Cancer cells release a covalent complex containing disulfide-linked domains from urinary plasminogen activator, neural cell adhesion molecule, and haptoglobin alpha and beta chains.
We have previously reported on the secretion of a family of high Mr plasminogen activators (PAs) by a human lung cancer cell line [Harvey et al. (1991) Biochim. Biophys. Acta 1078, 360-368]. We have now extended these studies to several human cancer cell lines and a human embryonic lung cell line. In the present study with HPL-SK-1 lung cancer, A431 epidermoid cancer, ovarian carcinoma, and embryonic lung cell lines, we show that the 900- and the 660-kDa PAs are disulfide-bonded multiprotein oligomeric complexes. They are functionally and immunologically related to human urinary PA (uPA). Their size and PA activity are not destroyed by strong denaturants such as 8 M urea or 2% sodium dodecyl sulfate (SDS), suggesting that the uPA moiety is covalently associated with the rest of the molecule. It is only under strong denaturing conditions with 1.4 M beta-mercaptoethanol and 2% SDS that the uPA moiety could be released as a 21- to 23-kDa fragment along with two major polypeptide chains of 70 and 40 kDa, respectively. The presence of the uPA active center in the reduced PA660 was demonstrated by [3H]diisopropylphosphorofluoridate labeling and by Western blot using a monoclonal antibody to uPA B chain. N-terminal amino acid sequencing of the 70- and 40-kDa polypeptides, respectively, showed homology to the neural cell adhesion molecule and the beta chain of haptoglobin. A minor fragment of 18 kDa obtained under strong reduction conditions was also sequenced and shown to share homology with the alpha chain of haptoglobin. Western blot analysis of the reduced PAs with monoclonal antibody to the neural cell adhesion molecule and rabbit anti-haptoglobin confirmed the homologies obtained by the sequence data. Further, immobilized monoclonal antibodies to the neural cell adhesion molecule, uPA B chain, and rabbit anti-haptoglobin bound the multiprotein complexes with uPA activity, from A431, ovarian cancer, and embryonic lung cell lines. The bound material, after dissociation, exhibited PA activity that was inhibited by monoclonal antibody to the uPA B chain. These data suggest that in tumor and embryonal cell lines, in addition to proper folding and assembly of proteins by intramolecular disulfide bond formation in the endomembrane compartment, intermolecular disulfide bonds could also occur, producing multiprotein oligomers as in the present case. Formation of such oligomers may have a selective advantage for such cells in the focalization of proteolytic activity through the interaction of the neural cell adhesion molecule domain with the extracellular matrix and in immunosuppression of lymphocytes by the haptoglobin portion of the complex.
Harvey
S R
SR
Department of Biological Sciences, State University of New York at Buffalo, 14260, USA.
Nayak
S K
SK
Markus
G
G
Ouhammouch
M
M
Hemperly
J J
JJ
Dillman
R O
RO
Doyle
D J
DJ
D001665:Binding Sites; D015153:Blotting, Western; D002277:Carcinoma / Q000378:metabolism; D015816:Cell Adhesion Molecules, Neuronal / Q000032:analysis* / Q000378:metabolism; D002846:Chromatography, Affinity; D004220:Disulfides / Q000032:analysis; D004795:Enzyme Stability; D005260:Female; D006242:Haptoglobins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D008875:Middle Aged; D009369:Neoplasms / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000378:metabolism; D010960:Plasminogen Activators / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017421:Sequence Analysis; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000378:metabolism
D015816:Cell Adhesion Molecules, Neuronal; D004220:Disulfides; D006242:Haptoglobins; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator