Macromolecular properties and polymeric structure of canine tracheal mucins

Abstract

Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.

Sulforhodamine B and exogenous surfactant effects on alveolar surface tension under acute respiratory distress syndrome conditions

“…Neither does Infasurf or SRB reduce T elevation by mucins, though Infasurf + ventilation appears close to having an effect. We speculate that the large, non-glycosylated hydrophobic domains of mucins (47,51) interfere with surfactant by hydrophobic interaction. For SRB there might, similarly, be a hydrophobic interaction between mucins and its xanthene rings (34).…”

Tracheal acid or surfactant instillation raises alveolar surface tension

“…Mucins have peptide cores with alternating anionic sialylated or sulfated glycosylated regions and bare hydrophobic cysteine-rich regions, and also cysteine-rich terminal regions (31,46,62,74,79). Disulfide bonding of terminal-region cysteines causes mucins to form oligomers.…”

Tailoring Formulations for Intranasal Nose-to-Brain Delivery: A Review on Architecture, Physico-Chemical Characteristics and Mucociliary Clearance of the Nasal Olfactory Mucosa

“…The cysteine region forms hydrophobic coils and it is thought to be the equivalent to the SEA domain in membrane bound mucins. Secreted mucins display several of those hydrophobic regions which are separated by recurring PTS domains [ 86 , 89 , 90 ]. The negative charge of glycans ensures hydrophilic properties of the mucins.…”