Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2–processing properties

Nyström, Eva; Arike, Liisa; Ehrencrona, Erik; Hansson, Gunnar C.; Johansson, Malin

doi:10.1074/jbc.ra119.009940

Cited by 39 publications

(28 citation statements)

References 43 publications

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“…As revealed by western blotting with an antibody to the murine mucin 2 (Muc2) D3 assembly, StcE cleavage reproducibly liberated colon Muc2 fragments similar in size to recombinant MUC2 D1D2D3 and to a greater extent D3 ( Figure 2 E), which were previously shown to be disulfide-bonded dimers ( Javitt et al., 2019 ). The appearance of a fragment close in size to dimeric D3 is consistent with the reported proteolysis of the mucin head between D2 and D3 ( Nyström et al., 2019 ).…”

Section: Resultssupporting

confidence: 85%

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Assembly Mechanism of Mucin and von Willebrand Factor Polymers

Javitt

Khmelnitsky

Albert

et al. 2020

Cell

100

111

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Summary The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

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Section: Resultssupporting

confidence: 85%

“…For clarity, D1/D2 assemblies are not shown. Analogous to the D1/D2 propeptide of VWF, D1/D2 may also be removed proteolytically from MUC2 ( Nyström et al., 2019 ). …”

Section: Discussionmentioning

confidence: 99%

Assembly Mechanism of Mucin and von Willebrand Factor Polymers

Javitt

Khmelnitsky

Albert

et al. 2020

Cell

100

111

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“…3A,B,E,F). GOB5, also known as Calcium-activated chloride channel regulator 1 (CLCA1), is a mucin-processing protein that is secreted with mucus 42 Consistent with previous studies 6,8,11 , the Eub signal is absent in the inner mucus layer closest to the epithelium (Fig. 3E,H, white arrow and Supp.…”

Section: Poloxamer Fixative Enables In Situ Analysis Of Colonic Microsupporting

confidence: 89%

Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

Macedonia

Drewes

Markham

et al. 2020

Preprint

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Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multi-modal analyses of both microbes and host cells in a spatially-resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatiallydefined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in Poloxamer-fixed samples. This in turn enabled us to integrate multi-modal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and crosslink at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.3 Introduction:

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“…3a, b, e, f, i, j ). GOB5, also known as calcium-activated chloride channel regulator 1 (CLCA1), is a mucin-processing protein that is secreted with mucus 42 . An intact mucus layer highlighted by antibody staining against GOB5 and WGA was observed with Carnoy’s solution, Methacarn, and Poloxamer fixative (Fig.…”

Section: Resultsmentioning

confidence: 99%

Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

Macedonia

Drewes

Markham

et al. 2020

npj Biofilms Microbiomes

View full text Add to dashboard Cite

Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

show abstract

Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2–processing properties

Cited by 39 publications

References 43 publications

Assembly Mechanism of Mucin and von Willebrand Factor Polymers

Assembly Mechanism of Mucin and von Willebrand Factor Polymers

Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

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