L4/5 Gut Mucosa Flashcards
What is mucus composed of
95% water
10% mucin glycoproteins
Siga
Proteins/lipids etc from dead cells
Explain the structure of mucin
Has a peptide core
On it has globular d domains with d1,2 and 3 on the n terminus before the vntr in the middle
C terminus has d4,b,c and cysteine knot ck on end
Which part is o glycosylated on ser/thr residues
Vntr
Why is mucin special
Can polymerise to form the gel part of mucus through disulfide bonding
Which mucin genes and what chr are part of the Mucins forming gel
11p15.5
Muc2(intestines)
Muc5ac
Muc6
Muc 5b
How is muc6 diff in structure
Has no d4,b or c on c terminal just has ck
Which protease can degrade these mucin polymers and into what
Pepsin
Into soluble glycoproteins which can’t form a gel
What are these further degraded by for their glycans
Gut commensals like b theta
Give a type of o-glycosylation
Galnac
What sort of cells line oesophagus
Non-keratinised stratified(many layers) Squamous epithelium (like mouth)
Give the 3 layers of these cells in the mucosa of oesophagus
Bottom basal layer above basal lamina is mitotic centre of the cells
Then prickle cell layer where cells spaced out with unknown glycoconjugate barrier
Then functional cell layer which can slowly slough/die and cells move to lumen of oesophagus
Is there a continuous layer of mucus in oesophagus and what releases it
No, only small amount released by submucosal gland
Why is it not needed
Because cells dying don’t need protected
Give a condition where excessive sloughing occurs
Sloughing esophagitis where acute or chronic inflam can occur if people get large fragments of squamous cells vomited or coughed up
What protects oesophagus from acid reflux
Hco secretion from both saliva and within esophageal cells
What 2 Mucins from saliva protect esophagus through antimicrobial mechanisms
Muc7 and muc5b
How is muc5b crucial in streptococcus mutans to prevent it from being antibiotic resistant
Muc5b glycosylated chains can prevent quorum signalling mechanisms of which can usually cause genetic transformation of bacteria into resistant
What is Barrett’s oesophagus
The change in terminal squamous cells of oe to intestinal like columnar cells with villi or gastric columnar cells both producing mucus usually not seen in oesophagus (intestinal / gastric meta plasia)
Is this precancerous
Yes
Which gastric mucosal columnar cells secrete mucus with mucins 5ac or 6
Apical (top of gastric pits) foveolar cells (5ac)
Or neck/base mucous secreting gland cells (6)
What else do foveolar secrete
Hco to neutralise gastric acid
What cells make gastric juice
Parietal cells produce hcl acid
Then pepsinogen released from chief cells then converted to pepsin (both in gastric pits)
What specialised cells release somatostatin to inhibit acid release for control
D cells
What do the cells in gastric pits sit above
Basal lamina and then below is the lamina propria (all part of gastric mucosa)
Explain the ph gradient on epi cell surface
In lumen of stomach ph is 1-2
HCO trapped by mucins near epithelial surface is 6-7 ph for protection
How does hcl and pepsin get out but can’t rediffuse back in through mucus to stop epi damage
At ph above 4 viscous fingers form which are channels of disrupted mucus to allow gastric juice to flow out helped by the hydrostatic pressure they cause in gastric glands
But when they reach lumen the low ph blocks reinvagination back = mucus protection
Which layer of mucus also helped block rediffusuon of acid back to epi cells
The firm inner layer of mucus which has polymeric mucin not the soluble outer layer because of the viscosity of the inner layer blocked acid flow
Explain gastric epithelial regeneration after ethanol damage as this freely diffuses through mucus (example of regeneration)
Dead cells and mucus will form a cohesive barrier with fibrin which is form converted fibrinogen released after damage
Also hco induced by prostaglandin released too which stops further acidic control
This barrier allows for new cells to migrate up gastric pits and sit on basal lamina to form new layer (stem cells from gastric pits)
How long does this reepitheliasation take
1 hour
Which type of aggressive pepsin replaces pepsin 3 as most abundant in gastric and duodenal peptic ulcer disease
Pepsin 1
What is this able to do
2fold greater mucin degradation in ph2 and 6 fold in ph4
Converts polymerised to soluble so reducing gel barrier
Molecular weight analysis in normal, gud or dud showed what % were soluble mucin forms
