1.C.111 The RegIIIγ (RegIIIγ) Family The mammalian intestine is home to ~100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host-bacterial relationships are maintained without eliciting potentially harmful immune responses. Here, we show that RegIIIγ, a secreted antibacterial lectin, is essential for maintaining a ~50-micrometer zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host-bacterial segregation in RegIIIγ(-/-) mice was coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Together, our findings reveal that RegIIIγ is a fundamental immune mechanism that promotes host-bacterial mutualism by regulating the spatial relationships between microbiota and host. The mammalian intestine harbors complex societies of beneficial bacteria that are maintained in the lumen with minimal penetration of mucosal surfaces. Microbial colonization of germ-free mice triggers epithelial expression of RegIIIγ which binds intestinal bacteria but lacks the complement recruitment domains present in other microbe-binding mammalian C-type lectins. Cash et al. (2006b) showed that RegIIIγ and its human counterpart, HIP/PAP, are direct antimicrobial proteins that bind their bacterial targets via interactions with peptidoglycan carbohydrate. They proposed that these proteins represent an evolutionarily primitive form of lectin-mediated innate immunity, and that they reveal intestinal strategies for maintaining symbiotic host-microbial relationships. Electron microscopic studies suggested that the protein inserts into the membrane and forms multimeric pores (Cash et al., 2006b; Cash et al., 2006). The mammalian intestine is home to ~100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host-bacterial relationships are maintained without eliciting potentially harmful immune responses. Li et al. (2011) showed that RegIIIγ maintains a ~50-micrometer zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host-bacterial segregation in RegIIIγ(-/-) mice was coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Thus, RegIIIγ provides a fundamental immune mechanism that promotes host-bacterial mutualism by regulating the spatial relationships between microbiota and host. Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate. Mukherjee et al. (2014) elucidated the mechanistic basis for RegIII bactericidal activity and showed that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. They derived a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and showed that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Their findings identified C-type lectins as mediators of membrane attack in the mucosal immune system and provided insight into an antibacterial mechanism that promotes mutualism with the resident microbiota (Cash et al., 2006b). The mammalian intestine is home to ~100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host-bacterial relationships are maintained without eliciting potentially harmful immune responses. Li et al. (2011) showed that RegIIIγ maintains a ~50-micrometer zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host-bacterial segregation in RegIIIγ(-/-) mice was coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Thus, RegIIIγ provides a fundamental immune mechanism that promotes host-bacterial mutualism by regulating the spatial relationships between microbiota and host. Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate. Mukherjee et al. (2014) elucidated the mechanistic basis for RegIII bactericidal activity and showed that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. They derived a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and showed that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Their findings identified C-type lectins as mediators of membrane attack in the mucosal immune system and provided insight into an antibacterial mechanism that promotes mutualism with the resident microbiota (Mukherjee et al. 2014).
References:
Bashirova, A.A., T.B. Geijtenbeek, G.C. van Duijnhoven, S.J. van Vliet, J.B. Eilering, M.P. Martin, L. Wu, T.D. Martin, N. Viebig, P.A. Knolle, V.N. KewalRamani, Y. van Kooyk, and M. Carrington. (2001). A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J Exp Med 193: 671-678.
Cash, H.L., C.V. Whitham, and L.V. Hooper. (2006). Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli. Protein Expr Purif 48: 151-159.
Cash, H.L., C.V. Whitham, C.L. Behrendt, and L.V. Hooper. (2006). Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313: 1126-1130.
Chen, C.H., H. Floyd, N.E. Olson, D. Magaletti, C. Li, K. Draves, and E.A. Clark. (2006). Dendritic-cell-associated C-type lectin 2 (DCAL-2) alters dendritic-cell maturation and cytokine production. Blood 107: 1459-1467.
Jiménez-Fernández, M., H. de la Fuente, P. Martín, D. Cibrián, and F. Sánchez-Madrid. (2023). Unraveling CD69 signaling pathways, ligands and laterally associated molecules. EXCLI J 22: 334-351.
Kim, D.K., J. Kabat, F. Borrego, T.B. Sanni, C.H. You, and J.E. Coligan. (2004). Human NKG2F is expressed and can associate with DAP12. Mol Immunol 41: 53-62.
Marshall, A.S., J.A. Willment, H.H. Lin, D.L. Williams, S. Gordon, and G.D. Brown. (2004). Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes. J. Biol. Chem. 279: 14792-14802.
Mukherjee, S., H. Zheng, M.G. Derebe, K.M. Callenberg, C.L. Partch, D. Rollins, D.C. Propheter, J. Rizo, M. Grabe, Q.X. Jiang, and L.V. Hooper. (2014). Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature 505: 103-107.
Plougastel, B. and J. Trowsdale. (1997). Cloning of NKG2-F, a new member of the NKG2 family of human natural killer cell receptor genes. Eur J Immunol 27: 2835-2839.
Vitry, J., G. Paré, A. Murru, X. Charest-Morin, H. Maaroufi, K.R. McLeish, P.H. Naccache, and M.J. Fernandes. (2021). Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region. Int J Mol Sci 22:.
Xu, Y., D. Song, W. Wang, S. Li, T. Yue, T. Xia, and Y. Shi. (2023). Clec12a inhibits MSU-induced immune activation through lipid raft expulsion. Life Sci Alliance 6:.
Yamuç, E., N.&.#.2.1.4.;. Barışık, S. Şensu, F. Tarhan, and C.C. Barışık. (2022). Correlation of REG1A, Claudin 7 and Ki67 expressions with tumor recurrence and prognostic factors in superficial urothelial urinary bladder carcinomas. Indian J Pathol Microbiol 65: 355-361.
Zafirova, B., F.M. Wensveen, M. Gulin, and B. Polić. (2011). Regulation of immune cell function and differentiation by the NKG2D receptor. Cell Mol Life Sci 68: 3519-3529.
Zuo, J., C.R. Willcox, F. Mohammed, M. Davey, S. Hunter, K. Khan, A. Antoun, P. Katakia, J. Croudace, C. Inman, H. Parry, D. Briggs, R. Malladi, B.E. Willcox, and P. Moss. (2017). A disease-linked polymorphism inhibits NKG2D-mediated target cell killing by enhancing the stability of NKG2D ligand binding. Sci Signal 10:.
Examples:
TC#	Name	Organismal Type	Example
1.C.111.1.1	Regenerating islet-derived protein 3-γ precursor, RegIIIγ (174aas)	Animals	*RegIIIγ of Mus musculus* (O09049)**

