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Neuraminidase 1–Mediated Desialylation of the Mucin 1

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Neuraminidase 1–Mediated Desialylation of the Mucin 1 ARTICLE cro Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection Received for publication, September 26, 2018, and in revised form, November 13, 2018 Published, Papers in Press, November 14, 2018, DOI 10.1074/jbc.RA118.006022 Erik P. Lillehoj‡1, Wei Guang‡, Sang W. Hyun§¶, Anguo Liu§¶, Nicolas Hegerle§ʈ, Raphael Simon§ʈ, Alan S. Cross§ʈ, Hideharu Ishida**, Irina G. Luzina§¶, Sergei P. Atamas§¶, and Simeon E. Goldblum§¶‡‡ From the Departments of ‡Pediatrics, §Medicine, and ‡‡Pathology and ʈInstitute for Global Health, University of Maryland School of Medicine, Baltimore, Maryland 20201, ¶U.S. Department of Veterans Affairs, Veterans Affairs Medical Center, Baltimore, Maryland 20201, and **Department of Applied Bio-organic Chemistry, Gifu University, Gifu 501-1193 Japan Edited by Gerald W. Hart Downloaded from Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, Epithelial cells (ECs)2 lining the airway express multiple that engages the mucin 1 (MUC1) ectodomain (ED) expressed receptors that recognize and respond to exogenous danger sig- on airway epithelia, increasing association of MUC1-ED with nals, including bacterial pathogens (1). Host-pathogen interac- neuraminidase 1 (NEU1) and MUC1-ED desialylation. The tions include engagement of microbial adhesins with their cog- MUC1-ED desialylation unmasks both cryptic binding sites nate receptors expressed on the EC surface (2). Such bacterial http://www.jbc.org/ for Pa and a protease recognition site, permitting its proteo- adhesion to ECs is often a prerequisite to establishment of inva- lytic release as a hyperadhesive decoy receptor for Pa. We sive infection (3). Pseudomonas aeruginosa (Pa) is a Gram-neg- found here that intranasal administration of Pa strain K ative, flagellated, opportunistic human pathogen that colonizes (PAK) to BALB/c mice increases MUC1-ED shedding into the and/or infects debilitated and immunocompromised patients bronchoalveolar compartment. MUC1-ED levels increased as (4). One Pa adhesin, flagellin, the structural protein of the at University of Wisconsin-Madison on February 18, 2019 early as 12 h, peaked at 24–48 h with a 7.8-fold increase, and flagellar filament (5), contributes to its virulence through decreased by 72 h. The a-type flagellin–expressing PAK increased motility, adhesion, and invasion (3), and inflamma- strain and the b-type flagellin–expressing PAO1 strain stim- tion through activation of Toll-like receptor (TLR) 5 (6). Pa ulated comparable levels of MUC1-ED shedding. A flagellin- flagellin also engages the membrane-tethered mucin, MUC1 deficient PAK mutant provoked dramatically reduced MUC1-ED (7), and this receptor-ligand interaction is coupled to intracel- shedding compared with the WT strain, and purified flagellin lular signaling (8). recapitulated the WT effect. In lung tissues, Pa increased associa- MUC1 is a noncovalently associated complex comprised of tion of NEU1 and protective protein/cathepsin A with MUC1-ED an NH2-terminal, heavily sialylated MUC1-ED coupled to a in reciprocal co-immunoprecipitation assays and stimulated C-terminal MUC1 cytoplasmic domain (MUC1-CD) (9, 10). MUC1-ED desialylation. NEU1-selective sialidase inhibition pro- The MUC1-ED contains a variable number of 20–amino acid tected against Pa-induced MUC1-ED desialylation and shedding. (aa) tandem repeats and a Gly-Ser protease recognition site 58 In Pa-challenged mice, MUC1-ED–enriched bronchoalveolar aa upstream of the transmembrane domain, within the proxi- lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to mal, extracellular, juxtamembranous region. The MUC1-ED human airway epithelia by up to 44% and flagellin-driven motil- exhibits a ␤-turn helix resulting from its high proline content ity by >30%. Finally, Pa co-administration with recombinant (11). This extended, rodlike conformation allows the MUC1-ED human MUC1-ED dramatically diminished lung and BALF bac- to protrude higher above the EC surface than other membrane- terial burden, proinflammatory cytokine levels, and pulmonary associated proteins, where it is strategically positioned to inter- leukostasis and increased 5-day survival from 0% to 75%. We act with flagellin-expressing Pa in the airway lumen. The conclude that Pa flagellin provokes NEU1-mediated airway MUC1-ED is up to 90% carbohydrate, almost entirely through shedding of MUC1-ED, which functions as a decoy receptor pro- Ͼ500 predicted O-linked glycosylation sites within its tandem tecting against lethal Pa lung infection. repeats (12). Approximately 80% of these O-glycan chains ter- 2 The abbreviations used are: EC, epithelial cell; 4-MU-NANA, 2Ј-(4-methylum- This work was supported by Maryland Technology Development Corpora- belliferyl)-␣-D-N-acetylneuraminic acid; C9-BA-DANA, C9-butyl-amide-2- tion Grant 0117-005 (to E. P. L.), U.S. Department of Veterans Affairs Grant deoxy-2,3-dehydro-N-acetylneuraminic acid; aa, amino