Actinia Tenebrosa: A Study Of Cnidarian Radial Symmetry And Mucus Immune Defense Mechanism

Characteristics of Actinia tenebrosa

Question:

Discuss about the Structure and Assembly of Secreted Mucins.

This coral reef cnidarian is radially symmetrical around the axis that is formed around the mouth, or the oral axis and the opposite end which is the aboral axis or the base. It has an opportunistic feeding habit and its radial symmetry allows it to detect prey and predators from any direction. It has a polyp form that has a cylindrical shape and its tentacles are arranged around the mouth. The mouth is surrounded by tentacles that are arranged in six circles.

The Cnidarians are radially symmetrical animals and the body develops from only two germinal layers, the inner endoderm and the outer ectoderm that are bound by a mesogleal, non-cellular matrix (Frazão, Vasconcelos, & Antunes, 2012). So, cnidarians are essentially diploblastic animals (Van der Burg, Prentis, Surm, & Pavasovic, 2016). The body is shaped like a tubular column that is red to brown in color, the tentacles are tapered and blood red in color (Stabili, Schirosi, Parisi, Piraino, & Cammarata, 2015). Apart from the function of feeding the tentacles have nematocysts that can paralyse the prey before it pushed into the mouth by the tentacles. Acrorhagi are blue colored structures that lie at the base of each tentacle and these play a role in offense (Harris, 1990).

When food is added to water surrounding , Actinia tenebrosa, pre-feeding behaviour can be observed. It is possible to find that some of the sessile polyps do not accept food or do not exhibit any pre-feeding behaviour. On the contrary they may close the oral opening completely when the food comes in contact with their tentacles. This is likely to happen in case of recent feeding or when embryos are being incubated in the coelenteron. On the contrary when Actinia tenebrosa are ready to feed, their tentacles spread out completely as the mouth to the oral cavity opens partially. The oral disc expands completely and a ridged actinopharynx can be observed as it protrudes to the exterior (Harris, 1990).

Actinia tenebrosa is an intertidal sea anemone usually is found attached to rocks and if it finds that the location that it is tethered to is unsuitable in some way, it can move to another location, albeit slowly. Fresh specimens when collected from the sea shore and put into an aquarium prefer to attach the base at some support. If the anemone happens to lie by its side, its substratum expands until it can touch base and the it stands erect again. But the process occurs very slowly and may take a few hours to upto two days. It also tends to move away from a source of light, when moving the column protrudes in the direction of the movement and the base is pulled by a millimeter or two in that direction due to the contraction of the basal muscles. Thus, Actinia tenebrosa can exhibit complex behaviour in response to light (Harris, 1990).

Radial Symmetry and opportunistic feeding habit

Mucus plays an important role in development of an innate immune response in cnidarians. Mucus has a lysozyme like activity which can help in killing bacteria. It creates a physical barrier between the lumen and the epithelial lining (Van der Burg, Prentis, Surm, & Pavasovic, 2016).  The typically sedentary habit of the actinarians and the lack of a protective outer covering, such as a shell, an exoskeleton or a cuticle keeps these animals exposed to a variety of biotic and abiotic threats in their ecosystem that includes microbes that can be pathogenic. The evolution of an immune defence mechanism that keeps these organisms protected consists of the antimicrobial peptides that are a component of the mucous secretions (Otero Gonzalez, et al., 2010).  Pattern recognition receptors work with the glycocalyx and strengthen the physicochemical barrier that keeps the organism protected. In Actinarians, the mucus is also known to possess cytotoxic activity and hemolytic activity (Stabili, Schirosi, Parisi, Piraino, & Cammarata, 2015). Together, these activities help the organisms to protect themselves throughhumoral immunity from microbial pathogens (Clare, 1995).

The mucin proteins are multi-domain proteins and may be either secreted mucins that are gel forming or membrane bound and their domain architectures are very different from one another. Mucins are known to play roles in important biological functions. Mucins can act as ligands for adhesion molecules, growth factors, lectins, chemokines and cytokines (Fong, et al., 2000). Mucin can bind water and thus determine the state of hydration. Mucins block the passage of bacteria and larger molecules and become a barrier for infectious organisms  (Pelaseyed, et al., 2014). Mucins have negatively charged oligosaccharides and so can retain positively charged particles when required (Becher, Waldorf, Merete, & Uldbjerg, 2009). Mucins play roles in several other biological functions, regulation of gene expression, signal transduction, cell proliferation, cell differentiation, embryogenesis, immunity and apoptosis. Mucins also play a role in carcinogenesis (Becher, Waldorf, Merete, & Uldbjerg, 2009).