1.C.111.1.10	C-type lectin 5 of 153 aas	Animals	Lectin of Azumapecten farreri

1.C.111.1.11	Lectin-like transmembrane protein of 273 aas and 1 N-terminal TMS		Lectin-like transmembrane protein of Mus musculus

1.C.111.1.12	Dendritic cell natural killer lectin group receptor 1, DNGR-1, of 238 aas and 1 N-terminal TMS. It is also called C-type lectin domain family 9 member A (Cle9a). It functions as an endocytic receptor on a small subset of myeloid cells specialized for the uptake and processing of material from dead cells (Sancho et al. 2009). It recognizes filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins, including spectrin, exposed when cell membranes are damaged. It mediates the cross-presentation of dead-cell associated antigens in a Syk-dependent manner (Sancho et al. 2009; ). DNGR-1 is a receptor expressed by certain dendritic cell (DC) subsets and by DC precursors in the mouse. It possesses a C-type lectin-like domain (CTLD) followed by a poorly characterized neck region coupled to a transmembrane region and short intracellular tail. The CTLD of DNGR-1 binds F-actin exposed by dead cell corpses and causes the receptor to signal and potentiate cross-presentation of dead cell-associated antigens by DCs.The neck region of DNGR-1 is an integral receptor component that senses receptor progression through the endocytic pathway and has evolved to maximize extraction of antigens from cell corpses (Hanč et al. 2016).		*DNGR-1 of Mus musculus* (Mouse)**

1.C.111.1.13	NKG2-D type II protein of 216 aas and 1 central TMS. Functions as an activating and costimulatory receptor involved in immunosurveillance upon binding to various cellular stress-inducible ligands displayed at the surface of autologous tumor cells and virus-infected cells. Provides both stimulatory and costimulatory innate immune responses on activated killer (NK) cells, leading to cytotoxic activity (Zafirova et al. 2011). It stimulates perforin-mediated elimination of ligand-expressing tumor cells; signaling involves calcium influx, culminating in the expression of TNF-alpha (Zuo et al. 2017).		NKG2-D of Homo sapiens

