SDS polyacrylamide gels under standard electrophoretic conditions, suggesting they contain a similar degree of intra‐chain disulphide bonding. Confirmatory evidence that the two components share a common polypeptide backbone is provided by the appearance of only the 20.5 kd component in lysates from SKOSC cells pulse‐labelled for 5 min in the presence of TM.