Monoclonal antibodies to human fibroblast procollagenase. Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme.
This study describes 11 monoclonal antibodies (Mabs) against human fibroblast collagenase that (i) inhibit the specific catalytic activity of the enzyme and/or (ii) react with one or more forms of the enzyme on Western blots. Each of the Mabs specifically immunoprecipitated the Mr 57,000/52,000 procollagenase from [35S]methionine-labeled culture medium. Five Mabs, designated VI-3, VI-4, 2C5, 4A2, and 7C2, inhibited the activity of fibroblast-type collagenase against soluble monomeric collagen and against reconstituted collagen fibrils but did not inhibit the genetically distinct human PMN leukocyte collagenase. The interstitial collagenase produced by human mucosal keratinocytes (SCC-25) was also inhibited, whereas the corresponding enzyme from rat was not. Assignment of epitopes to structural domains within the molecule based on immunoperoxidase staining of Western blots of collagenase and its autocatalytic fragments revealed that 9 of 11 epitopes, including those recognized by 4 inhibitory Mabs, were clustered in a 169-residue domain, which constitutes the NH2-terminal part of the Mr 46,000/42,000 active enzyme. One Mab (X-2a) specifically recognized the Mr 57,000/52,000 zymogen species and failed to react with the active Mr 46,000/42,000 form. The inhibitory Mab VI-3 was used for immunoaffinity purification of procollagenase from culture media with a recovery better than 80% and a yield of approximately 1.4 mg of enzyme/L of medium.
Birkedal-Hansen
B
B
Department of Oral Biology, University of Alabama, Birmingham 35294.
Moore
W G
WG
Taylor
R E
RE
Bhown
A S
AS
Birkedal-Hansen
H
H
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D017364:Collagenases; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification; D000939:Epitopes; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D000939:Epitopes; D010446:Peptide Fragments; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase