The molecular cloning and characterization of human heparanase cDNA and the immunochemical localization of heparanase in metastatic melanomas

Li Jin, The University of Texas Graduate School of Biomedical Sciences at Houston


Heparanase, an endo-$\beta$-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a M$\sb{\rm r}\sim 97,000$ protein upon SDS-polyacrylamide gel electrophoresis of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, metastatic human A375-SM and mouse B16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse organs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells, but not in adjacent normal tissues. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells, but not in adjacent connective tissues. Monoclonal antibodies directed against murine heparanase were developed and characterized. Monoclonal antibody 10E5, an IgM, precipitated and inhibitated the enzymatic activity of heparanase. A 2.6 kb cDNA was isolated from a human melanoma $\lambda$gt11 cDNA library using the monoclonal antibody 10E5. Heparan sulfate cleavage activity was detected in the lysogen lysates from E. Coli Y1089 infected with the $\lambda$gt11 cDNA and this activity was inhibited in the presence of 10-fold excess of heparin, a potent inhibitor of heparanase. The nucleotide sequence of the cDNA was determined and insignificant homology was found with the gene sequences currently known. The cDNA hybridized to a 3.2-3.4 kb mRNA in human A375 melanoma, WI-38 fibroblast, and THP-1 leukemia cells using Northern blots. Heparanase expression was examined using Western and Northern blots. In comparison to human A375-P melanoma cells, the quantity of 97,000 protein recognized by the polyclonal anti-heparanase antibodies doubled in the metastatic variant A375-SM cells and the quantity of 3.2-3.4 kb mRNA doubled in A375MetMix, a metastatic variant similar to A375-SM cells. In B16 murine melanoma cell, the intensity of the 97,000 protein increased more than 2 times comparing with B16-F1 cells. The extent in the increase of the protein and the mRNA levels is comparable to the change of heparanase activity observed in those cells. In summary, the studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; (c) heparanase antigens are localized in invasive and metastatic murine and human melanomas in vivo, but not in adjacent normal tissues; (d) heparanase molecule appeared to be differentially expressed at the transcriptional as well as at the translational level; and (e) the size of human heparanase mRNA is 3.2-3.4 kilobase.

Subject Area

Cellular biology|Molecular biology|Immunology

Recommended Citation

Jin, Li, "The molecular cloning and characterization of human heparanase cDNA and the immunochemical localization of heparanase in metastatic melanomas" (1992). Texas Medical Center Dissertations (via ProQuest). AAI9307237.