Numerous
anti-mucin 1 (anti-MUC1) antibodies that recognize O-glycan core structures have already been developed. However,
most of them show low specificities toward O-glycan
structures and/or low affinity toward a monovalent epitope. In this
study, using an MUC1 glycopeptide library, we established two novel
anti-MUC1 monoclonal antibodies (1B2 and 12D10) with designed carbohydrate
specificities. Compared with previously reported anti-MUC1 antibodies,
1B2 and 12D10 showed quite different features regarding their specificities,
affinities, and reactivity profiles to various cell lines. Both antibodies
recognized specific O-glycan structures at the PDT*R
motif (the asterisk represents an O-glycosylation
site). 1B2 recognized O-glycans with an unsubstituted O-6 position of the GalNAc residue (Tn, T, and 23ST), whereas
12D10 recognized Neu5Ac at the same position (STn, 26ST, and dST).
Neither of them bound to glycopeptides with core 2 O-glycans that have GlcNAc at the O-6 position of
the GalNAc residue. Furthermore, 1B2 and 12D10 showed a strong binding
to not only native MUC1 but also 20-mer glycopeptide with a monovalent
epitope. These anti-MUC1 antibodies should thus become powerful tools
for biological studies on MUC1 O-glycan structures.
Furthermore, the strategy of using glycopeptide libraries should enable
the development of novel antibodies with predesigned O-glycan specificities.
Matrix metalloproteinase-13 (MMP-13) is important in the pathology of osteoarthritis (OA). Although MMP-13 is considered a therapeutic target for OA, it is unclear how MMP-13 activity is regulated by the system that comprises various proteinases and their inhibitors. MMP-13 neutralizing antibodies could be a useful tool for investigating the involvement of MMP-13 in cartilage degeneration in OA-affected joints because antibodies possess high affinity and specificity compared with low-molecular weight chemical compounds. On the basis of three-dimensional structure and amino acid sequence information on MMP-13, we selected an appropriate antigen peptide and generated a neutralizing antibody by immunizing mice with the antigen. The most significant property of monoclonal antibody 14D10 was the specific binding to the active form of MMP-13, but not to the latent form, or other MMPs. With this property, active MMP-13 was measured selectively by an enzyme-linked immunosorbet assay. Furthermore, 14D10 suppressed the cleavage of type II collagen in human articular chondrocyte cultures, and 14D10 is thought to inhibit MMP-13 activity effectively. Thus, the highly selective MMP-13 neutralizing antibody (14D10) might be a useful tool for investigating the mechanism of type II collagen degradation in arthritic pathology.
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