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 activated forms of the enzymes. Activated human matrix metalloproteinase-9 (MMP-9, gelatinase B) was produced from the proenzyme by limited digestion with trypsin. The products of cleavage were characterised by SDS/PAGE and N-terminal sequencing. Trypsin treatment led to a stepwise removal 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 recognise the MMP-9 proenzyme or the active or proenzyme forms of matrix metalloproteinase-2 (MMP-2, gelatinase A) and do not react with unrelated proteins in an unfractionated tissue extract. The antibodies were used to detect, by immunohistochemistry, activated MMP-9 in formalin-fixed, wax-embedded sections from a series of oesophageal cancer cases previously shown to contain MMP-9. All of the tumours contained activated MMP-9 localised to tumour cells and macrophages. As the antibodies are effective in immunohistochemistry on formalin-fixed, wax-embedded sections, they should prove useful for the detection of activated MMP-9 in various disease processes.
Duncan
M E
ME
Department of Molecular and Cell Biology, University of Aberdeen, UK.
Richardson
J P
JP
Murray
G I
GI
Melvin
W T
WT
Fothergill
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D017364:Collagenases / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D004938:Esophageal Neoplasms / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D020780:Matrix Metalloproteinase 9; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014357:Trypsin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D014357:Trypsin; D017364:Collagenases; D020780:Matrix Metalloproteinase 9