New Monoclonal Antibodies to Non-Glycosylated Domains of the Secreted Mucins MUC5B and MUC7

Abstract

The separation and characterization of salivary mucins is not straightforward because of their large size, heterogeneity, and molecular interactions. The MUC5B and MUC7 mucins are major glycoprotein components of saliva that are thought to play a vital role in maintaining oral health. MUC5B is also a major component of respiratory mucus and is produced by the tracheal and bronchial glands, while MUC7 has a more limited pattern of expression in the bronchial tree. MUC5B is a gel-forming mucin and thus confers viscosity, whereas MUC7 is much smaller. MUC7 has anti-fungal activity, and both mucins interact with bacteria. The aim of this work was to produce new monoclonal antibodies that can be used to quantify and characterize these mucins by standard laboratory procedures. Peptide sequences in non-conserved and non-glycosylated regions were selected and monoclonal antibodies produced by an efficient immunization and cloning strategy, and screening against purified mucins. Three new antibodies-EU-MUC5Ba and EU-MUC5Bb (against MUC5B) and EU-MUC7a (against MUC7)-were isolated that do not show cross-reactivity with other gel-forming mucins. All work on immunohistochemistry can be used for semi-quantitative immunoblotting after agarose gel electrophoresis. These reagents are valuable tools to study changes in these mucins in oral and respiratory disease, and unlike other monoclonal antibodies to these mucins they recognize epitopes that are not affected by glycosylation.

Assay of mucins in human tear fluid

“…Our data using chemiluminescence detection and a newly developed antibody specific to a different region of human MUC5B (Rousseau et al, 2003) confirm our previous ELISA results that MUC5B protein, a mucin described in human lacrimal gland (Paulsen et al, 2004), is not detectable in human tears (Gipson et al, 2001), even when examining maximally loaded gels and 100-fold more total protein than necessary to detect MUC5B in a positive control secretion (purified human cervical mucin). Similarly, our use of 2 different MUC7-specific antibodies on maximally loaded gels, with 10-fold more total tear protein than necessary for detection in the control secretion (saliva), confirms the findings of Jumblatt et al (Jumblatt et al, 2003) that the small soluble mucin MUC7, which is produced by lacrimal gland epithelia, is not present in human tear film.…”

“…Their binding can be affected by posttranslational modifications (Rousseau et al, 2003) such as glycosylation or proteolysis. Thus, we used several antibodies per mucin (each recognizing a different epitope) on immunoblots of tear proteins pooled from up to 21 normal individuals to investigate the mucin's presence and whether the binding was to a protein of high molecular weight.…”

Salivary proteins as biomarkers in dental caries: In vivo study

“…Human saliva consists of atleast two distinct type of mucin; lower molecular weight MUC7 and high molecular weight MUC5B [28].…”

Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells

“…Specific antibodies were used as follows: monoclonal anti-human b-actin (1/1000, Sigma A5441), monoclonal anti-MUC2 (1/25, PMH1), monoclonal anti-MUC5AC (1/500, Novocastra 45M1, Le Perray, France), monoclonal anti-MUC5B (1/250, European consortium, BMH4-CT98-3222) (Rousseau et al, 2003) and monoclonal anti-MUC6 (1/10, CLH5). Secondary antibodies consisted of alkaline phosphatase-conjugated IgGs (Promega, for b-actin) or horseradish peroxidase-conjugated goat anti-mouse IgGs (Pierce, Perbio Sciences, Brebie`res, France, for mucins).…”