Secreted mucins contain several disulfide rich domains. The PTS tandem repeat domain is found in all mucins, the secreted and the membrane bound mucins. It is a serine, threonine and proline rich domain that carries the O-linked glycans. The signal sequence at the amino terminus is also found in all mucins and both the secreted and membrane bound mucins feature a signal peptide. It helps the transport of mucins to the endoplasmic reticulum and guides the insertion of the mucin into the unit membrane for secretion or insertion into membrane.

Cnidarian immunity: Mucus as a defense mechanism

The membrane associated mucins have a domain called the cytoplasmic tail which lies on the cytoplasmic side of the cell membrane and is likely involved in cell signalling. The SEA domain is also found in the membrane tethered mucins. This domain has protein binding property and has an autocatalytic cleavage site. The EGFdomains mediate interaction between different mucin subunits and the ERBB receptors. The transmembrane domain spans the membrane (Corfield, 2014).

The Cysteine rich domains, the Cysteine knot domain and the von Willebrand factor D domain are the structural features of the secreted mucins. The CYS domains are non-glycosated and multi-copy domains and lie between the tandem repeats, these perform the function of mucin-mucin interaction. The cysteine kno is a domain that is involved in dimerization and the von Willebrand factor D is a domain that mediates oligomerization at the C-terminus (Corfield, 2014).

Secreted mucins are those that are secreted by cells such as the goblet cells while the transmembrane mucins remain bound to the plasma membrane Secreted mucins are either the gel forming mucins or soluble mucins. The insoluble type of mucins are usually the gel forming mucins that include MUC2, MUC5AC, MUC5B, MUC6 while MUC19. MUC7, MUC8 and MUC9 belong to the category of soluble mucins (Rachagani, Torres, Moniaux, & Batra, 2009). On an evolutionary scale, the gel-forming mucins evolved before the membrane bound mucins. The location of their genes on chromosomes also gives clues to the evolutionary distance between the two types of secreted mucins (Gosalia, Leir, & Harris, 2013).  The differences also become evident when analysing the structure of their domains, their biosynthetic precursors and the posttranslational modifications of their polypeptide chains. The tandem repeats found in their domains and their functions point to the fact that they belong to the same family of proteins (Zaretsky & Wreschner, 2013).

Mucins are glycosylated proteins of large size and are essential components of the mucus that covers epithelial cells. Xenopus tropicalis has 26 mucin genes while most vertebrates have 5-6 mucin genes. Mucins form gel like structures and are provide protection to the epithelium from harmful substances and microorganisms (Lang, et al., 2016).

In corals, the mucocytes secrete the mucus and the chemical nature is sulphated glycoprotein polymers. The mucus in cnidarians performs the functions of adhesion, reducing drag, acts as a 'scaffolding' and aid in anchoring and protection of eggs, reduce loss of water and provide protection from infections, desiccation and several stressors from the environment (Stabili, Schirosi, Parisi, Piraino, & Cammarata, 2015).  The gel forming secreted mucins evolved in the metazoans and are found in all animals. Only insects do not have secreted mucins. The MUC proteins are numbered according to the sequence of their discovery. Secreted mucins are components of the mucous that coats the epithelial lining of the digestive tract, the respiratory passage and the urogenital tracts, in some amphibia, the moist skin is also coated with mucus (Lang, Alexandersson, Hansson, & Samuelsson, 2004). The presence of mucins in the mucous offers protection to the epithelium from desiccation, infection, physical or even chemical injury. Its lubricant effects helps the passage of material through the tracts. In higher animals, such as, vertebrates, lubrication and protection of the epithelial lining are the main functions of mucin. But in lower animals, such as, cnidarians, locomotion, mating, homing and capture of prey or food are the main functions of mucus (Denny, 1989).

