Mucin and Autism

The featured image is from a 2010 publication by Hansson and Johansson [1] They did not know back then what causes the stratification of the loose layer that bacteria can live in versus the tight, inner layer that excludes bacteria. They did mention the important role of protein thiol disulfide crosslinks. This post will not go into the hypotheses that autism is caused by defects in the mitochondia that can be found in the PubMed literature.What if defects in the mitochondria of the mucus secreting goblet cells can oxidize these all important thiols before they can form the all important disulfides?

The origin of the hypothesis

The work of Arthur Krigsman (pediatric gastroenterologist) Stephen J Walker (research professor) came recommended as a way to understand the GI symptoms of autism spectrum of disorders. A 2021 review does a good job of summarizing a bewildering volume of literature. The below images came from Figures 2 and 3 of the publication. [3] I have taken the liberty of zooming in and out of the white dots, or plumes seen in the proximal small bowel. In these regions decapitated villi are seen.

In the text these white dots are described as being composed of fibrous material. [2] My first thought was to a previous post on this site and the cysteine rich domains of mucins. Where they oxidized. This is summary figure 4 of the Krigsman and Walker review. [2] Note the emphasis on immune cells and inflammation. According to Wikipedia authors myoperoxidase is a hypochlorous acid (bleach) producing enzyme secreted by neurophils. This heme containing enzyme is what gives mucus its green color.

The following are some highlights of this review that pertain to the development of the hypothesis

1. Mucosal villi… Krigsman and Walker think that the inflammation lower in the crypts results in loss of villus tips, that leads to introduction of more antigens, for more inflammation.  They also mentioned fibrous plugs that immediately makes one think of collagen, fibrin, and the usual assortment of matrix proteins.  Krigsman and Walker cite a reference claiming reduced expression of tight junction proteins in GI epithelial cells. 
2. Lamina propria, has a high level of T and B lymphocytes. Krigsman and Walker discuss the immune system being hyper responsive in ASD, etiology unknown.  There is a high presence of T and B cells with IgG colocalization with complement c1q on the basolateral enterocyte membrane that is suggestive of autoimmunity Pro-inflammatory cytokines are also mentioned. 
3. Basement membrane, is thickened and fibrous  Krigsman and Walker mention thickening that obstructs lymphatic flow that gave impression that they hypothesis that this thickening leads to lymphangiectasia.
4. Sub mucosa  The submucosa plexus is part of the enteric nervous system is in this region
5. Muscularus mucosa has a circular and longitudinal layers.  In between these two layers is the myenteric plexus that arises from the Vagus nerve.
6. Serosa contains fibrous and lymphoid tissue
7. Fibrous plume arising from truncated villi  NOTE: this is reported for the proximal small bowel, the duodenum, not the colon.  The disacchridases are probably irrelevant for the colon.  However, the “white plume” affords an interesting insight into what is going on in the colon. 

Mucin, again

This image is recycled from a previous post on the terminal electron acceptor of Akkermansia muciniphila. In the previous post I was mostly interested in cysteine as a source of sulfur as a terminal electron acceptor. Thedisulfide rich von Willibrand domain was of interest to Bäckström and coauthors. These domains are found in von Willibrand factor and many other proteins. Bäckström and coauthors pointed out that there are three vWF domains at the N-terminus of secreted mucins and on at the C-terminus. There is a cysteine knot at the extreme C-terminus of MUC2. [3] The cysteine knot (CK) is another disulfide bond rich motif, as can be seen in the original figure when mucin was only being considered as a place to dump electron transport chain electrons.

Bäckström and coauthors were more concerned with what make the secreted mucins gel like. They used an expression system in which various domains had MUC2 is a protein of 5200 amino acids. The secreted form lacked the transmembrane region and the cytoplasmic tail. It should be poimnted out that more abundant MUC2 has only two Cys rich domains whereas MUC5AC and MUC5B have many more. [3] The Bäckström review suggested that the C-termi are disulfide cross-linked to each other. The vWD form disulfide cross-linked trimers. The first of two Cys rich domains of MUC2 form non covalent dimers. These interactions are more driven by hydrophobic interactions. [3] These cross links are needed for proper secreation from the Golgi. [3] My question is what happens if these thiols become oxidized to cysteine sulfenic or sulfonic acid by reactive oxygen species from the mitochondria that Pocketdenist.com has drawn right next to where all of these disulfide bonds should be forming.