1.C.111.1.14	NK cell receptor F, NKG2-F or NLRC4, of 158 aas and 1 central TMS. It may play a role as a receptor for the recognition of MHC class I HLA-E molecules by NK cells (Plougastel and Trowsdale 1997). It can associate with DAP12 (Kim et al. 2004).		NKG2-F of Homo sapiens

1.C.111.1.15	C-type lectin domain family 4 member M, CLEC4M or CD209L, of 399 aas and 1 N-terminal TMS. It is a probable pathogen-recognition receptor involved in peripheral immune surveillance in the liver, and may mediate the endocytosis of pathogens which are subsequently degraded in lysosomal compartments. It is a receptor for ICAM3, probably by binding to mannose-like carbohydrates (Bashirova et al. 2001).		CD209L of Homo sapiens

1.C.111.1.16	C-type lectin domain family 12 protein A, CLEC12A CLL7, DCAL2, MICL, of 265 aas and 1 TMS near its N-terminus. It is a cell surface receptor that modulates signaling cascades and mediates tyrosine phosphorylation of target MAP kinases in granulocytes and monocytes (Marshall et al. 2004). It alters dendrieic cell maturation and cytokine production (Chen et al. 2006). It is also a myeloid inhibitory receptor that negatively regulates inflammation in autoimmune and autoinflammatory arthritis (Vitry et al. 2021). Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. The stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation (Vitry et al. 2021). Clec12a inhibits MSU-induced immune activation through lipid raft expulsion (Xu et al. 2023). The transmembrane domain of Clec12a disrupts monosodium uric acid (MSU)-induced lipid raft recruitment and thus attenuates downstream signals. Single amino acid mutagenesis study showed the critical role of phenylalanine in the transmembrane region for the interactions between C-type lectin receptors and lipid rafts, which is critical for the regulation of MSU-mediated lipid sorting and phagocyte activation. This indicates the molecular mechanisms of solid particle-induced immune activation (Xu et al. 2023).		CLEC12A of Homo sapiens

1.C.111.1.17	Lithostathine-1-alpha, REG1A of 166 aas and 1 N-terminal TMS, possibly with one additional TMS centrally located. It might act as an inhibitor of spontaneous calcium carbonate precipitation, and may be associated with neuronal sprouting in brain, and with brain and pancreas regeneration. REG1A, Claudin 7 and Ki67 expressions correlate with tumor recurrence and prognostic factors in superficial urothelial urinary bladder carcinomas (Yamuç et al. 2022).		REG1A of Homo sapiens

1.C.111.1.18	CD69 of 199 aas and 1 TMS at residues 40 - 60. It is involved in lymphocyte proliferation and functions as a signal transmitting receptor in lymphocytes, natural killer (NK) cells, and platelets. A review provides a perspective on the molecular pathways, ligands and cellular functions known to be regulated by CD69 (Jiménez-Fernández et al. 2023).		CD69 of Homo sapiens

1.C.111.1.2	Regenerating islet-derived protein 3α, RegIIIα or Reg3A. Also called Proliferation-induced Protein 34, PAP or HIP of 157 aas. It is a C-type intestinal lectin and forms hexameric pores in Gram-positive bacterial membranes. The 3-d x-ray structure is known (Mukherjee et al. 2014). Lipopolysaccharides inhibit pore formation, and hence, Gram-negative bacteria are usually not susceptible to its killing action (Mukherjee et al. 2014).	Animals	RegIIIα of Homo sapiens

1.C.111.1.3	C-type lectin of 160 aas	Animals	Lectin of Morelia spilota (Carpet python)

1.C.111.1.4	SnEchinoidin of 192 aas	Animals	SnEchinoidin of Mesocentrotus nudus

1.C.111.1.5	Type II antifreeze protein of 192 aas	Animals	Antifreeze protein of Lates calcarifer

1.C.111.1.6	C-type lectin 1 of 155 aas	Animals	C-type lectin of Perca flavescens

1.C.111.1.7	Lactose-binding lectin I-2 of 181 aas	Animals	Lectin I-2 of Ictalurus furcatus

1.C.111.1.8	C-type lectin precursor of 177 aas	Animals	C-type lectin of Lachesis muta

1.C.111.1.9	C-type lecting domain family #4 member D of 205 aas	Animals	Lectin of Chelonia mydas