Multidomain structure of mucins

Even within an organism, a variety of mucins are secreted, which are structurally different from each other. At times, one type of mucin may be produced by more than one organ in an organism. The glycoprotein mucin may differ in size from a few hundred amino acids long to thousands of residues in length (Perez-Vilar & Hill, 1999). Mucins are rich in Serine, threonine , proline,  aspartate, glutamate and glycine residues and form one or more domains that attach the O-linked glycosides and are made of tandem repeats (Goffredo & Dubinsky, 2016). The dense arrangement of the oligosaccharides protects the mucin from proteolysis and keep the molecules in an extended conformation (Lang, Alexandersson, Hansson, & Samuelsson, 2004).

Since mucins are proteins their biosynthesis follows the general pathway of gene expression. And since the mucin glycoproteins are expressed by the goblet cells, the activated transcription factors cause an upregulation of the gene expression of MUC genes in the nucleus, where the activation of gene transcription leads to synthesis of mRNA. mRNA is then spiced and translated into the polypeptide which undergo the process of post-translational modification, and co-translational insertion into the endoplasmic reticulum. The O-linked glycosylation of the polypeptide backbone of mucin occurs in the Golgi bodies (Hanisch, 2001).  The enzyme N-acetyl galactosaminylpeptidyltransferase catalyses the transfer of GalNac to serine or threonine. The glycosylation causes the shape of the molecule to change from globular to linear. The completely glycosylated mucin molecules are stored in secretory granules and are secreted to the cell surface when required. The membrane tethered mucins get inserted into the membrane with the insertion of the transmembrane domain. The addition of the oligosaccharides to the polypeptide, during glycosylation, followed by folding into one or more domains and finally secretion into the lumen of the gut or airways through the goblet cells. The biosynthesis of the mucins, like all other proteins is preceded by the activation or up-regulation of mucin gene/s expression (Rose & Voynow, 2006).

Structures of mucins were elucidated by cloning and purifying mucins (Albone, Hagen, VanWuyckhuyse, & Tabak, 1994). The mucin proteins fold into certain characteristic domains, one of these is the von Willebrand D or the VWD domain. A domain architecture is encountered when studying the structures of MUC5AC, Muc5B and MUC2 that has the domains (VWD-C8-TIL)-( VWD-C8-TIL)-( VWD-C8-TIL)-PTS-( VWD-C8-TIL). (The C8 domain stands for eight conserved cysteines and TIL stands for "trypsin inhibitor-like cysteine rich domain). A similar domain structure is found in the mucin proteins MUC6 and MUC19except that these two mucins are devoid of the VWD-C8-TIL at the C-terminal. while the CysD domain is found in MUC2 or MUC5 type mucins. Typically mucins have a cystine knot at the C-terminal, while some mucins have a VWF domain. (Lang, et al., 2016).

Biosynthesis of mucins

The stimulation of gene expression of mucin genes can occur through inflammatory cytokines, such as tumor necrosis factor α, interleukins, interferons or cytokines, bacterial products, growth factors, environmental pollutants or chemicals or contact with miscellaneous chemicals that trigger intracellular signalling in the cells to active the gene expression of mucins (Thai, Loukoianov, Wachi, & Wu, 2008).

In several diseases that involve the respiratory airways, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease, hyper secretion of mucus is a common symptom (Angelis, et al., 2014). The inflammation of the epithelial linings and altered production of mucus in the airways is also a common symptom in asthma patients (Holgate, 2007). Increased expression of the mucin gene or genes is a natural precursor of increased mucus secretion.  Cytokines Interleukin -4, IL-9 and IL-13 have been associated with the diseases that involve airway inflammation (Schuijs, Willart, Hammad, & Lambrecht, 2013). When interleukins-1α, 1β,  2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18 and TNF α were used to treat cultures of human epithelial cells of the tracheo-bronchial region, it was found that mucin genes, MUC5B and MUC5AC could be stimulated by IL-6 and IL-17, while IL-4, IL-9 and IL-13 did not stimulate the expression of the mucin genes. The ERK signalling pathway played a role in stimulating the gene expression of the tested mucin genes (Chen, et al., 2003).

