Screening and identification of novel B cell epitopes in human heparanase and their anti-invasion property for hepatocellular carcinoma.
BACKGROUND
The aim of this study was to screen and identify novel B cell epitopes within the human heparanase protein and to investigate the impact of self-developed anti-heparanase polypeptide antibodies on growth and invasion of HCCLM6 human hepatocellular carcinoma cells in vitro.


METHODS
The flexible regions of secondary structure and the B cell epitopes of the human heparanase amino acid sequence were predicted by DNAStar and Bcepred software.The multiple antigenic peptides (MAP) of the epitopes were synthesized in eight-branched form. Rabbits were immunized with the eight-branched MAPs mixed with the universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acid 163-171). The immunogenicity of the synthesized peptides was evaluated by ELISA, western blot and immunohistochemistry. The impact of the self-developed rabbit anti-heparanase polyclonal antibodies on growth and invasion ability of HCCMLM6 cells were analyzed in a cell culture model. The cells were first treated with one of the three antibodies, respectively, and then measured by using MTT, flow cytometry, plate clone formation, invasion assay and heparan sulfate degrading enzyme assay.


RESULTS
The three amino acid sequences 1-15 (MAP1), 279-293 (MAP2), and 175-189 (MAP3) in the large subunit of the human heparanase protein were predicted as its most potential epitopes. ELISA, western blot and immunohistochemistry analysis showed that all three MAPs were capable to induce high titer of serum antibodies. Antibodies induced by MAP1 and MAP2 were high specific. Furthermore, anti-MAP2 antibodies showed the strongest avidity towards liver cancer tissues. Under the treatment with the three anti-heparanase antibodies, respectively, the growth, cell cycle and clone formation of the cells remained unchanged when compared with a treatment with normal rabbit IgG. However, an inhibition of cell invasiveness and heparanase activity could be detected under the treatment with anti-MAP1- or anti-MAP2-antibody (with a terminal concentration of 100 mug/ml). The cell invasiveness was decreased by 54 and 38%, respectively, the heparanase activity by 43 and 39%, respectively.


CONCLUSIONS
The multiple antigenic peptides MAP1 (AC 1-15) and MAP2 (AC 279-293) may be the dominant B cell epitopes in the human heparanase protein. The induced polypeptide antibodies can effectively inhibit the heparanase activity of HCCLM6 liver cancer cells and therefore influence their invasion ability, which provides a theoretic basis for the development of anti-heparanase antibodies and their clinical use as vaccine.
Yang
Jian-min
JM
Department of Gastroenterology, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang Province, China. jianminyang@hotmail.com
Wang
Hui-ju
HJ
Du
Ling
L
Han
Xiao-mei
XM
Ye
Zai-yuan
ZY
Fang
Yong
Y
Tao
Hou-quan
HQ
Zhao
Zhong-sheng
ZS
Zhou
Yong-lie
YL
D000818:Animals; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion; D002453:Cell Cycle / Q000276:immunology; D002465:Cell Movement; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D005434:Flow Cytometry; D005966:Glucuronidase / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D008113:Liver Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D000099175:Heparanase
D018985:Epitopes, B-Lymphocyte; D005966:Glucuronidase; D010446:Peptide Fragments; D000099175:Heparanase