• If thiols responsible forming the “three red circles” VWD3 trimer are oxidized to Cys sulfinic and sulfonic acid before the disulfide bonds are formed, less gel like structure would be predicted.
• If the C-terminus inter-chain cysteine knot (CK) “two pink ovals” disulfides are also oxidized to sulfinic and sulfonic acids…. less gel like structure would be predicted
• And finally, if the CydD non-covalent dimer thiols are are oxidized with big, bulky oxygen groups….

Another collaborative study between French and Belgian investigators created a transgenic mouse that secreted a protein containing 12 tandem repeat cysteine repeat domains. [4] These thiols do not form disulfide bonds in their dimers. [3] The transgenic mice exhibited an increase in the thickness of the mucus layer, more resistance to chemically induced colitis, more colonization of Lactobacillus species, and other alterations in the intestinal microbiome. [4]

Microbiome and mucus insights

The bulk of feces of an individual may be comprised of only 40 species that are stable over the course of five years.  Numbers may be temporarily knocked down in response to environmental factors, but then bounce back to previous numbers. Bile acids, lower pH, and shorter transit times are some factors thought to account for decreased numbers in the small intestine.  Anti-microbial peptides are also a factor in the small intestine.[5]

Donaldson and coauthors tell us that the mucus layer in the small intestine is composed but of a single, tightly attached layer whereas the colon has both a loosely attached outer layer and a tightly attached inner layer. [5] This review also mentions a species wide difference the characteristics of mucus secreted by the proximal colon and further down. [5] Biofilms of mucus are also mentioned. The biochemical mechanisms were not discussed.

The Paone and Cani review [6] fill in some points missing from the other reviews [3,5] I’ve taken the liberty to enlarge some of the text I think is important so that it can be read on this post. The reader is invited to explore the online version of the actual publication and click on the image.

CCFTR, cystic fibrosis transmembrane conductance regulator, is a Cl- channel that allows flow of chloride anions into the lumen of the intestine (and airways). Cations and water follow allowing the mucus to hydrate. Mutations in CFTR prevent proper hydration of airway mucus in cystic fibrosis patients. As an aside, high levels of reactive oxygen species can cause disulfide cross linking of these Cysteine Rich domains and an increase in the elasticity of the mucin. [7] Perhaps any condition that results in reactive oxygen species from immune cells, mitochondria, and environmental toxins can set off a vicious cycle that results in a compromised mucus polymeric structure…. that influences the bacteria that come in….

References

1. Hansson GC, Johansson ME. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Gut Microbes. 2010 Jan;1(1):51-54. PMC free article
2. Krigsman A, Walker SJ. Gastrointestinal disease in children with autism spectrum disorders: Etiology or consequence? World J Psychiatry. 2021 Sep 19;11(9):605-618. PMC free article
3. Bäckström M, Ambort D, Thomsson E, Johansson ME, Hansson GC. Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains. Mol Biotechnol. 2013 Jun;54(2):250-6. PMC free article
4. Gouyer V, Dubuquoy L, Robbe-Masselot C, Neut C, Singer E, Plet S, Geboes K, Desreumaux P, Gottrand F, Desseyn JL. Delivery of a mucin domain enriched in cysteine residues strengthens the intestinal mucous barrier. Sci Rep. 2015 May 14;5:9577. PMC free article
5. Donaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol. 2016 Jan;14(1):20-32. PMC free article
6. Paone P, Cani PD. Mucus barrier, mucins and gut microbiota: the expected slimy partners? Gut. 2020 Dec;69(12):2232-2243. PMC free article
7. Yuan S, Hollinger M, Lachowicz-Scroggins ME, Kerr SC, Dunican EM, Daniel BM, Ghosh S, Erzurum SC, Willard B, Hazen SL, Huang X, Carrington SD, Oscarson S, Fahy JV. Oxidation increases mucin polymer cross-links to stiffen airway mucus gels. Sci Transl Med. 2015 Feb 25;7(276):276ra27. PMC free article