Location of the secreted mucins of the gel forming type occur at several locations. The MUC2 gel-forming mucin is expressed in jejunum colon, ileum and endometrium. The MUC5AC is secreted in the respiratory tract, stomach, conjuctiva, endocervix and the endometrium (Ho, et al., 2004). The MUC5B is expressed in the respiratory tract, endocervix and in the submandibular glands (Thornton, Howard, Khan, & Sheehan, 1997). The mucin MUC6 forms part of the mucus in the stomach, ileum, gall bladder, endometrium and endocervix (Ho, et al., 2004). Among the non-gel-forming secreted mucins, MUC7 is expressed in the sublingual and submandibular glands while the mucin MUC8 is secreted in the respiratory tract, the endocervix, the endometrium and the uterus. MUC9 is expressed in the fallopian tubes.

As the name suggests the transmembrane mucin family includes mucins that remain bound to the membrane. The membrane bound mucins may have splice variants that may get secreted or these may undergo post translational processing that renders the mucin domains cleaved from the domains that are transmembrane in location. Examples of membrane bound mucins are MUC1, MUC4 AND MUC16 (Govindarajan & Gipson, 2010).

The generic structure of all transmembrane mucins has a large extracellular domain which has tandem repeat motifs that bind the O-linked oligosaccharides, a short domain that is transmembrane in location and a short cytoplasmic tail. The signal sequence at the N-terminal is encoded by mucin mRNA while translation occurs in the rough endoplasmic reticulum. For the addition of the oligosaccharides through glycosylation, the mucins are processed in the Golgi bodies. The oligosaccharides contribute to mucin functions of lubrication, anti-adhesion and hydration. If sialic acid and sulfation modifications are found, those can be associated with adhesion-promoting motifs and might even provide a surface for bacterial binding (Chaturvedi, Singh, & Batra, 2008). The domains that are characteristic of the membrane bound mucins include the SEA (Sea urchin sperm protein, enterokinase, agrin) domain (with the exception of MUC4), the epithelial growth factor like domain, the glycosylated internal repeat domain, von Willebrand factor domain, AMOP adhesion associated domain, PDZ binding motif and the NIDO domain (Van Putten & Strijbis, 2017).

The receptor like structure of the membrane bound mucins is similar to that of innate immune receptors. The extracellular domains perform barrier functions while the intracellular cytoplasmic domains can perform the function of signal transduction once they are phosphorylated. Proteolysis can lead to the shedding of the extracellular domain if they bind to bacteria and send signals for phosphorylation of the cytoplasmic domains. These changes can alter the cellular response to epithelial cell adhesion, inflammatory response, apoptosis and differentiation (Van Putten & Strijbis, 2017).

The transmembrane mucins are expressed on the surface of goblet cells and epithelial cells of the stomach the intestines, the respiratory system, the lacrimal and salivary glands, pancreas, gall bladder, liver and they differ among themselves based on the number of domains and the length. These proteins protect the epidermal surface from invasion by the pathogens and maintain the mucosal barrier function (Van Putten & Strijbis, 2017). The SEA domain may have several functions but one of them is the ability to protect the epithelial cells from mechanical force and acts as a cell protective device (Pelaseyed, et al., 2013). The shedding of the extracellular domains of mucins in the serum, intestinal lumen and in supernatants of cell cultures on further investigation has proved to be indicative of diseases such as inflammatory bowel disease (Buisine, et al., 2001), metastatic carcinoma (Smorodinsky, et al., 1996) and cystic fibrosis (Khatri, Ho, Specian, & Forstner, 2001). Transmembrane mucins are known to be overexpressed in various cancers, cystic fibrosis and even asthma. The domains in the mucins appear to play some role in cancer pathogenesis. The effects of mucins affect cell survival by altering the processes of cell growth, cell proliferation, apoptosis and autophagy. The intracellular domain of the MUC1 mucin serves a s a surface for the binding of signalling proteins (Bafna, Kaur, & Batra, 2010).

Membrane anchored mucins that are expressed are MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC20 AND MUC21, MUC1 is expressed ubiquitously while MUC16 is the largest protein in size. MUC 3, MUC12 and MUC13 are expressed in the gut epithelium while MUC1 and MUC16 are expressed in the epithelia of the female reproductive tract, the respiratory tract and the ocular surface (Gipson, Spurr-Michaud, Tisdale, & Menon, 2014). MUC16 is a large mucin that contains 22000 amino acids and is over expressed in ovarian cancer and some other types of cancer and is referred to as the CA125, which is often used to diagnose the cancer. The stimulation of expression occurs due to activation by interferon γ and tumor necrosis factor α. The over expression also occurs in breast and endometrial cancers (Morgado, Sutton, Simmons, Warren, & Lu, 2016).

