Periodic Reporting for period 1 - MOLECULAR MUCUS (Molecular Mechanisms for Construction of Protective Mucus Hydrogels)
Período documentado: 2023-05-01 hasta 2025-10-31
Though mucus is often considered an unpleasant product of the body, it is in fact a crucial element of the innate immune system, protecting exposed tissues from pathogens and other environmental hazards. On the macroscopic scale, mucus appears to be a sticky, amorphous substance. However, we hypothesize that, on the nanoscale, mucus hydrogels have an underlying molecular order that results from a complex and well-orchestrated bioassembly process. Understanding this process, i.e. the mechanisms by which robust, functional mucus is formed and maintained, will aid efforts to preserve the health of mucosal tissues in the gastrointestinal, respiratory, and reproductive tracts, and to restore them to homeostasis when damaged by disease.
The project MOLECULAR MUCUS addresses the mechanism by which “mucins,” the major glycoprotein components of mucus, polymerize and self-organize into three-dimensional, dynamic, and active hydrogels. In addition to providing practical information for the benefit of human health, revealing this mechanism contributes basic knowledge regarding the diversification of mucins during evolution to create mucus gels with the properties necessary to protect various tissue types and organs. Furthermore, the understanding we seek for how glycosylation (carbohydrate modification) affects mucin structural and interactive properties will shed light on how mucins could adapt in real time to acute environmental challenges. We aim to provide information that will eventually enable control of these putative rapid adaptations to enhance mucus barriers upon viral, bacterial, or fungal infection.
The project MOLECULAR MUCUS addresses the mechanism by which “mucins,” the major glycoprotein components of mucus, polymerize and self-organize into three-dimensional, dynamic, and active hydrogels. In addition to providing practical information for the benefit of human health, revealing this mechanism contributes basic knowledge regarding the diversification of mucins during evolution to create mucus gels with the properties necessary to protect various tissue types and organs. Furthermore, the understanding we seek for how glycosylation (carbohydrate modification) affects mucin structural and interactive properties will shed light on how mucins could adapt in real time to acute environmental challenges. We aim to provide information that will eventually enable control of these putative rapid adaptations to enhance mucus barriers upon viral, bacterial, or fungal infection.
As the first step of MOLECULAR MUCUS, we determined using cryo-electron microscopy the high-resolution structures of key segments of the respiratory mucins, MUC5B and MUC5AC. These structures supported the conclusion that secreted mucins share a fundamental mechanism for orchestrating intermolecular disulfide bonding to generate glycoprotein polymers. Specifically, key segments of all three mucins formed beaded filaments in vitro, at moderately low pH as found in the Golgi apparatus and secretory granules, consistent with the mechanism we discovered in the context of the intestinal mucin, MUC2, for aligning reactive cysteines at the center of the beads (Javitt et al., Cell 2020). However, we observed striking differences in the higher-order supramolecular assembly modes of the beaded filaments for the three mucins. We had previously observed that MUC2 forms isolated, elongated filaments (Javitt et al., Cell 2020). In the context of MOLECULAR MUCUS, we found that one of the lung mucins, MUC5AC, forms single or paired loose coils (Haberman et al., PNAS 2025), whereas the other lung mucin, MUC5B, forms bundles of elongated filaments (unpublished results). Analysis of the structural differences between the three mucins showed how slight changes in amino acid sequence during evolution dramatically altered supramolecular assembly, likely leading to differences in the properties of the respective mucus gels after secretion.
These structural studies revealed general principles of mucin organization. In particular, we identified three important regions of the mucins where diversification can be accommodated while preserving the fundamental polymerization mechanism: one is the packing angles between the domains that determine the relative orientation of adjacent beads in the filaments, the second is in segments known as CysD domains, which in some cases make intermolecular interactions stabilizing the beaded filament, and the third is the length of the first glycosylated, natively-disordered region of the mucins, which determines the reach of the adjacent CysD domain and directs its docking onto the beaded filament.
The two next steps of MOLECULAR MUCUS, which are already underway, are to determine the structural contribution of additional CysD domains, which are scattered along the lengths of the secreted mucin glycoproteins, and to quantify the extensibility of mucin glycosylated segments, which we hypothesize serve as “entropic spacers” between specific CysD adhesion domains.
These structural studies revealed general principles of mucin organization. In particular, we identified three important regions of the mucins where diversification can be accommodated while preserving the fundamental polymerization mechanism: one is the packing angles between the domains that determine the relative orientation of adjacent beads in the filaments, the second is in segments known as CysD domains, which in some cases make intermolecular interactions stabilizing the beaded filament, and the third is the length of the first glycosylated, natively-disordered region of the mucins, which determines the reach of the adjacent CysD domain and directs its docking onto the beaded filament.
The two next steps of MOLECULAR MUCUS, which are already underway, are to determine the structural contribution of additional CysD domains, which are scattered along the lengths of the secreted mucin glycoproteins, and to quantify the extensibility of mucin glycosylated segments, which we hypothesize serve as “entropic spacers” between specific CysD adhesion domains.
In the course of our research, we have broken ground in the study of mucus structure and assembly, but we have also identified a major need that must be met in the field. Knowledge of the structural and biophysical mechanisms of intracellular mucin polymer assembly is an important start, but what mucosal biologists and clinicians are truly seeking is a detailed, three-dimensional view of the molecular interactions that make up secreted mucus hydrogels. Scanning electron microscopy images of native dehydrated mucins in situ or in reconstituted mucus have been produced by multiple labs, but these images do not reveal the biophysical and biochemical basis of the structures observed, and therefore do not illuminate mucin construction principles. To ensure further impact and uptake, we must complement our detailed structural analyses of key mucin fragments with the development of novel methods to elucidate the three-dimensional organization, domain-domain interactions, and glycoprotein dynamics of native, intact mucin hydrogels.