HUMAN MATRIX METALLOPROTEINASE-9 - ACTIVATION BY LIMITED TRYPSIN TREATMENT AND GENERATION OF MONOCLONAL-ANTIBODIES SPECIFIC FOR THE ACTIVATED FORM

For many studies on matrix metalloproteinases in immunohistochemistry it is important to be able to distinguish between the zymogen and acti vated forms of the enzymes. Activated human matrix metalloproteinase-9 (MMP-9, gelatinase B) was produced from the proenzyme by limited dige stion with trypsin. The products of cleavage were characterised by SDS /PAGE and N-terminal sequencing. Trypsin treatment led to a stepwise r emoval of the propeptide domain and also caused cleavage within the C- terminal domain. Monoclonal antibodies specific for the activated form of human MMP-9 were raised by using a peptide corresponding to the N- terminus of the activated enzyme as immunogen. The antibodies do not r ecognise the MMP-9 proenzyme or the active or proenzyme forms of matri x metalloproteinase-2 (MMP-2, gelatinase A) and do not react with unre lated proteins in an unfractionated tissue extract. The antibodies wer e used to detect, by immunohistochemistry, activated MMP-9 in formalin -fixed, wax-embedded sections from a series of oesophageal cancer case s previously shown to contain MMP-9. All of the tumours contained acti vated MMP-9 localised to tumour cells and macrophages. As the antibodi es are effective in immunohistochemistry on formalin-fixed, wax-embedd ed sections, they should prove useful for the detection of activated M MP-9 in various disease processes.