The EGFdomains that occur as one of the extracellular domains in membrane bound mucins may interact with EGF receptors and thereby play a role in receptor signalling (Carraway, Ramsauer, Haq, & Carraway, 2003). The release of EGF domains perhaps has a role for them at a distant site, as in the case of cytokines. The mucin domains therefore communicate with the cellular environment within and outside the cells (Carraway, Ramsauer, Haq, & Carraway, 2003).

The regulation of gene expression of mucin genes occurs through the activation of MAPK pathways via cytokines, lipopolysaccharide from Gram negative bacteria and lipid constituents from Gram positive bacteria. Most of these pathways activate the NFκB upregulation (Thai, Loukoianov, Wachi, & Wu, 2008).  The Sp1transcription factors may also upregulate transcription of mucin genes (Thai, Loukoianov, Wachi, & Wu, 2008).

The expression of the membrane bound mucins occurs at several locations in the body. MUC1 is expressed in the breast duodenum, ileum, pancreas, colo, trachae, bronchii, cornea, conjunctiva, fallopian tubes, uterus, endometrium, endocervix, ectocervix and the  vagina (Corfield, 2014). MUC3A/B is associated with cell membranes in the small intestine, the colon and the gall bladder. The mucin MUC4 is expressed in the breast, the respiratory tract, small intestine, the colon, the conjunctiva, the cornea, the ectocervix, the vagina and the endometrium. MUC12 is expressed on the epithelial cell membranes of colon, pancreas, prostte and uterus. MUC13 is associated with the colon, kidney, trachaea and the small intestine (Corfield, 2014). MUC15 is expressed as a membrane tethered mucin in the colon, respiratory tract, small intestine and prostate. MUC16 is expressed in the ovary, cornea, conjunctiva, respitory tract and the endometrium (Corfield, 2014). the several functions of the mucus are inpart fulfilled by the mucins at different locations in the body.

Mucins can have applied uses as diagnostic markers, applications in immuno-therapy and gene therapy as delivery vehicles. Mucins have potential applications in the design of drug delivery systems, although mucin acts as a natural barrier in drug delivery because it can reduce drug permeation. Mucin's property of adhesion can be used in drug delivery systems, such as in design of encapsulated microspheres, or in nanoparticle based delivery. It can find applications in the design of mucoadhesive patches, gels or tablets.

It may be possible to utilize mucin networks for immobilization of nanomaterials or change the manner of particulate transfer because of its own properties of adhesion or employing the steric forces to use. If drug transmit time in the gastro intestinal tract is to be increased, mucin can be used effectively. If mucoadhesive polymers, such as, poly vinyl pyrrolidone, carboxymethyl cellulose or chitosan are used in conjunction with mucin, the residence time of drugs can be increased and this can enhance the chances of drug absorption (Authimoolam & Dziubla, 2016).

The role of mucin application in tissue engineering has also been explored by some researchers. Mucin can actually function as a functional scaffold because mucin has been found to associate with lectins and selectins,both carbohydrate binding proteins. At the time of embryo implantation, cervical mucus scaffolds can affect the binding of blastocysts which are rich in L-selectins on the surface. So, mucus-L-selectin binding can affect implantationof embryo to the uterine wall. Drug delivery and applications in regenerative medicine can therefore be based on layer by layer formation of scaffolds (Crouzier, Beckwitt, & Ribbeck, 2012).

References

Albone, E., Hagen, F., VanWuyckhuyse, B., & Tabak, L. (1994). Molecular cloning of a rat submandibular gland apomucin. The journal of biological chemistry, 269: 16845-16852.

Angelis, N., Porpodis, K., Zarogoulidis, P., Spyratos, D., Kioumis, I., Papaiwannou, A., & … Zarogoulidis, K. (2014). Airway inflammation in chronic obstructive pulmonary disease. . Journal of Thoracic Disease, 6(Suppl 1), S167–S172. https://doi.org/10.3978/j.

Authimoolam, S., & Dziubla, T. (2016). Biopolymeric Mucin and Synthetic PolymerAnalogs: Their Structure, Function andRole in Biomedical Applications. Polymers, 8(d71):oi:10.3390/polym8030071.

