Glycobiology and Extracellular Matrices
A previously uncharacterized O-glycopeptidase from Akkermansia muciniphila requires the Tn-antigen for cleavage of the peptide bondAkkermansia muciniphila is key member of the human gut microbiota that impacts many features of host health. A major characteristic of this bacterium is its interaction with host mucin, which is abundant in the gut environment, and its ability to metabolize mucin as a nutrient source. The machinery deployed by A. muciniphila to enable this interaction appears to be extensive and sophisticated, yet it is incompletely defined. The uncharacterized protein AMUC_1438 is encoded by a gene that was previously shown to be upregulated when the bacterium is grown on mucin.
Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinkingThe synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3.
Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferencesMucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation, and organ growth. O-Glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of GalNAc onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain two functional domains: a catalytic domain and a C-terminal ricin-like lectin domain comprised of three potential GalNAc recognition repeats termed α, β, and γ.
Tango1 coordinates the formation of endoplasmic reticulum/Golgi docking sites to mediate secretory granule formationRegulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Here, through high-resolution imaging of Tango1, COPII, Golgi, and secretory cargo (mucins) in Drosophila larval salivary glands, we found that Tango1 forms ring-like structures that mediate the formation of COPII rings rather than vesicles.
Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2–processing propertiesCalcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus.
The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretionMucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain.
Prospecting for microbial α-N-acetylgalactosaminidases yields a new class of GH31 O-glycanaseα-Linked GalNAc (α-GalNAc) is most notably found at the nonreducing terminus of the blood type–determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylgalactosaminidases are known. Here, to discover and characterize novel microbial enzymes that hydrolyze α-GalNAc, we screened small-insert libraries containing metagenomic DNA from the human gut microbiome.
Intestinal mucin activates human dendritic cells and IL-8 production in a glycan-specific mannerCross-talk between different components of the intestinal barrier and the immune system may be important in maintaining gut homeostasis. A crucial part of the gut barrier is the mucus layer, a cross-linked gel on top of the intestinal epithelium that consists predominantly of the mucin glycoprotein MUC2. However, whether the mucin layer actively regulates intestinal immune cell responses is not clear. Because recent evidence suggests that intestinal dendritic cells (DCs) may be regulated by the mucus layer, we purified intestinal mucin, incubated it with human DCs, and determined the functional effects.
Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramersMost MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1–D2 domains hooked into each other.
Loss of the mucosal barrier alters the progenitor cell niche via Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalingThe mucous barrier of our digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases such as Crohn’s disease, colitis, and colon cancer, but mechanistic insights into these processes and diseases are limited. We have previously shown that loss of a conserved O-glycosyltransferase (PGANT4) in Drosophila results in aberrant secretion of components of the peritrophic/mucous membrane in the larval digestive tract. Here, we show that loss of PGANT4 disrupts the mucosal barrier, resulting in epithelial expression of the IL-6–like cytokine Upd3, leading to activation of JAK/STAT signaling, differentiation of cells that form the progenitor cell niche, and abnormal proliferation of progenitor cells.
The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycansThe gut harbors many symbiotic, commensal, and pathogenic microbes that break down and metabolize host carbohydrates. Sialic acids are prominent outermost carbohydrates on host glycoproteins called mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota can promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, and Escherichia coli) that do not produce sialidases. O-Acetyl ester modifications of sialic acids help resist the action of many sialidases and are present at high levels in the mammalian colon.
N-Glycosylation affects the stability and barrier function of the MUC16 mucinTransmembrane mucins are highly O-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface.
Extrinsic Functions of Lectin Domains in O-N-Acetylgalactosamine Glycan BiosynthesisGlycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (β-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate.
The Reduction-insensitive Bonds of the MUC2 Mucin Are Isopeptide BondsThe main structural component of the mucus in the gastrointestinal tract is the MUC2 mucin. It forms large networks that in colon build the loose outer mucous layer that provides the habitat for the commensal flora and the inner mucous layer that protects the epithelial cells by being impenetrable to bacteria. The epithelial cells in mice lacking MUC2 are not adequately protected from bacteria, resulting in inflammation and the development of colon cancer as found in human ulcerative colitis. Correct processing of the MUC2 mucin is the basis for the building of these protective networks.
Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its MechanismBackground: The endo-α-d-N-acetylgalactosaminidase SpGH101 from Streptococcus pneumoniae hydrolyzes the O-linked T-antigen from proteins.Results: SpGH101 displays an unusual conformational change on substrate binding and a distinctive arrangement of its catalytic machinery.Conclusion: Substrate hydrolysis proceeds through a retaining mechanism with a proton shuttle.Significance: This is the first evidence of proton shuttle in a retaining glycoside hydrolase.
NEU1 Sialidase Regulates Membrane-tethered Mucin (MUC1) Ectodomain Adhesiveness for Pseudomonas aeruginosa and Decoy Receptor ReleaseBackgroundPseudomonas aeruginosa flagellin binds to the membrane-tethered mucin, MUC1. Results Flagellin drives NEU1 to desialylate MUC1, thereby increasing its adhesiveness for Pseudomonas aeruginosa and its shedding. ConclusionP. aeruginosa hijacks host NEU1 through its flagellin. SignificanceP. aeruginosa mobilizes NEU1 to enhance its pathogenicity, but the host retaliates by releasing MUC1 as a hyperadhesive decoy receptor.