65% for gastric was soluble
35% for normal
50% for duodenal disease
Explain the pathogenicity associated with h pylori infection
Can cause gastritis and if chronic can lead to gastric cancer
Also involved in peptic ulceration
What % of cases of gastric cancer from pre-existing infection of hp
90%
Where does it get nutrients from
The tj disruption it can induce itself or through mild inflammation eg releasing albumin proteins
How does it survive under the mucus layer
It needs to be protected from ph2 so before it enters mucus layer it uses it’s urease activity which converts urea to ammonia neutralising acid
Why is more soluble mucin found in h+
Urease can degrade mucin through ammonium
Which part of stomach does h pylori colonise best and why
Antrum because few parietal cells so less acid
Which adhesin on hp binds mucin 5ac Lewis b or cell surface Lewis b (muc1)
BabA
What does this cause
Interaction in the mucin but also can induce t4ss to deliver virulent proteins into cell
Give example of virulent fsctor injected by t4ss
CagA which can disrupt ecadherin and bcatenin complex interactions disrupting tj between cells
What else can be injected in via t4ss that can activate nod1 and inflammation
Peptidoglycan (dapi) of hp
How does hp structure help it enter mucus
Has 5/6 polar flagellate which can rotate and enter viscous mucus
How is flagella not recognised on hp by tlr5 confusing the immune system
The n terminus is different
How is lps activation of tlr4 by hp also downregulated
Express ‘self’ Lewis x antigen on the lps
Whcih enzymes do they have which stops ros killing through detoxifying ros
Catalase and sod
How does hp aswell as mucus which impedes movement of neutrophils affect neutrophils directly
It downregulated neutrophil receptors for chemoattractant il8
COLONIC MICOSA L5
.
Where is protein core / backbone synthesised and then travel to for glycosylation
Er then through Golgi network for glycosylation
Give example of n glycosylation being important too eg fucosylation or sialic acid addition
Muc1 on all epithelial has n-glycans and which help folding
Where is the bilayer existing from gastric down the thickest and thinnest
Thinnest in small intestine
Thickest in colon
What is the first step in mucin polymerisation and packaging in secretory vesicles
At ph7.4
D3 trimer forms where 3 d3 domains form disulfide bonds with eachother
At this point the c terminal Ck and n terminal d1/d2 is sticking out
What does the ph drop to in Golgi and secretory granules
6 then 5.2 respectively
What else happens at this stage
Ca levels increase
What does the lower ph and ca increase help form
Non covalent bonds between the d1/d2 domains and new d3 domains so that there is a 6 side ring with 6d3 trimers at each corner
This is how it’s stored
What happens to these non covalent bonds when mucin is secreted and why
They are disrupted so little rings dissolve with no d1/2 and d3 interactions
Because ca is exchanged for na and ph rises again
What happens next
Ck domains will start to become hinges (ck diners form) where the d3 trimer at 6 corners are linked with ck domains
Hexagonal Net formation where the corners are d3 trimers and sides are ck dimer links
Causes expansion of mucin and uptake of water forming a gel
Expansion by what fold
1000-3000
How does akkermansia feed off outer layer of mucus in colon
Has a o-glycopeptidase A (pfam13402 containing)
Cleaves backbone off mucin and help utilise glycans
Give 3 structures that help bacteria bind Mucins when they live on outer layer
Fimbriae (ecoli)
Flagella
Adhesins (lactobacillus binds non specific glycans)
How is it suggested bacteria can induce this bilayer formation from inner to outer (explains why colon has thickest)
Butyrate production and uptake by colonocytes causes their boxidation
The co2 from this converted to HCO from carbonic anhydrase which increases the ph and precipitates calcium
This as we know allows stratification of Mucins as the d1/2 and d3 interaction disrupted = wider net
Explains why low HCO in cf causes aggregation of mucus on surface
Which tf does butyrate induce action of on muc2 promoter
Ap-1
How can tlr stimulation by flagella or lps cause mucin secretion from secretory vesicles of colonic crypt goblet cells
Downstream signalling through myd88 adaptor protein on tlr receptor and subsequent ros generation causes nlrp6 inflammasome activation which stimulates muc2 exocytosis