acid; Ab, antibody; I01BX002352 (to S. E. G.), and National Institutes of Health Grants Ad, adenovirus; BALF, bronchoalveolar lavage fluid; BW, body weight; CD, AI-110627 (to R. S.), HL-086933 (to A. S. C.), and HL-126897 (to S. P. A.). The cytoplasmic domain; CFUs, colony forming units; DANA, 2-deoxy-2,3-de- authors declare that they have no conflicts of interest with the contents of hydro-N-acetylneuraminic acid; ED, ectodomain; EGFR, epidermal growth this article. The content is solely the responsibility of the authors and does factor receptor; Gal, galactose; i.n., intranasal; i.t., intratracheal; KC, kerati- not necessarily represent the official views of the National Institutes of nocyte chemoattractant; LB, Luria-Bertani; LPS, lipopolysaccharide; m.o.i., Health. multiplicity of infection; ODN, oligodeoxynucleotide; Pa, Pseudomonas This article contains Figs. S1–S4 and Table S1. aeruginosa; PAK, Pa strain K; PBS-T, PBS containing 0.05% Tween 20; PNA, 1 To whom correspondence should be addressed. Tel.: 410-706-3872; Fax: peanut agglutinin; qRT-PCR, quantitative RT-PCR; STm, Salmonella enterica 410-706-0020; E-mail: [email protected]. serovar Typhimurium; TNF, tumor necrosis factor. 662 J. Biol. Chem. (2019) 294(2) 662–678 Published in the U.S.A. MUC1-ED is a shed decoy receptor in vivo minate with sialic acid residues (13). There also are five pre- chyma compared with lung sections from the simultaneous dicted N-linked glycosylation sites within the juxtamembra- PBS controls (Fig. S1D). The 5-day survival of mice challenged nous region (9, 10). Pulse-chase studies have established that with 1.0 ϫ 105 CFUs/mouse was 100%, whereas for those chal- MUC1 is initially expressed on the cell surface as an incom- lenged with 2.0 ϫ 107 CFUs/mouse it was 0% (Fig. S1E). This in pletely sialylated glycoform that is subsequently sialylated vivo model of acute Pa lung infection and the airway responses through several rounds of internalization and recycling back to to Pa were used to study MUC1-ED shedding as a Pa-targeting, the surface (14). The relatively short, 72-aa MUC1 cytoplasmic host defense mechanism. domain contains binding sites for multiple cytosolic signaling molecules, including c-Src (15), epidermal growth factor recep- Flagellin-expressing Pa increases MUC1-ED shedding within tor (EGFR) (16), and ␥-catenin (17). murine airways Neuraminidase (NEU)1 has historically been studied in the The MUC1-ED is reportedly shed from the surface of context of its indispensable role in glycan catabolism (18). We extrapulmonary epithelia (21–24). In cultured human gastric previously established NEU1 as the predominant sialidase ECs, Helicobacter pylori–induced MUC1-ED shedding consti- expressed in human airway ECs, for which MUC1 is a NEU1 tutes an important component of the host response to limit substrate (19, 20). Moreover, in these same cells, Pa-expressed bacterial infection (24). We asked whether the MUC1-ED Downloaded from flagellin engages the MUC1-ED to rapidly increase its associa- might be shed from airway epithelia in vivo, and if so, whether tion with preformed pools of NEU1 and its intracellular chap- Pa might stimulate shedding. Mice were administered i.n. with erone and transport protein, protective protein/cathepsin A a WT PAK challenge of 1.0 ϫ 105 CFUs/mouse, an equivalent Ϫ (PPCA) (20). NEU1, in turn, desialylates the MUC1-ED, ren- challenge of the flagellin-deficient isogenic mutant, PAK/fliC , dering it hyperadhesive for Pa and its flagella. NEU1-mediated or an equivalent volume of the PBS vehicle alone. At increasing http://www.jbc.org/ desialylation of the MUC1-ED also unmasks the Gly-Ser prote- times postchallenge, BALF was collected and MUC1-ED quan- ase recognition site to permit its proteolytic release from the tified by enzyme-linked immunosorbent assay (ELISA) and EC surface into the airway lumen. The shed hyperadhesive normalized to total BALF protein. In the BALF of WT PAK- MUC1-ED disrupts adhesion of flagellin-expressing Pa to air- challenged mice, MUC1-ED levels increased as early as 12 h way ECs. In these same studies, NEU1 overexpression in TLR5- compared with the simultaneous controls, with further time- expressing cells failed to desialylate TLR5 or to increase its dependent increases through 24 h, after which it plateaued and at University of Wisconsin-Madison on February 18, 2019 adhesiveness for Pa. However, these studies were performed remained elevated through 48 h, and was decreasing at 72 h exclusively in cultured human airway ECs. Using an intact, (Fig. 1A). In BALF of mice challenged with the flagellin-defi- Ϫ physiologically relevant, in vivo murine model of Pa pneumo- cient PAK/fliC mutant, MUC1-ED levels modestly increased nia, we asked whether Pa-derived flagellin might stimulate at 24 h compared with the simultaneous controls, but were NEU1-dependent MUC1-ED desialylation to release a hyper- dramatically reduced at 24 h and 48 h postchallenge compared adhesive decoy receptor that provides a novel, protective host with WT PAK-challenged mice (Fig.
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