Published online Mar 8, 2017. doi: 10.4254/wjh.v9.i7.368
Peer-review started: November 23, 2016
First decision: December 15, 2016
Revised: December 19, 2016
Accepted: January 11, 2017
Article in press: January 14, 2017
Published online: March 8, 2017
To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan (HSPG), clone 1E4-1D9.
The antigen recognized by mAb 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3 (https://www.ebi.ac.uk/Tools/msa/clustalo/). The competitive reaction of mAb 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mAb 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mAb 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry.
Monoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by mAb 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350 (1478 amino acid) and gi60219551 (1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mAb 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of mAb 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti-glypican-3 could be detected by mAb 1E4-1D9 and vice versa. The recognition of antigens, on other solid tumor cell lines, by mAb 1E4-1D9 was studied. The results demonstrated that mAb 1E4-1D9 reacted with Huh7, HepG2, HT29, MCF7, SW620, Caco2, B16F1, U937, K562 and Molt4 cells. It was also found to be weakly positive to SW1353 and HL60 and negative to H460 and Hela cell lines.
All findings show that mAb 1E4-1D9 specifically recognizes glypican-3. Moreover, a new partner molecule of glypican-3, FYCO1 is proposed based on the results from co-precipitation studies.
Core tip: Heparan sulfate proteoglycan (HSPG) was isolated from human liver. Preliminary results showed that it was detected by rabbit anti-glypican. Monoclonal antibody, 1E4-1D9 was raised against human liver HSPG and its specific antigen was characterized. Amino acid sequence analysis revealed that the antigen recognized by mAb 1E4-1D9 specific molecule contained no transmembrane region. It has 15 cysteines and 11 putative glycosylation sites and 6 predicted N-glycosylation sites. The sequence matched to all PDZ domain proteins with an 85.6% match to glypican-3. Studies of co-expression and co-precipitation demonstrated that mAb 1E4-1D9 could compete with anti-glypican-3. It could also react with a various tumor cell lines including solid and hematopoietic cells. The findings suggested that the antigen recognized by 1E4-1D9 was glypican-3. Moreover, findings revealed that FYCO1 co-precipitated with glypican-3 using mAb 1E4-1D9, suggesting that FYCO1 is a partner molecule of glypican-3.