Explain the differences in bacteria eg in transcriptomics found by li 2015 in ecoli from colonise mice lumen or colonic mucus
Ecoli within the mucosal layer had adapted by increasing exp of enterobactin siderophore for better iron uptake for growth (low iron in mucus)
Also were found to increase proliferation rate from half life of 8 to 3hr to keep up with regeneration of mucus
Also since they can’t utilise mucinnglycans they use non specific carbon sources like phospholipids in the mucus layer
What other bacteria also were able to proliferate quicker in mucus layer and uprwgulsted Pul 78 for o-glycans and Pul 72 for n glycans
B theta
How is tolerance through lps kept in colon
Low exp levels of lps in colon
What did Shan 2013 hope to study
How muc2 in small intestine pp can induce dc to have anti inflammatory properties
How could dc from pp interact with glycosylated muc2 in combo with lps
Through galactin protein 3 on dc bind side chains
What sorts of cytokines were downregulated by this
Il8,il12, tnf
Which anti inflam cytokines were induced by the interaction
Il10 and tgfb = tregs
Which tf which blocks nfkb was upregualted by muc2 showing how it can modulate the cytokines
B catenin
What was the issue with the study
Used pig gastric mucin instead of muc2 human
Explain the effect wt shigella flexneri had on Mucins structure on surface and how this impacted its invasion ability
Wt flexneri was able to produce a lattice mucin layer on top of colonic epi cells which contained muc5ac in high amounts
It accumulated and was not able to spread from surface trapping the Mucins. As well as this wt flexneri was able to induce sulfation of the mucin side chains
Since it was found in vivo could invade cell better these mechanisms could be why
Why was this not backed up by expression effects the wt shigella had
It didn’t increase mucin exp eg of muc5ac only muc2 slightly. So doesn’t explain the large amounts of tight bound Mucins on surface
Where is 5b located (not down gi tract)
Airways
Where is susceptible to proteolysus by pepsin
Where glycosylation is sparse (same with iga) ie the protein cores
Explain the link between mucus shaping the Microbiota in uc
Loss of complex glycans, increases in sialyl rich rn antigen which increases strains with silaidade activity eg ecoli
Give the cross feeding interaction between hifidum and breve due to its exo sialidases
Can release sialic acid from mucin sialoglycans so that breve can then use the mucins
How often does gastric mucosa turnover occur
Every 72-96 hours
Why would nsaids cause PUD
Inhibition of cox1 so blocks production of protective prostaglandins (which usually if barrier injury will stimulate release of hco and mucus for repair)
Although babA binds Lewis B on muc5ac for interaction, how is muc1 a decoy
Also has it on cell surface and bc it’s c and n terminus are separated in er production it can be released to stop interactions with cells
How is disruption between b catenin and E cadherin also tumorigenic
B catenin sustains wnt signalling whcih causes continuous proliferation
How does toxin vacA stop an effective adaptive response
Downregulated of il2 to stop T cell effector proliferation
At what conc do mucins become gel
20mg/ml
Why are trefoil peptides important in mucus
Released can interact or cross link with mucins to help form a gel
In stomach found important for epithelial restitution after injury and mouse models with tff2 show resistance to nsaids damage
Why is ca important for mucin packaging in Golgi / secretory granules
Positive charge attracts negative mucin together to pack vs when drops in lumen the mucins will repel from eachother
Polymorphisms in which mucin are associated with stronger chronic gastritis by h pylori
Muc1 (steric hindrance and decoy binding are important )
In dss mouse model, which species of bacteria was able to reverse colitis via increased mucus thickness and tj expression
L. Reuteri (makes sense because of il22 effect)
Which disease has some mucin degrading r gnavus clades been associated with due to sialidases and fucosidases
Ibd
Which commensal has been shown to change glycosylation through fucosylatuon (fut2 enzyme) for their own nutrient advantage
B theta
Give a viral and bacterial example of adherence when there is too much fucosylation
Norovirus (associated with crohns) and salmonella
Which h pylori protein looks like iga proteases, induces inflam response but also blocks t helpers via il2 and DISRUPTS TJ (not adherens)
VacA
