Monoclonal antibodies to human fibroblast procollagenase. Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the amino-terminal end of the activated enzyme

Abstract

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.

Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage.

“…MMP-1 and MMP-13 degradation of type II collagen. Activated MMP-1 (2.5 g) was added to tubes containing increasing amounts of human type II collagen (4,5,6,7,8,9,10,11, and 12 g) and buffer (50 mM Tris, pH 7.5, 10 mM CaCl 2 , 100 mM NaCl) in a total volume of 50 l. The tubes were incubated at 25ЊC for 15 min, then reducing sample was added and the samples were boiled. The samples were then separated on an 8% polyacrylamide gel, stained with Coomassie blue, and destained in water.…”

“…Recombinant human MMP-1 and MMP-13 were purified from the culture medium of Sf 9 cells infected with recombinant baculovirus. MMP-1 was affinity purified using a monoclonal antibody column using methods essentially as described by Birkedal-Hansen et al (9). Human MMP-13 was purified using a combination of heparin-agarose and SP-Sepharose Fast Flow columns.…”

Integrin Fibronectin Receptors in Matrix Metalloproteinase-1–Dependent Invasion by Breast Cancer and Mammary Epithelial Cells

“…The role of MMP-1 in PHSRN-induced invasion was tested on SF SU-ECM invasion substrates in the presence of 100 ng/mL PHSRN peptide and increasing concentrations of blocking COMY-4A2 anti-MMP-1 mAb (29). As shown in Fig.…”

Expression of collagenase and IL‐1α in developing rat hearts

“…A polyclonal anti-human collagenase (Pab) (Birkedal-Hansen et al, 1988) and a monoclonal anti-rat collagenase (Mab LIPCPPCI-IID1-1D10) were used for the detection of collagenase in the embryo sections a t concentrations of 12.5 pg/ml and 2.5 pg/ml, respectively.…”