Heparan Sulfate Antibodies
10E4, 3G10, and JM403
Heparan sulfate is a highly sulphated polysaccharide, synthesized as the glycosaminoglycan component of heparan sulfate proteoglycans (HSPGs). It is widely distributed on the cell surfaces and basement membranes in mammals. It is involved in important biological processes due to it displaying specific interactions with many biologically active proteins. This includes examining of the dynamic distribution of HSPG in tissues and analysing HS structures for the elucidation of HSPG biological functions.
How 10E4, JM403, 3G10 antibodies and heparinase III enzymes work together
Clones 10E4 and JM403 recognize common epitopes on heparan sulfate (HS), which can be found on both basement membrane and cell-associated HS species. The 3G10 clone recognizes a neo-epitope exposed by digestion with heparinase III (HSase I) enzyme as seen in figure 3 (which destroys reactivity to 10E4 and JM403); so that the 3G10 clone can be used as control to 10E4 or JM403 (and vice versa).
The monoclonal antibody 10E4 (F58-10E4 clone), which recognises the 10E4 epitope containing N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. The 10E4 epitope also contains N-sulfated residues that are critical for the reactivity of the F58-10E4 antibody. The reactivity of heparan sulfate with this antibody is nearly nullified after the glycosaminoglycan has been treated with bacterial heparatinase I (Heparinase III) from Flavobacterium heparinum (EC 22.214.171.124). The 10E4 antibody does not react with hyaluronan, chondroitin sulfate, dermatan sulfate, keratan sulfate or DNA.
Examples of 10E4 Antibody Applications:
Anti-Heparan sulfate antibody (F69-3G10 clone) reacts with heparan sulfate neo-epitope 3G10, generated by digesting heparan sulfate with heparitinase I (Heparinase III) from Flavobacterium heparinum (EC 126.96.36.199). The desaturated hexuronate (glucuronate) located at the non-reducing end of the heparan sulfate fragments created by the enzyme is critical for the reactivity of the antibody. The 3G10 antibody does not react with intact heparan sulfate, oligosaccharides generated from chondroitin sulfates with bacterial chondroitinase ABC or AC, or generated from heparan sulfate with heparinase (Heparinase I): EC 188.8.131.52). There is only one 3G10 epitope on the HS stub which can only be recognized by one antibody at a time which allows these to be utilized as a tool for measuring how many chains are present per cell.
Heparan sulfate antibody (clone JM403) reacts with an epitope present in many types of heparan sulfate. The JM403 epitope includes (an) N-unsubstituted glucosamine residue(s) that are critical for the reactivity of the antibody. The reactivity of the JM403 antibody with most heparan sulfates is nearly completely abolished after treatment of the glycosaminoglycan with bacterial Heparitinase I (Heparinase III) from Flavobacterium heparinum (EC 184.108.40.206). The JM403 antibody does not react with heparin, hyaluronan, chondroitin sulfate, dermatan sulfate and keratan sulfate.