Bafna, S., Kaur, S., & Batra, S. (2010). Membrane-bound mucins: the mechanistic basis for alterations in the growth and survival of cancer cells. . Oncogene, 29(20), 2893–2904. https://doi.org/10.1038/onc.2010.87.

Becher, N., Waldorf, K., Merete, H., & Uldbjerg, N. (2009). The cervical mucus plug: Structured review of the literature. Acta Obstetricia et Gynecologica Scandinavica, 88(5), 502-513.

Buisine, M., Desreumaux, P., Leteurtre, E., Copin, M., Colombel, J., Porchet, N., & Aubert, J. (2001). Mucin gene expression in intestinal epithelial cells in Crohn's disease. Gut, 49(4):544-51.

Carraway, K., Ramsauer, V., Haq, C., & Carraway, C. (2003). Cell signaling through membrane mucins. Bioessays, 25(1):66-71.

Chaturvedi, P., Singh, A. P., & Batra, S. K. (2008). Structure, evolution, and biology of the MUC4 mucin. The FASEB Journal?: Official Publication of the Federation of American Societies for Experimental Biology, 22(4), 966–981. https://doi.org/10.1096/fj.

Chen, Y., Thai, P., Zhao, Y., Ho, Y., DeSouza, M., & Wu, R. (2003). Stimulation of Airway Mucin Gene Expression by Interleukin (IL)-17 through IL-6 Paracrine/Autocrine Loop*. Journal of biological chemistry, 278: 17036-17043.

Clare, A. (1995). Marine natural product antifoulants: Status and potential. Biofouling, 9(3): 211-229.

Corfield, A. (2014). Mucins; A Biologically Relevant Glycan Barrier in Mucosal Protection.. Biochimica et biophysica acta., 1850.10.1016/j.bbagen.2014.05.003. .

Crouzier, T., Beckwitt, C., & Ribbeck, K. (2012). Mucin multilayers assembled through sugar–lectin interactions. . Biomacromolecules, 13: 3401–3408.

Denny, M. (1989). Invertebrate mucous secretions: functional alternatives to vertebrate paradigms. Symposia of the society for experimental biology, 43:337-66.

Fong, A., Erickson, H., Zachariah, J., Poon, S., Schamberg, N., Imai, T., & Patel, D. (2000). Ultrastructure and Function of the Fractalkine Mucin Domain in CX3C Chemokine Domain Presentation. Journal of biological chemistry, 275, 3781-3786.

Frazão, B., Vasconcelos, V., & Antunes, A. (2012). Sea Anemone (Cnidaria, Anthozoa, Actiniaria) Toxins: An Overview. Marine Drugs, 10(8), 1812–1851. https://doi.org/10.3390/md10081812.

Gipson, I., Spurr-Michaud, S., Tisdale, A., & Menon, B. (2014). Comparison of the Transmembrane Mucins MUC1 and MUC16 in Epithelial Barrier Function. PLoS ONE , 9(6): e100393. https://doi.org/10.1371/journal.pone.0100393.

Goffredo, S., & Dubinsky, Z. (2016). The Cnidaria, Past, Present and Future. Springer.

Gosalia, N., Leir, S.-H., & Harris, A. (2013). Coordinate Regulation of the Gel-forming Mucin Genes at Chromosome 11p15.5. . The Journal of Biological Chemistry, 288(9), 6717–6725. https://doi.org/10.1074/jbc.M112.437400.

Govindarajan, B., & Gipson, I. K. (2010). Membrane-tethered mucins have multiple functions on the ocular surface. . Experimental Eye Research, 90(6), 655–663. https://doi.org/10.1016/j.exer.2010.02.014.

Hanisch, F. (2001). O-glycosylation of the mucin type. Biological chemistry, 382(2):143-9.

Harris, V. (1990). Sessile Animals of the Sea Shore. Springer Science & Business Media.

Ho, S., Takamura, K., Anway, R., Shekels, L., Toribara, N., & Ota, H. (2004). The adherent gastric mucous layer is composed of alternating l ayers of MUC5AC and MUC6 mucin proteins. Digestive diseases and sciences, 49:1598–1606.

Holgate, S. (2007). Epithelium dysfunction in asthma. The journal of allergy and clinical immunology, 120(6):1233–1244.

Khatri, I., Ho, C., Specian, R., & Forstner, J. (2001). Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis. American journal of physiology, gastrointesinal and liver physiology, 280(6):G1321-30.

Lang, T., Alexandersson, M., Hansson, G., & Samuelsson, T. (2004). Bioinformatic identification of polymerizing and transmembrane mucins in the puffer fish Fugu rubripes. Glycobiology.

Lang, T., Klasson, S., Larsson, E., Johansson, M., Hansson, G., & Samuelsson, T. (2016). Searching the Evolutionary Origin of Epithelial Mucus Protein. Mollecular Biology and Evolution, 33(8):1921–1936.

Morgado, M., Sutton, M., Simmons, M., Warren, C., & Lu, Z. (2016). Tumor necrosis factor-α and interferon-γ stimulate MUC16. Oncotarget, 7(12):14871-14884. .

Otero Gonzalez, A., Magalhaes, B., Garcia Villarino, M., Lopez Abarrategui, C., Sousa, D., Dias, S., & Franco, O. (2010). Antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control. . FASEB J, 24:1320–1334. .

Pelaseyed, T., Bergström, J. H., Gustafsson, J. K., Ermund, A., Birchenough, G. M., Schütte, A., & … Hansson, G. C. (2014). The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact. Immunilogical reviews, 260(1): 8–20.

Pelaseyed, T., Zäch, M., Petersson, A., Svensson, F., Johansson, D., & Hansson, G. (2013). Unfolding dynamics of the mucin SEA domain probed by force spectroscopy suggest that it acts as a cell-protective device. FEBS J. , 280(6):1491-501.

Perez-Vilar, J., & Hill, R. (1999). The Structure and Assembly of Secreted Mucins. Journal of Biological Chemistry, 274: 31751-31754.

Rachagani, S., Torres, M. P., Moniaux, N., & Batra, S. K. (2009). Current status of mucins in the diagnosis and therapy of cancer. . BioFactors (Oxford, England),, 35(6), 509–527. https://doi.org/10.1002/biof.64.

Rose, M., & Voynow, J. (2006). Respiratory Tract Mucin Genes and Mucin Glycoproteins in Health and Disease. Physiological Reviews Published 1 January 2006 Vol. 86 no. 1, 245-278, 86(1):245-278.

Schuijs, M., Willart, M., Hammad, H., & Lambrecht, B. (2013). Cytokine targets in airway inflammation. Current opinions in pharmacology, 13(3):351-61. doi: 10.1016/j.coph.2013.03.013. Epub 2013 May 2.

Smorodinsky, N., Weiss, M., Hartmann, M., Baruch, A., Harness, E., Yaakobovitz, M., . . . Wreschner, D. (1996). Detection of a secreted MUC1/SEC protein by MUC1 isoform specific monoclonal antibodies. Boichemical and biophysical research communications, 228(1):115-21.

Stabili, L., Schirosi, R., Parisi, M. G., Piraino, S., & Cammarata, M. (2015). The Mucus of Actinia equina (Anthozoa, Cnidaria): An Unexplored Resource for Potential Applicative Purposes. Marine Drugs, , 13(8), 5276–5296. https://doi.org/10.3390/md13085276.

Thai, P., Loukoianov, A., Wachi, S., & Wu, R. (2008). Regulation of Airway Mucin Gene Expression. Annual Review of Physiology, 70, 405–429. https://doi.org/10.1146/annurev.physiol.70.113006.100441.

Thornton, D., Howard, M., Khan, N., & Sheehan, J. (1997). Identi?cation of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a1097cysteine-rich sequence repeated within the molecule, J. Biol. Chem. 272. Journal of Biological chemistry, 272:9561-9566.

Van der Burg, C. A., Prentis, P. J., Surm, J. M., & Pavasovic, A. (2016). Insights into the innate immunome of actiniarians using a comparative genomic approach. . BMC Genomics , 17, 850. https://doi.org/10.1186/s12864-016-3204-2.

Van Putten, J. P., & Strijbis, K. (2017). Transmembrane Mucins: Signaling Receptors at the Intersection of Inflammation and Cancer. Journal of Innate Immunity, 9(3), 281–299. https://doi.org/10.1159/000453594.

Zaretsky, J., & Wreschner, D. (2013). Mucins – Potential Regulators of Cell Functions Volume Title: Gel-Forming and Soluble Mucins. Bentham books.